Linking Adhesive and Structural Proteins in the Attachment Plaque of Mytilus californianus

Abstract

The byssal attachment of California mussels Mytilus californianus provides secure adhesion in the presence of moisture, a feat that still eludes most synthetic polymers. Matrix-assisted laser desorption ionization mass spectrometry was used to probe the footprints of byssal attachment plaques on glass cover slips for adhesive proteins. Besides the abundant mcfp-3 protein family (Zhao, H., Robertson, N. B., Jewhurst, S. A., and Waite, J. H. (2006) J. Biol. Chem. 281, 11090 -11096), two new proteins, mcfp-5 and mcfp-6, with masses of 8.9 kDa and 11.6 kDa, respectively, were identified in footprints, partially characterized and completely sequenced from a cDNA library. mcfp-5 resembles mcfp-3 in its basic pI and abundant 3,4-dihydroxyphenyl-L-alanine (Dopa; 30 mol %), but is distinct in two respects: it is more homogeneous in primary sequence and is polyphosphorylated. mcfp-6 is basic and contains a small amount of Dopa (<5 mol %). In contrast to mcfp-3 and -5, tyrosine prevails at 20 mol %, and cysteine is present at 11 mol %, one-third of which remains thiolate. Given the oxidative instability of Dopa and cysteine at pH 8.2 (seawater), we tested the hypothesis that thiols serve to scavenge dopaquinones by adduct formation. Plaque footprints were hydrolyzed and screened for cysteine dopaquinone adducts using phenylboronate affinity chromatography. 5-S-Cysteinyldopa was detected at nearly 1 mol %. The results suggest that mcfp-6 may provide a cohesive link between the surface-coupling Dopa-rich proteins and the bulk of the plaque proteins.Mussels inhabit wind and wave swept rocky seashores. Such habitats are deathtraps for exposed organisms lacking a secure attachment. Accordingly, mussels have evolved a robust holdfast known as the byssus, which is essentially a specialized 4 -5-cm long bundle of collagenous fibers that is proximally rooted in the mussel and distally bonded to foreign surfaces underwater by flattened attachment plaques (1).Given that engineering durable adhesive bonds between minerals and organic polymers in the presence of moisture remains a serious technological challenge, fundamental insights into the mechanism of holdfast adhesion in mussels and other sessile marine organisms represent a potential data base of bio-inspired solutions to the moisture problem (1). One popular technique for improving "wet" adhesion on siliceous substrates involves the application of surface-coupling agents or adhesion promoters (2). Organosilanes are the best known synthetic adhesion promoters and typically designed with specific moieties for silica ligation at one end and reactivity toward the organic polymer at the other (2).The use of surface-coupling agents to promote adhesion resonates with the adhesive biochemistry of byssal plaques made by mussels. A recent investigation of plaque footprints in Mytilus californianus has revealed a family of protein variants (mcfp-3) with a 3,4-dihydroxyphenyl-L-alanine (Dopa) 2 content that approaches 25 mol % (3, 4). With its many Dopa residues, mcfp-3 has been compared wi...

Recent approaches in designing bioadhesive materials inspired by mussel adhesive protein

“…1(D)] 14. Although the total phenolic residues content in Mfp‐6 is similar to those found in Mfp‐3 and 5, the tyrosine residues in Mfp‐6 are not efficiently converted to Dopa.…”

“…14. These proteins are characterized by a basic isoelectric point (pI) due to a high content of cationic amino acids.…”

Three intrinsically unstructured mussel adhesive proteins, mfp‐1, mfp‐2, and mfp‐3: Analysis by circular dichroism

“…10,15,16 Mfp-1 used for this study was prepared from M. californianus feet, whereas mfp-2 and mfp-3 were prepared from M. edulis feet. Live M. californianus were collected from Goleta Pier on Sandspit Road (Goleta, CA); flash-frozen M. edulis feet were purchased from Northeast transport (Waldoboro, ME).…”

“…Mfp-1 (from Mytilus californianus) is the only protein known to be present in the byssal cuticle remarkable for its combination of high stiffness and high extensibility. 6 Mfp-1 has a mass between 88 and 92 kDa, a basic isoelectric point (8)(9)(10), and 60 or more tandem repeats of a decapeptide sequence (PKISYO*OTY*K in which Y* is 3,4-dihydroxyphenyl-L-alanine (Dopa), O is trans-4-hydroxproline, and O* denotes trans-2,3-cis-3,4-dihydroxyproline (diHyp). 15 Mfp-2 (from M. edulis) is an abundant protein in the plaques ($25% of adhesive plaque by dry weight) with a mass of 45 kDa and consists of 11 tandem repeats of a 40-res long epidermal growth factor (EGF) motif.…”

“…Mfp-3 is the most polymorphic of all known plaque proteins with more than 30 different known sequence variants ranging in mass from 5 to 7 kDa. 10 Mfp-3 has 10-20 mol % of Dopa and $4 mol % of 4-hydroxyarginine. Our main purpose here is to compare and contrast the secondary structure of three dominant mfps, mfp-1, mfp-2, and mfp-3, under identical solution conditions.…”