Primary structure of N‐linked carbohydrate chains of a human chimeric plasminogen activator K2tu‐PA expressed in Chinese hamster ovary cells

Abstract

A recombinant human plasminogen activator hybrid variant K₂tu‐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‐N⁴‐(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‐NH₂, and analysed by 500‐MHz ¹H‐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 K₂tu‐PA were analysed. The oligosaccharides attached to Asn12 are less processed than those attached to Asn247. Comparison of the glycosylation pattern of K₂tu‐PA with that of tissue‐type plasminogen activator from different biological sources showed significant differences. Profiling studies on different K₂tu‐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.