Characterization of a Cystine-Rich Polyphenolic Protein Family from the Blue Mussel Mytilus edulis L

Abstract

Marine bivalve mollusks synthesize, in the phenol and accessory glands of the foot, proteins that integrate the post-translationally hydroxylated amino acid 3,4-dihydroxyphenylalanine (DOPA) into their primary sequence. These polyphenolic proteins serve as structural and adhesive components of the byssal threads which form the extraorganismic holdfast. One family of byssal precursors, previously characterized in a number of mytiloid species, consists of proteins between 70-130 kDa containing 8-18 mol % DOPA. The high molecular weight precursor isolated from the foot of the blue mussel (Mytilus edulis Linnaeus, 1758) is here designated as Mefp-1. We now present evidence for the occurrence, in M. edulis, of a second, structurally unrelated, family of DOPA proteins (Mefp-2) of about 42-47 kDa. These novel proteins contain 2-3 mol % DOPA and, in startling contrast to Mefp-1, are also enriched in the disulphide-containing amino acid cystine (6-7 mol %). Consideration of the amino acid compositions of Mefp-1 and 2 and of the terminal adhesive plaques of byssal threads suggests that Mefp-2 makes up about 25% of plaque protein, whereas Mefp-1 content is about 5%. The Mefp-2 family exhibits electrophoretic microheterogeneity, but members share similar N- and C-terminal amino acid sequences. Analysis of peptides isolated after tryptic hydrolysis suggests that the primary sequence of Mefp-2 is tandemly repetitive, with at least three types of motif. The sequence degeneracy of the motifs is greater than in Mefp-1. Mefp-2 has minimal sequence homology with known structural proteins and may be a structural element of the plaque matrix.

Coating Proteins: Structure and Cross-Linking in fp-1 from the Green Shell Mussel Perna canaliculus,

“…The Cys residues in the EGF domains of mefp-2 (Mytilus) are all disulfide-bonded (24). Reductive alkylation of pcfp-1 with DTT and iodoacetate followed by hydrolysis and amino acid analysis showed that total cysteine (detected as carboxymethylcysteine) is present at 2.7 mol %.…”

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).…”

“…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. 16,17 Mfp-3 (from M. edulis) is present at the interface with the substratum as a protein family where it is presumed to function as an adhesive primer. 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.…”

Adhesive strength and curing rate of marine mussel protein extracts on porcine small intestinal submucosa

“…From the reported procedure, extractions for the DOPA-containing proteins can be estimated to be ∼80% Mefp-1 protein and ∼20% Mefp-2 [30]. The extinction coefficient was calculated from the sequences of Mefp-1 [56] and Mefp-2 [57] using standard methods [58,59]. Estimates of the coefficients were 223, 500 M -1 cm -1 for Mefp-1 and 19, 140 M -1 cm -1 for Mefp-2, and these were combined to reflect an 80/20 mixture giving a composite value of 182,600 M -1 cm -1 .…”