Isolation and structure determination of the intact sialylated N‐linked carbohydrate chains of recombinant human follitropin expressed in Chinese hamster ovary cells

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Abstract
Biologically active recombinant human follitropin has been expressed in Chinese hamster ovary cells. The carbohydrate chains of the recombinant glycoprotein hormone were enzymatically released by peptide-N4-(N-acetyl-.beta.-glucosaminyl)-asparagine amidase F. The oligosaccharides were separated from the N-deglycosylated protein by gel-permeation chromatography on Bio-Gel P-100, and fractionated by a combination of FPLC on Mono Q and HPLC on Lichrosorb-NH2. The structures of the carbohydrate chains were determined by 500- or 600-MHz 1H-NMR spectroscopy. The following types of carbohydrates occur: monosialylated diantennary (10%), disialylated diantennary (43%), disialylated tri-antennary (5%), trisialylated tri-antennary (13%), trisialylated triantennary (8%), and tetrasialylated tetraantennary (12%) N-acetyllactosamine type of carbohydrate chains, all bearing exclusively .alpha.2-3-linked N-acetylneuraminic acid (Neu5Ac). Previously, for pituitary follitropin mono-, di-, tri-, tri''-, and tetra-antennary oligosaccharides containing .alpha.2-3- as well as .alpha.2-6-linked Neu5Ac residues were reported. The bisecting GlcNAc residues prssent in native follitropin were not detected in the recombinant glycoprotein. Of the oligosaccharides 29% have an .alpha.1-6 linked Fuc residue at the asparagine-bound GlcNAc, whereas this amount is about 50% in pituitary follitropin. In some of the tri-, tri''- and tetra-antennary oligosaccharide fractions small amounts (< 5%) of compounds were detected having one or more additional N-acetyllactosamine units.

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