Individual Glycosylation Sites Research Articles

Glycosylation of glycoproteins 52 and 65 encoded by the polycythemia-inducing strain of Friend spleen focus-forming virus. Isolation of glycopeptides containing individual glycosylation sites

The primary envelope gene product of the polycythemia-inducing strain of Friend spleen focus-forming virus, glycoprotein 52 (gp52), as well as its processed form, glycoprotein 65 (gp65), were isolated from virus-infected normal rat kidney cells metabolically labeled with [2-3H]mannose. Following digestion with trypsin, glycopeptides containing individual N-glycosylation sites were obtained by gel filtration and subsequent reversed-phase high-performance liquid chromatography. N-terminal amino acid sequencing of the glycopeptides demonstrated that only asparagine residues 11 and 26, located in the N-terminal domains of gp52 and gp65, carry carbohydrate substituents, while the potential N-glycosylation sites in the C-terminal portions of the molecules are not used. Carbohydrates attached were liberated by treatment with endo-beta-N-acetylglucosaminidase H or peptide: N-glycosidase F and characterized by high-performance liquid chromatography. The results demonstrated that gp52 carries similar patterns of oligomannosidic glycans in both positions. In gp65, however, asparagine residue 11 is almost exclusively substituted by complete, fucosylated N-acetyllactosaminic oligosaccharides, whereas asparagine residue 26 carries oligomannosidic or truncated N-acetyllactosaminic glycans.

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

Site specificity of the chorionic gonadotropin N-linked oligosaccharides in signal transduction

The role of the human chorionic gonadotropin (hCG) N-linked oligosaccharides in receptor binding and signal transduction was analyzed using site-directed mutagenesis and transfection studies. hCG derivatives with alterations at individual glycosylation sites were expressed in Chinese hamster ovary cells. Receptor binding studies showed that absence of any or all of the hCG N-linked oligosaccharides had only a minor effect on the receptor affinity of the derivatives. Similarly, absence of the N-linked oligosaccharides from the beta subunit or a single oligosaccharide from Asn-78 of alpha had no effect on the production of cAMP or on steroidogenesis. However, the absence of carbohydrate at Asn-52 of alpha decreases both the steroidogenic and cAMP responses. Furthermore, absence of this critical oligosaccharide unit on alpha unmasks differences in the two N-linked oligosaccharides on beta; the beta Asn-13 oligosaccharide but not the beta Asn-30 oligosaccharide plays a more important role in steroidogenesis. Dimers containing deglycosylated beta subunit and an alpha subunit lacking either the Asn-52 oligosaccharide or both oligosaccharides fail to stimulate cAMP or steroid formation. Moreover, these derivatives bind to receptor and behave as competitive antagonists. The use of site-directed mutagenesis was critical in uncovering site-specific functions of the hCG N-linked oligosaccharides in signal transduction and reveals the importance of the Asn-52 oligosaccharide in this process.

Differential Susceptibility toN-Glycanase at the Individual Glycosylation Sites of Mouse Thyrotropin and Free α-Subunits*

We have studied the differential susceptibility to N-glycanase (peptide-N4-[N-acetyl-beta-glucosaminyl]asparagine amidase) of oligosaccharides at the individual glycosylation sites of mouse TSH and free alpha-subunits. Mouse thyrotropic tumor tissue or hypothyroid pituitary tissue were incubated with D-[2-3H]mannose for 6 h. [3H]Mannose-labeled TSH or free alpha-subunits were obtained from homogenates using specific antisera and were digested with N-glycanase in their native state or after heat denaturation and reduction in the absence or presence of detergents. Tryptic fragments of the digestion products were then analyzed by reverse phase HPLC so that the effects of N-glycanase at the individual glycosylation sites could be determined. N-Glycanase treatment of native molecules did not cleave oligosaccharides efficiently at Asn56 of alpha-subunits and Asn23 of TSH beta, whereas oligosaccharides at Asn82 of alpha-subunits were more susceptible regardless of whether the alpha-subunits were combined with TSH beta. Heat denaturation, reduction, and the presence of detergents did not substantially increase the cleavage by N-glycanase of the protected oligosaccharides, suggesting that the primary structures of the TSH subunits influenced efficiency at specific sites. Pretreatment of free alpha-subunits with trypsin failed to enable N-glycanase to work fully, as oligosaccharides at Asn56 were cleaved less effectively than those at Asn82. Thus, the susceptibility to N-glycanase differs at the individual glycosylation sites of mouse TSH and free alpha-subunits, and these differences may result from effects of the primary structures of the TSH subunits.

Rates of Processing of the High Mannose Oligosaccharide Units at the Three Glycosylation Sites of Mouse Thyrotropin and the Two Sites of Free α-Subunits*

We have determined the structures of high mannose (Man) oligosaccharide units at individual glycosylation sites of mouse TSH. Mouse thyrotropic tumor tissue was incubated with D-[2-3H]Man with or without [14C]tyrosine ([14C] Tyr) for 2, 3, or 6 h, and for a 3-h pulse followed by a 2-h chase. TSH heterodimers or free alpha-subunits were obtained from homogenates using specific antisera. After reduction and alkylation, subunits were treated with trypsin. The tryptic fragments were then loaded on a reverse phase HPLC column to separate tryptic fragments bearing labeled oligosaccharides. The N-linked oligosaccharides were released with endoglycosidase-H and analyzed by paper chromatography. Man9GlcNac2 and Man8GlcNac2 units predominated at each time point and at each specific glycosylation site, but the processing of high Man oligosaccharides differed at each glycosylation site. The processing at Asn23 of TSH beta-subunits was slower than that at Asn56 or Asn82 of alpha-subunits. The processing at Asn82 was slightly faster than that at Asn56 for both alpha-subunits of TSH heterodimers and free alpha-subunits. The present study demonstrates that the early processing of oligosaccharides differs at the individual glycosylation sites of TSH and free alpha-subunits, perhaps because of local conformational differences.

Differential effects of oncogenic transformation on N-linked oligosaccharide processing at individual glycosylation sites of viral glycoproteins.

Hamster sarcoma virus (HSV) transformation of Nil-8 fibroblasts is associated with an increase in the average size of N-acetyllactosamine (complex) type N-linked glycans due to an increase in both the average number of branches/chain and in the fraction of N-linked glycans containing poly(GlcNAc(beta 1,3) Gal-(beta 1,4)) (polylactosaminylglycan) chains. Analysis of glycopeptides from the envelope glycoproteins of Sindbis virus and vesicular stomatitis virus (VSV) grown in Nil-8 and Nil/HSV cells indicated that the transformation-associated shift to larger N-linked oligosaccharides selectively affects some glycosylation sites far more than others. Glycosylation of the Sindbis virus glycoproteins and of Asn-179 of VSV G was similar in Nil-8 and Nil/HSV cells; oligosaccharide processing generally did not proceed beyond the biantennary complex stage. In contrast, Asn-336 of VSV G carried primarily biantennary complex glycans in Nil-8-grown virus (ratio, triantennary, and larger to biantennary complex glycans (tri+/bi) = 0.5) but more highly branched structures in Nil/HSV-grown virus (tri+/bi = 8.1). All of the triantennary or larger oligosaccharides from Asn-336 of Nil/HSV-grown VSV G bound to leukoagglutinating phytohemagglutinin-agarose, indicating the presence of a branch attached to the Man3GlcNAc2 core via a beta 1,6-linked GlcNAc residue and suggesting that increased UDP-GlcNAc:alpha-D-mannoside beta 1,6-N-acetylglucosaminyl transferase V (GlcNAc transferase V) activity accompanied transformation. At least 20% of these leukoagglutinating phytohemagglutinin-binding oligosaccharides were sensitive to an enzyme specific for polylactosaminylglycan chains, Escherichia freundii endo-beta-galactosidase.

Oligosaccharide processing at individual glycosylation sites on MOPC 104E immunoglobulin M. Differences in alpha 1,2-linked mannose processing.

Processing of the asparagine-linked oligosaccharides at the known glycosylation sites on the mu-chain of IgM secreted by MOPC 104E murine plasmacytoma cells was investigated. Oligosaccharides present on intracellular mu-chain precursors were of the high mannose type, remaining susceptible to endo-beta-N-acetylglucosaminidase H. However, only 26% of the radioactivity was released from [3H]mannose-labeled secreted IgM glycopeptides, consistent with the presence of high mannose-type and complex-type oligosaccharides on the mature mu-chain. [3H]Mannose-labeled cyanogen bromide glycopeptides derived from mu-chains of secreted IgM were isolated and analyzed to identify the glycopeptide containing the high mannose-type oligosaccharide from those containing complex-type structures. [3H]Mannose-labeled intracellular mu-chain cyanogen bromide glycopeptides corresponding to those from secreted IgM were isolated also, and the time courses of oligosaccharide processing at the individual glycosylation sites were determined. The major oligosaccharides on all intracellular mu-chain glycopeptides after 20 min of pulse labeling with [3H]mannose were identified as Man8GlcNAc2, Man9GlcNAc2, and Glc1Man9GlcNAc2. Processing of the oligosaccharide destined to become the high mannose-type structure on the mature protein was rapid. After 30 min of chase incubation the predominant structures of this oligosaccharide were Man5GlcNAc2 and Man6GlcNAc2 which were also identified on the high mannose-type oligosaccharide of the secreted mu-chain. In contrast, processing of oligosaccharides destined to become complex type was considerably slower. Even after 180 min of chase incubation, Man7GlcNAc2 and Man8GlcNAc2 were the predominant structures at some of these glycosylation sites. The isomeric structures of Man8GlcNAc2 obtained from all of the glycosylation sites were identical. Thus, the different rates of processing were not the result of a different sequence of alpha 1,2-mannose removal.

Carbohydrates of influenza virus. Structural elucidation of the individual glycans of the FPV hemagglutinin by two-dimensional 1H n.m.r. and methylation analysis.

The structures of the oligosaccharides of the hemagglutinin of fowl plague virus [influenza A/FPV/Rostock/34 (H7N1)] have been elucidated by one- and two-dimensional 1H n.m.r. spectroscopy at 500 MHz and by microscale methylation analysis. N-Glycosidic oligosaccharides of the oligomannosidic (OM) and of the N-acetyllactosaminic type have been found, the latter type comprising biantennary structures, without (A) or with (E) bisecting N-acetylglucosamine, and triantennary (C) structures. Analysis of the tryptic and thermolytic glycopeptides of the hemagglutinin allowed the allocation of these oligosaccharides to the individual glycosylation sites. Each attachment site contained a unique set of oligosaccharides. Asn12 contains predominantly structures C and E which are highly fucosylated. Asn28 contains OM and A structures that lack fucose and sulfate. Asn123 shows A that has incomplete antennae but is highly fucosylated and sulfated. Asn149 has fucosylated A and E. Asn231 shows fucosylated A and E with incomplete antennae. Asn406 has OM oligosaccharides. Asn478 has A and E with little fucose. Localization of the oligosaccharides on the three-dimensional structure of the hemagglutinin revealed that the oligomannosidic glycans are attached to glycosylation sites at which the enzymes responsible for carbohydrate processing do not have proper access. These observations demonstrate that an important structural determinant for the oligosaccharide side chains is the structure of the glycoprotein itself. In addition, evidence was obtained that the rate of glycoprotein synthesis also has an influence on carbohydrate structure.

Isolation of glycopeptides containing individual glycosylation sites of friend murine leukemia virus glycoprotein: Studies of glycosylation by methylation analysis

Glycopeptides containing individual N-glycosylation sites of the glycoprotein from Friend murine leukemia virus were isolated by digestion of the viral glycoprotein with protease of S. aureus (V8) or with trypsin followed by fractionation of the resulting (glyco)peptides by gel filtration and reversed-phase, high-performance liquid chromatography at pH 6. Isolated glycopeptides were assigned to the known amino acid sequence of the protein by amino acid analysis and by determination of the NH 2-termini. The carbohydrate moieties of each glycosylation site were analysed by methylation analysis. A high selectivity of the glycoprotein glycosylation was found with regard to the distribution of oligomannosidic, mixed, and N-acetyl-lactosaminic oligosaccharides.

Carbohydrates of influenza virus. V. Oligosaccharides attached to individual glycosylation sites of the hemagglutinin of fowl plague virus

The carbohydrate side chains of the hemagglutinin of fowl plague virus (A/FPV/Rostock/34 (H7N1)) have been localized by a procedure involving fragmentation of the polypeptide with cyanogen bromide and various proteases. The positions of the fragments were determined by radioactive labeling of the sugars and of specific amino acids. Side chains of the complex type I are attached to asparagine residues 12, 28, 123, 149, and 478. A mannose-rich (type II) side chain is linked to asparagine 406. Asparagine 231 is not glycosylated. The side chains attached to asparagine residues 12, 123, 149, and 478 contain sulfate. Glycopeptides derived by Pronase digestion from the individual attachment sites have been analyzed by their affinity to concanavalin A and Lens culinaris agglutinin. The results indicate that each glycosylation site has a typical set of heterogeneous oligosaccharides. Comparison of the glycosylation patterns of the hemagglutinins of FPV and other influenza A viruses reveals that the glycosylation sites at asparagine residues 12, 28, and 478, which are located at the base of the spike, are highly conserved. Mannose-rich side chains appear to be located preferentially at interfaces between the three monomers of a spike or between the globular and fibrous domains of a monomer.

Stable oligosaccharide microheterogeneity at individual glycosylation sites of a murine major histocompatibility antigen derived from a B-cell lymphoma.

The H-2Kk glycoprotein has been isolated by monoclonal antibody affinity chromatography, and an analysis of the asparagine-linked oligosaccharides present at the two major glycosylation sites has been performed. Antigen obtained from the AKTB-1b B-cell lymphoma that had been labeled with [2,6-3H]mannose for 5 or 21 h or for 5 h followed by a 5-h chase was digested exhaustively with trypsin. Each glycosylation site was then isolated by reverse phase high performance liquid chromatography using a C18 column. After removal from the peptide backbone by the almond emulsin peptide: N-glycosidase, the oligosaccharides from each isolated site were analyzed by gel filtration, ion exchange chromatography, concanavalin A affinity chromatography, and glycosidase treatment to assess the contribution of sialic acid and branching patterns of the oligosaccharide backbones to the overall microheterogeneity. The glycosylation of the H-2Kk antigen derived from several different AKTB-1b tumor preparations was examined during a period covering 1 year, during which time the tumor was passaged continuously in vivo in 2-week cycles. Our results conclusively demonstrate that the pattern of oligosaccharide microheterogeneity at the two glycosylation sites of the H-2Kk antigen derived from AKTB-1b cells is stable and that each site differs as to the specific array of oligosaccharide types found on the fully processed glycoprotein. In addition, this report describes an analytical scheme employing reverse phase high performance liquid chromatography to follow oligosaccharide processing and hydrolysis of the N-glycosidic bond by the peptide: N-glycosidase.

Host-dependent variation of asparagine-linked oligosaccharides at individual glycosylation sites of Sindbis virus glycoproteins.

We examined the Asn-linked oligosaccharides at individual glycosylation sites of the two envelope glycoproteins of Sindbis virus, E1 and E2. The analysis was done by separating tryptic glycopeptides by reverse phase high performance liquid chromatography and analyzing the oligosaccharides from isolated glycopeptides by gel filtration chromatography. Both E1 and E2 have two glycosylation sites each in virus grown in chick embryo fibroblasts, baby hamster kidney cells, and Chinese hamster ovary cells. Our results indicate that previously observed host-dependent variation in the oligosaccharides of Sindbis virus glycoproteins are probably attributable to differences in oligosaccharide composition at individual sites of E1 and E2 and not to differences in the frequency of glycosylation of these sites. One of the two glycosylation sites in E1 has exclusively complex type oligosaccharides regardless of the host cell type. However, the second glycosylation site in E1 has high mannose oligosaccharides in virus grown in chick embryo fibroblasts, complex oligosaccharides in virus from baby hamster kidney cells, and both complex and high mannose oligosaccharides in virus from Chinese hamster ovary cells. E2 has one glycosylation site which has primarily high mannose oligosaccharides and one site which has primarily complex type oligosaccharides. Certain sites which have predominantly complex type glycans also have a small number of two specific classes of oligosaccharides which are cleaved by endo-beta-N-acetylglucosaminidase H and may be high mannose type. Our findings are discussed with regard to regulation of oligosaccharide processing in animal cells.

Selective cleavage by endo-beta-N-acetylglucosaminidase H at individual glycosylation sites of Sindbis virion envelope glycoproteins.

Selective cleavage by endo-beta-N-acetylglucosaminidase H at individual glycosylation sites of Sindbis virion envelope glycoproteins.