STRUCTURAL STUDIES OF A NOVEL TYPE OF PENTAANTENNARY LARGE GLYCAN UNIT IN THE FERTILIZATION-ASSOCIATED CARBOHYDRATE-RICH GLYCOPEPTIDE ISOLATED FROM THE FERTILIZED-EGGS OF ORYZIAS-LATIPES

In a previous report (Kitajima, K., Inoue, S., and Inoue, Y. (1989) De v. Biol. 132, 544-553), we found the presence of a heavily glycosylate d polyprotein, ''H-hyosophorin,'' isolated from the unfertilized eggs of Oryzias latipes. We now report our detailed analysis of the structu re of the N-glycan chain in L-hyosophorin, the smallest repeating unit of H-hyosophorin, which was isolated from the fertilized eggs of O. l atipes and formed from H-hyosophorin upon fertilization. The N-glycan structures were defined by a combination of compositional analysis, me thylation analysis, selective chemical degradation (i.e. mild methanol ysis, periodate-Smith degradation, and hydrazinolysis nitrous acid dea mination), enzymatic (endo-beta-galactosidase, peptide:N-glycanase and Newcastle disease virus sialidase) digestion, and instrumental analys es (one- and two-dimensional proton nuclear magnetic resonance spectro scopy and fast atom bombardment mass spectrometry) which revealed nove l and unique features: (a) the presence of highly branched poly-N-acet yllactosamino pentaantennary structures; (b) the presence of a beta-ga lactosylated Lewis X antigenic epitope, Gal beta 1 --> 4 Gal beta 1 -- > 4 (Fuc alpha 1 --> 3)GlcNAc beta 1 -->; (c) the presence of a beta-g alactosylated sialyl Lewis X structure, Gal beta 1 --> 4(Neu5Ac alpha 2 --> 3)Gal beta 1 --> 4(Fuc alpha 1 --> 3)GlcNAc beta 1 -->; (d) the presence of Gal beta 1 --> 4Gal beta 1 --> and Gal beta 1 -->; 4Gal be ta 1 --> 4Gal beta 1 --> as the major and minor groupings, respectivel y; and (e) the presence of the branched Gal residues, --> 4GlcNAc beta 1 --> 3(Gal beta 1 --> 4) Gal beta 1 -->. This study represents the f irst detailed investigation regarding the nature of highly branched co mplex asparagine-linked pentaantennary glycans in glycoproteins. The u nique expression of such bulky multiantennary glycan units on proteins could be essential during early embryogenesis.