Open AccessIsolation and structure determination of the intact sialylated N‐linked carbohydrate chains of recombinant human follitropin expressed in Chinese hamster ovary cells
Author(s) -
HÅRD Karl,
MEKKING Albert,
DAMM Jan B. L.,
KAMERLING Johannis P.,
BOER Willem,
WIJNANDS Robert A.,
VLIEGENTHART Johannes F. G.
Publication year - 1990
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1990.tb19332.x
Subject(s) - chinese hamster ovary cell , chemistry , biochemistry , glycoprotein , recombinant dna , oligosaccharide , asparagine , glycosylation , carbohydrate conformation , glycan , carbohydrate , residue (chemistry) , enzyme , polysaccharide , gene , receptor
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‐ N 4 ‐( 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‐NH 2 . The structures of the carbohydrate chains were determined by 500‐ or 600‐MHz 1 ‐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.