Open AccessPrimary structure of N‐linked carbohydrate chains of a human chimeric plasminogen activator K 2 tu‐PA expressed in Chinese hamster ovary cells
Author(s) -
BERGWERFF Aldert A.,
OOSTRUM Jan,
ASSELBERGS Fred A. M.,
BÜRGI Rolf,
HOKKE Cornelis H.,
KAMERLING Johannis P.,
VLIEGENTHART Johannes F. G.
Publication year - 1993
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1993.tb17702.x
Subject(s) - chinese hamster ovary cell , sialic acid , chemistry , biochemistry , glycosylation , glycan , plasminogen activator , peptide sequence , fast protein liquid chromatography , amidase , carbohydrate conformation , glycoprotein , microbiology and biotechnology , biology , enzyme , polysaccharide , gene , receptor , endocrinology
A recombinant human plasminogen activator hybrid variant K 2 tu‐PA, expressed in Chinese hamster ovary cells, is partially glycosylated at Asn12 (A chain, kringle‐2 domain) and completely glycosylated at Asn247 (B chain, protease domain). After release of the N‐linked carbohydrate chains by peptide‐ N 4 ‐( N ‐acetyl‐β‐glucosaminyl)asparagine amidase F, the oligosaccharides were separated from the protein by gel permeation chromatography, then fractionated by FPLC on Mono Q, followed by HPLC on Lichrosorb‐NH 2 , and analysed by 500‐MHz 1 H‐NMR spectroscopy. The following types of carbohydrates occur: monosialylated diantennary (8%), disialylated diantennary (45%), disialylated tri‐ and trí‐antennary (1%), trisialylated tri‐ and trí‐antennary (28%), and tetrasialylated tetra‐antennary (18%) structures, all having fucose in α(1‐6)‐linkage at the Asn‐bound N ‐acetylglucosamine. Sialic acid occurred exclusively in α(2‐3)‐linkage to galactose, and consisted of N ‐acetylneuraminic acid (94%), N ‐glycolylneuraminic acid (3%), and N ‐acetyl‐9‐ O ‐acetylneuram‐inic acid (3%). In addition, glycopeptide fragments corresponding with the A or B chain of K 2 tu‐PA were analysed. The oligosaccharides attached to Asn12 are less processed than those attached to Asn247. Comparison of the glycosylation pattern of K 2 tu‐PA with that of tissue‐type plasminogen activator from different biological sources showed significant differences. Profiling studies on different K 2 tu‐PA production batches demonstrated that the structures of N‐linked oligosaccharides were identical, but that relative amounts vary with the applied isolation procedure of the chimeric glycoprotein.