The structural characterization of fucan sulfate from sea urchin by NMR spectroscopy and methylation analysis

The sea urchin egg fertilization system is an important model in reproductive biology. Numerous studies have implicated the egg jelly fucan sulfate polysaccharides in a variety of functions in the fertilization process. These include species-specific signaling, adhesion, and structural organization of the complex egg jelly layer. Nevertheless, their biological function is still largely unknown, and to date their structural characterization is limited. We have employed methylation linkage analysis and two-dimensional NMR spectroscopy to define the glycosyl and sulfate linkage structure of the fucan sulfates. The fucan sulfates are homofucans characterized by molecular weights of approximately 10{dollar}sp6{dollar} g/mol and sulfate substitution of 0.95-1.1(mol sulfate/mol fucose). Comparative methylation analysis of the linkage sites in each sulfated fucan and those in its desulfated analog unambiguously distinguishes glycosyl linkage sites from sulfate linkage sites. Strongylocentrotus franciscanus and S. purpuratus share a linear (1 {dollar}to{dollar} 3)-{dollar}alpha{dollar}-L-fucopyranan chain structure, while each is characterized by unique sulfate substitution. The former is comprised of 75% 2-monosulfated-{dollar}alpha{dollar}-L-fucose residues while the latter is comprised of 73% 4-monosulfated residues. Two-dimensional NMR experiments, including DQF-COSY, HMQC, and HOHAHA-2D further establish the sulfate and glycosyl positions and corroborate results from methylation analysis. Solvolytic desulfation and hydrolytic conditions are described for NMR sample preparation. Complete {dollar}sp1{dollar}H and {dollar}sp{lcub}13{rcub}{dollar}C assignments are presented for a desulfated S. franciscanus derivative, while partial assignments are reported for sulfated samples due to the complexity of their NMR spectra.