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

K. Hard, A. Mekking, J.B.L. Damm, J.P. Kamerling, W. De Boer, R.A. Wijnands, J.F.G. Vliegenthart

    Research output: Contribution to journalArticleAcademicpeer-review

    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-β-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 tri'-antennary (8%), and tetrasialylated tetraantennary (12%) N-acetyllactosamine type of carbohydrate chains, all bearing exclusively α2-3-linked N-acetylneuraminic acid (Neu5Ac). Previously, for pituitary follitropin mono-, di-, tri-, tri'-, and tetra-antennary oligosaccharides containing α2-3 as well as α2-6 linked Neu5Ac residues were reported. The bisecting GlcNAc residues present in native follitropin were not detected in the recombinant glycoprotein. Of the oligosaccharides 29% have an α1-6-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.
    Original languageEnglish
    Pages (from-to)263-271
    Number of pages9
    JournalEuropean Journal of Biochemistry
    Volume193
    Issue number1
    Publication statusPublished - 15 Jul 1990

    Keywords

    • carbohydrate
    • follitropin
    • recombinant protein
    • animal cell
    • article
    • cell culture
    • CHO cell line
    • hamster
    • nonhuman
    • priority journal

    Fingerprint

    Dive into the research topics of 'Isolation and structure determination of the intact sialylated N-linked carbohydrate chains of recombinant human follitropin expressed in Chinese hamster ovary cells'. Together they form a unique fingerprint.

    Cite this