©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Mussel Adhesive Plaque Protein Gene Is a Novel Member of Epidermal Growth Factor-like Gene Family (*)

(Received for publication, September 9, 1994; and in revised form, January 4, 1995)

Koji Inoue (§) Yasuhiro Takeuchi Daisuke Miki Satoshi Odo

From the Kamaishi Laboratories, Marine Biotechnology Institute, Heita, Kamaishi, Iwate 026, Japan

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

A mussel (Mytilus galloprovincialis) cDNA encoding Mgfp2, a major component of the adhesive plaque that anchors mussels tightly to underwater surfaces was isolated. It encoded a protein mainly consisted of epidermal growth factor-like repeats, containing tyrosine residues that will be converted to 3,4-dihydroxyphenylalanine near C and N termini. Amino acid residues important for cell-cell interaction in other epidermal growth factor-like proteins were, however, not conserved in the structure of Mgfp2. RNA blot analysis on adult tissues showed foot-specific expression of this gene, while the analysis on developing larvae showed that the expression starts with formation of the foot. These results suggest that the function of Mgfp2 has been specialized to form the adhesive plaque.


INTRODUCTION

Mussels inhabit the turbulent and inhospitable niches in the intertidal zone. They adhere tightly to underwater surfaces using the adhesive holdfast, the byssus, which is a bundle of threads that terminate in an adhesive plaque(1) . The role of the adhesive plaque is to anchor byssal threads tightly to wet and irregular surfaces of substrates with enough strength to withstand turbulent waves. Major components of the plaque are two types of protein both of which incorporate varying proportions of (DOPA) (^1)into their primary structures(1) . One component, foot protein 1, is an adhesive protein containing more than 60 tandem repeats of a ``decapeptide'' motif (AKPSYPPTYK) (2, 3) and is insolublized through a series of process promoted by catechol oxidase existing in the foot, which catalyzes the conversion of peptidyl DOPA residues into peptidyl DOPA-quinones and into quinone tanning(1) . Tyrosine residues are converted to peptidyl DOPA residues in an earlier intracellular process involving a putative protein tyrosyl hydroxylase. The other major component, foot protein 2, is a cystine-rich structural element of the plaque matrix, but little is known about it because of its insoluble and proteolysis-resistant nature. Recently, the foot protein 2 was purified from the foot, the organ that synthesizes the byssus, and sequences of peptide fragments obtained by trypsinization following reductive alkylation were determined(4) . Three types of peptide fragments were found to be present, but neither the full sequence nor distribution of each fragment in the whole protein was determined. In this study, we isolated a cDNA clone encoding the foot protein 2 from the mussel, Mytilus galloprovincialis to derive the whole structure. It was found that it encodes a peptide containing epidermal growth factor (EGF)-like repeats. The tissue specificity and developmental stage specificity of the expression were also examined.


MATERIALS AND METHODS

Mussels and Their Larvae

Adult mussels (M. galloprovincialis) about 4 cm in shell length were sampled at Heita Bay, Iwate Prefecture, Japan. Mature mussels were purchased from an aquaculture company in Mie prefecture, Japan. They were placed in seawater at room temperature until spawning occurred. Fertilized eggs were collected and maintained at 9-15 °C in several 100-liter polycarbonate tanks. (^2)

RNA Isolation

Total RNA was isolated from adult tissues and larvae using the total RNA separator kit (Clontech Laboratories). Poly(A) RNA was isolated from the foot total RNA using the mRNA separator (Clontech Laboratories). cDNA was synthesized using the cDNA synthesis kit plus (Amersham Corp.).

PCR Amplification

The oligonucleotide primers, GA(T/C)GA(T/C)GA(A/G)GA(T/C)GA(T/C)TA(T/C)AC encoding a part of the N-terminal peptide (DDEDDYT) and (T/C)TC(A/G)TC(A/G)TC(A/G)TC(A/G)TT(A/G)TA corresponding to antisense strand of the sequence encoding a part of the putative C terminus peptide (YNDDDE) previously reported (4) were prepared, and a cDNA pool obtained from the foot was screened by polymerase chain reaction. The reaction buffer containing 6 ng each of these primers, 1 times Tth reaction buffer (Toyobo), 200 µM dNTP, 1 µg cDNA, and 1 unit of Tth DNA polymerase (Toyobo) was amplified for 30 cycles using the DNA thermal cycler 480 (Perkin-Elmer). Each cycle consisted of 30 s at 95 °C, 30 s at 42 °C, and 4 min at 70 °C.

Plaque Hybridization

The foot cDNA library of M. galloprovincialis(3) was screened with the polymerase chain reaction fragment labeled with [P]dCTP. The cDNA obtained was subcloned into a plasmid vector pBluescriptII SK (Stratagene).

Sequencing

Nucleotide sequences were determined using PRISM DyeDeoxy sequencing kit and 373A DNA sequencer (Applied Biosystems).

Computer Analysis

Sequence analysis was carried out with Genetyx Mac version 24.0.0 (Software Development Co. Tokyo).

RNA Blot Hybridization

Ten µg of total RNA obtained from tissues of adult mussels or from larvae was electrophoresed on a 1% agarose gel, transferred onto a nylon membrane, and hybridized with whole cDNA labeled with [P]dCTP.


RESULTS AND DISCUSSION

Cloning of the Foot Protein 2 cDNA

As a result of the screening of foot cDNA pool by polymerase chain reaction, an about 1.2-kilobase amplified fragment containing all the three types of peptides previously reported (4) was obtained. The foot cDNA library of M. galloprovincialis(3) was screened using the fragment as a probe and a clone carrying an insert of approximately 1.5-kilobase DNA was isolated.

Structure of the Foot Protein 2

The sequence analysis indicated that the clone contains an open reading frame of 1422 nucleotides ending with a stop codon (Fig. 1). A stop codon was also found preceding the first ATG codon by a 5` rapid amplification of cDNA ends experiment (data not shown). The open reading frame encoded a polypeptide of 473 amino acids, which is hereafter referred to as Mgfp2 (M. galloprovincialis foot protein 2). The first 17 residues probably represent the signal peptide because of the hydrophobicity of this region and because of the N-terminal sequence of the mature protein reported previously(4) . The expected cleavage position is consistent with the rule of von Heijne (5) . No other distinct hydrophobic region, which was likely to be a transmembrane domain, was observed in the sequence. The mature sequence was classified into three distinct regions: the short N-terminal region, the long central repetitive region (37-40 amino acids in length), and the short C terminus region (Fig. 2). The N- and C-terminal regions mainly consisted of polar residues and several proline and tyrosine residues. These regions contained characteristic acidic sequences, DDEEDD and DDDE, respectively. It is interesting that YKPPVYKP is identical to a repeat motif of proline-rich cell wall protein of soybean (6) not to mention the homology to the repeat consensus of Mgfp1, AKPSYPPTYK(P)(1, 2, 3) . The repetitive region, which occupies more than 90% of the mature peptide, consists of 11 repeats of the EGF-like motif in which 6 cysteine residues were arranged at a characteristic spacing (Fig. 2). Although the length of the repeats was not constant, the positions of cysteine and glycine residues in each repeat were highly conservative. Tyrosine residues were also well conserved in each repeat, although some of them were replaced with functionally similar phenylalanine residues as observed in other EGF-like peptides(7) . Positions of proline, lysine, and asparagine residues were also common to most of 11 repeats (Fig. 2). The molecular mass of the mature Mgfp2 calculated from the predicted amino acid sequence was 49.8 kDa, slightly larger than that roughly estimated from peptide fragments in the previous study in Mytilus edulis (42-47 kDa)(4) .


Figure 1: Nucleotide sequence and predicted amino acid sequence of Mgfp2 cDNA. Nucleotides are numbered above the sequence. The termination codon is indicated by an asterisk. Underlined is a possible N-glycosylation site. Underlineditalic letters indicate the possible hydroxylation sites of asparagine/aspartic acid.




Figure 2: Amino acid sequence of Mgfp2 deduced from cDNA sequence. Signal peptides, N terminus region, repetitive region and C terminus region were shown separately. In the repetitive region, sequences of all the repeats were aligned according to the positions of conserved cysteine residues. Consensus amino acids (>50% conservation) of the sequence repeats are boxed. Tyrosine residues that are expected to be converted to DOPA co- or post-translationally are underlined. The position within the total Mgfp2 sequence of the first amino acid of each line was indicated.



Homology of Mgfp2 to Other EGF-like Proteins

A considerable number of proteins were found to be significantly homologous to the EGF-like motif of Mgfp2 by computer analysis. The proteins revealing highest homologies, shown in Fig. 3, were other members of EGF family including differentiation factors(8, 9, 10, 11, 12, 13, 14) , coagulation factors(15, 16) , and extracellular matrix components(17, 18) . Positions of cysteine, glycine, and tyrosine/phenylalanine residues were shared with most of other members, but total similarities were moderate. EGF domains are supposed to have important roles for cell-cell interaction, but its functional mechanisms are not yet completely understood. Several characteristic elements in EGF-like structure have recently been identified in some EGF-like proteins. N-glycosylation sites and the hydroxylation sites of asparagine/aspartic acid have often been found in the EGF family of proteins. Possible modification sites were also present in Mgfp2: position 93 for N-glycosylation and positions 283 and 442 for aspartic acid and asparagine hydroxylation sites (Fig. 1). Some residues responsible for calcium binding have been determined in coagulation factors, e.g. aspartic acid or asparagine residues at positions 1 and 3 (numbering starts at the left in Fig. 3)(19) . Amino acid residues important for receptor binding and/or biological activities have also been identified in some members of EGF-family, e.g. the arginine residue at position 41, tyrosine residue at position 37, and several residues outside the region shown in Fig. 3(20) . These amino acid residues are, however, not conserved in the EGF-like motif of Mgfp2. In addition, Mgfp2 contains no possible transmembrane domain. Thus, no amino acid residues reminiscent of cell-cell interaction were found in EGF-like motif of Mgfp2.


Figure 3: Alignment of Mgfp2 with other EGF-like proteins. The consensus sequence of Mgfp2 is shown in this figure. The letter X indicates a non-consensus amino acid. Other sequences were obtained from Swiss Plot data base. Hyphens indicate gaps introduced to maximize homology. Residues homologous with the Mgfp2 consensus sequence are boxed. Numbers at the beginning of every sequence indicate the position of the first amino acids of the sequences within the total protein sequences.



Positions of DOPA Residues

The unique structural feature of Mgfp2, which is not observed in any other EGF-like proteins, is that it has DOPA residues in its primary structure. It is possible to identify positions of tyrosine residues that will be converted co- or post-translationally to DOPA by aligning the previously reported DOPA-containing peptides (4) with the whole sequence. It is interesting that putative DOPA residues are clustered in both ends of the sequence (Fig. 2). This finding suggests that both ends are located at the surface of the molecule in the tertiary structure where DOPA residues will be accessible to catechol oxidase, the conversion enzyme(2) . The DOPA residues at the surface of the molecule may play a role in interacting with other Mgfp2 molecules or with other protein species including the foot protein 1. Thus, it is presumed that the basic conformation of Mgfp2, which is relatively resistant to proteolytic degradation, is formed mainly by the EGF-like structure and DOPA-containing terminal regions to promote the interaction among molecules and insolublization through cross-linking processes of DOPA residues.

Expression of Mgfp2 Gene in Developing Larvae

It is important to know the expression site of the gene to estimate its function. To examine whether Mgfp2 gene is expressed in other tissues, RNA blot analysis on major tissues was performed. As the result, it was shown that Mgfp2 gene is transcribed only in the foot, not in other organs examined (Fig. 4a). RNA blot analysis on developing larvae was also performed to examine the stage specificity of the expression of Mgfp2 (Fig. 4b), because some EGF-like genes known to promote the cell differentiation are expressed in specific stages of development(21) . It was found that Mgfp2 is undetectable during free swimming stages but became detectable at the pediveliger, foot formation stage.


Figure 4: RNA blot analysis on adult tissues (a) and developing larvae (b) of M. galloprovincialis. 10 µg of total RNA extracted from the foot, mantle, adductor muscle, and gill of adult mussels (a) or that from fertilized eggs within 3 h after fertilization, 3-day-old trochophore, 4-day-old veliger, 10-day-old veliger, 32-day-old pediveliger, 42-day-old pediveliger, and the foot of the young adult that had attached to the wall of tanks with the byssus (b) was electrophoresed in a formaldehyde gel, transferred onto a nylon membrane and hybridized with whole Mgfp2 cDNA probe. Arrowheads indicate the positions of 28 and 18 S rRNA.



Mgfp2 Is a Novel Class of EGF Family

The structural features of Mgfp2 indicated that Mgfp2 is a secreted protein containing DOPA residues but has no amino acid residues implicated in the role of cell-cell interaction. RNA blot analysis on adult tissues indicated that the expression of the Mgfp2 gene is foot-specific (Fig. 4a). Similar analysis on developing larvae indicated that Mgfp2 gene expression starts with the foot formation (Fig. 4b). These structural and expression data suggest that Mgfp2 functions only as the matrix protein of the adhesive plaque and not as a growth or differentiation factor. It is supposed that Mgfp2 and other EGF family genes are derived from the same ancestral gene and only Mgfp2 has acquired the function to form an insoluble extraorganismic material by incorporating DOPA-containing sequences into its structure. Thus it seems appropriate to classify Mgfp2 as a novel class of EGF-like protein family.


FOOTNOTES

*
This work was performed as a part of the Industrial Science and Technology Frontier Program supported by New Energy and Industrial Technology Development Organization. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) D43794[GenBank].

§
To whom correspondence should be addressed: Kamaishi Laboratories, Marine Biotechnology Inst. 3-75-1 Heita, Kamaishi, Iwate 026 Japan. Tel.: 81-193-26-6544; Fax: 81-193-26-6592.

(^1)
The abbreviations used are: DOPA, 3,4-dihydroxyphenylalanine; EGF, epidermal growth factor.

(^2)
K. Inoue, Y. Takeuchi, D. Miki, and S. Odo, manuscript in preparation.


ACKNOWLEDGEMENTS

We thank Drs. J. H. Waite and S. Harayama for critical reading the manuscript, Dr. S. Miyachi for support in this study, and S. Dobashi, M. Nozawa, and Y. Mito for technical assistance.


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©1995 by The American Society for Biochemistry and Molecular Biology, Inc.