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Mar Drugs
2023 Feb 24;213:. doi: 10.3390/md21030145.
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Glycoproteins Involved in Sea Urchin Temporary Adhesion.
Ventura I, Harman V, Beynon RJ, Santos R.
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Biomedical adhesives, despite having been used increasingly in recent years, still face a major technological challenge: strong adhesion in wet environments. In this context, biological adhesives secreted by marine invertebrates have appealing characteristics to incorporate into new underwater biomimetic adhesives: water resistance, nontoxicity and biodegradability. Little is still known about temporary adhesion. Recently, a transcriptomic differential analysis of sea urchin Paracentrotus lividus tube feet pinpointed 16 adhesive/cohesive protein candidates. In addition, it has been demonstrated that the adhesive secreted by this species is composed of high molecular weight proteins associated with N-Acetylglucosamine in a specific chitobiose arrangement. As a follow-up, we aimed to investigate which of these adhesive/cohesive protein candidates were glycosylated through lectin pulldowns, protein identification by mass spectroscopy and in silico characterization. We demonstrate that at least five of the previously identified protein adhesive/cohesive candidates are glycoproteins. We also report the involvement of a third Nectin variant, the first adhesion-related protein to be identified in P. lividus. By providing a deeper characterization of these adhesive/cohesive glycoproteins, this work advances our understanding of the key features that should be replicated in future sea urchin-inspired bioadhesives.
Figure 1. Glycoproteins pulldown assay from Paracentrotus lividus tube feet discs protein extracts using lectin-bounded agarose beads. Lectin blot of pulldowns using non-inhibited (A,C,E,G) and inhibited (B,D,F,H) lectin-bounded agarose beads. Abbreviations: E—eluted fraction; M—molecular weight markers; NE—non-eluted fraction; UB- unbound fraction; Wash 1–4—wash fractions. The dashed line indicates the separation between stacking and resolving gel. GSL II, Griffonia simplicifolia lectin II; WGA, Wheat germ agglutinin; LEL, Lycopersicon esculentum lectin; and SBA, Soybean agglutinin.
Figure 2. Lectin inhibition comparative analysis. Enzyme-linked lectin assay comparing the obtained absorbance values for non-inhibited and inhibited lectins. Assay performed in a 96 wells plate coated with Paracentrotus lividus tube feet discs protein extracts. The legend shows the tested inhibiting sugar or sugar solution. Black bars are controls with no bound protein. GSL II was used to detect the presence of N-acetylglucosamine; WGA and LEL to detect chitobiosis and SBA to detect N-acetylgalactosamine in the extracts. E-5100 glycoprotein eluting solution is comprised of GlcNAc and chitin, while E-2100 contains galactose and GalNAc. Each bar represents the mean and the standard deviation (N = 8). Parametric T-tests were performed to compare absorbance values of each lectin; * p < 0.05 and ** p < 0.001. Abbreviations: GSL II, Griffonia simplicifolia lectin II; Gal, galactose; GalNAc, N-acetylgalactosamine; GlcNAc, N-acetylglucosamine; LEL, Lycopersicon esculentum lectin; SBA, Soybean agglutinin; WGA, Wheat germ agglutinin.
Figure 3. Global protein composition by functional groups of LEL-pulldown eluted fraction. Experimental approach used to identify proteins pulldown with LEL-bounded agarose beads (A) and the correspondent GO annotation analysis of Paracentrotus lividus disc proteins conjugated with GlcNAcβ(1,4)GlcNAc (B).
Figure 4. Adhesion-related and uncharacterized Paracentrotus lividus tube feet disc glycoproteins. Percentage of adhesion-related and uncharacterized proteins recovered in the different glycoprotein pulldown assays (A) and their conjugated glycans (B).
Figure 5. Summary of the characterization of glycosylated adhesive/cohesive protein candidates. Abbreviations: A2M, alpha macroglobulin domain; A2M_BRD, alpha macroglobulin bait region; A2M_recep, alpha macroglobulin receptor binding domain; A2M_TED, alpha macroglobulin receptor binding domain; Ala, alanine; Arg, arginine; EGF, EGF-like calcium-binding domain; FA58C, discoidin domains; GalNAc, N-acetylgalactosamine; GlcNAc, N-acetylglucosamine; GlcNAcβ(1,4)GlcNAc, N-acetylglucosamine in a specific chitobiose arrangement; Gly, glycine; Glu, glutamine; Leu, leucine; Ser, serine; SUEL_Lectin, SUEL lectin domain; Val, valine; vWF D, von Willebrand factor type D domain.
Figure 6. Comparative structural analysis of Paracentrotus lividus Nectin variants. Structural prediction of Nectin tertiary structure (first line) using Alphafold, with a close up on each of the six discoidin like domains (second and third line). Secondary structures are represented as arrows (beta-sheets), spirals (alpha-helix) and disorganized regions (random coils). Non-conservative amino acid substitutions are represented in red, while conservative substitutions maintain the color of the domain in which they occur. The cysteine residue responsible for Nectin homodimerization is indicated as a grey sphere and the LTD motif is identified as black spheres.
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