Glycosylated Hydroxytryptophan in a Mussel Adhesive Protein from Perna viridis

Abstract

The 3,4-dihydroxyphenyl-L-alanine (Dopa)-containing proteins of mussel byssus play a critical role in wet adhesion and have inspired versatile new synthetic strategies for adhesives and coatings. Apparently, however, not all mussel adhesive proteins are beholden to Dopa chemistry. The cDNA-deduced sequence of Pvfp-1, a highly aromatic and redox active byssal coating protein in the green mussel Perna viridis, suggests that Dopa may be replaced by a post-translational modification of tryptophan. The N-terminal tryptophan-rich domain of Pvfp-1 contains 42 decapeptide repeats with the consensus sequences ATPKPW 1 TAW 2 K and APPPAW 1 TAW 2 K. A small collagen domain (18 Gly-X-Y repeats) is also present. Tandem mass spectrometry of isolated tryptic decapeptides has detected both C 2 -hexosylated tryptophan (W 1 ) and C 2 -hexosylated hydroxytryptophan (W 2 ), the latter of which is redox active. The UV absorbance spectrum of W 2 is consistent with 7-hydroxytryptophan, which represents an intriguing new theme for bioinspired opportunistic wet adhesion.The amino acid 3,4-dihydroxyphenyl-L-alanine (Dopa) 3 occurs in many proteins of the mussel holdfast or byssus (1, 2) and has recently been incorporated into mussel-inspired synthetic polymers with versatile adhesive consequences (3-7). One byssal protein in particular, mussel foot protein-1 (Mfp-1), has been investigated from over 15 mussel byssi, where it protectively coats compliant collagen-like proteins in the thread core (8 -11). Mfp-1s typically contain 10 -15 mol % Dopa in a highly conserved repeating peptide structure (8). In the blue mussel Mytilus edulis fp-1 (Mefp-1), for example, the consensus decapeptide AKPSYPPTYK is repeated over 70 times in tandem, and much of the tyrosine (Y) is converted to Dopa (Y*) (Fig. 1). In stark contrast to this, only trace levels of Dopa were detected in Pvfp-1 from the green mussel Perna viridis (Linnaeus 1758) (12), a notoriously invasive fouling species originally from the Indo-Pacific region (13). Because P. viridis fp-1 (Pvfp-1) and its homologue, Mefp-1, are both strongly aromatic, quinogenic, and composed of highly polar decapeptide repeats (12), identifying the Dopa-mimetic substitutes in Pvfp-1 has been a matter of considerable interest. MATERIALS AND METHODSProtein Isolation from Mussel Feet-Green mussels (P. viridis) were collected from Tampa harbor in Florida. The feet were severed from about 100 freshly shucked mussels and stored at Ϫ80°C. Protein extraction from mussel feet was adapted from a previous report (12). Frozen mussel feet (in lots of 10 g of wet weight) were thawed and depigmented by scraping with a scalpel. The depigmented feet were homogenized in a glass tissue grinder with 50 ml of 5% acetic acid and two protease inhibitors (pepstatin A and leupeptin, both 30 mM). The homogenate was centrifuged (15,000 ϫ g, 4°C, 30 min), and the recovered supernatant (S1) was chilled in an ice bath. 70% perchloric acid was added dropwise with stirring to a final perchloric acid concentration of 1.4% (v/v). The mixtur...

N-Formylkynurenine as a Marker of High Light Stress in Photosynthesis

“…The oxindolalanine and hydroxy-indole derivatives were proposed to be intermediates produced during oxidative cleavage to give kynurenine (22). None of these species are expected to show an absorption maximum at 325 nm (53,54), as observed here for the NFK-containing peptides. NFK can be formed as a stable intermediate during the production of kynurenine (Fig.…”

“…Some oxidative tryptophan products, such as hydroxytryptophan, dioxyindolylalanine, and oxyindolylalanine, absorb near 295 nm, only slightly red-shifted from the tryptophan absorption band (53,54). However, other PTMs of tryptophan, including NFK and kynurenine (Fig.…”

Profiling of adhesive-related genes in the freshwater cnidarianHydra magnipapillataby transcriptomics and proteomics

“…Several adhesive proteins performing either permanent or temporary adhesion have been found to be glycosylated in other systems, such as in the marine sea stars, barnacles and green mussels (Ohkawa et al 2004; Zhao et al 2009; Hennebert et al 2010; Kamino et al 2012), in freshwater zebra mussels (Rzepecki & Waite 1993), and also in the stick threads secreted by terrestrial velvet worms (Graham et al 2013). Both N- and O-glycosylation were reported, as is the case of N-glycosylation of cp52 k in barnacles, and O-linked glycans primarily to threonine residues in green and zebra mussels Pvfp-1 and Dpfp-1 (Rzepecki & Waite 1993; Ohkawa et al 2004; Zhao et al 2009). They were also reported for C-mannosylated tryptophan, and C-mannosylated hydroxyl-tryptophan (Ohkawa et al 2004; Zhao et al 2009).…”

“…Both N- and O-glycosylation were reported, as is the case of N-glycosylation of cp52 k in barnacles, and O-linked glycans primarily to threonine residues in green and zebra mussels Pvfp-1 and Dpfp-1 (Rzepecki & Waite 1993; Ohkawa et al 2004; Zhao et al 2009). They were also reported for C-mannosylated tryptophan, and C-mannosylated hydroxyl-tryptophan (Ohkawa et al 2004; Zhao et al 2009). The sea star adhesive was proposed to contain at least two glycoproteins, Sfp-290 which contains mainly N-linked oligosaccharides, and Sfp-210 containing mainly O-linked oligosaccharides (Hennebert et al 2010).…”

Recombinant mussel coating protein fused with cell adhesion recognition motif enhanced cell proliferation

“…Recently, a thin cuticle, coating around the byssal thread of Mytilus species with the thickness of 2-5 µm, has attracted public attention due to its exceptionally high stiffness and toughness [16]. The cuticle has similar hardness and stiffness to epoxies, robust engineered coating material, while its extensibility is ~70-100% which more than twenty times higher than that of epoxies.…”