The influence of carbohydrate structure on the clearance of recombinant tissue-type plasminogen activator.

Modification of the carbohydrate structures of recombinant tissue-type plasminogen activator (rt-PA) can increase or decrease its rate of clearance in rabbits. When rt-PA was treated with sodium periodate to oxidize carbohydrate residues, the rate of clearance was decreased from 9.6 +/- 1.9 ml min-1 kg-1 to 3.5 +/- 0.6 ml min-1 kg-1 (mean +/- SD, n = 5). A similar change in the clearance of rt-PA was introduced by the use of endo-beta-N-acetyl-glucosaminidase H (Endo-H), which selectively removes high mannose asparagine-linked oligosaccharides; the clearance of Endo-H-treated rt-PA was 5.0 +/- 0.5 ml min-1 kg-1. A mutant of rt-PA was produced with an amino acid substitution at position 117 (Asn replaced with Gln) to remove a potential glycosylation site that normally contains a high mannose structure. The clearance of this material was also decreased, similar to the periodate and Endo-H-treated rt-PA. Conversely, when rt-PA was produced in the CHO 15B cell line, which can produce only high mannose oligosaccharide structures on glycoproteins, the clearance was increased by a factor of 1.8. These results demonstrate that the removal of rt-PA from the blood depends significantly upon the nature of its oligosaccharide structures.

Carbohydrate structures of recombinant soluble human CD4 expressed in Chinese hamster ovary cells.

Infection of T-lymphocytes and macrophages by human immunodeficiency virus (HIV) is mediated by the binding of the HIV envelope glycoprotein to the cell-surface receptor glycoprotein CD4. A soluble, recombinant CD4 molecule (rCD4), produced by expression of a truncated CD4 gene in Chinese hamster ovary (CHO) cells [Smith et al. (1987) Science 238, 1704-1707], is in clinical trials as a potential therapeutic agent in the treatment of acquired immunodeficiency syndrome (AIDS). In the present study, the structures of the Asn-linked oligosaccharides of soluble rCD4 have been elucidated. The rCD4 molecule has two potential sites for N-glycosylation, Asn-271 and Asn-300. Tryptic glycopeptides containing either of the sites were purified by reversed-phase HPLC, and their oligosaccharides were released enzymatically. The structures of the oligosaccharides were determined by methylation analysis, high-pH anion-exchange chromatography, fast-atom bombardment mass spectrometry, and 1H NMR spectroscopy at 500 MHz. Asn-271 was found to carry diantennary N-acetyllactosamine-type ("complex") oligosaccharides, of which 8% were asialo, 55% were monosialyl, and 37% were disialyl. Approximately 18% of these structures contained fucose alpha(1-->6) linked to the reducing GlcNAc residue. Two different hybrid structures were found to account for 34% of the oligosaccharides attached to Asn-300. The remainder of the oligosaccharides attached to Asn-300 were diantennary N-acetyllactosamine-type, of which 10% were asialo, 61% were monosialyl, and 29% were disialyl. Approximately 9% of the hybrid structures and 40% of the N-acetyllactosamine structures at Asn-300 were found to contain fucose alpha(1-->6) linked to the innermost GlcNAc residue.

Tissue plasminogen activator has an O-linked fucose attached to threonine-61 in the epidermal growth factor domain.

An unusual type of glycosylation has been observed for tissue plasminogen activator (t-PA). The monosaccharide fucose is glycosidically linked to threonine-61 in the epidermal growth factor region of t-PA. The presence of O-linked fucose was demonstrated by carbohydrate analysis and mass spectrometry of tryptic and chymotryptic peptides that contain this site. The susceptibility of the fucose residue to alpha-fucosidase indicated that it was in the alpha-anomeric configuration. Fucosylation of threonine-61 was observed in t-PA isolated from the Bowes melanoma cell line and from recombinant expression systems using Chinese hamster ovary or human embryonic kidney cells. Fucosylation of the homologous residue in prourokinase has also been reported recently. Our results indicate that this novel type of glycosylation may be common to the epidermal growth factor domains found in coagulation and fibrinolytic proteins and, therefore, suggest that the modification may have functional significance.

Assignment of intrachain disulfide bonds and characterization of potential glycosylation sites of the type 1 recombinant human immunodeficiency virus envelope glycoprotein (gp120) expressed in Chinese hamster ovary cells.

This report describes the structural characterization of the recombinant envelope glycoprotein (rgp120) of human immunodeficiency virus type 1 produced by expression in Chinese hamster ovary cells. Enzymatic cleavage of rgp120 and reversed-phase high performance liquid chromatography were used to confirm the primary structure of the protein, to assign intrachain disulfide bonds, and to characterize potential sites for N-glycosylation. All of the tryptic peptides identified were consistent with the primary structure predicted from the cDNA sequence. Tryptic mapping studies combined with treatment of isolated peptides with Staphylococcus aureus V8 protease or with peptide:N-glycosidase F followed by endoproteinase Asp-N permitted the assignment of all nine intrachain disulfide bonds of rgp120. The 24 potential sites for N-glycosylation were characterized by determining the susceptibilities of the attached carbohydrate structures to peptide:N-glycosidase F and to endo-beta-N-acetylglucosaminidase H. Tryptic mapping of enzymatically deglycosylated rgp120 was used in conjunction with Edman degradation and fast atom bombardment-mass spectrometry of individually treated peptides to determine which of these sites are glycosylated and what types of structures are present. The results indicate that all 24 sites of gp120 are utilized, including 13 that contain complex-type oligosaccharides as the predominant structures, and 11 that contain primarily high mannose-type and/or hybrid-type oligosaccharide structures.

Carbohydrate structures of human tissue plasminogen activator expressed in Chinese hamster ovary cells.

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

Study of the primary structure of recombinant tissue plasminogen activator by reversed-phase high-performance liquid chromatographic tryptic mapping.

Two high-resolution tryptic maps have been developed for recombinant tissue plasminogen activator (rt-PA) that separate the expected 51 tryptic peptides. The trypsin digestion was performed after reduction and S-carboxymethylation of the protein. The high-performance liquid chromatographic separation of the tryptic peptides used a Nova-Pak C18 (5 microns) column with a mobile phase that contained 0.1% aqueous trifluoroacetic acid (TFA) or 50 mM sodium phosphate (pH 2.85) and a linear gradient of acetonitrile. A TFA solvent system was also used for re-purification and for characterization of the peptides isolated from the phosphate-based separation. All of the isolated peptides had compositions consistent with the sequence proposed for rt-PA. The identities of the glycopeptides were confirmed by lectin chromatography on concanavalin A-Sepharose. The mixture of tryptic peptides was also treated with endo-beta-N-acetylglucosaminidase H and peptide:N-glycosidase F to locate the position of either high mannose or complex oligosaccharides. These studies demonstrated that a high mannose oligosaccharide is attached to Asn-117 while complex carbohydrate side-chains are attached to Asn-184 and Asn-448. The residue Asn-184 is the site of optional glycosylation that results in the formation of two rt-PA variants that contain either two or three oligosaccharides.