A PRECISE STRUCTURAL-ANALYSIS OF A FERTILIZATION-ASSOCIATED CARBOHYDRATE-RICH GLYCOPEPTIDE ISOLATED FROM THE FERTILIZED-EGGS OF EURYHALINE KILLI FISH (FUNDULUS-HETEROCLITUS) - NOVEL PENTA-ANTENNARY N-GLYCAN CHAINS WITH A BISECTING N-ACETYLGLUCOSAMINYL RESIDUE

A novel carbohydrate-rich sialoglycopeptide of apparent molecular mass similar to 6 kDa was isolated from the fertilized eggs of Fundulus heteroclitus (euryhaline killi fish). This glycopeptide is a member of the L-hyosophorin family, characterized by its high content of carbohydrate (80-90% by weight) and formed by depolymerization of the precursor glycopoly-protein (H-hyosophorin) upon fertilization, The structures of the N-glycan chains were unambiguously established by a combination of compositional analysis, methylation analysis, selective chemical degradation (periodate oxidation-Smith degradation and hydrazinolysis-nitrous acid deamination), enzymatic (peptide:N-glycosidase F, several beta-galactosidases, beta-hexosaminidase and alpha-galactosidase) digestions and instrumental analyses (H-1-NMR and fast atom bombardment mass spectrometry) to have the novel and unique carbohydrate sequences, Gal alpha 1-->3(Gal beta 1-->4) Gal beta 1-->4GlcNAc beta 1--> and Gal alpha 1-->3(+/-GalNAc beta 1-->4GlcNAc beta 1-->3Gal beta 1-->4)Gal beta 1-->4GlcNAc beta 1-->. This study represents the first detailed investigation of the nature of bulky complex asparagine-linked penta-antennary glycans with a bisecting GlcNAc residue in glycoproteins. Expression of such bulky multiantennary glycan units on proteins may be essential during early embryogenesis.