Carbohydrate Structures of Human Tissue Plasminogen Activator Expressed in Chinese Hamster Ovary Cells

M W Spellman,L J Basa,C K Leonard,J A Chakel,J V O'Connor,S Wilson,H Van Halbeek

doi:10.1016/s0021-9258(18)71649-9

Abstract

Recombinant human tissue plasminogen activator (rt-PA), produced by expression in Chinese hamster ovary cells, is a fibrin-specific plasminogen activator which has been approved for clinical use in the treatment of myocardial infarction. In this study, the structures of the Asn-linked oligosaccharides of Chinese hamster ovary-expressed rt-PA have been elucidated. High mannose and hybrid oligosaccharides were released from the protein by endoglycosidase H digestion, whereas N-acetyllactosamine-type ("complex") oligosaccharides were released by peptide:N-glycosidase F digestion. The oligosaccharides were fractionated by gel permeation chromatography and anion exchange high performance liquid chromatography (HPLC), and their structures were analyzed by composition and methylation analysis, high pH anion exchange chromatography, fast atom bombardment-mass spectrometry (FAB-MS), and 500-MHz 1H NMR spectroscopy. High mannose oligosaccharides were found to account for 38% of the total carbohydrate content of rt-PA and consisted of Man5GlcNAc2, Man6GlcNAc2, and Man7GlcNAc2 in the ratio 1.8:1.7:1. Two hybrid oligosaccharides were identified and accounted for 3% of the carbohydrate of rt-PA. The N-acetyllactosamine-type oligosaccharides were found to comprise diantennary (34% of total carbohydrate), 2,4-branched triantennary (11%), 2,6-branched triantennary (9%), and tetraantennary (5%) structures. Sialylation of these oligosaccharides was by alpha (2----3) linkages to galactose. Most (greater than 90%) of the N-acetyllactosamine-type structures contained fucose alpha (1----6) linked to the Asn-linked N-acetylglucosamine residue. The distribution of oligosaccharide structures at individual glycosylation sites (Asn residues 117, 184, and 448) was also determined. rt-PA exists as two variants that differ by the presence (type I) or absence (type II) of carbohydrate at Asn-184. Tryptic glycopeptides were isolated by reversed phase high performance liquid chromatography and treated with peptide:N-glycosidase F. The oligosaccharides released from each glycosylation site were analyzed by high pH anion exchange chromatography. By this analysis, Asn-117 was demonstrated to carry exclusively high mannose oligosaccharides. When glycosylated, Asn-184 carried diantennary, 2,4-branched triantennary, 2,6-branched triantennary, and tetraantennary N- acetyllactosamine oligosaccharides in the ratio 9.0:4.5:1.4:1. Asn- 448 carried the same types of oligosaccharides, but in the ratio 7.5:1.6:2.1:1. The distributions of Asn-linked oligosaccharides at positions 117 and 448 were found not to be affected by the presence or absence of carbohydrate at position 184. The relevance of the

Highlights

Asnwhich has been approved for clinical use in the treat- 184 carried diantennary, 2,l-branched triantennary, ment of myocardial infarction.In this study, the struc-2,6-branchedtriantennarya,ndtetraantennary tures of the Asn-linkedoligosaccharides of Chinese N-acetyllactosamineoligosaccharidesintheratio hamster ovary-expressed rt-PA havbeeen elucidated. 9.0:4.5:1.4:1
The oligosaccharides were fractionated by The relevance of the structural analysisof the carbogel permeation chromatography and anion exchange hydrate moieties of rt-PA is evident from the obserhigh performance liquid chromatography (HPLC), anvdation that thespecific activity of type I1 tissue plastheirstructureswereanalyzed by composition and minogen activator (t-PA)is 30-50% greater than that methylation analysis, high pH anion exchange chro- of type I t-PA (Einarsson,M., Brandt, J., and Kaplan, matography, fast atom bombardment-mass spectrom- L. (1985)Biochim
Pohl et al [16] structurally characterized the carbohydrate chains of three glycopeptides from melanoma-derived t-PA and found that Asn-117 carried a mixture of high mannose oligosaccharides, Asn-184 a diantennary N-acetyllactosamine-type of oligosaccharide, and Asn-448 a tri- or tetraantennary N-acetyllactosamine-typeof carbohydrate chain

Summary

Introduction

The disleased fromthe proteinby endoglycosidase H digestion, tributions of Asn-linked oligosaccharides at positions whereas N-acetyllactosamine-type(“complex”) oligo- 117 and 448 were found not to be affected by the saccharides were released by peptide:N-glycosidase F presence or absence of carbohydrate at position 184. Pohl et al [16] structurally characterized the carbohydrate chains of three glycopeptides from melanoma-derived t-PA and found that Asn-117 carried a mixture of high mannose oligosaccharides, Asn-184 a diantennary N-acetyllactosamine-type of oligosaccharide, and Asn-448 a tri- or tetraantennary N-acetyllactosamine-typeof carbohydrate chain.

Results

Conclusion