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Expression sites of two byssal protein genes of Mytilus galloprovincialis.

Introduction

Marine sessile invertebrates such as mussels and barnacles adhere tightly to wet and irregular surfaces in the sea. They have evolved specific adhesive mechanisms to resist the impact of waves. For example, barnacles secrete cement proteins (Saroyan et al., 1970) and mussels secrete byssus threads. These substances act as glues that becomes insoluble after secretion in water and attach the animal to the substrate.

The byssus of mussels, which has a high bonding strength (Allen et al., 1976), is a bundle of threads, each consisting of a fibrous collagenous core coated by adhesive proteins. The protein components of the byssus have been studied extensively in Mytilus edulis (Waite, 1987, 1992, for reviews). A major component, foot protein 1 (fp-1), is an adhesive 130-kDa protein containing a high proportion of 3,4-dihydroxyphenylalanine (DOPA) residues (Waite and Tanzer, 1981; Waite, 1983). Its primary sequence exhibits tandem repeats of the decapeptide motif Ala-Lys-Pro-Ser-Tyr-diHyp-Hyp-Thr-DOPA-Lys, where diHyp and Hyp are 3,4-dihydroxyproline and 4-hydroxyproline, respectively (Waite et al., 1985; Taylor and Waite, 1994). Another component, foot protein 2 (fp-2), is also a DOPA-containing protein that is structurally unrelated to fp-1. It is a plaque matrix protein of 42-47 kDa in size; it contains a smaller proportion of DOPA residues than fp-1 and is rich in the disulfide-containing amino acid cystine (Rzepecki et al., 1992).

Partial sequences of cDNA and genomic DNA encoding fp-1 have been reported in M. edulis (Strausberg et al., 1989; Filpula et al., 1990; Laursen, 1992). Recently, cDNA sequences encoding the whole coding region of the two foot proteins of M. galloprovincialis, another mussel species closely related to M. edulis, have been determined (Inoue and Odo, 1994; Inoue et al., 1995a). The cDNA of M. galloprovincialis fp-1 (Mgfp-1) encodes 62 repeats of the same decapeptide motif as that of M. edulis, but lacks the hexapeptide motif (Inoue and Odo, 1994; Inoue et al., 1995b). Sequencing of the M. galloprovincialis fp-2 (Mgfp-2) cDNA has shown that the Mgfp-2 protein consists mainly of repeats resembling those of epidermal growth factor (EGF) (Inoue et al., 1995a).

Still, many questions remain unresolved. The phenol gland is an apparent site of fp-1 synthesis and storage, so the apparent localization of fp-2 to the phenol gland is a little ambiguous (Rzepecki et al., 1992; Waite, 1983; Benedict and Waite, 1986). In this study, we examined expression sites of the Mgfp-1 and Mgfp-2 genes by northern blots and in situ hybridization. We found that tissues expressing the Mgfp-1 and Mgfp-2 genes were located along the ventral groove of the foot in an arrangement appropriate for byssus formation.

Materials and Methods

Isolation of total RNA

Mussels (M. galloprovincialis) of about 6 cm in shell length were purchased from an aquaculture company in Rikuzen-Takada, Iwate prefecture, Japan. The foot, mantle, gill, and adductor muscle were isolated from mussels, and about one quarter of the distal part, which contains the phenol gland, and the remainder of the foot, which contains the accessory gland, were separated with a scalpel. Total RNA was extracted by using the Total RNA Separator Kit (Clontech Laboratories, Palo Alto, CA).

Northern blot hybridization

Ten micrograms of total RNA obtained from mussel tissues was electrophoresed on a 0.8% agarose gel containing 2.2 M formaldehyde and 0.02 M 3-(N-morpholino) propanesulfonic acid and transferred onto a nylon membrane (Hybond-[N.sup.+], Amersham). Hybridization was performed with cDNA encoding the whole coding region of Mgfp-1 and Mgfp-2 (Inoue and Odo, 1994; Inoue et al., 1995) labeled with [[[Alpha].sup.-32]P]dCTP in accordance with the instructions for the Random Primer DNA Labeling Kit (Takara Shuzo Co., Ltd. Kyoto, Japan.) The hybridization buffer contained 5X standard saline citrate 5X Denhardt's mediums, 0.1% sodium dodecyl sulfate, 0.1% sodium pyrophosphate, and 100 [[micro]gram]/ml sonicated salmon sperm DNA. After prehybridization, denatured probe was added and the nylon membrane was incubated at 60 [degrees] C for 16 h. The nylon membrane was washed to a final stringency of 0.1X standard saline citrate at 60 [degrees] C. The radioactive signals were detected by exposing the membrane for 1 h to X-ray film at -80 [degrees] C with an intensifying screen.

In situ hybridization

M. galloprovincialis foot was fixed with 4% (w/v) paraformaldehyde dissolved in phosphate-buffered saline (PBS) at 4 [degrees] C for 4 h. The fixative was replaced with 30% sucrose dissolved in PBS at 4 [degrees] C for 2 h. Then the foot was embedded in OTC compound (Miles, Inc.), frozen at -20 [degrees] C, and cut into 13-[[micro]meter] thick sections. Sections were attached to slide-glass coated with gelatin, immediately dried for 1 h, and further dried in an oven at 45 [degrees] C for 2 h. Digoxigenin-labeled sense- and antisense-RNA probes were prepared according to the instructions for the DIG RNA labeling kit (Boehringer Mannheim Biochemica). Sense and antisense probes were synthesized with T3- and T7-RNA polymerases, and full-length cDNA of Mgfp-1 and Mgfp-2 was subcloned into the EcoRI-XhoI sites of a plasmid vector, BluescriptIISK + (Stratagene), as a template. The template plasmids were digested with HindIII/SpeI and ClaI/SpeI before transcription by T3- and T7-RNA polymerases, respectively. RNA probes were degraded into about 150 bases by alkaline treatment at 60 [degrees] C for 8 and 2.8 min, respectively. Hybridization and washing were performed, as described by Yokouchi et al. (1991). Detection was carded out according to the instructions of the DIG Detection kit (Boehringer Mannheim Biochemica).

Results

Northern blot hybridization

To identify tissues expressing the Mgfp-1 and Mgfp-2 genes, northern blot hybridization was performed using RNA from major organs - i. e., foot, gill, mantle, and adductor muscle. The Mgfp-1 and Mgfp-2 genes were detected only in the foot, not in other organs [ILLUSTRATION FOR FIGURE 1 OMITTED!. To estimate the expression sites of the two genes in the foot, RNA was isolated separately from the distal end and from the remainder of the foot and was also analyzed by northern blotting. The distal end of the foot consisted mainly of the phenol gland, but included a small fraction of the accessory gland, because there is a hairpin turn of the accessory gland interdigitated with the underlying phenol gland (Waite, 1992). As a result, Mgfp-1 mRNA was detected in both the distal end and the remainder of the foot, whereas Mgfp-2 mRNA was detected primarily in the distal end and was undetectable in the remainder of the foot. Moreover, the level of Mgfp-1 mRNA accumulation was higher in the remainder of the foot than in the distal end [ILLUSTRATION FOR FIGURE 2 OMITTED!. These results indicated that the Mgfp-1 and Mgfp-2 genes were expressed at different sites in the foot. The length of Mgfp-1 cDNA was about 2.5 kb (Inoue and Odo, 1994). This size was consistent with the position of the bands detected in Figures 1A and 2A. The length of Mgfp-2 cDNA was about 1.5 kb (Inoue et al., 1995), and this size was also consistent with the position of the bands detected in Figures 1B and 2B. Mgfp-1 mRNA accumulated so much that there were degraded bands below the major band [ILLUSTRATION FOR FIGURES 1A, 2A OMITTED].

In situ hybridization

The foot is an organ that has many functions. It contains various types of tissues and glands (Waite, 1992). To determine exact sites expressing the Mgfp-1 and Mgfp-2 genes in the foot, in situ hybridization was performed. The foot was dissected longitudinally and the sections were hybridized with sense- and antisense-RNA probes. It was shown that the Mgfp-1 transcripts accumulate in cells around the ventral groove of the foot corresponding to the accessory gland, and that the transcript were barely detectable in the phenol gland [ILLUSTRATION FOR FIGURE 3 OMITTED]. The Mgfp-2 transcripts, in contrast, were detectable only in the distal phenol gland [ILLUSTRATION FOR FIGURE 4 OMITTED!.

Discussion

Around the ventral groove of the mussel foot are various glands - such as the accessory gland, the phenol gland, and the collagen gland (Waite, 1992) - from which thread-forming materials are secreted. In this study, we determined the expression sites of two foot proteins of M. galloprovincialis, Mgfp-1 and Mgfp-2. We showed that Mgfp-1 and Mgfp-2 mRNA accumulated in the foot and not in other tissues [ILLUSTRATION FOR FIGURE 1 OMITTED!. We also showed that the two types of mRNA accumulated at different sites of the foot [ILLUSTRATION FOR FIGURE 2 OMITTED]. By in situ hybridization analysis of the foot, Mgfp-1 mRNA was found in the accessory gland around the ventral groove [ILLUSTRATION FOR FIGURE 3 OMITTED!, and Mgfp-2 mRNA was found in the phenol gland [ILLUSTRATION FOR FIGURE 4 OMITTED!.

It is known that Mgfp-1 forms a protective coat on the collagenous core of byssal threads (Brown, 1952; Pujol, 1967; Vitellaro-Zuccarello, 1981), and that the collagenous core of the threads and the polyphenolic protein protecting it are secreted by the collagen gland and the accessory gland, respectively (Brown, 1952; Pujol, 1967; Vitellaro-Zuccarello, 1980, 1981). The distal plaque matrix of byssal threads consists of Mefp-2 (Rzepecki et al., 1992). The plaque matrix is formed by secretion from the phenol gland at the distal depression (Rzepecki et al., 1992), and Mefp-2 is detectable only in extracted foot tip (Waite, 1992).

By comparing the location of the proteins and the sites of accumulation of the mRNA for the proteins, we conclude that the expression sites are located around the ventral groove in an arrangement appropriate for byssus formation [ILLUSTRATION FOR FIGURE 5 OMITTED]. Since Mgfp-2 was detected exclusively in the phenol gland in the previous study (Rzepecki et al., 1992), the phenol-gland-specific expression of Mgfp-2 gene was expected [ILLUSTRATION FOR FIGURE 4 OMITTED!. However, the very low level expression of Mgfp-1 in the phenol gland was unexpected [ILLUSTRATION FOR FIGURE 3 OMITTED] because it has been reported that an antibody to Mefp-1 binds strongly to the phenol gland as well as to the accessory glands (Benedict and Waite, 1986) and that the phenol gland is an apparent site of Mefp-1 storage (Waite, 1983; Benedict and Waite, 1986). Furthermore, Mefp-1 has been isolated from dissected phenol glands as well as from the accessory gland (Waite, 1983, 1992). We thus interpret our results to suggest that the Mgfp-1 gene is transcribed in the accessory gland and the translated protein is delivered to and stored in the phenol gland. We also postulate that Mgfp-1 and Mgfp-2 are synthesized as raw materials in the accessory and phenol glands, respectively, and that Mgfp-1 is stored in both the accessory and phenol glands, but Mgfp-2 only in the phenol gland. Mussels may then secrete each protein as needed when thread is formed. We also have another interpretation for the presence of Mefp-1 in the phenol gland region. The accessory gland makes a hairpin turn around the distal depression, and the phenol gland is just before it (Waite, 1992); the two glands may have an overlap there. We suggest that phenol gland dissections include an overlap between the accessory and phenol glands, so an antibody to Mefp-1 binds to the phenol gland and Mefp-1 can be isolated from dissected phenol gland.

Mgfp-2 is a protein consisting mainly of EGF-like repeats (Inoue et al., 1995). Generally, EGF-like proteins promote cell-to-cell interactions (Massague, 1990). It is, therefore, interesting to ask whether Mgfp-2 has a function in addition to its structural role in the plaque matrix. In this study, we found that the Mgfp-2 gene is expressed primarily in a limited part of the foot - i.e., at the distal phenol gland [ILLUSTRATION FOR FIGURE 4 OMITTED!. This observation strongly suggests that Mgfp-2 has a specialized function, to form the matrix of the secreted protein, but does not act to promote cell-to-cell interaction.

Acknowledgments

The authors express their sincere thanks to Drs. J. Herbert Waite and Shigeaki Harayama for critical reading of the manuscript. We also thank Drs. Shigetoh Miyachi and Tadashi Maruyama for support in this study. This work was a part of the Industrial Science and Technology Frontier Program supported by the New Energy and Industrial Technology Development Organization.

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Author:Miki, Daisuke; Takeuchi, Yasuhiro; Inoue, Koji; Odo, Satoshi
Publication:The Biological Bulletin
Date:Apr 1, 1996
Words:2566
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