Evidence for a Repeating 3,4-Dihydroxyphenylalanine- and Hydroxyproline-containing Decapeptide in the Adhesive Protein of the Mussel, Mytilus edulis L.*

Previous work has shown that the permanent adhe- sive of the marine mussel Mytilus edulis is a protein containing large amounts of hydroxyproline (13%) and 3,4-dihydroxyphenylalanine (Dopa, 11%). “he protein also known as the polyphenolic protein is produced and stored in the exocrine phenol gland of the mussel and deposited onto marine surfaces by the animal’s foot during the formation of new adhesive plaques. The adhesive protein has been purified by a combination of ion exchange on sulfonylpropyl-Sephadex and gel filtration on low surface energy chromatographic media. Polyacrylamide gel electrophoresis of the protein at acidic pH shows it to consist of two components having a molecular weight of about 130,000. Treatment of the protein with clostridial collagenase reduced the molecular weight by less than 108. The collagenase-resietant fragment contains most or all of the Hyp and Dopa. Trypsin treatment of the polyphenolic protein results in extensive degradation. The major tryptic peptide (80%) contains 10 amino acids including Hyp and Dopa and was shown by sequence analysis to be HZN-Ala-Lys-Pro-Ser-Qr-Hyp-Hyp-Thr-Dopa-Lys-COOH. Cal- culations suggest that this and related sequences may be repeated as often as 75 times in the polyphenolic protein.

Previous work has shown that the permanent adhesive of the marine mussel Mytilus edulis is a protein containing large amounts of hydroxyproline (13%) and 3,4-dihydroxyphenylalanine (Dopa, 11%). "he protein also known as the polyphenolic protein is produced and stored in the exocrine phenol gland of the mussel and deposited onto marine surfaces by the animal's foot during the formation of new adhesive plaques. The adhesive protein has been purified by a combination of ion exchange on sulfonylpropyl-Sephadex and gel filtration on low surface energy chromatographic media. Polyacrylamide gel electrophoresis of the protein at acidic pH shows it to consist of two components having a molecular weight of about 130,000. Treatment of the protein with clostridial collagenase reduced the molecular weight by less than 108. Permanently sessile invertebrates in the sea have necessarily evolved adhesive strategies to resist the impact of waves and the buoyant effect of water (1,2). The common mussel Mytilus edulis secures itself to solid substrates through a complex array of plaque-tipped collagenous byssal threads. On glass, the attachment plaques exhibit a mean adhesive tensile strength of IO6 newton.rneter-* although maximal values often exceed lo' newton .meter-' (2). The substance in the plaque mediating adhesion between the collagenous threads and the substrate is the polyphenolic protein (3-6). The polyphenolic protein is attracting much attention as an adhesive since, unlike most synthetic adhesives, its performance, polymerization, and longevity are not adversely affected by the presence of water. Although the reason for this resistance to water remains unknown, it is very likely related to the unusual chemical composition of the polyphenolic protein; Waite and Tanzer (6) reported that prior to polymerization the polyphenolic protein consists of a rather large polypeptide * This work was supported by Grant DE 05956 from the National Institutes of Health, Grant PCM-8206463 from the National Science Foundation, and Grant 35-084 from the Connecticut Research Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "aduertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact, chain (Mr = 130,000) in which seven amino acids, lysine, hydroxyproline, alanine, serine, threonine, tyrosine, and Dopa', account for about 80% of all the residues. The occurrence of Dopa and 3-and 4-hydroxyprolines seems particularly odd. Dopa is only rarely encountered as a component of naturally occuning proteins (7), and hyclroxyprolines are primarily associated with collagens in which every third residue is glycine (8). The very low glycine content (3%) of the polyphenolic protein tends to militate against a resemblance to collagen.
The objective of this study was to describe a purification of the polyphenolic protein and to characterize a Dopa-containing peptide prepared from the polyphenolic protein by enzymatic digestion. The results suggest that the adhesive polyphenolic protein may consist in large part of a repeated Dopacontaining sequence.

RESULTS
The purification of the polyphenolic protein from dissected phenol glands is outlined in Table I. The rather low solubility of the polyphenolic protein at neutral pH can be exploited by first extracting extraneous proteins with large amounts of neutral salt buffer followed by gentle centrifugation and reextraction of insoluble materials with dilute acetic acid. The neutral salt buffer contains various protease inhibitors to prevent premature degradation of polyphenolic protein and cyanide to avoid enzymatic oxidation of Dopa residues. The gentle first centrifugation is critical to prevent the irreversible coalescence of insoluble proteins in the pellet. Developing a high yield purification of polyphenolic protein has been difficult due to extensive adsorption of the protein to surfaces. Considerable improvements are necessary. Ion exchange on sulfonylpropyl-Sephadex ( Fig. 1) provides the single most effective step; however, up to 70% of the applied polyphenolic protein is not recovered (even at 6 M guanidine hydrochloride). Gel fitration of polyphenolic protein using a variety of ehromatographic materials (Bio-Gel P, Sepharose, Sephacryl-S and Fractogel-Merck) and buffers has resulted in very low or Repeating Dopa-and Hyp-containing Decapeptide negligible yields. Yields are improved on Sephadex if an elution buffer with low pH (2-4) and a cationic detergent is used (Fig. 2). Recovery from phenyl-Sepharose 4B is excellent although the limited fractionation range of this material is not particularly helpful (Fig. 3). Using the Dopa/protein ratio as an index of purity, the highest Dopa/protein ratios observed were 0.155. As indicated in Table 11, this corresponds to about 110 residues of Dopa/1000 residues of amino acids.
As noted earlier (6), extracts of polyphenolic protein from the phenol gland contain two species (A and B) which migrate closely on polyacrylamide gels (Fig. 4). These two species are co-purified using all of the methods presented. In extractions from individual mussel glands, the ratio of A to B seems to vary (with B 3 50%) without known reason from one individual to the next. A separation of the two has not yet been achieved for comparison. The apparent molecular weight of the polyphenolic protein as determined by polyacrylamide gel electrophoresis in the presence of cetylpyridinium bromide is estimated to be 130,000 & 10,000 (Fig. 5). The amino acid composition is shown in Table I. Collagenase treatment of polyphenolic protein results in a limited degradation (Fig. 6). Only about 8% of the protein was attacked, leaving entirely intact a fragment with M , = 120,000. This fragment has an amino acid composition similar to that of the original protein but is noticeably reduced in glycine and proline (Table 11). Clostridial collagenase selectively cleaves  -ProVGly-X-Y-where X and Y can be any amino acid (22).
Trypsin digestion of polyphenolic protein is extensive and rapid. Under the conditions used, polyphenolic protein completely disappeared within 5 min of the addition of trypsin as determined by gel electrophoresis. Fractionation of the tryptic digest was achieved using gel filtration on Sephadex LH-60 ( Fig. 7), which removes trypsin from the peptides, followed by ion exchange on Sephadex SP-25 (Fig. 8) with a pyridine acetate gradient (the latter method being recommended for basic aromatic peptides (16)). Fig. 8 illustrates that 75-80s of the ninhydrin-positive material eluting from SP-Sephadex can be ascribed to the Dopa-containing peak. Moreover, nearly 95% of the Dopa and 80% of the protein as measured by Waite and Tanzer (10) and Hartree (9), respectively, originally applied to SP-Sephadex are recovered in the major Dopa-rich peak. This material was further purified by passage through Sephadex LH-20 (Fig. 9). The tryptic peptide resembles the polyphenolic protein in containing the same group of amino acids that predominate in the latter, namely Hyp, Thr, Ser, Pro, Ala, Dopa, Tyr, and Lys. In the tryptic peptide, however, Dopa and Hyp are significantly enriched, whereas Lys, Pro, and Tyr are lower (Table 11). The tryptic peptide was homogeneous on 12% acrylamide gels in 3 M urea and 5% acetic acid but was visualized by Dopa staining since it could not be fmed for protein staining. Molecular weight in cetylpyridinium bromide gel electrophoresis was estimated to be about 6500 (Fig. 10). Peptide homogeneity was also suggested by thin layer chromatography on cellulose in 1.5% formic acid and thin layer electrophoresis in 5% acetic acid (Table 111). Since borate strongly complexes Dopa a t pH 7-9, it has the property of introducing additional negative charges into the peptide (17). Heterogeneity of the Dopa peptide (2 spots) in borate (Table 111) suggests variation in the degree of Tyr to Dopa conversion.
Sequenator analysis (35 cycles) of the tryptic Dopa peptide (Table IV) revealed it to be a decapeptide (molecular weight of about 1400), in contrast to expectations based on the estimated molecular weight of 6500. The amino acid sequence is given in Fig. 11, and features 2 lysines, 2 hydroxyprolines, 2 (Dopa + tyrosine), 1 proline, 1 serine, 1 threoniv, and 1 alanine. Lys 2 is clearly protected from tryptic proteolysis by the presence of Pro 3. Dopa is detected mostly penultimate to the carboxyl terminus although significant amounts are also  Hyp 7. present with Tyr 5. The 2 Hyps are located next to one another at positions 6 and 7; additional Hyp is present with Pro 3. 3-Hyp as well as 4-Hyp occurs at position 7.

DISCUSSION
The adhesive polyphenolic protein of the mussel M. edulis contains a large number of hydroxylated amino acids (500 residues/1000). Among these, Dopa and 3-/4-hydroxyproline are presumably derived post-translationally from tyrosine and proline, respectively. Although hydroxyproline is erroneously assumed to be unique to collagenous proteins, in the polyphenolic protein most or all of the hydroxyproline remains associated with the glycine-deficient collagenase-resistant fragment. The collagenase-labile moiety may contain a collagen-like domain such as that in Torpedo acetylcholinesterase (23), alveolar glycoprotein, (24) and others, but this remains to be demonstrated. Dopa is a relative newcomer to protein biochemistry. Waite et al. (7) detected Dopa in another protein (periostracin) also from Mytilus, but there are no other known reports of its natural occurrence in proteins. The role of Dopa in proteins is still a matter of speculation (25). Two recent studies are worth mentioning in light of their relevance to adhesion: 1) o-diphenols (including Dopa) and o-quinones can irreversibly displace water from a surface (26); 2) o-diphenols are readily oxidized to o-quinones which undergo nucleophilic addition reactions with primary amines such as lysine (27). In order to make intimate contact with a surface, the polyphenolic protein needs first to displace water molecules from that surface. Whether it relies on Dopa residues to accomplish this remains to be shown. With regard to the second point, some kind of protein cross-linking in the adhesive plaque is suggested by the insolubility of material in all but hydrolytic solvents (28). Although the formation of Dopa quinones is known to be catalyzed by a Dopa oxidase in the foot and byssal secretion of the mussel (3,29,30), Dopa lysine cross-links have yet to be isolated. Formation of covalent cross-links in the polyphenolic protein could serve to improve the cohesive performance of the substance.
The tryptic digestion of polyphenolic protein, comparison of the amino acid composition of the tryptic peptide and polyphenolic protein, and the sequence of the tryptic peptide all suggest that the polyphenolic protein may consist primarily of repeating Dopa-containing decapeptides. The decapeptide appears to be chromatographically homogeneous, although some heterogeneity in the extent of tyrosine hydroxylation to Dopa is implied by thin layer electrophoresis in borate. The occurrence of Dopa at Tyr 5 as well as at Dopa 9 lends support to this. Variations in Pro hydroxylation are also suggested. If the molecular weight estimate of 130,000 is a reliable one and 80% of the polyphenolic protein is indeed converted to a decapeptide following trypsin digestion, then up to 75 repeats of the decapeptide are possible in the intact protein. It is not yet possible to say whether the repeats are in a run or are separated by spacer peptides. Peptide repeats in proteins (particularly structural proteins) are not new. The Gly-Pro-Hyp tripeptide of collagen is well known. Elastin contains as many as 15 repeating hexapeptides and 11 pentapeptides in a run (31); silk fibroin has 50 repeats of a sequence 59-amino acids long (32); and, more recently, the proline-rich submaxillary protein of saliva was found to contain 4 repeats of a heptapeptide (33).
A predictive analysis of the secondary structure of the tryptic decapeptide by methods of Chou and Fasman (34) is of limited value. There are residues (Ala, Lys, and Dopa (35)) with strong a-helix potential, and others (Thr, Tyr, and Ser) with P-sheet propensities. The high frequency of imino acids, however, is theoretically capable of breaking either of the above conformations or imposing a poly(L-Pro) type-I1 helix. The noncollagenous hydroxyproline-rich glycoproteins from the cell walls of algae (36, 37), plants (38), and in some bacterial capsules (39) probably do have some poly(L-Pro) type-I1 helical structure (40). Tryptic peptides from one of these (extensin) reflect a much greater clustering of Hyp residues, however, i.e. Ser 1-Hyp 2-Hyp 3-Hyp 4-Hyp 5-Ser 6-Hyp 7-Lys 8 (41), than that observed in the tryptic decapeptide from the polyphenolic protein.
Perhaps future structural studies with synthetic analogues of the Dopa decapeptide will shed some light on what conformations are adopted in solution and how these conformations might be related to the adhesive function of the polyphenolic protein.