Structure of the acidic N‐linked carbohydrate chains of the 55‐kDa glycoprotein family (PZP3) from porcine zona pellucida

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Abstract
N-linked carbohydrate chains of the major 55-kDa family, PZP3, of porcine zona pellucida glycoproteins are composed of neutral (28%) and acidic (72%) complex-type chains. The structures of the main components of the neutral chain have been established [Noguchi, S., Hatanaka, Y., Tobita, T. & Nakano, M. (1992) Eur. J. Biochem. 204, 1089–1100]. Here we report the structures of the acidic chains. Only two kinds of acidic fragments were released from PZP3 by endo-β-galactosidase digestion following β-elimination of O-linked chains. 500-MHz one-dimensional and two-dimensional 1H-NMR spectroscopy revealed their structures to be Siaα(2–3)Galβ(1–4)[HSO3-6]GlcNAcβ(1–3)Gal and HSO3-6GlcNAcβ(1–3)Gal, showing that the sulfate-containing acidic chains are constructed with non-branched N-acetyllactosamine repeats which have sialic acid(s) at the non-reducing end(s) and sulfate at the C-6 position of GlcNAc residues. The acidic N-linked chains obtained from PZP3 by hydrazinolysis were separated into diantennary chains (34%) and tri- and tetra-antennary chains (66%) by concanavalin-A–agarose gel chromatography. The diantennary chains and their sialidase digests were fractionated by DEAE-HPLC. From the analyses of the endo-β-galactosidase digests of each fraction, structures of the diantennary acidic chains were determined. They are classified into four groups. The first group is the sialylated chains without the sulfated N-acetyllactosamine repeating unit. The other three groups have the chains of various lengths differing in the number of monosulfated N-acetyllactosamine unit. These chains are extended from the Manα(1–3) branch of the trimannosyl core in the second group, from the Manα(1–6) branch in the third group, and from both branches in the fourth group. The structural features of the tri- and tetra-antennary acidic chains are also presented.