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Mechanisms in protein O-glycan biosynthesis and clinical and molecular aspects of protein O-glycan biosynthesis defects: a review.

The human proteome, originating from expression of the protein-coding genes of the genome, comprises ~30 000 proteins (1), a surprisingly low number considering that the genome of the nematode Caenorhabditis elegans comprises 20 000 genes (2). However, a higher order of complexity of protein products in humans arises from pretranslational events, such as alternative splicing, and posttranslational modifications, such as phosphorylation and glycosylation. Glycosylation, the enzymatic addition of carbohydrates to proteins or lipids, is the most common and most complex form of posttranslational modification. This is illustrated by the estimation that 1% of human genes are required for this specific process (3). Furthermore, more than one half of all proteins are glycosylated, according to estimates based on the SwissProt database (4). In humans, protein-linked glycans can be divided into 3 categories: N-linked (linkage to the amide group of Asn), O-linked [linkage to the hydroxyl group of Ser, Thr, or hydroxylysine (hLys)[3]], and C-linked (linkage to a carboxyl group of Trp) (5).

Initially, the study of glycoproteins and their role in human congenital diseases focused on N-linked glycans. The diseases in this pathway have collectively been referred to as congenital disorders of glycosylation (CDG). N-Glycans share a common protein-glycan linkage and have a common biosynthetic pathway that diverges only in the late Golgi stage. Endoglycosidases are available that can cleave intact N-glycans from the protein backbone, making it relatively easy to study alterations of N-glycosylation in health and disease. In contrast, O-glycans are built on different protein glycan linkages and have extremely diverse structures; in addition, there is no endoglycosidase available for the release of intact O-glycans. However, methods for the chemical release of O-glycans have been developed and have enabled the generation of structural information for O-glycans, making it more feasible to study alterations in O-glycosylation in relation to health and disease. This review focuses on the biosynthesis of O-glycans and the human congenital disorders of O-glycosylation and their screening.

Structures of O-Linked Glycans

The O-glycosylation process produces an immense multiplicity of chemical structures. Each monosaccharide has 3 or 4 attachment sites for linkage of other sugar residues and can form a glycosidic linkage in an [alpha] or [beta] configuration, allowing glycan structures to form branches. Glycans therefore have a larger structural diversity in contrast to other cellular macromolecules such as proteins, DNA, and RNA, which form only linear chains. Theoretically, the 9 common monosaccharides found in humans could be assembled into more than 15 million possible tetrasaccharides, all of which would be considered relatively simple glycans (6).

The 7 different types of O-linked glycans found in humans are summarized in Table 1. O-Linked glycans are classified on the basis of the first sugar attached to a Ser, Thr, or hLys residue of a protein. The mucin-type O-glycan, with N-acetylgalactosamine (GalNAc) at the reducing end, is the most common form in humans. In total, 8 mucin-type core structures can be distinguished, depending on the second sugar and its sugar linkage, of which cores 1-6 and core 8 have been described in humans (summarized in Table 2) (7). In addition to the 7 core structures, the Tn (GalNAc[alpha]1-Ser/Thr) and sialyl Tn [NeuAc[alpha]2-6GalNAc[alpha]1-Ser/ Thr; where NeuAc is N-acetylneuraminic acid (sialic acid)] epitopes can be distinguished. The core structures can be further modified; for example, by the addition of an N-acetyllactosamine unit (Gal[beta]1-4GlcNAc; where GlcNAc is N-acetylglucosamine), also seen on N-glycans. The N-acetyllactosamine unit may be branched by a GlcNAc[beta]1-6 residue or form repeating N-acetyllactosamine units, called poly N-acetyllactosamine extensions. It can also attach to the blood group determinants (A, B, and H) and the type 2 Lewis determinants [[Le.sup.x], sialyl [Lewis.sup.x] ([sLe.sup.x]), and [Le.sup.y]]. N-Acetyllactosamine elongations are seen mainly on core 2 O-glycans. Sugars occurring at the nonreducing termini include NeuAc, Fuc, GlcNAc, and GalNAc. GlcNAc and Gal residues can be modified at position 6 or at positions 3 and/or 6, respectively, by sulfation (8), and NeuAc residues can be further modified at positions 4, 7, 8, and 9 with O-acetyl ester groups (9). This gives rise to several hundreds of different mucin-type O-glycan structures, of which core 1 and 2 are most abundant (7).

Another common type of O-glycosylation with large structural diversity involves the glycosaminoglycans (GAGs). Proteoglycans are proteins containing GAG chains. GAGs are attached to a Ser residue of a protein via the linker tetrasaccharide GlcA[beta]1-3Gal[beta]1-3Gal[beta]1-4Xyl, except for keratan sulfate, which is linked to proteins either through N- or core 1 O-glycans. GAGs are long, unbranched polysaccharides containing a disaccharide repeat that consists of either a GalNAc or GlcNAc residue combined with a glucuronic acid (GlcA) or a Gal residue. Three different types of GAGs can be distinguished on the basis of the composition of the disaccharide repeat: (a) dermatan sulfate and chondroitin sulfate (GlcA + GalNAc); (b) heparin/heparan sulfate (GlcA + GlcNAc); and (c) keratan sulfate (Gal + GlcNAc). GlcA in dermatan sulfate and heparin/heparan sulfate can be epimerized to iduronate. The heterogeneity of GAGs results from variable O-sulfation at defined locations (10). An extra modification step occurs in heparin and heparan sulfate by the deacetylation and N-sulfation of GlcNAc residues. Regions in which the hexosamine units are acetylated remain (almost) unmodified and consist of disaccharide repeats with GlcA, whereas regions with deacetylated hexosamine units become highly sulfated and exist as disaccharide repeats with iduronate. Heparin is a highly and uniformly sulfated GAG, whereas heparan sulfate is highly sulfated only in defined blocks (11).

The structures of the other 5 O-glycan types seem to show less variability, and they occur mostly in one conformation. A frequently occurring O-linked glycan is the single GlcNAc linked to nuclear and cytosolic proteins. This posttranslational modification is more analogous to phosphorylation than to classical complex O-glycosylation because it is a reversible process catalyzed by the enzymes O-GlcNAc transferase and O-GlcNAcase, respectively (12), and the "normal glycosylation machinery" is not implicated (12, 13).

O-Galactosyl glycans have been found only on collagen domains. Gal or Glc[alpha]1-2Gal residues are covalently linked to hLys residues found in collagens, but not all hLys residues become glycosylated. The collagen 3-dimensional structure depends on the extent of this posttranslational modification. The quantities and types of O-galactosyl glycans vary considerably not only among the different types of collagen, but also among the same collagen type from different tissues and even the same collagen type from different areas of the same type of tissue (14, 15).

O-Mannosyl glycans are a less common type of protein modification, present on a limited number of glycoproteins in the brain, nerves, and skeletal muscle. The best known O-mannosylglycosylated protein is [alpha]-dystroglycan, which is a skeletal muscle extracellular matrix protein (16). To date, only the NeuAc[alpha]2-3Gal[beta]1-4GlcNAc[beta]1-2Man structure has been found in humans. [alpha]-Dystroglycan containing Gal[beta]1-4(Fuc[alpha]1-3)GlcNAc[beta]1-2Man has been found in sheep brain (17, 18), and the O-mannosyl glycan HSO3-3GlcA[beta]1-3Gal[beta]1-4GlcNAc[beta]1-2Man has been detected in rat brain (18, 19). Studies have also shown that mammalian N-acetylglucosaminyltransferase IX acts on the GlcNAc[beta]1,2-Man[alpha]1-Ser/Thr moiety, suggesting that 2,6-branched O-mannosyl glycan structures are formed in the brain (20). It is therefore likely that structural diversity of O-mannosyl glycans will also be present in humans.

O-Glucosyl and O-fucosyl glycans are also rare types of protein glycosylations that have been found in the epidermal growth factor homology regions (EGF modules) of some human proteins. An EGF module is a common structural motif found in several secreted and cell-surface proteins that is often involved in mediating protein-protein interactions. The EGF repeat is typically 30-40 amino acids long and is characterized by 6 conserved Cys residues participating in 3 disulfide bridges. Glc is linked to the Ser residue in proteins in the putative consensus sequence [C.sup.1]XSXP[C.sup.2] (where [C.sup.1] and [C.sup.2] are the first and second conserved cysteines of the EGF module, S is the modified Ser residue, and X can be any amino acid) (21). O-Linked Glc can be further elongated with 1 or 2 [alpha]1-3 linked xyloses and is found on proteins such as human factor VII, factor IX, and protein Z (22, 23). All O-fucosylated glycoproteins are modified with a single O-linked Fuc residue (e.g., urinary-type plasminogen activator, tissue-type plasminogen activator, and coagulation factors VII and XII) except for coagulation factor IX, which contains O-linked Fuc that is elongated to the tetrasaccharide NeuAc[alpha]2-6Gal[beta]1-4GlcNAc[beta]1-3Fuc[alpha]1-Ser/ Thr. Most O-Fuc modifications on EGF repeats are found on the consensus site [C.sup.2][X.sub.3-5]S/T[C.sup.3] (where [C.sup.2] and C3 are the second and third conserved cysteines of the EGF repeat, S/T is the modified Ser/Thr residue, and X can be any residue) (22). A second type of O-fucosylation has been identified. On thrombospondin type 1 repeats (TSRs), a disaccharide form of O-fucosyl glycans (Glc[beta]1-3Fuc[alpha] 1-Ser/Thr) is found on the human extracellular matrix protein "thrombospondin-1" (24). TSRs are found in many extracellular proteins. A single TSR is ~60 amino acids long and is characterized by conserved Cys, Trp, Ser, and Arg residues. The putative consensus sequence site for this modification is W[X.sub.5]C[X.sub.2/3]S/TC[X.sub.2]G (22).


For most O-glycosylation types, a recognition consensus sequence for the attachment of the first sugar residue remains unknown. The exceptions are the O-Glc and O-Fuc modifications, for which putative consensus sites have been described [see above and Refs. (21, 22)]. The lack of a consensus sequence can arise from the coexistence of multiple transferases with overlapping but different substrate specificities, as seen, e.g., in mucin-type O-glycosylation, or is the result of a nonlimited consensus sequence, as seen, e.g., in O-GlcNAc modifications. Statistical studies yielded some general rules for mucin-type O-glycans and O-GlcNAc modifications, leading to the development of algorithms for the prediction of these 2 O-glycan types. These O-glycosylation prediction sites are available on the Internet. The NetOglyc 3.1 prediction server correctly predicts 76% of the glycosylated residues and 93% of the nonglycosylated residues in any protein (25).

Biosynthesis of O-Glycans

The main pathway for the biosynthesis of complex N- and O-linked glycans is located in the endoplasmic reticulum (ER) and Golgi compartments, the so-called secretory pathway. Glycosylation is restricted mainly to proteins that are synthesized and sorted in this secretory pathway, which includes ER, Golgi, lysosomal, plasma membrane, and secretory proteins. There is one exception; nuclear and cytosolic proteins can be modified with a single O-linked GlcNAc (12). Proteins synthesized by ribosomes and sorted in the secretory pathway are directed to the rough ER by an ER signal sequence in the N[H.sub.2] terminus (26, 27). After protein folding is completed in the ER, these proteins move via transport vesicles to the Golgi complex. The biosynthesis of O-glycans is initiated after the folding and oligomerization of proteins either in the late ER or in one of the Golgi compartments (28-31). Intriguingly, for the biosynthesis of glycans, no template is involved; whereas DNA forms the template for the sequence of amino acids in a protein, there is no such equivalent for the design of glycans. The biosynthesis of glycans can be divided into 3 stages. In the first stage, nucleotide sugars are synthesized in the cytoplasm. In the second stage, these nucleotide sugars are transported into the ER or the Golgi. In the third stage, specific glycosyltransferases attach the sugars to a protein or to a glycan in the ER or Golgi. An additional prerequisite for proper glycosylation is Golgi trafficking. Recently, it was discovered that a defect in a protein involved in Golgi traffic secondarily caused abnormal N- and O-glycans in 2 patients with CDG-IIe. For this reason, Golgi traffic will be discussed briefly in this section.


Monosaccharides used for the biosynthesis of nucleotide sugars derive from dietary sources and salvage pathways. Glucose (Glc) and fructose (Fru) are the major carbon sources in humans from which all other monosaccharides can be synthesized (Fig. 1). Series of phosphorylation, epimerization, and acetylation reactions convert them into various high-energy nucleotide sugar donors (see Fig. 1). Nucleotide sugar biosynthesis takes place in the cytosol, except for CMP-NeuAc, which is synthesized in the nucleus (32).


As observed in patients with CDG-Ia and CDG-Ib, aberrant glycosylation results from insufficient availability of GDP-Man. The availability of nucleotide sugars is tightly regulated. UDP-GlcNAc, for example, inhibits glutamine-fructose-6P-transaminase, which catalyzes the first step in the biosynthetic pathway of UDP-GlcNAc (33), and CMP-NeuAc inhibits UDP-GlcNAc-2-epimerase/ N-acetylmannosamine kinase (GNE/MNK), which catalyzes the first 2 biosynthetic steps of CMP-NeuAc (34). Although much is known about the nucleotide sugar biosynthesis pathways and their feedback regulators, the actual cytosol and Golgi steady-state concentrations of most nucleotide sugars are unknown at present. Furthermore, because of the interconnected pathways of nucleotide sugar metabolism, the results of an individual enzyme deficiency are difficult to predict.

Several steps in the biosynthesis of nucleotide sugars require ATP; therefore, the metabolic state of the cell influences the availability of the nucleotide sugars. The tight regulation of the biosynthesis of nucleotide sugars means that alterations in a single nucleotide sugar can significantly impair glycosylation.


The nucleotide sugars are biosynthesized in the cytosol, and their monosaccharides must be translocated into the lumen of the ER and/or Golgi before they can be used for the glycosylation process. Because nucleotide sugars cannot cross the membrane lipid bilayer, specific transport mechanisms are responsible for their translocation. Two transport mechanisms for the generation of monosaccharide donors in the ER/Golgi can be distinguished (Fig. 2). The first mechanism is the entrance of Man and Glc through binding to the lipid carrier dolichol phosphate (Dol-P). To date, this transport system has been described only in the ER. Cytosolic Dol-P-Man and Dol-P-Glc synthases link GDP-Man and UDP-Glc to the cytosolic site of Dol-P by cleaving off the nucleotide moiety. A hypothetical "flippase" then mediates the turnover of the Dol-P monosaccharide from the cytoplasmic leaflet to the lumenal leaflet of the ER. Subsequently, the monosaccharides can be used by ER-located glycosyltransferases (Fig. 2A) (35). As observed in patients with CDG-Ie who are deficient for Dol-P-Man synthase and in patients with CDG-If who have mutations in the MPDU1 gene, known to be required for efficient use of Dol-P-Man and Dol-PGlc as donor substrates, abnormal glycosylation results from diminished Dol-P-monosaccharide transport (36, 37). In CDG-If patients, it was observed that the mannosylation of N-glycans, glycosylphosphatidylinotisol anchors, and C-mannosyl glycans was defective. Although O-mannosylation was not studied, it is likely that this is also aberrant in these patients.

The second mechanism is the transport of nucleotide sugars through specific nucleotide sugar transporters (NSTs). NSTs belong to solute carrier family 35 and reside in the Golgi and/or ER membranes with their C- and N-terminal regions exposed to the cytosol. These NSTs are antiporters in which nucleotide sugar entry into the ER/Golgi is coupled to equimolar exit of the corresponding nucleoside monophosphate from the ER/Golgi lumen (38). The nucleotide moiety of the nucleotide sugar is the recognition feature required for initial binding to the NST, whereas the attached monosaccharide finally determines whether the entire nucleotide sugar is translocated. After entrance of the nucleotide sugar into the ER/Golgi lumen, a glycosyltransferase will transfer the monosaccharide to a glycan by cleaving off the nucleotide part. The nucleoside diphosphates are converted to dianionic nucleoside monophosphates (used for the antiporter) and inorganic phosphate by a nucleoside diphosphatase. It is postulated that inorganic phosphate exits the ER/Golgi lumen via a specific transporter (Fig. 2B) (38). Nucleoside di- and monophosphates can inhibit the nucleotide sugar transport process and the activity of glycosyltransferases.


Some NSTs transport more than one substrate: for example, the UDP-Gal/UDP-GalNAc transporter (hereafter referred to as UDP-Gal transporter) (39), the UDPGlcA/ UDP-GalNAc/UDP-GlcNAc transporter (hereafter referred to as UDP-GlcA transporter) (40, 41), and the recently described UDP-Xyl/UDP-GlcNAc transporter (hereafter referred to as UDP-Xyl transporter) (42). In contrast, the CMP-NeuAc (43), GDP-Fuc (44), and UDP-GlcNAc transporters (45) are monospecific.

In general, the transport of a nucleotide sugar occurs in the organelle in which the corresponding glycosyltransferase is localized. Some nucleotide sugars enter only the lumen of Golgi vesicles, others enter the lumen of ER-derived vesicles, and a few enter both. It has been shown that the CMP-NeuAc, GDP-Fuc, UDP-GlcNAc, and UDP-Xyl transporters have a strict Golgi membrane localization (38, 42, 45), whereas the UDP-GlcA transporter is localized in the ER membrane (40). Experiments investigating the intraorganelle availability of nucleotide sugars have shown that UDP-Xyl and UDP-Glc can also be found in the ER, whereas UDP-GlcA and UDP-Glc can be found in the Golgi (38), suggesting that the corresponding NSTs are yet to be identified.

Galactosylceramide is galactosylated by a galactosyltransferase (UDP-galactose:ceramide galactosyltransferase) found exclusively in the ER, whereas the UDP-Gal transporter has mainly a Golgi localization. This galactosyltransferase is produced only in specialized cells, such as myelinating cells, spermatogonia, and in various epithelial cell types. The question of how an ER-resident glycosyltransferase could function without a source of substrate was answered by showing that the galactosyltransferase forms a complex with the UDP-Gal transporter (46). This led to the presence of a fraction of the UDP-Gal transporter in the ER. It is not clear whether this is attributable to the retention of the UDP-Gal transporter by the galactosyltransferase or to recycling of the UDP-Gal transporter through the cis-Golgi. In this way, a biosynthetic pathway can be established only when required (46). Recently, a second active mechanism has been found for the ER localization of the UDP-Gal transporter. The UDP-Gal transporter is produced in 2 splice forms, UGT1 and UGT2. UGT1 has a strict Golgi localization, whereas UGT2 shows dual localization in both the ER and Golgi caused by a dilysine motif (KVKGS) in its COOH terminus (47).

As observed in the case of patients with CDG-IIc who have a deficient GDP-Fuc transporter (FUCT) (48) and in a patient with CDG-IIf who has a deficient CMP-NeuAc transporter (49), abnormal glycosylation results from diminished NST function. In addition, in Chinese hamster ovary (CHO) lec8 and lec2 cells defective in UDP-Gal and CMP-NeuAc transport, respectively, 70%-90% of the glycans lacked that particular monosaccharide (38). It was also shown that the nucleotide sugar transport process depends on the continuous production of nucleoside monophosphates. Abeijon et al. (50) showed that in vitro transport of GDP-Man into the Golgi is severely decreased in a Saccharomyces cerevisiae guanosine diphosphatase-null mutant. All glycoproteins and glycolipids showed impaired mannosylation (50). This results indicates that NSTs are critical components of glycosylation pathways.


O-Glycans are assembled by the sequential action of several specific, membrane-bound glycosyl-, O-acetyl-, and sulfotransferases in a highly controlled fashion (8). The pathways of O-glycosylation are determined by the distinct substrate specificities of glycosyltransferases, sulfotransferases, and O-acetyltransferases. Transferases involved in O-glycan biosynthesis are localized mainly in the Golgi. Although many of these enzymes catalyze similar reactions, there is a surprisingly limited sequence homology among different classes. The Golgi glycosyltransferases described to date are all type II transmembrane proteins, with a short N-terminal cytoplasmic domain, a single hydrophobic membrane-spanning domain, and a large C-terminal catalytic domain localized in the lumen of the Golgi.

The activity of glycosyltransferases can be influenced by different factors. It is known, for example, that some of the glycosyltransferases require divalent cations, such as [Mn.sup.2+] and/or [Mg.sup.2+], for optimal action. In contrast to the reactions involving UDP- and GDP-nucleotide sugars, the biosynthetic steps involving CMP-NeuAc do not require these cations (51). Petrova et al. (52) showed that divalent cations react strongly with nucleotide sugars in solution, thus altering their conformation.

Furthermore, it was recently discovered that human core 1 [beta]3-galactosyltransferase (core 1 [beta]3-Gal-T), which is involved in the formation of core 1 (and core 2) mucintype O-glycans, requires a molecular chaperone for its functioning. This molecular chaperone is called core 1 [beta]3-Gal-T-specific molecular chaperone (Cosmc) and is an ER-localized type II transmembrane protein that appears to be required for the proper folding of the core 1 [beta]3-Gal-T enzyme. In the absence of functional Cosmc, core 1 [beta]3-Gal-T is degraded in the proteosome (53). This raises the question of whether additional chaperones specific for other glycosyltransferases exist.

A third factor that might influence glycosyltransferase activity is the structure of the protein substrate. It is thought that the protein structure contains information for the action of specific transferases. This is seen, for example, in proteoglycans, in which the core protein dictates whether it will receive a heparan sulfate or a chondroitin sulfate chain (54), or in lysosomal enzymes, in which GlcNAc-phosphotransferase recognizes subtle motifs in the secondary structure and selectively phosphorylates the N-glycans on proteins that should reach the lysosome (55, 56). However, how proteins are recognized by glycosyltransferases remains largely unknown. Finally, glycosyltransferase activity can be dependent on heterocomplex formation. O-Mannosyltransferase activity, for example, is generated only when the genes POMT1 and POMT2 (both encoding mannosyltransferases) are coexpressed (57).

Golgi transferases can recognize a single sugar residue, a sugar sequence, or a peptide moiety, leading to variable specificity. With very few exceptions, each type of transferase is regio- and stereospecific. Glycosyltransferases involved in the linkage of monosaccharides to the protein backbone and those involved in the core processing of mucin-type O-glycans are specific and not involved in other classes of glycoconjugates, whereas most glycosyltransferases involved in the elongation, branching, and termination of glycans are not specific for one glycoconjugate class. For example, the ubiquitous [alpha]2,6-sialyltransferase ST6Gal I recognizes the N-acetyllactosamine unit and catalyzes the formation of an [alpha]2,6 linkage to terminal N-acetyllactosamine structures found on N-glycans, O-glycans, and glycosphingolipids, whereas the [beta]1,4-galactosyltransferase (Gal-T1) galactosylates any terminal Glc- NAc residue.

The attachment of UDP-GalNAc in an [alpha] linkage to the hydroxyl residue of Ser or Thr in mucin-type O-glycans is a complex and as yet not fully understood process. This transfer is catalyzed by specific UDP-GalNAc: polypeptide N-acetylgalactosaminyltransferases (EC The mammalian family of UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferases (pp-GalNAc-Ts) comprises 15 members, the 15th being discovered only recently (58, 59). It is estimated that at least 24 unique human pp-GalNAc-Ts exist on the basis of sequence homology (59). The different pp-GalNAc-Ts have overlapping but different specificities and are tissue specific (8, 59). It seems that mucin-type O-glycosylation proceeds in a hierarchical manner, because some of the characterized pp-GalNAc-Ts glycosylate only peptides that are already partly glycosylated (59). Currently, no consensus sequence has been formulated because every pp-GalNAc-T has its own specific attachment site. Only Ser and Thr residues that are exposed on the protein surface will be glycosylated, as O-glycosylation is a postfolding event. Therefore, O-glycosylation takes place mainly in coil, turn, and linker regions. Furthermore, all attachment sites have high Ser, Thr, and Pro content (25).

The biosynthesis of GAG structures differs from the "small O-linked glycans" in 2 significant ways: (a) the transferases required are all specific and not involved in other glycoconjugate classes, with the exception of chondroitin 6-sulfotransferase, keratan sulfate Gal-6-sulfotransferase, and the GlcNAc 6-O-sulfotransferase that also sulfates N-acetyllactosamine extensions (60); and (b) the mechanism of GAG chain elongation is different. Chondroitin/ dermatan sulfate and heparin/heparan sulfate are synthesized on the common tetrasaccharide linker (GlcA[beta]1-3Gal[beta]1-3Gal[beta]1-4Xyl). Chondroitin/dermatan sulfate is synthesized when GalNAc is transferred to the linkage region, whereas heparin/heparan sulfate is synthesized if GlcNAc is added first. It has been demonstrated that the human exostoses-like family (EXTL1, -2, and -3) is responsible for the heparin/heparan sulfate chain initiation with the attachment of the first and second GlcNAc residues and that the exostoses enzymes extosin-1 and -2 are the copolymerases that elongate the GAG chain with [(GlcA[beta]1-4GlcNAc[alpha]1-4).sub.n] (10). Recently, chondroitin GalNAc transferases I and II and chondroitin synthetase were discovered (61-63). Chondroitin GalNAc transferases I and II are responsible for the initiation of the chondroitin/dermatan sulfate GAG chain with the attachment of the first few GalNAc residues to the linker region, whereas chondroitin synthetase acts as a copolymerase and is responsible for the elongation of chondroitin/ dermatan sulfate with [(GalNAc[beta]1-4GlcA[beta]1-3).sub.n].


The Golgi apparatus consists of several cisternae, starting from the nucleus with the cis-Golgi network, through the cis-, medial-, and trans-Golgi compartments, and ending with the trans-Golgi network, which are organized in the form of a stack. The Golgi position and organization within a cell are sustained largely through the combined efforts of a complex cytoskeletal matrix composed of microtubules, an actin-spectrin network, and intermediate filaments. The interaction between these filament systems and Golgi membranes is mediated by mechanochemical enzymes such as dyneins, kinesins, myosins, and dynamin and different structural proteins (64).


A schematic overview of the transport route from ER through the different Golgi compartments is shown in Fig. 3 [for reviews, see Refs. (65, 66)]. The journey of proteins between these compartments starts with the exit from 100-200 export sites on the ER in COPII-coated vesicles. Coatamer proteins (COPs) recognize transport signals present in the cytoplasmic tail of cargo membrane proteins for their incorporation in COPII vesicles. Three classes of ER export signals have been described to date. Most type I membrane proteins have a diacidic or dihydrophobic motif, and the type II glycosyltransferases have a [RK(X)RK] motif proximal to the transmembrane domain (67). Signals that direct soluble cargo into ER-derived vesicles are less well defined. It is thought that soluble proteins are exported from the ER in 2 ways: (a) through a passive bulk flow process; and (b) through an active receptor-mediated process relying on receptor-like proteins that attach proteins to the inner membrane of the coated vesicle (68).

Different COPII-coated vesicles fuse to form the ER-Golgi intermediate compartment (ERGIC). From here, escaped ER proteins or misfolded proteins are transported back to the ER via COPI-coated vesicles. The ERGIC elements are transported to and fused with the cis-Golgi network. From here, both anterograde and retrograde transport is mediated via COPI-coated vesicles. Three major protein families regulate vesicle transport. The ARF and Sar1 family GTPases are involved in COPI and COPII vesicle formation, which starts with the activation of ARF and Sar1 by a nucleotide exchange factor into ARF-GTP and Sar1-GTP. ARF-GTP and Sar1-GTP recruit many additional components for the synthesis of the vesicle coat. Subsequently, the Rab family GTPases mediate vesicle targeting. The mammalian Rab protein family includes at least 63 isoforms. All cytosolic Rab proteins form a complex with the Rab guanine nucleotide dissociation inhibitor chaperone, which transports the Rab proteins to the membrane of specific Golgi compartments, where they become activated to the GTP state. Activated Rabs mediate vesicle motility and the tethering of transport intermediates to their target membranes. The third family consists of SNARE proteins, which direct vesicle fusion. Each type of transport vesicle carries a specific vesicle-SNARE (v-SNARE), which binds to a tethering-SNARE (t-SNARE) on the target membrane, producing the trans-SNARE complex. After fusion, the cargo is transferred to that specific compartment (65, 66).

The Golgi is a very dynamic organelle; it has the capacity to transform in response to specific stimuli or cellular changes. For example, the Golgi or any Golgi-like structures fragment into numerous tubular and vesicular structures when cells undergo mitosis: the ER export sites disappear, the Golgi integral membrane proteins are trapped in the ER, and Golgi peripheral proteins are retargeted to the ER or cytoplasm. After mitosis is complete, the Golgi is readily re-formed by outgrowth from the ER and fusion of the tubular and vesicular structures (69).

Until recently, the Golgi was seen as a static organelle. In this model, Golgi enzymes are retained within one Golgi cisterna, and cargo (the proteins that are transported to and processed in the Golgi) are transported through the different Golgi compartments in the anterograde direction via COPI vesicles. However, at present, the cisternal maturation model is favored. It is now believed that cargo remains in one cisterna and that this Golgi compartment traffics in the anterograde direction, whereas the Golgi enzymes traffic backward by COPI vesicles. This model is based on the experimental observations that cargo indeed remains in a specific cisterna and that COPI vesicles are enriched with Golgi enzymes (70, 71).

Given the sequential and competing nature of glycosyltransferases, the precise localization of these enzymes within the Golgi is of great importance. It is thought that glycosyltransferases are arranged in an assembly line in the Golgi, whereas early-acting transferases are localized in the cis-Golgi, intermediate-acting transferases in the medial-Golgi, and terminating transferases in the trans-Golgi. A signal targeting glycosyltransferases to a specific Golgi localization has not yet been described. Studies have indicated that glycosyltransferases from a certain Golgi compartment form high-molecular-mass complexes (72). The presence of multienzyme complexes is likely to be functionally relevant in the regulation of glycosylation and contribute to the maintenance of the steady-state localization of the Golgi glycosyltransferases (72). When Nilsson and Warren (73) re-directed a Golgi resident glycosyltransferase to the ER, another Golgi enzyme also was retained in the ER. Not all glycosyltransferases form complexes; in particular, those found in the trans-Golgi network seem to be unbound. Another factor that is likely to play a role in the targeting of glycosyltransferases is the thickness of the lipid bilayer, which increases en route to the plasma membrane. The fact that Golgi proteins have shorter transmembrane domains than do plasma membrane proteins suggests that cisternae of a specific compartment can accommodate glycosyltransferases with a transmembrane domain of matching length. However, it has been shown that some soluble forms of glycosyltransferases, which have lost their transmembrane domain, are retained in the Golgi probably as a result of being associated in complexes (70, 71). It is likely that more independent signals act together to mediate efficient Golgi localization.


Recently, 2 patients were identified to have a defect in subunit 7 of the conserved oligomeric Golgi complex (COG7); the patients were classified as CDG type IIe (74). The mammalian COG complex contains 8 subunits, of which COG1 through -4 form lobe A and COG5 through -8 form lobe B with COG4 as the core component linking the 2 lobes (75).

Mutations in COG subunits (COG1 through -8) of CHO, yeast, and Drosophila melanogaster sperm cells have been shown to affect the structure and function of the Golgi, producing defects in glycoconjugate biosynthesis, intracellular protein sorting, protein secretion, and in some cases, cell growth. In the recessive COG1- and COG2-null CHO mutants, for example, the Golgi showed an abnormal morphology with dilated cisternae and pleiotropic defects in several medial- and trans-Golgi-associated reactions affecting N-linked, O-linked, and lipid-linked glycoconjugates (76). The COG complex thus seems to play a role in determining and maintaining Golgi structure and morphology. Furthermore, COG works in concert with COPI. Their function is to retrograde transport several Golgi resident proteins to the appropriate Golgi compartment where they reside. Evidence came from the work of Oka et al. (77), who investigated the consequences of the loss and overexpression of COG on a set of Golgi resident type II transmembrane proteins, including members of the SNARE, Rab, and golgin protein families. The expression and localization of some proteins were COG-dependent, whereas for others this was not the case. The COG-sensitive proteins are referred to as "GEARs".

Functions of O-Linked Glycans

Various functions have been described for O-linked glycans. Only the main roles are given here; for details, the reader is referred to other reviews (8, 78). In general, O-linked glycans have been found to function in protein structure and stability, immunity, receptor-mediated signaling, nonspecific protein interactions, modulation of the activity of enzymes and signaling molecules, and protein expression and processing. The biological roles of oligosaccharides appear to span the spectrum from those that are trivial to those that are crucial for the development, growth, function, or survival of an organism. A particular glycan may mediate diverse functions at distinct locations at specific times within a single organism (79).

Just like N-glycans, O-glycans can influence the secondary protein structure: the glycan can break the [alpha]-helicity of peptides (80); can have a role in the tertiary protein structure [seen, e.g., on the porcine filamentous-shaped submaxillary mucin, in which release of an O-glycan leads to a globular shape (81)]; and in the quaternary protein structure and protein aggregation [seen, e.g., in ovine submaxillary mucin, which only forms aggregates when it is O-glycosylated (82)]. Subsequently, O-linked glycans maintain protein stability, heat resistance, hydrophilicity, and protease resistance by steric hindrance (8).

Additionally, mucin-type O-glycans are important for the binding of water. Mucins are proteins that are heavily glycosylated with mucin-type O-glycans and are often present at outer surfaces lacking an impermeable layer, such as the surfaces of the digestive, genital, and respiratory system tracts. These mucins bear clusters of sialylated glycans, which produce regions with a strong negative charge. This gives mucins the capacity to bind large amounts of water and form mucus. The gels observed in nasal secretions, for example, are formed by secreted MUC2 polypeptides linked together to form long, cross-linked polymers holding water. The primary function of these viscous mucin solutions and gels is to form a protective coating with antibacterial properties (83). Like mucin-type O-glycans, GAGs bind large volumes of water via the strong negative charge of the sulfate groups, providing resilience or resistance to compression rather than lubrication or reduction of friction. GAGs are found in extracellular matrices. In structural tissues, such as the cartilage of joints, GAGs can act as shock breakers by the slow reduction of their water content under high pressure.

Another important function of O-linked sugars is to mediate recognition between proteins. Glycan structures can be substrates for nonenzymatic sugar-binding proteins, known as lectins. By interacting with lectins, glycans influence the targeting of the proteins to which they are attached. Examples of glycan-mediated recognition of glycoproteins are ubiquitous. For example, selectins and galectins, representing 2 classes of lectins located in the leukocyte-vascular system, bind to carbohydrate epitopes that induce cellular signaling, which in turn influences many crucial cellular processes, including cell growth, apoptosis, endocytosis, cell-cell interactions, cell-matrix interaction, matrix network assembly, and oocyte fertilization (84). Additionally, sialylated O-mannosyl glycan serves as binding ligand for laminin in the dystroglycan complex, which is important in muscle and brain development (18). Moreover, O-linked glycans are known to have an effect on immunologic recognition; for example, the ABO blood group antigens and recognition of glycopeptides by the MHC complex or by antibodies (85).

Subsequently, it is known that GAGs have a role in nonspecific protein interactions. Cell surface proteoglycans, for example, adhere to soluble polypeptide growth factors through electrostatic interactions mediated by their GAGs, preventing the growth factors from diffusing. GAG interactions increase and stabilize concentration gradients of growth factors (78).

The effects of O-linked glycosylation on the bioactivity of many signaling molecules, particularly hormones and cytokines, and a relatively small number of enzymes, have been described. In most cases, the influence is not very strong (a difference of 2- or 3-fold), but rather provides a fine regulation mechanism. Effects leading to both an increase and a decrease in biological activity have been described. For example, a mucin-type O-linked glycan decreases the biological activity of interleukin-5 (86), whereas it induces a higher enzymatic activity of human lactase phlorizin hydrolase (87). The influence of unusual carbohydrate modifications on the activity of signaling molecules appears often to be crucial and specific. For example, O-Fuc on urinary-type plasminogen activator was shown to be required for activation of its receptor, and the presence of O-fucosyl glycans seems to be required for proper Notch function (22). Another example is the dynamic O-GlcNAc modification that seems to have an important role in a variety of signaling pathways, such as transcriptional regulation, proteasome-mediated protein degradation, insulin, and cellular stress signaling. Recently, it was found that O-GlcNAc modulates the activity of critical intermediates involved in the regulation of neutrophil motility (88). Some very specific GAG structures are known to act as co-receptors, allowing activation of the primary receptor necessary for the activation of growth factors. The fibroblast growth factor, for example, must interact with the heparan sulfate chain of the proteoglycan syndecan to activate the primary fibroblast growth factor receptor (89).

Finally, O-linked glycosylation is essential for the expression and processing of particular proteins. Glycophorin A, for example, is a heavily glycosylated protein present on the surface of human erythrocytes. It has been shown that O-linked sugars are necessary for cell surface expression of this glycoprotein (90). The influence of O-glycans in the processing of proteins is, for example, seen in pro-insulin-like growth factor II, which is cleaved into IGF-II only when [Thr.sub.75] contains an O-linked sugar (91).

As O-glycans are involved in numerous processes, it is inevitable that defects in O-glycan biosynthesis might lead to severe abnormalities for cellular functioning.

Congenital Disorders in the Biosynthesis of O-Glycans in Humans

CDG form a group of autosomal recessive metabolic disorders caused by defects in the biosynthesis of protein-linked glycans. To date, mainly genetic defects in Nglycan biosynthesis have been classified as CDG. The division of CDG into types I and II is based on the location of the defect in the N-glycan biosynthetic pathway. CDG-I includes all defects in the early N-glycan pathway in the cytoplasm or the ER and covers all steps until the transfer of the glycan to the protein. CDG-II includes all defects localized in the processing of N-glycans on the glycosylated protein. These are situated mainly in the Golgi compartment. At present, most defects in the biosynthesis of protein O-linked glycans are not included in this CDG classification and still have "popular" names and/or "biochemical" names that are informative about the nature of the disease. Some O-glycosylation disorders affect only a particular O-glycan type, certain disorders affect more O-glycan types, and others also affect the biosynthesis of other glycoconjugates. It is becoming increasingly evident that the primary defect of these disorders is not necessarily localized in one of the glycan-specific transferases, but can likewise be found in the biosynthesis of nucleotide sugars, their transport to the ER/Golgi, and in Golgi trafficking. The clinical variations within a disorder and among the different inborn errors of O-glycan metabolism are enormous. Defects can lead to a severe autosomal recessive multisystem syndrome with neurologic involvement, whereas some defects, for example, those in persons with the Bombay blood group or the Lewis-null blood group, do not produce a clinical phenotype. As O-glycosylation biosynthesis is a very complex process with an enormous number of genes involved, it is obvious that the disorders described to date are just the tip of the iceberg. This novel area of inborn errors of metabolism still needs further exploration.

This section discusses the clinical, molecular genetic, laboratory, and biochemical aspects of the known congenital disorders in the biosynthesis of O-glycans. The human congenital disorders that affect the biosynthesis of protein O-linked glycans are summarized in Table 3A, whereas the human congenital disorders with a defect affecting the biosynthesis of both N- and O-glycans are summarized in Table 3B. Both parts of Table 3 also list the group(s) who first discovered the genetic defects in the disorders.

DEFECTS IN MUCIN-TYPE O-GLYCAN BIOSYNTHESIS UDP-GalNAc transferase 3 (polypeptide N-acetylgalactosaminyltransferase 3) deficiency

The GALNT3 gene encodes UDP-GalNAc transferase 3 (GalNT3; EC, which transfers UDP-GalNAc to Thr/Ser of a protein backbone. GALNT3 is expressed in organs that contain secretory epithelial glands. It is highly expressed in human pancreas, skin, kidney, and testis and weakly expressed in prostate, ovary, intestine, and colon (92). Patients with familial tumoral calcinosis (FTC) can have mutations in the GALNT3 gene.

FTC. FTC is an autosomal recessive progressive metabolic disorder that manifests with massive calcium deposits in the skin and subcutaneous tissues and unresponsiveness to parathyroid hormone (93). At present, FTC is the only syndrome with an isolated defect in mucin-type O-glycan biosynthesis (94). The syndrome can be treated with phosphate-binding antacids (aluminum hydroxide) and a low-phosphorus diet combined with calcium deprivation, which reduces and prevents the recurrence of calcific masses (95).

Laboratory findings: Hyperphosphatemia has been described accompanied by inappropriately normal or increased concentrations of parathyroid hormone and 1,25-dihydroxyvitamin D3, two essential regulators of phosphate metabolism. Serum calcium is within reference values, and 25-hydroxycholecalciferol is decreased (94).

Diagnosis: Recently immunostaining with a monoclonal antibody against GalNT3 revealed that the protein was absent in a frozen skin biopsy from a patient with FTC, whereas GalNT3 was strongly expressed in the epidermis of a healthy individual. This suggests that immunostaining of skin biopsy samples for GalNT3 might be a useful tool in the diagnosis of this disorder (96). The diagnosis can be confirmed at the molecular genetic level (94).


[beta]-1,4-Galactosyltransferase 7 deficiency

The B4GALT7 gene encodes [beta]-1,4-galactosyltransferase 7 (B4GalT7; EC, which transfers Gal to the Xyl-Ser linkage in the linker region of proteoglycans (97). B4GALT7 is expressed in human heart, pancreas, liver, and to a lesser extent, in placenta, kidney, brain, skeletal muscle, and lung. Patients with the autosomal recessive progeroid variant of Ehlers-Danlos syndrome have mutations in the B4GALT7 gene.

Progeroid variant of Ehlers-Danlos syndrome. To date, 5 patients have been described with the progeroid variant of Ehlers-Danlos. Characteristic clinical features are a premature aging phenotype with a loose, elastic skin, failure to thrive, joint laxity, psychomotor retardation, hypotonia, and macrocephaly. Because proteoglycans are important structural components of the extracellular matrix of connective tissue, these patients suffer from skin, cartilage, and bone problems.

Laboratory findings: Thyroid, kidney, and liver function test results are within reference values. Urine organic acids, amino acids, mucopolysaccharides, and oligosaccharides were all within reference values. In addition, nitroprusside test results, chromosomal studies, serum creatine kinase concentrations, and growth hormone concentrations were all normal (98).

Diagnosis: This disorder can be diagnosed at the enzyme level by use of an assay for galactosyltransferase I in human fibroblasts (99). Mutations can be found in the corresponding gene (97).

Deficiencies of extosin-1, -2, and -3

The EXT1 and EXT2 genes encode the proteins extosin-1 and -2, respectively, which oligomerize with the copolymerases (EC and responsible for the elongation of the heparin and heparan sulfate chains. The exact function of extosin-3 is not known. The EXT genes are ubiquitously expressed in human tissue. Patients with hereditary multiple exostoses (HME) have a mutated EXT1, EXT2, or EXT3 gene.

HME (types I, II, and III). HME is a genetically heterogeneous autosomal dominant disorder characterized by the development of multiple cartilage-capped benign bone tumors (exostoses) located mainly on the long bones. This disorder is often accompanied by skeletal deformities and short stature. In many cases, the exostoses transform to malignant tumors. Mutations in EXT1 and EXT2 account for 44%-66% and 30% of HME patients, respectively, whereas EXT3 appears to be the minor locus (100). For a review on hereditary multiple exostoses, please see the article by Wicklund et al. (101).

Laboratory findings: No laboratory findings have been reported that may aid in diagnosis.

Diagnosis: Genetic confirmation of the diagnosis can be obtained by mutation analysis of EXT1, EXT2, and EXT3 (100, 102, 103).


N-Acetylglucosamine-6-O-sulfotransferase deficiency

The CHST6 gene encodes human GlcNAc-6-O-sulfotransferase (EC 2.8.2.-) that transfers sulfate to the 6-O position of GlcNAc and Gal residues in the poly-N-acetyllactosamine extensions in keratan sulfate. GlcNAc-6-O-sulfotransferase is produced in human cornea, brain, spinal cord, and trachea. Macular corneal dystrophy (MCD) is caused by distinct mutations in the gene CHST6. MCD (types I and II). MCD is a progressive autosomal recessive disease in which minute, gray, punctuate opacities in the cornea lead to bilateral loss of vision. Onset of clinical signs occurs in the first decade of life. Most patients have painful attacks with photophobia, foreign body sensations, and recurrent corneal erosions. MCD is characterized by nonsulfated (MCD type I) or low-sulfated (MCD type II) keratan sulfate (104).

Laboratory findings: MCD patients have an accumulation of GAGs in corneal fibroblasts (105, 106). Keratan sulfate in serum and cartilage is nonsulfated or low-sulfated (107, 108). The defect is thus not restricted to cells in the cornea. No abnormalities have been described in the total amount of urinary GAGs or in GAG subfractions. In addition, the electrophoretic migration of urinary GAG subfractions was normal.

Diagnosis: In earlier days, histopathologic diagnosis of MCD was based on the fact that GAG deposits stain positively with Hale's colloidal iron, alcian blue, periodic acid-Schiff, and metachromic dyes (109). Currently, MCD can be diagnosed with an ELISA that makes use of a monoclonal antibody specific for a sulfated epitope on keratan sulfate (110). The subdivision of MCD into types I and II is based on the results of this ELISA. The diagnosis can be confirmed at the molecular genetic level (104).

Chondroitin 6-sulfotransferase 1 deficiency

The CHST3 gene encodes chondroitin 6-sulfotransferase 1 (EC, which catalyzes the sulfation of the 6-O position of GalNAc residues in chondroitin sulfate chains. CHST3 is widely expressed in adult tissues. It is expressed in the human heart, placenta, skeletal muscle, and pancreas, but also in various immune tissues such as the spleen, lymph nodes, and thymus. Recently, it was found that mutations in the CHST3 gene cause autosomal recessive spondyloepiphyseal dysplasia (SED), Omani type (111).

SED type Omani. Patients with SED Omani type are normal in length at birth but show growth retardation later, and are short in stature in adulthood (110-130 cm). Severe progressive kyphoscoliosis, severe arthritic changes with joint dislocations, rhizomelic limbs, genu valgum, cubitus valgus, mild brachydactyly, camptodactyly, and microdontia occur in this disease (112).

Laboratory findings: Laboratory investigations revealed hematologic indices and biochemistry results within reference values, normal results for routine metabolic investigations, and normal karyotype. Urinary excretion of mucopolysaccharides was normal. Thyroid function was normal, and growth hormone, insulin-like growth factor 1, follicle-stimulating hormone, and prolactin concentrations were within reference values (112).

Diagnosis: Chondroitin 6-sulfotransferase 1 activity can be measured in human fibroblasts (111). Mutations can be found in the corresponding gene (111).

Diastrophic dysplasia sulfate transporter deficiency

The DTDST gene (also called SLC26A2) encodes the diastrophic dysplasia sulfate transporter (DTDST), which is a sulfate/chloride antiporter. The primary source of sulfur for the sulfation pathway of proteoglycans is free S[O.sub.4.sup.2-], which is transported to the cytoplasm mainly by DTDST. Mutations in the sulfate transporter lead to undersulfation of the GAGs. DTDST is ubiquitously expressed. Mutations in the DTDST gene are the cause of diastrophic dysplasia (DTD), achondrogenesis type 1B (ACGB1), atelosteogenesis type II (AO-II), and multiple epiphyseal dysplasia 4 (EDM4). The clinical features in the DTDST skeletal dysplasia family range from a relatively mild condition to severe conditions incompatible with life and are subdivided into the 4 syndromes listed above. The disorders have autosomal recessive inheritance. The severity of the phenotype correlates with the underlying DTDST mutation; mutations leading to stop codons or transmembrane domain substitutions mostly lead to the most severe phenotype (ACGB1), whereas other structural or regulatory mutations usually lead to one of the less severe phenotypes (113). The classification of DTD, AO-II, or EDM4, and thus of the severity of the disease, depends on residual sulfate uptake capacity and the extent of proteoglycan undersulfation (114). For a review, see the article by Rossi and Superti Furga (115). ACGB1. ACGB1 is among the most severe skeletal disorders in humans. The disease is characterized by severe hypodysplasia of the spine, the rib cage, and the extremities. ACGB1 is always lethal immediately after and sometimes even before birth (113).

AO-II. AO-II is a lethal chondrodysplasia caused by collapse of the airways, resulting from abnormalities in the tracheal, laryncheal, and bronchial cartilage. Phenotypically, AO-II is the severe variant of DTD. In addition to the clinical features described for DTD, AO-II is characterized by severe and progressive kyphosis, horizontal sacrum, and a gap between the first and the second toes (116).

DTD. DTD is a skeletal dysplasia associated with short stature [adult height, 100-140 cm (117)], joint contractures, cleft palate, scoliosis, bilateral clubfeet, and characteristic clinical signs such as the so-called "hitchhiker thumb" and cystic swelling of external ears. Phenotypic variability is wide (115, 118).

EDM4. Patients with EDM4 have a condition with clubfoot, scoliosis, mild finger deformity, and mildly short or normal stature, but without palatal clefting, ear swelling, or thumb deviation (119).

Laboratory findings: Histochemical studies revealed that cartilage proteoglycans of ACGB1 patients were quantitatively decreased and do not stain with toluidine blue. Impaired synthesis of sulfated proteoglycans was observed in fibroblast cultures of an ACGB1 patient (120). There have been no published studies on the quantitative excretion or the composition of urinary GAGs, the catabolic products of proteoglycans.

Diagnosis: Sulfate transport within cells can be measured in human fibroblasts (121). The diagnosis of DTDST deficiency can be confirmed at the molecular genetic level (122-125).

3'-Phosphoadenosine 5'-phosphosulfate synthase 2 deficiency

The ATPSK2 gene encodes the enzyme 3'-phosphoadenosine 5'-phosphosulfate synthase 2 (PAPSS2; EC (126). PAPSS2 is a bifunctional enzyme that activates cytoplasmic S[O.sub.4.sup.2-] into a high-energy form in 2 enzymatic steps: (a) its ATP-sulfurylase uses ATP and S[O.sub.4.sup.2-] to synthesize adenosine 5'-phosphosulfate (APS) and (b) its APS kinase catalyzes the phosphorylation of APS to 3'-phosphoadenosine 5'-phosphosulfate (PAPS). PAPS is the universal sulfate donor for posttranslational protein sulfation. Defective PAPSS2 thus leads to undersulfation of GAGs. PAPSS2 is produced in human cartilage. In a large Pakistani family, comprising 8 generations, a mutation was found in the ATPSK2 gene that leads to SED, Pakistany type.

SED, Pakistany type. The clinical features of SED, Pakistany type, include short stature evident at birth; short, bowed lower limbs; a mild, generalized brachydactyly, kyphoscoliosis, an abnormal gait, and early-onset degenerative joint disease in the hands and knees. Radiographs showed delayed epiphyseal ossification, especially of the hips and knees, and platyspondyly. Inheritance of the disease is autosomal recessive (127).

Laboratory findings: No laboratory findings have been reported that may aid in diagnosis (128).

Diagnosis: PAPSS2 activity can be measured in human liver biopsy samples (128). Diagnosis can be confirmed at the molecular genetic level (126).


Lysyl hydroxylase-1 deficiency

The PLOD gene encodes lysyl hydroxylase-1 (EC Lysyl hydroxylase-1 catalyzes the formation of hLys in collagens and other proteins with collagen-like amino acid sequences by the hydroxylation of Lys residues. hLys serves as attachment site for O-galactosyl glycans and is essential for the formations of collagen cross-links, contributing to collagen structure and stability. Lysyl hydroxylase-1 deficiency indirectly leads to an O-glycosylation defect. The function of the O-galactosyl glycans is unclear, although it is suggested that they may play a role in recognizing and activating collagen receptors in the cell membrane (129). Subsequently, it has been shown that there is a relationship between cross-link content and the degree of collagen glycosylation (130). Lysyl hydroxylase is produced in human liver, heart, lung, skeletal muscle, brain, and placenta (131). Patients with Ehlers-Danlos syndrome type VIa have mutations in the PLOD gene.

Ehlers-Danlos syndrome type VIa. Patients with Ehlers- Danlos type VIa are characterized clinically by neonatal kyphoscoliosis, generalized joint laxity, skin fragility, and severe muscle hypotonia at birth. Arterial rupture has caused death in some patients (132). The inheritance of the syndrome is autosomal recessive.

Laboratory findings: Urinary amino acids were within reference values. Amino acid analysis of dermal collagen showed a marked decrease in hLys content (133).

Diagnosis: The activity of lysyl-protocollagen hydroxylase can be measured in cultured skin fibroblasts (133). The diagnosis can be further confirmed by mutation analysis in the corresponding gene (134).


O-Mannosyl glycan biosynthesis disorders are characterized by an abnormal [alpha]-dystroglycan glycosylation. [alpha]-Dystroglycan is an essential component of the dystropin- glycoprotein complex, which is produced in human tissues such as muscle, brain, nerve, and heart. The dystrophin-glycoprotein complex is a multimeric transmembrane complex, providing a tight connection between the cytoskeleton and the extracellular matrix. Dystroglycan is generated from a single gene (DAG1) and is subsequently cleaved into 2 subunits: transmembrane [beta]-dystroglycan and peripheral [alpha]-dystroglycan. In muscle, the intracellular side of transmembranic [beta]-dystroglycan binds to a variety of cytoplasmic molecules, such as dystropin, which in turn interacts with the cytoskeleton of cells. The extracellular side of [beta]-dystroglycan binds noncovalently to [alpha]-dystroglycan, which in turn binds to extracellular matrix proteins such as laminin (135, 136). Different mammalian glycan sequencing studies have revealed that [alpha]-dystroglycan is heavily glycosylated with O-linked Man chains (~70%) and to a lesser extent with mucin-type O-glycans (~30%), which mediate proteinprotein interactions (137, 138). For the screening of defects in O-mannosyl glycan biosynthesis, the immunohistochemical staining of [alpha]-dystroglycan is used on muscle biopsies of patients. At present, [alpha]-dystroglycan is the only known substrate for this type of glycosylation in mammals. Often the monoclonal antibodies VIA4-1 and IIH6, which recognize an unknown carbohydrate epitope in [alpha]-dystroglycan, are used. Antibodies against the core structures of [alpha]- and [beta]-dystroglycan serve as controls in such experiments (139).

Protein O-mannosyltransferase deficiency

Coexpression of the POMT1 and the POMT2 genes is necessary for the enzymatic activity of protein O-mannosyltransferase [EC (57)]. Mannosyltransferase catalyzes the attachment of Man residues to Thr/Ser amino acids of a protein. POMT1 is highly expressed in human testis, heart, and pancreas, whereas expression is lower in kidney, skeletal muscle, brain, placenta, lung, and liver. Walker-Warburg syndrome (WWS) and limbgirdle muscular dystrophy type 2K (LGMD2K) can be caused by mutations in the POMT1 gene. POMT2 is highly expressed in testis, with expression lower in most tissues. Recently, it was discovered that mutations in the POMT2 gene also cause WWS (140).

WWS. In 20% of WWS patients, a mutation is found in the POMT1 gene. The incidence of POMT2 mutations is in the same range as that of POMT1 (140). The phenotype seen in the WWS patients with a POMT2 mutation is indistinguishable from that of patients with POMT1 mutations. Patients with this rare autosomal recessive disorder have a life expectancy of <3 years (mean, 0.8 years). WWS patients have malformations of the muscle, eye, and brain. Typical brain anomalies include hydrocephalus, cerebellar hypoplasia, absent corpus callosum and cerebellar vermis, cobblestone cortex, and fusion of the hemispheres. Additionally, WWS patients can have numerous eye anomalies, such as cataracts, microphthalmia, persistent hyperplastic primary vitreous, and Peters anomaly. WWS patients have little motor activity because of severe muscle dystrophy (141). For a review, see van Reeuwijk et al. (141).

LGMD2K. Patients with LGMD2K have progressive muscle weakness involving the proximal muscles of the shoulder and pelvic girdles. These patients also have a slow, progressive limb-girdle muscular dystrophy, a mild microcephaly, and severe mental retardation, but normal brain imaging. Onset of the autosomal recessive disorder is in the first decade of life (142, 143).

Laboratory findings: In both WWS and LGMD2K patients, serum creatine kinase was increased and staining of [alpha]-dystroglycan by the VIA4-1 and/or IIH6 monoclonal antibodies was abnormal (142, 144, 145). Decreased staining of the laminin [alpha]2-chain (merosin) has been observed in WWS, although patients have been reported with normal amounts of merosin (146, 147).

Diagnosis: The activity of protein O-mannosyltransferase can be measured in human kidney cells (57). The diagnosis of POMT1 deficiency can be confirmed by mutation analysis of the gene (142, 148).

O-Mannosyl-[beta]1,2-N-acetylglucosaminyltransferase-1 deficiency

The POMGNT1 gene encodes the enzyme O-mannosyl-[beta]1, 2-N-acetylglucosaminyltransferase-1 (EC This enzyme catalyzes the second step, the linkage of a GlcNAc residue to protein-bound Man, in the O-mannosyl glycan core structure. The enzyme O-mannosyl-[beta]1,2-N-acetylglucosaminyltransferase-1 appears to be present in all tissues. Muscle-eye-brain disease (MEB) is caused mainly by mutations in the POMGNT1 gene.

MEB. MEB is a muscular dystrophy/neuronal migration disorder with a phenotype similar to, but less severe than, that of WWS patients. The life expectancy of MEB patients is 10-30 years (144). Clinically, MEB is differentiated from WWS mainly on the basis of the presence of a normal or thin corpus callosum and of pronounced cerebellar cysts, which are both absent in WWS patients (144). The inheritance of the disorder is autosomal recessive. For a review, see Diesen et al. (149).

Laboratory findings: MEB patients have increased serum creatine kinase and abnormal staining of [alpha]-dystroglycan by VIA4-1 and/or IIH6 monoclonal antibodies (144, 150). Staining of the laminin [alpha]2-chain (merosin) is generally normal (150).

Diagnosis: The activity of O-mannosyl-[beta]1,2-N-acetylglucosaminyltransferase- 1 can be measured in human muscle biopsies (151). The diagnosis can be further confirmed by molecular genetic analysis of the corresponding gene (152).


Fukutin deficiency

The FCMD gene encodes the protein fukutin. The function of fukutin is not known, but it is suggested to be a glycosyltransferase (153). Fukutin is produced in many parts of the body, with the highest amounts in the brain, heart, pancreas, and skeletal muscle. Fukuyama-type congenital muscular dystrophy (FCMD) and WWS can be caused by mutations in the FCMD gene. Deficiencies in fukutin are putative defects in O-mannosyl glycan biosynthesis. These deficiencies might be reclassified depending on the function of fukutin.

FCMD. FCMD is an autosomal recessive disorder in which patients manifest with generalized muscle weakness, severe hypotonia, mental retardation, and brain malformations. Brain malformations are very similar to those reported in WWS and MEB and include cerebral and cerebellar micropolygyria, hydrocephalus, fibroglial proliferation of the leptomeninges, focal interhemispheric fusion, and hypoplasia of the corticospinal tracts (154, 155). Compared with MEB and WWS patients, the eye involvement in patients with FCMD is more variable, ranging from myopia to retinal detachment, persistent vitreous bodies, persistent hyaloid artery, or microphthalmia (156). For a review, see Toda et al. (157).

WWS. Severe mutations in the FCMD gene lead to WWS (158, 159).

Laboratory findings: In both FCMD and WWS patients, serum creatine kinase was increased. Subsequently, abnormal staining of [alpha]-dystroglycan by the VIA4-1 and/or IIH6 monoclonal antibodies and decreased staining of laminin [alpha]2-chain were observed in all cases investigated (146, 160, 161).

Diagnosis: As the function of fukutin is unknown at present, the diagnosis can be confirmed only at the molecular genetic level (158, 162).

Deficiencies in fukutin-related protein

The FKRP gene encodes the fukutin-related protein (FKRP), the function of which is undefined at present. FKRP is expressed in skeletal muscle, placenta, and heart and weakly in brain, lung, liver, kidney, and pancreas. FKRP is predicted to be a tissue-specific glycosyltransferase involved in the O-mannosylation of [alpha]-dystroglycan (163). Mutations in the FKRP gene with autosomal recessive inheritance have been found in congenital muscular dystrophy type 1C (MDC1C), LGMD2I, WWS, and MEB, but also in asymptomatic cases (164). Deficiencies in FKRP are putative defects in O-mannosyl glycan biosynthesis. These deficiencies might be reclassified depending on the function of FKRP.

MDC1C. The onset of the characteristic clinical features of MDC1C occurs at birth or within the first 6 months of life; these features include severe weakness and wasting of the shoulder-girdle muscles, hypertrophy and weakness of the leg muscles with an inability to walk, and a severe restrictive pulmonary disease leading to respiratory failure in the second decade of life. Cardiomyopathy has been observed in several patients (163). The brain is not always involved in this disorder. Severe mutations can lead to structural cerebellar changes (165) or more extensive structural brain and eye involvement similar to that seen in MEB and WWS (166).

LGMD2I. In LGMD2I, the age at onset of clinical signs ranges from 6 months to 40 years. The disorder presents as hypotonia, weakness in the hip and shoulder-girdle muscles, and hypertrophy of the calf muscles. The spectrum ranges from infants with early presentation and a Duchenne-like disease course, including cardiomyopathy, to milder phenotypes with a long-term outcome. Moreover, these patients lack structural brain or eye involvement (167, 168).

WWS and MEB. Recently, a patient diagnosed with WWS and a patient diagnosed with MEB were found to have a mutated FKRP gene (169).

Asymptomatic carriers for homozygous FRKP mutations. De Paula et al. (164) investigated 86 Brazilian LGMD genealogies and identified 4 persons with novel homozygous FKRP gene mutations who were asymptomatic.

Laboratory findings: In patients with the 4 disorders, serum creatine kinase was increased. In all MDC1C cases investigated, staining [alpha]-dystroglycan by VIA4-1 and/or IIH6 monoclonal antibodies was abnormal, and staining of laminin [[alpha].sub.2]-chain was decreased, whereas the LGMD2I cases studied showed a variable decreases in VIA4-1 and/or IIH6 staining and often (but not always) a laminin [[alpha].sub.2]-chain deficiency was found (163, 167, 170).

Diagnosis: As the function of FKRP is unknown at present, the diagnosis can be confirmed only at the molecular genetic level (163, 167, 169).

N-Acetylglucosaminyltransferase-like protein deficiency

The LARGE gene encodes the N-acetylglucosaminyltransferase-like protein (LARGE), a homolog of mammalian [beta]1,3-N-acetylglucosaminyltransferase. The gene is ubiquitously expressed, with the highest expression in the heart, brain, and skeletal muscle (171). Mutations in the LARGE gene have been found in a patient with MDC1D. Barresi et al. (172) showed that gene transfer of LARGE not only restores the [alpha]-dystroglycan function in a LARGE-deficient mouse and in an MDC1D patient, but also in patients with FCMD, MEB, WWS, LGMD2I, and other glycosyltransferase-deficient muscular dystrophies. Kanagawa et al. (173) showed that the N-terminal domain of [alpha]-dystroglycan serves as an intracellular recognition site for LARGE, which initiates subsequent functional glycosylation of [alpha]-dystroglycan. This glycosylation is essential for ligand binding and cell surface laminin/perlecan organization. Thus, LARGE is the key determinant of the functional expression of [alpha]-dystroglycan (173). Recently, it was shown that LARGE is localized in the Golgi, where it stimulates glycosylation. In a patient with MDC1D, LARGE was mislocalized and thus failed to have an effect on [alpha]-dystroglycan glycosylation (174). Inducing LARGE expression or activity could be an attractive target for the design of therapeutics for glycosyltransferase-deficient congenital muscular dystrophies (172). LARGE deficiency is a putative defect in O-mannosyl glycan biosynthesis. This deficiency might be reclassified depending on the function of LARGE.

MDC1D. One patient with MDC1D was identified with a compound heterozygous mutation in the LARGE gene (166). The LARGE-deficient patient presented with congenital muscular dystrophy, profound mental retardation, white matter changes, and subtle structural brain abnormalities (166).

Laboratory findings: The MDC1D patient has increased serum creatine kinase. In addition, staining of [alpha]-dystroglycan by the VIA4-1 and/or IIH6 monoclonal antibodies was abnormal, whereas staining of the laminin [[alpha].sub.2]-chain was normal (166).

Diagnosis: Because the function of LARGE is unknown at present, the diagnosis can be confirmed only at the molecular genetic level (166).


UDP-N-Acetylglucosamine 2 epimerase/N-acetylmannosamine kinase deficiency

The GNE gene encodes the enzyme GNE/MNK (EC GNE/MNK is a bifunctional enzyme that catalyzes the first 2 steps of the biosynthesis of the nucleotide sugar CMP-NeuAc. GNE/MNK activity is highest in the liver and placenta, and it s also found in the heart, brain, lung, kidney, skeletal muscle, and pancreas. Although CMP-NeuAc is incorporated in both N- and O-glycans, it seems that defects in GNE/MNK influence only the sialylation of O-linked glycans and not of N-glycans (175). Mutations in GNE/MNK have been described in hereditary inclusion body myopathy (hIBM), in distal myopathy with rimmed vacuoles (DMRV), and in sialuria.

hIBM and DMRV. Patients with hIBM and DMRV (also known as Nonaka myopathy) have biallelic missense mutations in the epimerase and/or kinase domains of the GNE gene. hIBM and DMRV are autosomal recessive forms of inclusion body myopathy that typically cause progressive, severe, noninflammatory muscle disease, leading to myopathic weakness and atrophy of the limb muscles. The quadriceps, however, is nearly always spared. The syndromes manifest in early adult life. For a review, see Darvish (176).

Laboratory findings: Histologically, the muscle fibers degenerate and form rimmed vacuoles in hIBM and DMRV, especially in the atrophic areas (177, 178). Creatine kinase was within reference values or moderately increased (179, 180). In most hIBM patients investigated, hypoglycosylation of [alpha]-dystroglycan was found with the VIA4-1 and/or IIH6 monoclonal antibodies, whereas staining of the laminin [[alpha].sub.2]-chain was normal (181, 182). Arachis hypogaea peanut agglutinin lectin, which recognizes unsialylated core 1 O-glycans, reacts strongly with sarcolemmal glycoproteins and with [alpha]-dystroglycan in DMRV patients, but not in controls. The sialic acid content of O-glycans was decreased to 60%-80% of control values in DMRV patients (175).

Sialuria. Sialuria, formerly called French-type sialuria, is an autosomal dominant inborn error of metabolism in which the feedback control mechanism in the biosynthesis of CMP-NeuAc is lost. This is caused by mutations in codons 263 to 266 of GNE (183), which eliminates feedback inhibition of this enzyme by CMP-NeuAc. To date, 7 patients have been reported with sialuria; presenting clinical features included mild psychomotor delay, coarse face, recurrent upper respiratory tract infections, and hepatomegaly (183).

Laboratory findings: Highly increased concentrations of urinary free sialic acid (NeuAc) and of fibroblast free sialic acid have been found in patients with sialuria (184, 185). Hypersialylated core 1 O-glycans were observed, whereas the sialylation of N-glycans was normal (Wopereis S, unpublished data).

Diagnosis: The epimerase and kinase activities can both be measured in human lymphoblastoid cells (186), and total GNE activity can be measured in human leukocytes (187). The diagnosis can be confirmed by molecular genetic approaches (188-191).

Golgi CMP-sialic acid transporter deficiency

The SLC35A1 gene encodes the Golgi CMP-NeuAc transporter, which is responsible for the transport of cytoplasmic CMP-NeuAc into the Golgi. The SLC35A1 gene is likely to be ubiquitously expressed. Recently, a patient classified as having CDG-IIf was found to have mutations in the SLC35A1 gene.

CDG-IIf. The patient presented initially with spontaneous massive bleeding of the posterior chamber of the right eye and cutaneous hemorrhages. The clinical phenotype worsened to more severe hemorrhages, respiratory distress syndrome, and opportunistic infections. Because of graft-vs-host disease after bone marrow transplantation, the patient died at the age of 37 months (49).

Laboratory findings: The patient had macrothrombocytopenia, neutropenia, and complete lack of sialyl [Le.sup.x] antigen on polymorphonuclear cells. Severe thrombocytopenia and abnormalities of the megakaryocyte morphology were found, including small mononuclear or hyposegmented megakaryocytes, vacuolated cells, and abnormal fragmentation of megakaryocyte cytoplasm into large platelet masses. Coagulation and the enzyme activities of [alpha]1,3-fucosyl- and [alpha]2,3-sialyltransferase were all normal (49). Unsialylated core 1 O-glycans were detected with peanut agglutinin lectin staining. Plasma N-glycosylated proteins appeared to have a normal sialylation pattern apparent from the normal serum transferrin isoform profile (49).

Diagnosis: The activity of the Golgi CMP-NeuAc transporter can be determined by cloning of the human cDNA alleles into a recombinant adeno-associated virus for gene complementation of lec2-deficient cells. Lec2-deficient cells are commercially available CHO cells that have a deficient Golgi CMP-NeuAc transporter (49). CMP-NeuAc transporter activity can also be determined in human fibroblasts (74). The diagnosis can be confirmed by mutation analysis of the SLC35A1 gene (49).


Deficiencies in fucosyltransferase 1 and 2

The FUT1 and FUT2 genes encode fucosyltransferase 1 (FUT1) and 2 (FUT2), respectively (EC FUT1 and -2 catalyze the addition of Fuc to a Gal residue in an [alpha]1,2 linkage, which is also known as the H determinant and is the essential precursor for the A and B antigens. The A, B, and H blood group determinants are linked to the N-acetyllactosamine unit or to Gal[beta]1-3GlcNAc structures of N- and mucin-type O-glycans, found on erythrocytes or on secreted proteins in saliva and other secretions. It is thought that FUT1 and FUT2 are expressed in a tissue-specific manner, with the expression is restricted to cells of mesodermal or endodermal origin. Human FUT2 is expressed in the small intestine, colon, and lung, whereas which tissues express human FUT1 is unknown at present. Individuals with the Bombay, para-Bombay, and non-secretor blood groups have mutations in the FUT1 or FUT2 gene.

Bombay, para-Bombay, and non-secretor blood groups. The para-Bombay blood group is caused by a deficient FUT1 (191), whereas the non-secretor blood group is caused by a deficient FUT2 (192). The Bombay blood group can be caused by either a deficient FUT1 or a deficient FUT2 (193). Inheritance of the 3 conditions is autosomal recessive. Individuals with deficiencies in one of the FUT enzymes have no clinical phenotype.

Laboratory findings: Individuals with the Bombay blood group lack the H determinant in all tissues, and individuals with the para-Bombay blood group synthesize H determinants in soluble form but not on erythrocytes. Individuals with the non-secretor blood group lack the H determinants in soluble form, but still have their erythrocyte antigens.

Diagnosis: The diagnosis of FUT1 or FUT2 deficiency can be confirmed by mutation analysis of the genes (191-193).

Galactoside 3(4)-fucosyltransferase deficiency

The FUT3 gene encodes a galactoside 3(4)-fucosyltransferase (FUT3; EC FUT3 can use both type 1 and type 2 carbohydrate chains as substrate, producing either an [alpha]1,3 or [alpha]1,4 linkage of Fuc to the Gal residue. Type 1 Lewis determinants ([Le.sup.a] and [Le.sup.b]) are linked to Gal[beta]1-3GlcNAc structures of N- and mucin-type O-glycans, whereas the type 2 Lewis determinants ([Le.sup.x], [sLe.sup.x], and [Le.sup.y]) are linked to the N-acetyllactosamine unit of N- and mucin-type O-glycans. FUT3 is highly expressed in the stomach, colon, small intestine, lung, and kidney and to a lesser extent in the salivary glands, bladder, uterus, and liver. Individuals with a Lewis-negative blood group have mutations in the FUT3 gene.

Lewis-null ([Le.sup.[alpha]/[beta]]) blood group. Individuals with the Lewis-null blood group have no clinical phenotype (194). Inheritance is autosomal recessive.

Laboratory findings: Persons with a Lewis-negative blood group do not have the type 1 Lewis determinants. Because other fucosyltransferases (FUT4, FUT6, FUT7, or FUT9) catalyze the Fuc [alpha]1,3 and [alpha]1,4 linkages to Gal residues of type 2 glycans, the [Le.sup.x], [sLe.sup.x], and [Le.sup.y] determinants are expressed in persons with mutated FUT3.

Diagnosis: The diagnosis of FUT3 deficiency can be confirmed by mutation analysis of the gene (194).

Golgi GDP fucose transporter deficiency

The FUCT1 gene encodes a FUCT. The FUCT is likely to be ubiquitous and has a strict Golgi localization. Fuc is present in both N-linked and mucin-type O-linked glycoproteins, mainly as a constituent of N-glycans, the antigenic determinants [Le.sup.a], [Le.sup.b], [Le.sup.x], [sLe.sup.x], [Le.sup.y], and the blood group determinants A, B, and H linked to the N-acetyllactosamine unit. Additionally, Fuc is present in O-fucosyl glycans. Patients with CDG-IIc, formerly called leukocyte adhesion deficiency type II, have mutations in the FUCT1 gene.

CDG-IIc. CDG-IIc is a rare autosomal recessive syndrome characterized by recurrent infections, typical dysmorphic features, and severe growth and psychomotor retardation. In some cases, CDG-IIc can be treated with Fuc, depending on the nature of the mutation (195).

Laboratory findings: Patients with CDG-IIc have neutrophilia, the Bombay blood group, the Lewis-negative blood group, and a lack of [sLe.sup.x] on the polymorphonuclear cells. Cell binding to E- and P-selectin is severely impaired (196). Mutations in FUCT lead to a lack of Fuc residues in N- and mucin-type O-glycans, whereas the biosynthesis of O-fucosyl glycans is normal (197). This is explained by the fact that protein O-fucosyltransferase 1, responsible for O-fucosylation of proteins, is localized in the ER (198). Serum of patients with CDG-IIc gives normal results with transferrin isoelectric focusing (IEF).

Diagnosis: The activity of Golgi FUCT can be determined in human fibroblasts (199). The diagnosis can be confirmed at the molecular genetic level in the corresponding gene (44, 48).

B4GalT1 deficiency

The B4GALT1 gene encodes B4GalT1 (EC, which catalyzes the binding of UDP-Gal in a [beta]1,4 linkage to GlcNAc residues. B4GalT1 is ubiquitous, but it is found only at very low concentrations in the fetal and adult brain. B4GalT1 is involved in the formation of the N-acetyllactosamine unit. CDG-IId is caused by mutations in the B4GALT1 gene.

CDG-IId. To date, only 1 patient with CDG-IId has been described, and in that patient, only the N-glycans were studied (200). However, in B4GALT1-knockout mice, both N- and O-glycans from erythrocyte membrane glycoproteins were found to have abnormal structures (201). It may be anticipated that O-glycan biosynthesis is also impaired in patients with CDG-IId. The child with CDG-IId has mental retardation, macrocephaly attributable to a Dandy-Walker malformation with progressive hydrocephalus, myopathy, and blood clotting defects (202). The inheritance is autosomal recessive.

Laboratory findings: The patient has increased serum creatine kinase, prolonged activated partial prothrombin time, and an abnormal serum transferrin pattern by IEF (200). Apolipoprotein C-III (apoC-III) IEF results are normal in this patient, which is as expected because B4GalT1 is not involved in the biosynthesis of core 1 O-glycans (203, 204).

Diagnosis: B4GalT1 activity can be determined in human fibroblasts (200). The diagnosis can be confirmed by mutation analysis of the corresponding gene (200).

COG7 deficiency

The COG7 gene encodes subunit 7 of the COG complex. It is thought that the COG complex has a role in the regulation, compartmentalization, transport, and activity of several Golgi enzymes. It is not known in which human tissues COG7 is located, but it is likely to be a ubiquitous protein. CDG-IIe is caused by mutations in the COG7 gene.

CDG-IIe. To date, only 2 siblings have been described with perinatal asphyxia and dysmorphia, including low-set dysplastic ears, micrognathia, a short neck, and loose, wrinkled skin. Generalized hypotonia, hepatosplenomegaly, and progressive jaundice developed shortly after birth. Both siblings developed severe epilepsy and died from recurrent infections and cardiac insufficiency within the first 10 weeks of life (74).

Laboratory findings: Multiple lysosomal enzymes were increased and coagulation factors were decreased in the serum of the 2 CDG-IIe patients. Transferrin and apoC-III both showed abnormal IEF patterns, and peanut agglutinin lectin staining was increased in the fibroblasts. Increased amounts of CMP-NeuAc were detected, whereas total amounts of serum NeuAc were decreased. The transport rates of the CMP-NeuAc and UDP-Gal NSTs were reduced to 30% of reference values. The activities of the core 1 galactosyltransferase and the [alpha]2,3-sialyltransferase acting on mucin-type O-glycans were decreased. Peripheral blood indices, serum electrolytes, urea, and creatinine were within reference values, whereas the liver enzymes and bilirubin were increased in serum. Metabolic investigations of urine from these patients showed increased amounts of galactitol, Gal, and Tyr metabolites, but no succinylacetone in one of the patients. Urinary organic acids, oligosaccharides, and mucopolysaccharides were within reference values (74, 205).

Diagnosis: The COG subunits can be identified in human fibroblasts with rabbit polyclonal antibodies against the different COG subunits (74, 76). The diagnosis can be confirmed at the molecular genetic level in the corresponding gene (74).

GTP-binding protein (SAR1b) deficiency

The SARA2 gene encodes the GTP-binding protein SAR1b (SAR1b-GTPase; EC This enzyme is involved in the ER-to-Golgi transport of proteins, where it has a function in protein cargo selection and in the assembly of the coat of COPII vesicles. Sar1b is present in many tissues, including the small intestine, liver, muscle, and brain. Mutations in the SARA2 gene lead to chylomicron retention disease (CMRD), Anderson disease, and CMRD with Marinesco-Sjogren syndrome. CMRD, Anderson disease, and CMRD with Marinesco-Sjogren syndrome are all autosomal recessive disorders.

CMRD and Anderson disease. Phenotypically, CMRD and Anderson disease present as a malabsorption syndrome with severe diarrhea with steatorrhea, failure to thrive, and growth retardation. Mild neurologic disturbances occur, including mental deficiency, loss of deep tendon reflexes, decreased vibratory sensation, axonal neuropathy, mild deficits of color perception, nystagmus, and action tremor. There is little acanthocytosis (206, 207). The distinction between CMRD and Anderson disease has been made on the basis of the apparent differences between the partitioning of chylomicrons and of lipid droplets between membrane-bound and cytosolic compartments.

CMRD with Marinesco-Sjogren syndrome. Two brothers have been diagnosed with the Marinesco-Sjogren syndrome combined with CMRD. Marinesco-Sjogren syndrome is a rare form of cerebellar ataxia associated with congenital cataracts, mental deficiency, brisk tendon reflexes, skeletal anomalies, and cerebellar atrophy (208).

Laboratory findings: Apoprotein B is absent in the intestine and liver of patients with CMRD. Chylomicrons cannot be synthesized, and VLDL and LDL are undetectable in the plasma. Patients have low blood cholesterol and a deficiency in fat-soluble vitamins (209). [[sup.14]C]Mannose incorporation is evidently decreased in the total protein fraction of chylomicrons in CMRD patients compared with controls, pointing toward deficient N-glycan biosynthesis (210). Unfortunately, the effect of Sar1b-GTPase deficiency on O-glycosylation has not been studied, but it is likely to be abnormal as well.

Diagnosis: The diagnosis of this disorder can be confirmed by mutation analysis of the SARA2 gene (211).


The first individuals who were found to have a defect in the biosynthesis of O-linked glycans were diagnosed with known clinical syndromes, such as multiple exostoses, the progeroid form of Ehlers-Danlos syndrome, and WWS. In these patients, the gene defect was discovered earlier than the underlying abnormality in the biochemical pathway. In most of these syndromes, there is genetic heterogeneity, and not all forms are familial. For example, only 10% of the patients with multiple exostoses have the hereditary form of the syndrome, and only 20% of the patients with WWS have a defect in the POMT1 gene.

Defects in the biosynthesis of protein-linked O-glycans lead to a highly heterogeneous group of diseases. The majority of patients with "classical CDG" have a defect in N-glycan biosynthesis. They have common symptoms such as muscle hypotonia, central nervous system abnormalities, growth delay, feeding problems, coagulation defects, and liver disease, and frequently show specific signs such as abnormal fat distribution and inverted nipples, which help with the early clinical diagnosis. In contrast, patients with O-glycosylation disorders commonly have involvement of only one organ or one organ system and do not have the general symptoms that are suggestive for an inborn error of metabolism. For example, patients with a defect in the biosynthesis of GAGs often have cartilage problems leading to skeletal malformations, whereas patients with a defect in the biosynthesis of O-mannosyl glycans present with abnormalities in the musculo-cerebral system. Most of the disorders of O-glycan biosynthesis seem to have very specific tissue expression, whereas N-glycans are expressed ubiquitously. Another remarkable difference between N- and O-glycan deficiencies is that N-glycan deficiencies generally have recessive inheritance, whereas in some of the O-glycan biosynthesis diseases, such as sialuria and HME, inheritance is autosomal dominant.

Mucin-type O-glycans are more or less an exception among the O-glycans because they are expressed ubiquitously. Intriguingly, GalNTs, which are responsible for the attachment of the first GalNAc residue to the protein, are tissue specific. This becomes obvious in patients with FTC (GalNT3 deficiency), who present only with massive calcium deposits in the skin and subcutaneous tissues. These are the tissues in which the activity of this specific transferase is particularly high. Most of the other transferases involved in the biosynthesis of mucin-type O-glycans, such as galactosyl- and sialyltransferases, have a broad tissue distribution. It is therefore to be expected that defects in mucin-type O-glycosylation that are not localized in one of the tissue-specific GalNTs will produce a phenotype in which more than one organ/organ system is involved.

Patients with combined defects in protein N- and O-glycosylation often have a phenotype that is a mixture of the features of inborn errors in combination with congenital malformations. For example, patients diagnosed with CDG-IIe (COG7 deficiency) have a phenotype with central nervous system involvement, hypotonia, and hepatopathy combined with severe congenital heart malformations, limb malformations, and skin abnormalities. This new group of metabolic diseases, presenting with combined defects in N- and mucin-type O-glycosylation, is growing continuously. In most cases described to date, the genetic background is not yet known. Wopereis et al. (204) showed in a recent investigation that 75% of the 12 CDG-IIx patients examined also had abnormal biosynthesis of core 1 O-glycans. Clinically, these patients presented with widely variable clinical features ranging from a patient with only hepatic dysfunction to patients having a unique phenotype with congenital cutis laxa and congenital brain malformations in association with skeletal anomalies (204).

In summary, the phenotypes of patients with a congenital defect in O-glycosylation are a continuum. This ranges from patients who have a defect in the biosynthesis of O-glycans affecting only a few proteins and therefore have only one or two tissues involved (for example, patients with a defect in O-mannosyl glycan biosynthesis), to patients who have a defect in the biosynthesis of O-glycans disturbing many proteins and thus have more than one organ/organ system involved (probably defects in the biosynthesis of mucin-type O-glycans not localized in a GalNT), to patients who have combined defects in the biosynthesis of N- and O-glycans, who have a typical multisystem disease (for example patients with CDG-IIe).

Screening Methods for Unraveling Defects in O-Glycosylation Biosynthesis

Most of the congenital defects in the biosynthesis of O-glycans have been found by genetic approaches. O-Glycans are very complex and heterogeneous structures because of their variable composition, linkage, and branching. In view of the estimated number of genes involved in O-glycosylation processes, it is likely that the currently described congenital disorders of O-glycosylation form only a small part of a much larger group of defects. This area of inborn errors of metabolism still needs further research, beginning with the development of screening techniques to identify defects in the biosynthesis of the various types of O-glycans.

N-Linked glycans have a common protein-glycan linkage and a common biosynthetic pathway that diverges only in the late stages. There thus is limited variability in the design of plasma protein N-glycans. This explains the success of transferrin IEF as a screening method to identify defects in N-glycan biosynthesis. It will, however, be impossible to screen for all defects in O-glycan biosynthesis with just one assay. Recently, a first approach to screen for defects in the biosynthesis of the abundant mucin-type core 1 O-glycan has been developed. An IEF assay of apoC-III was used for this purpose (Fig. 4) (203). ApoC-III is a plasma protein with a single core 1 O-glycan at position [Thr.sub.94]. Three isoforms of apoC-III ([apoC-III.sub.0], [apoC-III.sub.1], and [apoC-III.sub.2]) can be distinguished. They differ in the number of NeuAc residues attached to the O-glycan core. Because NeuAc has a negative charge, it is possible to separate the 3 isoforms by IEF. In normal human plasma, [apoC-III.sub.1] is the most abundant form, accounting for ~50%, followed by [apoC-III.sub.2] with ~45%, whereas [apoC-III.sub.0] accounts for ~5% of total apoC-III. The ratio of the 3 isoforms in normal human plasma varies with age as the degree of apoC-III sialylation decreases. When the biosynthesis of core 1 O-glycans is disturbed, an abnormal apoC-III isoform ratio is found (203). In combination with transferrin IEF, this technique has been useful for the detection of combined defects in N- and O-glycosylation (204, 212). In addition, apoC-III IEF profiling should be capable of identifying most diseases in which mucin-type core 1 O-glycan but not N-glycan biosynthesis is affected. Familial tumoral calcinosis, the only genetically defined defect in the biosynthesis of mucin-type O-glycans at present, may turn out to be an exception. ApoC-III IEF profiling has not yet been performed in this disease. It is expected to give a normal result because GalNT3, the defective enzyme in FTC, is not expressed in liver tissue, where apoC-III is synthesized (94).

For the screening of defects in O-mannosyl glycan biosynthesis, immunohistochemical staining of [alpha]-dystroglycan with the monoclonal antibodies VIA4-1 and IIH6 is used on muscle biopsy specimens from patients. VIA4-1 and IIH6 recognize a carbohydrate epitope on [alpha]-dystroglycan. At present, [alpha]-dystroglycan is the only known substrate for this type of glycosylation in mammals (139). Some pitfalls of this technique are that it is not clear which carbohydrate epitope(s) is recognized by the antibodies and that batches of the same antibody differ in quality. In the report by Huizing et al. (181), for example, batches of VIA4-1 and IIH6 antibodies were found to give variable results. It is known that [alpha]-dystroglycan carries O-mannosyl glycans as well as mucin-type O-glycans and Nglycans (137, 138). Thus, abnormal staining of [alpha]-dystroglycan may occur in defects of N- or mucin-type O-glycan biosynthesis. For future diagnostics, the challenge is to find a secreted protein with 1 or 2 O-mannosyl glycans attached to it. This would allow the development of a less invasive screening technique using plasma samples to detect defects in O-mannosyl glycan biosynthesis.


In general, it would be advantageous to have a technique that can give an overview of all O-glycans present in a sample. The development of such a holistic approach is hampered by the fact that it is difficult to remove all O-glycans from their protein backbones. A general endoglycosidase for release of all O-glycans remains to be discovered. Alternatively, a chemical cleavage method, such as hydrazinolysis or (non)reductive [beta]-elimination may be useful. O-Glycan profiles have been published for the human glycoproteins glycophorin A, serum and secretory IgA, and neutrophil gelatinase B (213). A future challenge will be to test such methods on human blood samples, isolated cells, or biopsy materials. A first study on profiling of total serum O-glycans described alterations in the glycans of patients with sialuria (Wopereis S, unpublished data).

Similarly, no screening method is available to identify defects in GAG biosynthesis. Substantial amounts of GAGs occur physiologically in urine. These GAGs derive mainly from limited proteolysis of proteoglycans from the glomerulus, the renal tubule, and the urinary tract. They are released into the extracellular environment without passing the lysosomes and end up in urine having the same sugar structure as in vivo. The largest proportion of urinary GAG is chondroitin sulfate (62%-77%), whereas heparan sulfate accounts for ~25% and dermatan sulfate for only ~5% (214). The mucopolysaccharidoses, a group of inborn errors in GAG catabolism, can be diagnosed by measuring increased urinary GAGs. Reliable quantitative methods for urinary GAGs, such as the dimethyl-methylene blue assay, are available (215). Theoretically, patients with defects in the GAG biosynthesis pathway would be expected to have decreased GAG excretion in the urine. As assays for urinary GAG measurement have been developed for diagnosing mucopolysaccharidoses, most published studies have concentrated on the upper reference limit. Most laboratories using this assay therefore do not have a lower reference limit and would disregard any abnormally low value. Another limitation of this approach to finding GAG biosynthesis disorders is the age dependency of urinary GAG excretion. Patients older than 15 years of age excrete only limited amounts of GAGs in the urine. The lower reference limit would thus be close to zero. This approach would therefore allow the identification of only patients younger than 15 years with GAG biosynthesis defects.

Because chondroitin sulfate is the main GAG constituent in the urine, defects in chondroitin sulfate biosynthesis may be found by measuring urinary GAGs. It may require dedicated methods to measure urinary GAG subspecies to detect defects of heparan sulfate and dermatan sulfate biosynthesis. New ways may be found by studying the GAG composition in tissue samples. This may be accomplished by releasing the GAGs from the proteoglycans or by applying mass spectrometric techniques to peptide digests.

We thank Jack Fransen for assistance in the sections on the Golgi and Kristopher Clark for improving the English of this review. This work was supported by the European Commission [contract QLG-CT2000-0047 (Euroglycan) and contract 512131 (Euroglycanet)].

Received November 2, 2005; accepted January 24, 2006.

Previously published online at DOI: 10.1373/clinchem.2005.063040


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* Address correspondence to this author at: Laboratory of Pediatrics and Neurology (830), Institute of Neurology, Radboud University Nijmegen Medical Center, Geert Grooteplein 10, 6525 GA Nijmegen, The Netherlands. Fax 31-24-3540297; e-mail

[1] Laboratory of Pediatrics and Neurology and [2] Department of Pediatrics, Radboud University Nijmegen Medical Center, Nijmegen, The Netherlands. [3] Nonstandard abbreviations: hLys, hydroxylysine; CDG, congenital disorders of glycosylation; GalNAc, N-acetylgalactosamine; NeuAc, N-acetylneuraminic acid (sialic acid); GlcNAc, N-acetylglucosamine; [sLe.sup.x], sialyl [Lewis.sup.x] antigen; GAG, glycosaminoglycan; GlcA, glucuronic acid (or glucuronate); EGF, epidermal growth factor; TSR, thrombospondin type-1 repeat; ER, endoplasmic reticulum; GNE/MNK, UDP-GlcNAc 2 epimerase/N-acetylmannosamine kinase; Dol-P, dolichol phosphate; NST, nucleotide sugar transporter; CHO, Chinese hamster ovary; FUCT, GDP-Fuc transporter; [beta]3-Gal-T, [beta]3-galactosyltransferase; Cosmc, core 1 [beta]3-Gal-T-specific molecular chaperone; pp-GalNAc-T, polypeptide N-acetylgalactosaminyltransferase; EXTL exostoses-like; COP, coatomer protein; ERGIC, endoplasmic reticulum-Golgi intermediate compartment; SNARE, soluble N-ethylmaleimide-sensitive fusion attachment protein receptor; COG, conserved oligomeric Golgi complex; GalNT, N-acetylgalactosyltransferase; FTC, familial tumoral calcinosis; B4GalT, [beta]-1,4-galactosyltransferase; HME, hereditary multiple exostoses; MCD, macular corneal dystrophy; SED, spondyloepiphyseal dysplasia; DTDST, diastrophic dysplasia sulfate transporter; DTD, diastrophic dysplasia; ACGB1, achondrogenesis type 1B; AO-II, atelosteogenesis type II; EDM4, multiple epiphyseal dysplasia 4; PAPSS2, 3'-phosphoadenosine 5'-phosphosulfate synthase 2; APS, adenosine 5'-phosphosulfate; PAPS, 3'-phosphoadenosine 5'-phosphosulfate; WWS, Walker-Warburg syndrome; LGMD2, limbgirdle muscular dystrophy type 2; MEB, muscle-eye-brain disease; FCMD, Fukuyama-type congenital muscular dystrophy; FKRP, fukutin-related protein; MDC, congenital muscular dystrophy; LARGE, N-acetylglucosaminyllike protein; hIBM, hereditary inclusion body myopathy; DMRV, distal myopathy with rimmed vacuoles; FUT, fucosyltransferase; IEF, isoelectric focusing; apoC-III, apolipoprotein C-III; and CMRD, chylomicron retention disease.
Table 1. Different types of O-linked glycans in humans.

Type of O-
linked glycan Structure and peptide linkage Glycoprotein

Mucin-type (R)-GalNAc[alpha]1-Ser/Thr Secreted +
 plasma membrane
GAG (R)-GlcA[beta]1-3Gal[beta]1- Proteoglycans
O-linked GlcNAc GlcNAc[beta]1-Ser/Thr Nuclear and
O-linked Gal Glc[alpha]1-2[+ or -] Collagens
O-linked Man NeuAc[alpha]2-3Gal[beta]1- [alpha]-Dystroglycan
O-linked Glc Xyl[alpha]1-3Xyl[alpha]1- EGF protein domains
 3[+ or -]Glc[beta]1-Ser
O-linked Fuc NeuAc[alpha]2-6Gal[beta]1- EGF protein domains
 4GlcNAc 1-3 Fuc 1-Ser/Thr
 Glc 1-3Fuc 1-Ser/Thr TSR repeats

Type of O-
linked glycan Reference(s)

Mucin-type (8)

GAG (216, 217)

O-linked GlcNAc (218)
O-linked Gal (219)
O-linked Man (16)
O-linked Glc (220)
O-linked Fuc (220)

Table 2. Diversity of mucin-type O-linked glycans.

Core Structure Human tissue Reference(s)

1 Gal[beta]1-3GalNAc Most cells and (7)
 secreted proteins
2 Gal[beta]1-3 All blood cells (221)
3 GlcNAc[beta]1-3GalNAc Colon and saliva (222, 223)
4 GlcNAc[beta]1- Mucin-secreting (221)
 3(GlcNAc[beta]1-6)GalNAc cell types
5 GalNAc[alpha]1-3GalNAc Meconium (224)
6 GlcNAc[beta]1-6GalNAc Ovarian tissue (225)
7 GlcNAc[alpha]1-6GalNAc
8 Gal[alpha]1-3GalNAc Bronchia (226)

Table 3. Congenital disorders of glycosylation.

A. Human congenital disorders of O-glycosylation.

Name OMIM Gene

Defects in mucin type O-glycan biosynthesis
 FTC 211900 GALNT3
Defects in GAG biosynthesis
 Progeroid variant of Ehlers--Danlos 130070 B4GALT7
 HME type I 133700 EXT1
 HME type II 133701 EXT2
 HME type III 600209 EXT3
Defects in GAG sulfation
 MCD 217800 CHST6
 SED type Omani 608637 CHST3
 ACGB1B 600972 DTDST
 AO-II 256050 DTDST
 Diastrophic dysplasia 222600 DTDST
 EDM4 226900 DTDST
 SED Pakistany 603005 ATPSK2
Defects in O-galactosyl glycan biosynthesis
 Ehlers--Danlos syndrome type VI 225400 PLOD
Defects in O-mannosyl glycan biosynthesis
 WWS 236670 POMT1, POMT2,
 LGMD2K 609308 POMT1
 MEB 253280 POMGNT1 or FKRP
Putative defects in O-mannosyl glycan
 FCMD 253800 FCMD
 CMD1C 606612 FKRP
 LGMD2I 607155 FKRP
 CMD1D 608840 LARGE
Defects in O-glycan sialylation
 hIMB 600737 GNE
 DMRV 605820 GNE
 Sialuria 269921 GNE

B. Human combined congenital disorders of N- and O-glycosylation.

Bombay blood group 211100 FUT1 or FUT2
Para-Bombay blood group 211100 FUT1
Non-secretor blood group 182100 FUT2
Lewis-negative blood group 111100 FUT3
CDG-IIc 266265 FUCT1

CDG-IId 607091 B4GALT1
CDG-IIe 608779 COG7
CMRD 246700 SARA2
Anderson disease 607689 SARA2
CMRD with Marinesco--Sjogren syndrome 607692 SARA2

A. Human congenital disorders of O-glycosylation.

 O- Refe-
Name Glycan type rence(s)

Defects in mucin type O-glycan biosynthesis
 FTC Mucin-type (94)
Defects in GAG biosynthesis
 Progeroid variant of Ehlers--Danlos GAG (97)
 HME type I Heparan/ (102)
 HME type II Heparan/ (103)
 HME type III Heparan/ (100)
Defects in GAG sulfation
 MCD Keratan (104)
 SED type Omani Chondroitin (111)
 ACGB1B Sulfated GAGs (122)
 AO-II Sulfated GAGs (123)
 Diastrophic dysplasia Sulfated GAGs (124)
 EDM4 Sulfated GAGs (125)
 SED Pakistany Sulfated GAGs (126)
Defects in O-galactosyl glycan biosynthesis
 Ehlers--Danlos syndrome type VI O-linked Gal (134)
Defects in O-mannosyl glycan biosynthesis
 WWS O-linked Man (148,
 LGMD2K O-linked Man (142)
 MEB O-linked Man (152,
Putative defects in O-mannosyl glycan
 FCMD O-linked Man? (162)
 CMD1C O-linked Man? (163)
 LGMD2I O-linked Man? (167)
 CMD1D O-linked Man? (166)
Defects in O-glycan sialylation
 hIMB Sialylated (188)
 DMRV Sialylated (189)
 Sialuria Sialylated (190)
 CDG-IIf Sialylated (49)

B. Human combined congenital disorders of N-
and O-glycosylation.

Bombay blood group A, B, H blood (193)
Para-Bombay blood group A, B, H blood (191)
Non-secretor blood group A, B, H blood (192)
Lewis-negative blood group Le (a) and Le (194)
 determinants (44, 48)
CDG-IIc Fucosylated N-
 and mucin-type
CDG-IId N-Acetyl- (200)
CDG-IIe N- + O-glycans (74)
CMRD N-glycans; (211)
Anderson disease N-glycans; (211)
CMRD with Marinesco--Sjogren syndrome N-glycans;
 O-glycans? (211)
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Author:Wopereis, Suzan; Lefeber, Dirk J.; Morava, Eva; Wevers, Ron A.
Publication:Clinical Chemistry
Date:Apr 1, 2006
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