Printer Friendly

Secretion of glycosylated pro-B-type natriuretic peptide from normal cardiomyocytes.

B-type natriuretic peptide (BNP) [3] is a key regulatory hormone that increases sodium excretion, lowers blood pressure, suppresses the renin-angiotensin-aldosterone system, and inhibits cardiomyocyte hypertrophy and proliferation of cardiac fibroblasts (1-3). Human BNP is produced as a 108 amino acid prohormone, proBNP1-108 (proBNP108), which is converted to a biologically active peptide BNP1-32 (BNP32) and an inactive N-terminal (NT)-proBNP through proteolytic cleavage (4, 5). A cardiac serine protease, corin, and a ubiquitous serine protease, furin, are currently proposed as possible convertases (6-8). Low concentrations of BNP32 and NT-proBNP have been detected as circulating forms of BNP in healthy individuals, whereas increased concentrations of unprocessed proBNP108 circulate in patients with congestive heart failure (CHF) (9-14). These observations have established the concept that the normal heart releases a proteolytically processed BNP32 and NT-proBNP, whereas the diseased heart secretes a high amount of inactive, inappropriately processed proBNP108. It should be noted, however, that detection of proBNP in healthy individuals has been problematic because commonly used detection assays are directed against BNP32 or NT-proBNP that in part cross-react with proBNP (9). Recently, Giuliani et al. used a novel proBNP detection assay, which uses an antibody specific to the junction between NT-proBNP and BNP32 but not to the NT-proBNP or BNP32 itself, and detected proBNP in 50 adults without clinical evidence of cardiovascular disease (12). We used the same proBNP detection assay for 1939 individuals and demonstrated circulating proBNP in all healthy individuals (15). These observations indicated that unprocessed proBNP is released from the normal heart. The identification of circulating proBNP in plasma samples of healthy individuals changes the current model of proBNP processing in the heart before release into circulation.

Elucidation of proBNP intracellular trafficking, secretion, and maturation/processing is vital to enhance our understanding of how the active form of BNP is processed in healthy individuals and explain why high concentrations of immunoreactive BNPs (irBNPs) in CHF patients have such impaired biological activity. However, the precise mechanisms underlying proBNP trafficking, maturation and secretion remain to be determined. Therefore to study these mechanisms, we used BNP-domain mutants and determined the molecular mechanisms underlying the secretion of proBNP.



HEK 293T cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% calf serum, 50 000 U/L penicillin, and 50 mg/L streptomycin. Amurine atrial cardiomyocyte cell line, HL-1 (16), was kindly provided by Dr. William C. Clay-comb (Louisiana State University Medical Center) and cultured in Claycomb's medium with 10% FBS, 100 [micro]mol/L norepinephrine, and 4 mmol/L L-glutamine on 0.02% gelatin/fibronectin-coated flasks or plates. Normal human cardiomyocytes (from a 48-year-old female, white, ventricle-derived) were purchased from Promocell and maintained according to the manufacturer's guidelines. The corin-expressing plasmid (6) was kindly provided by Dr. Qingyu Wu (Cleveland Clinic).


HIV-based lentiviral vectors were generated by 3-plasmid transfection in 293T cells (for more details see the supplementary materials in the Data Supplement that accompanies the online version of this article at vol57/issue6).


FuGene6 (Roche) was used for transfection. Antibodies used in this study, including anti-BNP32 and anti-proBNP antibodies, are summarized in Table 1. Detailed protocols are described in the online Data Supplement.



We first examined the intracellular trafficking of BNP using green fluorescent protein (GFP)-tagged constructs. Our data demonstrated that the signal peptide controls the supranuclear accumulation of intracellular BNP, and that the supranuclear BNP-GFP signals colocalized frequently with Golgi marker signals (see online Supplementary Fig. S1). Because GFP tagging could cause artifacts due to artificial protein structures, we evaluated the intracellular trafficking of unlabeled BNP using constructs expressing preproBNP1-108. Because of the low transfection efficiency of murine cardiomyocyte HL-1 cells, we introduced the preproBNP1-108 expression cassette through lentiviral vector transduction. We generated a lentiviral vector construct, pSIN-BNP-UbEm, which expresses the wild-type preproBNP protein as well as a modified GFP Emerald independently, and a vector pSIN-BNP, which encodes a wild-type preproBNP alone (Fig. 1A). After infection of HL-1 cells with the SIN-BNP-UbEm vector, more than 90% of treated cells became Emerald-GFP positive, indicating efficient lentiviral transduction of HL-1 cells. When BNP expression was verified by an anti-BNP32 monoclonal antibody (mAb), 24C5, we found 11-kDa proBNP signals as a dominant intracellular form in HL-1 cells expressing preproBNP (Fig. 1B). We also observed glycol-sylated, high molecular weight (HMW) proBNP, but failed to detect a BNP32 signal in the cells. After verifying expression of the preproBNP construct, we examined the intracellular localization of irBNPs in the cells. When the preproBNP-expressing HL-1 cells were analyzed by immunostaining with an anti-proBNP mAb 15F11, clear supranuclear localization of irBNPs (red) was found in the cells that showed diffuse cyto-plasmic Emerald GFP signals (Fig. 1C). Using antiBNP32 polyclonal antibodies, we also observed regular supranuclear irBNPs as well as discrete cytoplasmic body signals (red) in SIN-BNP-infected HL-1 cells (Fig. 1D), confirming the results observed with GFP-tagged BNP proteins.


We next assessed the subcellular localization of proBNP in primary human cardiomyocytes, which were stained positive for a-actinin, a cardiomyocyte-specific marker (Fig. 1E). When the GFP-tagged full-length preproBNP construct was transfected, proBNP-GFP signals were observed in the supranuclear region, which was also positive for a Golgi protein, giantin, and in cytoplasmic bodies (Fig. 1F). When human cardio-myocytes were infected with the preproBNP-expressing lentiviral vector SIN-BNP and periodically observed for subcellular BNP localization, strong supranuclear signals of irBNPs were observed at 36 h after vector infection, and prominent cytoplasmic body signals were detected at 48 h after vector infection (Fig. 1G).


We first examined the stability of BNP32 in 293T cell lysates and supernatants. Cell lysates prepared in radio-immunoprecipitation assay buffer containing protease inhibitor cocktail (Clontech) were mixed with synthesized BNP32 and incubated at 0, 22, or 37 [degrees]C for 0, 10, or 30 min, and then analyzed by immunoblotting with anti-BNP32 mAb 24C5. BNP32 was rapidly degraded at 37 [degrees]C in 293T lysates, even in the presence of protease inhibitors, but not at 0 [degrees]C (Fig. 2A, left panel). When synthesized BNP32 was incubated with 293T culture supernatant, BNP32 was stable even at 37 [degrees]C (Fig. 2A, right panel). To avoid degradation of proBNP and BNP32 during sample collection, we processed BNP-expressing cells and supernatants at 0 [degrees]C in the following experiments.

We then analyzed the molecular forms of BNP in preproBNP-expressing cells and in the supernatants. We generated mammalian expression plasmids, which encoded full-length human preproBNP or a preproBNP mutant with a deletion in the NT-proBNP region [signal peptide (SP)-BNP] (Fig. 2B). 293T cells were transfected with the preproBNP-expression construct, and intracellular forms of irBNPs were determined by use of 3 antibodies: polyclonal anti-proBNP and 2 monoclonal anti-BNP32 antibodies, 24C5 and 50E1. Nonglycosylated proBNP (11 kDa) was identified as the predominant intracellular form of BNP (Fig. 2C). Similar results were observed with anti-BNP32 polyclonal antibodies (not shown). Overexpression of corin did not enhance the processing of nonglycosylated 11-kDa proBNP (Fig. 2, C and D). Although furin is a ubiquitously expressed protease, we did not see the proteolytically cleaved, mature BNP32 signal in cell lysates (Fig. 2C). When we examined the secreted forms of BNP in the culture supernatants, we found HMW forms (17-22 kDa) of proBNP but not nonglycosylated proBNP (Fig. 2C). Incubation of the HMW forms of proBNP with furin did not lead to any cleaved form or reduction of HMW proBNP (Fig. 2E). These data demonstrated that glycosylated proBNP is efficiently secreted from preproBNP-expressing cells and is resistant to furin cleavage.

Transfection of the SP-BNP construct resulted in expression of intracellular BNP32, and additional corin expression did not affect the mobility of the SP-BNP signal (Fig. 2C). No irBNP was detected in the supernatants of SP-BNP-expressing cells. When we analyzed the concentrations of irBNPs in the culture supernatants by using BNP32 ELISA (Phoenix), we found 6.2 [micro]g/L and 106 [micro]g/L of irBNPs (means of 4 independent experiments) in the SP-BNP- and wild-type preproBNP-transfected supernatants, respectively, suggesting the critical role of NT-proBNP region in efficient BNP secretion.




Because the lack of detection of extracellular nonglycosylated proBNP could be due to weak secretory signal activity of the BNP signal peptide sequence, we tested whether introduction of a potent secretory signal from Gaussia luciferase (17) could accelerate secretion of nonglycosylated proBNP108 or BNP32. Gaussia luciferase secretory signal (GSS)-proBNP, a preproBNP mutant with the GSS (Fig. 2B), demonstrated the same phenotype as the wild-type preproBNP: efficient secretion of HMW proBNP but no detectable nonglycosylated proBNP in the supernatants (Fig. 2F). We concluded that the lack of detectable nonglycosylated proBNP in the supernatant was not due to the weak secretory signal activity of preproBNP signal peptide.


We further examined the identity of immunoreactive, HMW proBNP. Treatment of secreted HMW proBNP with a mixture of O-linked deglycosylation enzymes (O-glycosidase, [beta]-galactosidase, glucosaminidase, peptidyl N-glycosidase F) and sialidase reduced the molecular weights of immunoreactive proBNP to 11-13-kDa species. Because there is no N-linked glycosylation site in preproBNP, our data indicate that secreted HMW proBNP is O-glycosylated (Fig. 3A). When irBNPs were enriched by immuno-precipitation with 2 anti-BNP32 polyclonal antibodies, glycosylated and nonglycosylated proBNP were observed in preproBNP-expressing 293T cell lysates. In contrast, these antibodies could pull down glycosylated proBNP, but not nonglycosylated proBNP, from the supernatants (see online Supplemental Fig. S2). When the mAb 24C5 was used to enrich irBNPs in the preproBNP-expressing 293T supernatants, glycosylated proBNP and BNP32, but not nonglycosylated proBNP, were efficiently pulled down (Fig. 3B). The anti-proBNP mAb 15F11 also enriched the HMW, glycosylated proBNP in the supernatants, but failed to pull down nonglycosylated proBNP, verifying the secretion of glycosylated proBNP from preproBNP-expressing 293T cells (Fig. 3B). In addition, when immunoprecipitation-enriched HMW BNP (18-22 kDa) were analyzed by tandem mass spectrometry, we found 6 proBNP-derived peptides (Fig. 3C, 4 peptides from NT-proBNP region and 2 peptides from BNP32 region), further demonstrating the secreted, immunoreactive HMW BNPs are of proBNP.




We examined the extracellular forms of BNP using primary human cardiomyocytes. To enrich low concentrations of endogenous BNP expression in the culture supernatants of primary human cardiomyocytes, we used antibodies 24C5 and 15F11 for immunoprecipitation. As a positive control, we used the culture supernatants from primary human cardiomyocytes, which were transduced with the preproBNP-expressing lentiviral vector, SIN-BNP. Immunoprecipitation with the antibody 24C5 detected the glycosylated proBNP in the SIN-BNP-transduced cardiomyocyte supernatant (Fig. 3D, left panel). A faint signal for the glycosylated proBNP was also seen in the untreated sample (Fig. 3D, left panel). When the proBNP mAb 15F11 was used for immunoprecipitation, we could detect glycosylated proBNP, but not nonglycosylated proBNP, in the supernatants harvested from vector-infected and untreated cardiomyocytes (Fig. 3D, right panel). Extra-cellular BNP32 was undetectable in this assay. These observations demonstrate that human cardiomyocytes also secrete glycosylated proBNP.


Semenov et al. previously reported that prevention of O-glycosylation of proBNP at the T71 amino acid residue resulted in enhanced proBNP processing (18). Because the dominant extracellular form of BNP was unprocessed and glycosylated (Fig. 2 and 3), we hypothesized that the glycosylation at the T71 residue also plays the key role in the secretion of glycosylated proBNP. We generated an expression plasmid encoding a preproBNP with a T71A amino acid substitution by extension PCR (Fig. 4A). Transfection of 293T cells with the T71A mutant resulted in intracellular accumulation of nonglycosylated proBNP, which was comparable to the wild-type preproBNP construct (Fig. 4B). Ablation of T71 glycosylation resulted in reduced abundance of glycosylated forms of proBNP in the cells (Fig. 4B). Although other glycosylation sites in the NT-proBNP region remained intact, notably reduced concentrations of glycosylated proBNP were observed in the supernatants of cells transfected with the T71A mutation (Fig. 4B). When compared with the wild-type preproBNP construct, the T71A mutant showed increased levels of BNP32 in the supernatants (Fig. 4B). These observations suggest 2 possibilities: the essential role of T71 glycosylation in efficient proBNP secretion, or efficient secretion but rapid degradation of the T71A mutant. To address these possibilities, we analyzed the concentrations of extracellular irBNPs. The ELISA assay for irBNPs (Phoenix) detected comparable levels of BNPs in the supernatants producing wild-type preproBNP or T71A mutant (wild-type, 122 [micro]g/L; T71A, 112 [micro]g/L). These data suggest that the secretion efficiency of irBNPs is similar between wild-type preproBNP and the T71A mutant, whereas stability of extracellular proBNP is impaired by ablation of T71A glycosylation.

Despite the increased BNP32 in the supernatant, BNP32 was not detectable in the cell lysates of T71A mutant-expressing cells. The ubiquitous endogenous convertase furin or overexpressed corin did not result in accumulation of intracellular BNP32 (Fig. 4B). These observations suggest that neither furin nor corin cleaved intracellular proBNP, even when the T71 glycosylation was ablated. Given the detection of BNP32 in supernatants but not in the producer cells, it is plausible that cleavage of proBNP with T71A mutant occurred at the late stage of BNP secretion or immediately after secretion. In addition, we noted the increased concentration of an undefined, 8-kDa form of intracellular BNP in the T71A mutant-transfected cells. It is possible that the lack of glycosylation of proBNP at T71 exposed a cryptic protease-recognition site for some other cellular protease, resulting in an accelerated cleavage of proBNP in the middle of the NT-proBNP region.

We also examined the molecular forms of intracellular and extracellular BNP with or without T71A mutation in a cardiomyocyte cell line. When preproBNP-expressing HL-1 cells were used for IP with proBNP mAb 15F11, we could detect nonglycosylated proBNP and glycosylated proBNP in the cell lysates and the culture supernatant, respectively (Fig. 4C, left panel). Similarly, IP with the anti-BNP32 antibody 24C5 detected nonglycosylated proBNP in cell lysates and glycosylated proBNP in the supernatant of wild-type preproBNP-expressing HL-1 cells (Fig. 4C, left panel). Although nonglycosylated proBNP was detected in the preproBNP-T71A-transfected cell lysates, secretion of glycosylated proBNP was not observed with the T71A mutant. Extracellular BNP32 was below the detectable concentration, indicating that glycosylation of preproBNP at residue T71 also controlled the concentrations of extracellular proBNP in cardiomyocyte cultures.


Our study of intracellular BNP trafficking demonstrated that synthesized proBNP first accumulates in the Golgi apparatus for posttranslational modification and is then trafficked to the cell surface as cytoplasmic secretory vesicles. Although many proteins are known to be transported in a Golgi-independent manner through nonconventional secretory pathways [for the review see (19)], our results demonstrated that proBNP108 trafficking is through the conventional endoplasmic reticulum-Golgi-secretory pathway.

We demonstrate detection of O-glycosylated proBNP as the dominant extracellular form of irBNPs. This observation confirms the recent findings of O-glycosylated proBNP secretion in preproBNP transfected cell lines (18, 20). Our multiple attempts failed to detect nonglycosylated form of extracellular proBNP. Moreover, the T71A mutant, which ablates one of the O-glycosylation sites near the proBNP cleavage site (71TLRAPR [down arrow] S, with T71 glycosylation site and RXXR 2 [down arrow] protease recognition/cleavage sequence), resulted in severe reduction in the extracellular proBNP. These data demonstrate that the glycosylation at the T71 residue is essential to stabilize extracellular proBNP. Intriguingly, a similar finding was reported for the secretion of fibroblast growth factor 23 (FGF23) (21). The O-glycosylation of FGF23 precursor protein at the threonine residue adjacent to the convertase cleavage site (171TPIPRRHTR [down arrow] S, with T171 O-glycosylation site and RXXR [down arrow] S convertase cleavage sequence) was essential to obtain high levels of extracellular FGF23 precursor protein (21). Similar to the O-glycosylation of proBNP at residue T71, where the glycosylation controls cleavage of proBNP [Fig. 4 and (18)], the glycosylation of FGF23 at T171 residue also blocks FGF23 processing by a convertase (21). These observations suggest the existence of a universal, glycosylation-controlled mechanism, which stabilizes extracellular pro-hormone or cytokine precursors and prevents premature hormone activation by convertase processing. Indeed, through screening of the human protein database, we found several secretory proteins that have similar T(or S)-X(n)-RXXR [down arrow] sequences, including endothelin 1 pre-prohormone (40TPSPPWRLRRSIR 2 C) and von Willebrand factor preprotein (768SHRSKR [down arrow] S). It is possible that O-glycosylations near the convertase recognition sites also play a role in stabilization of extra-cellular endothelin 1 preprohormone or von Wille-brand factor preproteins.

A ubiquitous cellular protease, furin, has been proposed as a BNP convertase (7, 22). Semenov et al. reported that deglycosylation of proBNP or disruption of the O-glycosylation at T71 were necessary for furin to process proBNP, suggesting that glycosylation of proBNP at T71 masks the furin recognition site (18). We also found that extracellular, glycosylated proBNP was resistant to furin cleavage. We therefore hypothesized that intracellular nonglycosylated proBNP, but not glycosylated proBNP, would be efficiently processed by endogenous furin in BNP-producing cells. However, repeated attempts failed to detect intracellular BNP32 in the 293T overexpression system, where nonglycosylated proBNP was a dominant intracellular form. Similarly, the T71A mutant, which should be more accessible for furin recognition, did not show-increased intracellular BNP32 accumulation. Instead, we found an increase in an alternatively processed form of BNP (approximately 8-kDa, Fig. 4B) in the T71A mutant-expressing cells. These observations suggest that intracellular proBNP is inherently resistant to intracellular furin cleavage. It is plausible that synthesized proBNP is compartmentalized in subcellular localizations that intracellular furin cannot easily access. In addition to furin, atrial NP convertase corin is also proposed as a BNP convertase (6). However, over-expression of corin in proBNP-expressing cells showed little effects on the concentrations of intracellular or extracellular BNP32. We did not find proBNP processing into BNP32 in HL-1 cells, which express endogenous corin (23). These observations suggest that corin may not process intracellular proBNP, and that proBNP is released as prohormone.

Detection of unprocessed or glycosylated proBNP in CHF patients (12, 13, 24) has established the concept that plasma proBNP occurs due to a defect in cardiac proBNP processing, with the spillover of unprocessed proBNP into the plasma (9,25,26). Our observations of circulating proBNP in healthy individuals (15) and the efficient secretion of glycosylated proBNP upon expression of preproBNP provide a novel model of BNP secretion, in which glycosylated proBNP is a dominant form of BNP released from the normal heart. Because glycosylated proBNP shows a biological activity 6- to 8-fold less than that of BNP32 in vitro (13), it is possible that a subset of glycosylated proBNP function without proteolytic processing in vivo. However, because previous studies have demonstrated circulating NT-proBNP and BNP32 in human circulation, it is likely that glycosylated proBNP is processed into BNP32 in the circulation or on the target cells. This newer model provides an interesting possibility of no difference in BNP secretory process between diseased and normal hearts. It is plausible that CHF patients may have defects in processing the circulating proBNP. In this context, further understanding of the BNP maturation mechanism is critical.

The issues that remain to be determined include (a) where the proBNP processing occurs after secretion, (b) what protease works as a convertase for the secreted proBNP, (c) how the BNP convertase can access the recognition site in proBNP that is likely masked by glycosylation, and (d) whether CHF patients have any defect in the processing steps of secreted proBNP. As for the problem of glycosylation masking the convertase recognition site, the study by Seferian et al. may provide an important clue (27). The authors have demonstrated that the central region (residues 28-56), but not the C-terminal region (residues 61-76), of circulating NT-proBNP in human blood is glycosylated. Given that proBNP is secreted as a fully glycosylated form, their observation may suggest the presence of deglycosylation process before convertase recognition. Since a circulating glycosidase is known to play a role in serum vitamin D3-binding protein deglycosylation (28), it is conceivable that a glycosidase in the circulation, or on the target cells, may remove the O-glycosylation at T71 of proBNP, which facilitates the proteolytic processing of proBNP for local BNP32 activation. Indeed, we have recently shown that nonglycosylated proBNP can be processed in plasma in vitro (8). Further studies on the molecular forms of BNPs in healthy and diseased hearts and the precise mechanism of glycosylated proBNP processing in vivo would provide critical information for diagnostic and therapeutic BNP applications.

Author Contributions: All authors confirmed they have contributed to the intellectual content of this paper and have met the following 3 requirements: (a) significant contributions to the conception and design, acquisition of data, or analysis and interpretation of data; (b) drafting or revising the article for intellectual content; and (c) final approval of the published article.

Authors' Disclosures or Potential Conflicts of Interest: Upon manuscript submission, all authors completed the Disclosures of Potential Conflict of Interest form. Potential conflicts of interest:

Employment or Leadership: None declared. Consultant or Advisory Role: None declared. Stock Ownership: None declared. Honoraria: None declared. Research Funding: A. Cataliotti, NIH grant RO1 HL098502-01A1; J. Burnett, Jr., NIH grants RO1 HL36634 and PO1 HL76611; Y. Ikeda, NIH grant RO1 HL098502-01A1, Marriott Individualized Medicine Award, Bernard and Edith Waterman Pilot Grant, and Mayo Foundation. Expert Testimony: None declared.

Role of Sponsor: The funding organizations played no role in the design of study, choice of enrolled patients, review and interpretation of data, or preparation or approval of manuscript.


(1.) Yamamoto K, Burnett JC Jr, Redfield MM. Effect of endogenous natriuretic peptide system on ventricular and coronary function in failing heart. Am J Physiol 1997;273:H2406-14.

(2.) Tamura N, Ogawa Y, Chusho H, Nakamura K, Nakao K, Suda M, et al. Cardiac fibrosis in mice lacking brain natriuretic peptide. Proc Nat Acad SciUSA 2000;97:4239-44.

(3.) Ogawa Y, Itoh H, Tamura N, Suga S, Yoshimasa T, Uehira M, et al. Molecular cloning of the complementary DNA and gene that encode mouse brain natriuretic peptide and generation of transgenic mice that overexpress the brain natriuretic peptide gene. J Clin Invest 1994;93:1911-21.

(4.) Suzuki E, Hirata Y, Kohmoto O, Sugimoto T, Hayakawa H, Matsuoka H, et al. Cellular mechanisms for synthesis and secretion of atrial natriuretic peptide and brain natriuretic peptide in cultured rat atrial cells. Circ Res 1992;71:1039-48.

(5.) Minamino N, Kangawa K, Matsuo H. Isolation and identification of a high molecular weight brain natriuretic peptide in porcine cardiac atrium. Biochem Biophys Res Commun 1988;157: 402-9.

(6.) Yan W, Wu F, Morser J, Wu Q. Corin, a trans-membrane cardiac serine protease, acts as a pro-atrial natriuretic peptide-converting enzyme. Proc Nat Acad SciUSA 2000;97:8525-9.

(7.) Steinhelper ME. Structure, expression, and genomic mapping of the mouse natriuretic pep tide type-B gene. Circ Res 1993;72:984-92.

(8.) Ichiki T, Huntley BK, Heublein DM, Sandberg SM, McKie PM, Martin FL, et al. Corin is present in the normal human heart, kidney, and blood, with pro-B-type natriuretic peptide processing in the circulation. Clin Chem 2011;57:40-7.

(9.) Heublein DM, Huntley BK, Boerrigter G, Cataliotti A, Sandberg SM, Redfield MM, Burnett JC Jr. Immuno-reactivity and guanosine 3',5'-cyclic monophosphate activating actions of various molecular forms of human B-type natriuretic peptide. Hypertension 2007;49:1114-9.

(10.) Yandle TG, Richards AM, Gilbert A, Fisher S, Holmes S, Espiner EA. Assay of brain natriuretic peptide (BNP) in human plasma: evidence for high molecular weight BNP as a major plasma component in heart failure. J Clin Endocrinol Metab 1993;76:832-8.

(11.) Shimizu H, Masuta K, Aono K, Asada H, Sasakura K, Tamaki M, et al. Molecular forms of human brain natriuretic peptide in plasma. Clin Chim Acta 2002;316:129-35.

(12.) Giuliani I, Rieunier F, Larue C, Delagneau JF, Granier C, Pau B, et al. Assay for measurement of intact B-type natriuretic peptide prohormone in blood. Clin Chem 2006;52:1054-61.

(13.) Liang F, O'Rear J, Schellenberger U, Tai L, Lasecki M, Schreiner GF, et al. Evidence for functional heterogeneity of circulating B-type natriuretic peptide. J Am Coll Cardiol 2007;49:1071-8.

(14.) Schellenberger U, O'Rear J, Guzzetta A, Jue RA, Protter AA, Pollitt NS. The precursor to B-type natriuretic peptide is an O-linked glycoprotein. Arch Biochem Biophys 2006;451:160-6.

(15.) Macheret F, Boerrigter G, McKie PM, Costello-Boerrigter L, Lahr B, Heublein D, et al. Pro-B-type natriuretic peptide1-108 circulates in the general community: plasma determinants and detection of left ventricular dysfunction. J Am Coll Cordiol 2011;57:1386-95.

(16.) Claycomb WC, Lanson NA Jr, Stallworth BS, Egeland DB, Delcarpio JB, Bahinski A, Izzo NJ Jr. HL-1 cells: a cardiac muscle cell line that contracts and retains phenotypic characteristics of the adult cardiomyocyte. Proc Nat Acad Sci USA 1998;95: 2979-84.

(17.) Wen B, Deng Y, Guan J, Yan W, Wang Y, Tan W, Gao J. Signal peptide replacements enhance expression and secretion of hepatitis C virus envelope glycoproteins. Acta Biochim Biophys Sin (Shanghai) 2011;3:96-102.

(18.) Semenov AG, Postnikov AB, Tamm NN, Seferian KR, Karpova NS, Bloshchitsyna MN, et al. Processing of pro-brain natriuretic peptide is suppressed by O-glycosylation in the region close to the cleavage site. Clin Chem 2009;55: 489-98.

(19.) Nickel W, Rabouille C. Mechanisms of regulated unconventional protein secretion. Nat Rev Mol Cell Biol 2009;10:148-55.

(20.) Jiang J, Pristera N, Wang W, Zhang X, Wu Q. Effect of sialylated O-glycans in pro-brain natriuretic peptide stability. Clin Chem 2010;56:959-66.

(21.) Kato K, Jeanneau C, Tarp MA, Benet-Pages A, Lorenz-Depiereux B, Bennett EP, et al. Polypeptide GalNAc-transferase T3 and familial tumoral calcinosis. Secretion of fibroblast growth factor 23 requires O-glycosylation. J Biol Chem 2006; 281:18370-7.

(22.) Sawada Y, Suda M, Yokoyama H, Kanda T, Sakamaki T, Tanaka S, et al. Stretch-induced hyper trophic growth of cardiocytes and processing of brain-type natriuretic peptide are controlled by proprotein-processing endoprotease furin. J Biol Chem 1997;272:20545-54.

(23.) Gladysheva IP, Robinson BR, Houng AK, Kovats T, King SM. Corin is co-expressed with pro-ANP and localized on the cardiomyocyte surface in both zymogen and catalytically active forms. J Mol Cell Cardiol 2008;44:131-42.

(24.) Hammerer-Lercher A, Halfinger B, Sarg B, Mair J, Puschendorf B, Griesmacher A, et al. Analysis of circulating forms of proBNP and NT-proBNP in patients with severe heart failure. Clin Chem 2008;54:858-65.

(25.) Hawkridge AM, Heublein DM, Bergen HR 3rd, Cataliotti A, Burnett JC Jr, Muddiman DC. Quantitative mass spectral evidence for the absence of circulating brain natriuretic peptide (BNP-32) in severe human heart failure. Proc Nat Acad Sci USA 2005;102:17442-7.

(26.) Seferian KR, Tamm NN, Semenov AG, Mukhary-amova KS, Tolstaya AA, Koshkina EV, et al. The brain natriuretic peptide (BNP) precursor is the major immunoreactive form of BNP in patients with heart failure. Clin Chem 2007;53:866-73.

(27.) Seferian KR, Tamm NN, Semenov AG, Tolstaya AA, Koshkina EV, Krasnoselsky MI, et al. Immuno-detection of glycosylated NT-proBNP circulating in human blood. Clin Chem 2008;54:866-73.

(28.) Yamamoto N, Naraparaju VR, Moore M, Brent LH. Deglycosylation of serum vitamin D3-binding protein by alpha-N-acetylgalactosaminidase detected in the plasma of patients with systemic lupus erythematosus. Clin Immunol Immunopathol 1997;82:290-8.

Jason M. Tonne, [1] Jarryd M. Campbell, [1] Alessandro Cataliotti, [2] Seiga Ohmine, [1] Tayaramma Thatava, [1] Toshie Sakuma, [1] Fima Macheret, [2] Brenda K. Huntley, [2] John C. Burnett, Jr., [2] and Yasuhiro Ikeda [1] *

[1] Department of Molecular Medicine, Mayo Clinic, College of Medicine, Rochester, MN; [2] Cardiorenal Research Laboratory, Division of Cardiovascular Diseases, Departments of Medicine and Physiology, Mayo Clinic and Mayo Clinic College of Medicine, Rochester, MN.

* Address correspondence to this author at: Department of Molecular Medicine, Mayo Clinic, 200 First St. SW, Rochester MN 55905. Fax 507-266-2122; e-mail

Received October 1, 2010; accepted March 24, 2011.

Previously published online at DOI: 10.1373/clinchem.2010.157438

[3] Nonstandard abbreviations: BNP, B-type natriuretic peptide; proBNP108, 108 amino acid prohormone proBNP1-108; BNP32, biologically active peptide BNP1 32; NT, N-terminal; irBNP, immunoreactive BNP; CHF, congestive heart failure; GFP, green fluorescent protein; mAb, monoclonal antibody; SP, signal peptide; HMW, high molecular weight; GSS, Gaussia luciferase secretory signal; DAPI, diaminopyrolylindole 4,6-diamino, 2-pyrolylindole.
Table 1. Antibodies used in this study.

Name Company Species Detection BNP Epitope

mAb 24C5 Abcam Mouse BNP32 BNP11-22
mAb 50E1 Abcam Mouse proBNP108 proBNP77-108
mAb 15F11 Abcam Mouse proBNP108 proBNP13-27
Polyclonal Abcam Rabbit BNP32 BNP32
Polyclonal Phoenix Rabbit BNP32 BNP32
Polyclonal Phoenix Rabbit proBNP108 proBNP22-46
Polyclonal Abcam Rabbit Corin spacer
Polyclonal Cell Signaling Rabbit EEA1
Polyclonal Cell Signaling Rabbit AIF
mAb Sigma Mouse [beta]-Actin
mAb Abcam Mouse Giantin
mAb Sigma Mouse [alpha]-Actinin
COPYRIGHT 2011 American Association for Clinical Chemistry, Inc.
No portion of this article can be reproduced without the express written permission from the copyright holder.
Copyright 2011 Gale, Cengage Learning. All rights reserved.

Article Details
Printer friendly Cite/link Email Feedback
Title Annotation:Proteomics and Protein Markers
Author:Tonne, Jason M.; Campbell, Jarryd M.; Cataliotti, Alessandro; Ohmine, Seiga; Thatava, Tayaramma; Sak
Publication:Clinical Chemistry
Date:Jun 1, 2011
Previous Article:Diagnostic accuracy of holotranscobalamin, methylmalonic acid, serum cobalamin, and other indicators of tissue vitamin [B.sub.12] status in the...
Next Article:Prognostic utility of ST2 in patients with acute dyspnea and preserved left ventricular ejection fraction.

Terms of use | Copyright © 2018 Farlex, Inc. | Feedback | For webmasters