Subunit-specific sulphation of oligosaccharides relating to chargeheterogeneity in porcine lutrophin isoforms.

Lutrophin (LH) consists of an array of isoforms with different charges and bioactivities. This study was undertaken to clarify specifically how oligosaccharides of α and β subunits contribute to LH isoform charges. Porcine LH (pLH) was separated into four isoforms by isoelectric focusing (IEF), followed by subunit isolation. Their oligosaccharides were released by hydrazinolysis, labelled by reduction with NaBH, and fractionated by HPLC with a Mono Q column into five populations differing in the number of sulphate (S) and sialic acid (N) residues, designated as Neutral, N-1, S-1, S-N and S-2. Oligosaccharides were predominantly sulphated (S-1 and S-2) and infrequently sialylated (N-1 and S-N). Further analysis, including concanavalin A (Con A) affinity chromatography, desialylation, desulphation, sequential exoglycosidase digestion and methylation, clarified the structures of the acidic oligosaccharides. All were of the biantennary complex type. Their two peripheral branches were SO-4GalNAcβ1–4Glc-NAc and GalNAcβ1–4GlcNAc or GlcNAc in S-1, SO4-GalNAcβ1–4GlcNAc and Siaα2–6Galβ1–4GlcNAc in S-N, and (SO-4GalNAcB1–4G1cNAc)2 in S-2 (where GalNAc is N-acetylgalactosamine and G1cNAc is -acetylglucosamine). Ten percent of S-1 and of S-N had a bisecting GlcNAc residue. Sulphate residues occurred in nearly the same amount for both subunits; however, the α and β subunits were sulphated differently. S-1 predominated in the α subunit, while S-1 and S-2 were major components in the β subunit. In pLH isoforms, the amount of sulphate residue changed equally in both subunits, with a maximum two-fold difference. Thus, it seems most likely that subunit-specific sulphated oligosaccharides contribute primarily to charge heterogeneity in pLH isoforms. [ABSTRACT FROM PUBLISHER]