Regulation of glycosylation. The influence of protein structure on N-linked oligosaccharide processing.

The Sindbis virus glycoproteins, E1 and E2, comprise a useful model system for evaluating the effects of local protein structure on the processing of N-linked oligosaccharides by Golgi enzymes. The conversion of oligomannose to N-acetyllactosamine (complex) oligosaccharides is hindered to different extents at the four glycosylation sites, so that the complex/oligomannose ratio decreases in the order E1-Asn139 greater than E2-Asn196 greater than E1-Asn245 greater than E2-Asn318. The processing steps most susceptible to interference were deduced from the oligosaccharide compositions at hindered sites in virus from baby hamster kidney cells (BHK), chick embryo fibroblasts (CEF), and normal and hamster sarcoma virus (HSV)-transformed hamster fibroblasts (Nil-8). Persistence of Man6-9GlcNAc2 was taken to indicate interference with alpha 2-mannosidase(s) I (alpha-mannosidase I), Man5GlcNAc2, with UDP-GlcNAc:alpha-D-mannoside beta 1----2-N-acetylglucosaminyltransferase I (GlcNAc transferase I), and unbisected hybrid glycans, with GlcNAc transferase I-dependent alpha 3(alpha 6)-mannosidase (alpha-mannosidase II). Taken together, the results indicate that all four sites acquire a precursor oligosaccharide with equally high efficiency, but alpha-mannosidase I, GlcNAc transferase I, and alpha-mannosidase II are all impeded at E2-Asn318 and, to a lesser extent, at E1-Asn245. In contrast, sialic acid and galactose transfer to hybrid glycans (in BHK cells) is virtually quantitative even at E2-Asn318. E2-Asn318 carried no complex oligosaccharides, but the structures of those at E1-Asn245 indicate almost complete GlcNAc transfer by UDP-GlcNAc:alpha-D-mannoside beta 1----2-N-acetylglucosaminyltransferase II (GlcNAc transferase II), galactosylation, and sialylation. Because the E2-Asn318 and E1-Asn245 glycans have previously been shown to be less accessible to a steric probe than those at E2-Asn196 or E1-Asn139, a simple explanation for these results would be that alpha-mannosidase I, GlcNAc transferase I, and alpha-mannosidase II are more susceptible to steric hindrance than are the later processing steps examined. Finally, in addition to these site-specific effects, the overall extent of viral oligosaccharide processing varied with host and cellular growth status. For example, alpha-mannosidase I processing is more complete in BHK cells compared to CEF, and in confluent Nil-8 cells compared to subconfluent or HSV-transformed Nil-8 cells.

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.

Synthesis of the N-linked oligosaccharides of glycoproteins. Assembly of the lipid-linked precursor oligosaccharide and its relation to protein synthesis in vivo.

The asparagine-linked oligosaccharides of chick embryo fibroblast glycoproteins were previously shown to derive from a common lipid-linked precursor, Glc3Man9GlcNAc2. The formation of this precursor oligosaccharide was examined in intact chick embryo fibroblasts, NIL-8 cells, and Chinese hamster ovary cells. The labeling kinetics and compositions of the lipid-linked oligosaccharides were examined, and the results indicate that lipid-linked Man5GlcNAc2 is rapidly assembled (< 1.5 min) and then extended (< 2.5 min) to Glc3Man9GlcNAc2 via the intermediate Man8GlcNAc2. Chain elongation from Man5GlcNAc2- to Man8GlcNAc2-lipid probably occurs by addition of single mannose residues. The pool of lipid-linked Glc3Man9GlcNAc2 turns over with a half-time of 3.5 to 6 min; since there is little if any degradation (the mannose residues do not turn over), this reflects the rate at which completed chains are transferred to acceptor proteins. The same intermediates and similar kinetics were observed in all three cell types. Oligosaccharide-lipid assembly was also examined in cells in which protein synthesis was decreased (using actinomycin D to depress levels of mRNA) or abolished (using cycloheximide). The results indicate that the rate of oligosaccharide-lipid synthesis is proportional to the rate of protein synthesis. The regulated step is prior to the Man5GlcNAc2 stage, and we suggest that the most likely control mechanism is limitation of available oligosaccharide carrier lipid.

Glycerophospholipid variation in choline and inositol auxotrophs of Neurospora crassa. Internal compensation among zwitterionic and anionic species.

The glycerophospholipids of cultures of Neurospora crassa were extracted, deacylated, and analyzed. In addition to a wild-type strain, several auxotrophic mutant strains were examined: chol-1 (defective S-adenosylmethionine: phosphatidylethanolamine methyltransferase), chol-2 (defective S-adenosyl methionine:phosphatidylmonomethylethanolamine (dimethylethanolamine) methyltransferase), and inos (defective myoinositol-1-phosphate phosphatase). In addition, a double mutant strain, chol-1;chol-2, was constructed. Cultures of the mutant strains grown with concentrations of supplement(s) just adequate to support growth had bizarre phospholipid compositions. By appropriate choice of mutant and supplement(s), it was possible to vary the relative level of every phospholipid of the organism, with the exception of cardiolipin. The maximum ranges encountered for the zwitterionic species, expressed as per cent of total phospholipid phosphorus, were lecithin (0.9 to 53.1%), phosphatidyldimethylethanolamine (0.0 to 55.5%), phosphatidylmonomethylethanolamine (0.0 to 53.9%), and phosphatidylethanolamine (9.8 to 43.3%). For the anionic species, the ranges were phosphatidylserine (1.7 to 10.4%) and phosphatidylinositol (3.6 to 25.1%). Despite the wide variation of the relative proportions of the individual phospholipid species, five quantities remained constant: the cardiolipin content, the total phospholipid content, the total content of the zwitterionic species, the total content of the anionic species, and the ratio of the zwitterionic to anionic totals. The data suggest the existence of an internal compensation mechanism, the net effect of which is maintenance of a fairly constant contribution by the phospholipid components to the over-all membrane charge.

HLA-DR antigens of autologous melanoma and B lymphoblastoid cell lines: differences in glycosylation but not protein structure.

The HLA-DR antigen expressed on the surface of the human melanoma cell line SK-MEL-37 was characterized and compared with the HLA-DR antigen from the MU B lymphoblastoid cell line originating from the same individual. The HLA-DR heavy chain from SK-MEL-37 cells had an apparent mobility on SDS-PAGE slightly slower than that isolated from MU cells. In contrast, the HLA-DR light chains and the HLA-A,-B heavy chains from the two cell lines had identical mobilities. Double-labeled tryptic peptide mapping and limited N-terminal sequencing showed that the SK-MEL-37 HLA-DR antigen, like all previously examined B lymphoblastoid cell HLA-DR antigens, was homologous to the murine I-E/C subregion antigens and that the mobility difference of the SK-MEL-37 HLA-DR heavy chain was not attributable to differences in the primary structure of the polypeptide. Treatment of the cells with tunicamycin abolished the m.w. difference, suggesting that it was due to a change in glycosylation in SK-MEL-37. This was confirmed by analysis of the glycopeptides from pronase-digested HLA-DR light and heavy chains and HLA-A,B heavy chains purified from the two cell types. The results suggest 1) there is a difference in asparagine-linked oligosaccharide processing in the two cell types, with more of the larger complex glycans synthesized in the melanoma cells than in the B lymphoblastoid cells, 2) the effect is more pronounced with HLA-DR heavy chains than with HLA-DR light chains or HLA-A,B heavy chains, and 3) the oligosaccharide size difference is not solely due to sialic acid content.

Enrichment of insertional mutants following retrovirus gene trap selection.

The present study has investigated the use of gene trap retroviruses as insertional mutagens. A gene trap vector (U3Hygro) was used to target single-copy thymidine kinase (tk) genes, present at different sites in the genome. Cell populations isolated by gene trap selection contained a higher proportion of insertional mutants as compared with nonselected cells containing randomly integrated viruses. The number of integration events required to observe loss of gene function was reduced from 8-40 x 10(6) to 2-10 x 10(4), an overall enrichment of 100- to 1000-fold. The feasibility of targeting normally diploid genes was also demonstrated in hypodiploid Chinese hamster ovary cells. The cellular gene encoding GlcNAc transferase I was disrupted in one wheat germ agglutinin resistant clone selected from a total of 5 x 10(4) gene trap events. The clone was nullizygous for GlcNAc transferase I, indicating that the allele opposite the provirus was lost as a result of preexisting hemizygosity or by loss of heterozygosity. Finally, the total number of genes in the genome that could activate the expression of retrovirus gene traps was estimated at between 2 x 10(4) and 10(5), suggesting that most expressed genes can be mutagenized by gene trap selection.

Generation of Chinese hamster ovary cell glycosylation mutants by retroviral insertional mutagenesis. Integration into a discrete locus generates mutants expressing high levels of N-glycolylneuraminic acid.

Retroviral insertional mutagenesis can both generate somatic cell mutants and pinpoint the genomic locus associated with a mutant phenotype. In the present study, this approach was applied to Chinese hamster ovary cells (CHO) made susceptible to Moloney murine leukemia virus (MoMuLV) infection by stable expression of an ecotropic retrovirus receptor. These CHO cells were infected with a replication incompetent MoMuLV construct with a promoterless hygromycin phosphotransferase (hygro) gene inserted into the U3 region of the long terminal repeat and a second selectable marker, neomycin phosphotransferase (neo), expressed from an internal promoter. CHO clones containing integrated proviruses were selected with hygromycin or G418, and the subset of these with reduced cell surface Neu5Ac were then selected with wheat germ agglutinin (WGA). The majority of the resulting clones had a phenotype not previously described for WGA-resistant CHO mutants arising spontaneously or from chemical mutagenesis: Neu5Ac was almost completely replaced by Neu5Gc. We have provisionally termed these clones SAP mutants, for sialic acid phenotype. Southern analysis of HindIII digested DNA from four SAP mutants revealed that the MoMuLV provirus is present in a 10.4-kilobase (kb) fragment. Probing with a flanking CHO sequence resulted in equivalent hybridization to a 4.6-kb fragment and the 10.4-kb provirus-containing fragment in all four cases, while uninfected parental cells and non-SAP glycosylation mutants generated in the same retrovirus insertional mutagenesis experiments yielded only the 4.6-kb fragment. Sequencing of the 3'-flanking DNA revealed that each of the four SAP mutants had a unique provirus integration site falling within a 796 bp region of the CHO genome. The frequency with which SAP mutants arise suggests that this may be a preferred site for retrovirus integration.

Arrangement of glucose residues in the lipid-linked oligosaccharide precursor of asparaginyl oligosaccharides.

The lipid-linked oligosaccharide synthesized in vitro, in the presence of 1.0 microM UDP-[3H]Glc, GDP-[14C]Man, and UDP-GlcNAc has been isolated and the structure of the oligosaccharide has been analyzed. The oligosaccharide contains 2 N-acetylglucosamine, 9 mannose, and 3 glucose residues. The N-acetylglucosamine residues are located at the reducing terminus. The 3 glucose residues are arranged in a linear order at one of the nonreducing termini in the sequence Glc 1,2--Glc 1,3--Glc--(Man)9 (GlcNAc)2. The structural analysis was made possible largely by the availability of glucosidase preparations of fungal anad microsomal origin which remove glucose residues from the oligosaccharide without releasing mannose residues.

alpha-D-Mannosidases of rat liver Golgi membranes. Mannosidase II is the GlcNAcMAN5-cleaving enzyme in glycoprotein biosynthesis and mannosidases Ia and IB are the enzymes converting Man9 precursors to Man5 intermediates.

Current evidence indicates that the trimming of mannosyl residues from intermediates in the biosynthesis of the N-linked oligosaccharides of glycoproteins occurs in the Golgi complex. We now present evidence that mannosidase II (Tulsiani, D. R. P., Opheim, D. J., and Touster, O. (1977) J. Biol Chem. 252, 3227-3233) is the Golgi enzyme that converts GlcNAc Man5 species to GlcNAcMan3 species in completing the mannosyl trimming process required in the biosynthesis of complex type glycoproteins. GlcNAc([3H]Man)5GlcNAc-mannosidase and p-nitrophenyl alpha-D-mannosidase activities copurify throughout the preparative procedure and show the same properties. In addition to mannosidase IA (Tabas, I., and Kornfeld, S. (1979) J. Biol. Chem. 254, 11655-11663), a second alpha-1,2-mannosidase (mannosidase IB) can be prepared from Golgi membranes which is effective in converting Man9GlcNAc to Man5GlcNAc. The two alpha-1,2-mannosidases are very similar in catalytic properties, but they are also distinguishable by several criteria. Although these two enzymes have not been extensively purified, several lines of evidence lead to the tentative conclusion that they are distinct enzymes. They appear to be present in comparable activities in the Golgi membranes and together account for the alpha-1,2-mannosidase activity of these membranes. The particular role of each alpha-1,2-mannosidase remains to be determined.

Glycosylation of the T-cell antigen-specific receptor and its potential role in lectin-mediated cytotoxicity.

Cytotoxic T lymphocytes (CTLs) normally destroy only those cells ("target cells") whose surface antigens they recognize. However, in the presence of lectins such as Con A, CTLs destroy virtually any cell, regardless of its antigens. The oligosaccharides of the T-cell antigen-specific receptor, a dimeric surface glycoprotein composed of disulfide-linked alpha and beta subunits, are of interest because of their potential involvement in this lectin-dependent cytotoxic activity. We report here that three or four asparagine-linked oligosaccharides could be enzymatically removed from each of the receptor subunits expressed by a cloned line of murine CTLs (clone 2C), consistent with the presence of glycosylation sites deduced from cDNA sequences of the alpha and beta genes expressed in this clone. All the N-linked glycans on the alpha subunit were of the complex type (i.e., resistant to endoglycosidase H), but the beta subunit carried two or three endoglycosidase H-sensitive (high-mannose) oligosaccharides. High-mannose glycans can bind tightly to Con A and, indeed, this lectin was found to bind specifically to solubilized 2C T-cell receptor. The Con A-dependent cytotoxic activity of clone 2C, but not of other CTL clones, was inhibited by a monoclonal antibody (1B2) that is specific for the T-cell receptor of clone 2C. Antibody 1B2 also inhibited clone 2C cytotoxicity mediated by phytohemagglutinin, lentil-lectin, and wheat-germ agglutinin. These results suggest that, although lectin-dependent lysis of target cells by CTLs is antigen nonspecific, the cytolytic activity can be triggered by binding of the lectin to the T-cell antigen-specific receptor.

A phosphorylated, disulfide-linked membrane protein in murine cytotoxic T lymphocytes.

The previously determined sequence of the murine T-cell gamma gene and its transcription in cloned T lymphocytes suggests that the polypeptide encoded by this gene is generally present in cytotoxic T cells as a 33-kDa monomer in a disulfide-bonded dimer. The gamma chain is also expected to be phosphorylated because a sequence in its cytoplasmic domain is homologous to an active site for serine phosphorylation in the regulatory subunit of cAMP-dependent protein kinase. We describe here a cytotoxic-T-cell-associated phosphorylated protein, many of whose properties suggest that it may be the product of the T-cell gamma gene. Its phosphorylation is greatly enhanced by interleukin 2 stimulation.

Genes for immunodominant protein antigens are highly homologous in Mycobacterium tuberculosis, Mycobacterium africanum, and the vaccine strain Mycobacterium bovis BCG.

The relatedness of immunodominant protein antigens in Mycobacterium tuberculosis, M. africanum, and M. bovis BCG was investigated by comparing the genes that encode major protein antigens in M. tuberculosis with their counterparts in the other two mycobacteria. Genes encoding homologs of M. tuberculosis major protein antigens were isolated from M. africanum and M. bovis BCG by constructing lambda gt11 recombinant DNA expression libraries and screening them with murine monoclonal antibodies and DNA probes. The antibodies were directed against four major protein antigens of M. tuberculosis with molecular masses of 71, 65, 19, and 14 kilodaltons. The isolated M. africanum and M. bovis BCG DNA clones were mapped with restriction endonucleases, and the maps of the mycobacterial genes were confirmed by Southern analysis of mycobacterial genomic DNA. The restriction maps of DNA containing the four genes in M. tuberculosis, M. africanum, and M. bovis BCG are identical, indicating that the immunodominant proteins that they encode are highly homologous in the three mycobacteria. Thus, the immunity against tuberculosis engendered by M. bovis BCG vaccination could be provided, at least in part, by the immune response to these homologous antigens.