Shrimp thrombospondin (TSP): presence of O-β1,4 N-acetylglucosamine polymers and its function in TSP chain association in egg extracellular matrix

We characterized the existence of O-β(1,4)-GlcNAc polymers (β1,4GNP) that were anchored on the O-linked glycosylation sites of shrimp thrombospondin (pmTSP-II). There were five putative β1,4GNP linkages on the epithelial growth factor-like domain of pmTSP-II. Antibody against O-β-GlcNAc (CTD110.6) was used to prove the existence of linear and complex β1,4GNP. The antibody well reacted with linear chito-triose, -tetraose and -pentaose conjugated with phosphatidylethanolamine lipid. The immunoreactivity could also be detected with a complex β1,4GNP within pmTSP-II (at MW > 250 kDa). Upon denaturing the protein with SDS-PAGE buffer, the size of pmTSP-II was shifted to be 250 kDa, approximately 2.5 folds larger than the deduced molecular mass of pmTSP-II (110 kDa), suggesting additional association of pmTSP-II apart from its known disulfide bridging. This was confirmed by chitinase digestion on pmTSP-II protein leading to the subsequent smaller protein bands at 110–170 kDa in time- and concentration-dependent manners. These bands well reacted with CTD110.6 antibody and disappeared after extensive chitinase hydrolysis. Together, we believe that β1,4GNP on pmTSP-II serve the function in an inter-chain association to provide structural architecture of egg extracellular matrix, a novel function of pmTSP-II in reproductive biology.