High Mannose Oligosaccharide Units Research Articles

Biosynthesis and processing of human immunodeficiency virus type 1 envelope glycoproteins: effects of monensin on glycosylation and transport

When human immunodeficiency virus type 1 envelope glycoproteins were expressed in 293 cells by using a recombinant adenovirus expression vector, the envelope precursor (gp160) was initially glycosylated by cotranslational addition of N-linked high-mannose oligosaccharide units to the protein backbone and then cleaved to gp120 and gp41. The subunits gp120 and gp41 were then further modified by the addition of fucose, galactose, and sialic acid, resulting in glycoproteins containing a mixture of hybrid and complex oligosaccharide side chains. A fraction of glycosylated gp160 that escaped cleavage was further modified by the terminal addition of fucose and galactose, but the addition of sialic acid did not occur, consistent with the notion that it is compartmentalized separately from the gp120 envelope protein. Processing and transport of gp160 were blocked by the monovalent ionophore monensin, which at high concentrations (25 microM and above) was a potent inhibitor of the endoproteolytic cleavage of gp160; at lower concentrations (1 to 10 microM), it selectively blocked the secondary glycosylation steps so that smaller products were produced. Monensin (1 microM) treatment also resulted in a reduction in syncytium formation, which was observed when recombinant infected cells were cocultivated with CD4-bearing HeLa cells. The infectivity of human immunodeficiency virus type 1 was also reduced by monensin treatment, a decrease that may be due to incompletely glycosylated forms of gp120 that have a lower affinity for the CD4 receptor.

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.

The high mannose oligosaccharides of Dictyostelium discoideum glycoproteins contain a novel intersecting N-acetylglucosamine residue.

Previous studies have shown that the majority of the asparagine-linked high mannose oligosaccharide units present on the lysosomal enzymes of the slime mold Dictyostelium discoideum contain mannose 6-sulfate and mannose 6-phosphate residues, the latter being diesterified to methyl groups. Using a variety of techniques, including NMR spectroscopy and fast atom bombardment mass spectrometry, we have found that many of these oligosaccharides also contain an N-acetylglucosamine residue linked beta 1-4 to the mannose residue linked alpha 1-6 to the beta-linked core mannose. (Formula: see text) As far as we are aware, this is the first description of an intersecting N-acetylglucosamine residue in this position on a high mannose oligosaccharide.

The removal of carbohydrates from ricin with endoglycosidases H, F and D and α-mannosidase

Recently, several investigators have explored the possibility of targetting ricin to designated cell types in animals by its linkage to specific antibodies. There is evidence, however, that the mannose-containing oligosaccharide chains on ricin are recognised by reticuloendothelial cells in the liver and spleen and so cause the immunotoxins to be removed rapidly from the blood stream. In the present study we analysed the carbohydrate composition of ricin and examined enzymic methods for removing the carbohydrate. The carbohydrate analysis ricin A-chain revealed the presence of one residue of xylose and one of fucose in addition to mannose and N-acetylglucosamine which had been detected previously. The B-chain contained only mannose and N-acetylglycosamine. Ricin A-chain is heterogeneous containing two components of molecular weight 30 000 and 32 000. Strong evidence was found that the heavier form of the A-chain contains an extra carbohydrate unit which is heterogeneous with respect to concanavalin A binding and sensitivity to endoglycosidase H. The lower molecular weight form of A-chain did not bind concanavalin A and was insusceptible to endoglycosidases. Only one of the two high mannose oligosaccharide units on the isolated B-chain could be removed by endoglycosidases H or F, whereas both were removable after denaturation of the polypeptide by SDS. Both the isolated A- and B-chains were sensitive to α-mannosidase. Intact ricin was resistant to endoglycosidase treatment and was only slightly sensitive to α-mannosidase. The addition of SDS allowed endoglycosidase H to remove both of the B-chain oligosaccharides from intact ricin and increased the toxin's sensitivity to α-mannosidase. In conclusion, extensive enzymic deglycosylation of ricin may only be possible if the A- and B-chains are first separated, treated with enzymes and then recombined to form the toxin.

Lysosomal enzyme phosphorylation. Recognition of a protein-dependent determinant allows specific phosphorylation of oligosaccharides present on lysosomal enzymes.

We have investigated the basis for the specific recognition of lysosomal enzymes by UDP-GlcNAc:lysosomal enzyme N-acetylglucosaminylphosphotransferase. This enzyme is responsible for the selective phosphorylation of mannose residues on lysosomal enzymes. Two mammalian lysosomal enzymes, cathepsin D and uteroferrin, and two nonlysosomal glycoproteins were treated with endo-beta-N-acetylglucosaminidase H to remove those high mannose oligosaccharide units which are accessible on the native protein. These proteins were then tested as inhibitors of three different glycosyltransferases. The endo H-treated lysosomal enzymes were shown to be specific inhibitors of the phosphorylation of intact lysosomal enzymes. Proteolytic fragments of cathepsin D, including the entire light chain and heavy chain, did not retain the ability to be recognized by the N-acetylglucosaminylphosphotransferase. These findings indicate that the intact protein portion of lysosomal enzymes contains a specific recognition determinant which leads to high-affinity binding to the N-acetylglucosaminylphosphotransferase. The expression of this determinant appears to be dependent on the conformation of the protein.

Glycosylation of Thyroid-Stimulating Hormone in Pituitary Tumor Cells: Influence of High Mannose Oligosaccharide Units on Subunit Aggregation, Combination, and Intracellular Degradation

We have studied the glycosylation of TSH in cell culture and have examined the influence of carbohydrate on subunit aggregation, intracellular degradation, and combination. Dispersed mouse thyrotropic tumor cells were labeled by pulse-chase methods with [35S]methionine and various 3H-labeled carbohydrates; cell lysates and media were precipitated with antisera to TSH alpha and TSH beta, and the products were analyzed by sodium dodecyl sulfate gradient gel electrophoresis without or with preexposure to Endoglycosidase (Endo) H. At early pulses, both intracellular alpha and beta were mainly composed of one Endo H-sensitive (high mannose) carbohydrate unit and a small amount of nonglycosylated forms; alpha only had the posttranslational addition of a second high mannose unit. With increasing chase times up to 18 h, intracellular subunits showed a slow but progressive increase in Endo H-resistant (complex) forms, and media subunits were completely resistant. Preincubation of cells with tunicamycin caused production of nonglycosylated subunits that showed a high degree of aggregation, especially after heating at 37 C under nonreducing conditions. Unlike glycosylated subunits, which were not degraded, nonglycosylated subunits were 50-65% degraded intracellularly before secretion; the degradation caused by tunicamycin was specific for TSH subunits and not noted for other 35S-labeled proteins. Incubation of various 35S-labeled alpha forms with excess unlabeled TSH beta showed high combining activity for intracellular alpha with two high mannose units, intermediate activity for media alpha with two complex units, and low activity for intracellular alpha with one high mannose unit or nonglycosylated media alpha. These data suggest that the initial glycosylation with high mannose carbohydrate units prevents intracellular aggregation and degradation of TSH subunits and enhances attainment of the conformation necessary for alpha- and beta-subunit combination.

Mucolipidosis III beta-N-acetyl-D-hexosaminidase A. Purification and properties.

Mucolipidosis III acid hydrolases possess an altered carbohydrate recognition marker needed for their lysosomal localization. As a result of this alteration, a portion of these enzymes is secreted from the cell to the extracellular spaces. The structural changes that may have occurred to one of these secreted enzymes, beta-N-acetyl-d-hexosaminidase A (EC 3.2.1.52) were investigated. Normal and mucolipidosis III urinary beta-N-acetyl-d-hexosaminidase A were purified to apparent homogeneity by using affinity [Sepharose-2-acetamido-N-(epsilon-aminocaproyl)-2-deoxy-beta- d-glucopyranosylamine] and ion-exchange (DEAE- and CM-cellulose) chromatography. Sodium dodecyl sulphate/polyacrylamide-slab-gel electrophoresis showed that both enzymes had similar subunit patterns consisting of apparent mol.wts. of 68000, 60000-58000, 55000 and 29000. Differences, however, were noted in the relative proportions of the protein bands where the normal urinary beta-N-acetyl-d-hexosaminidase A contained predominantly the smaller subunits, whereas the mucolipidosis III enzyme had a predominance of the larger subunits. The binding of mucolipidosis III beta-N-acetyl-d-hexosaminidase A to Ricinus communis lectin and concanavalin A with and without endo-beta-N-acetyl-d-glucosaminidase H treatment indicated that the mutation leads to a modification of a portion of the normally occurring high-mannose-type oligosaccharide units to the complex-type. This was further supported by carbohydrate compositional analysis, which revealed a mannose/galactose ratio of 2.1 for the mucolipidosis III beta-N-acetyl-d-hexosaminidase A compared with a ratio of 3.5 for the normal enzyme. Our results indicate that as a result of their inability to be properly localized to the lysosome the majority of the mucolipidosis III lysosomal hydrolase high-mannose oligosaccharide units are further processed to the complex-type before secretion of predominantly higher-molecular-weight subunits from the cell.

Lysosomal enzyme targeting. N-Acetylglucosaminylphosphotransferase selectively phosphorylates native lysosomal enzymes.

Lysosomal enzymes contain 6-phosphomannosyl moieties which mediate their translocation to lysosomes. This recognition marker is synthesized by the sequential action of UDP-N-acetylglucosamine:lysosomal enzyme N-acetylglucosamine-1-phosphotransferase and alpha-N-acetylglucosaminyl phosphodiesterase. A new assay for the N-acetylglucosaminylphosphotransferase, using alpha-methylmannoside as acceptor, is presented. Using this assay, we partially purified the transferase and examined its substrate specificity. The transferase exhibited a very high affinity toward lysosomal enzymes (apparent Km values of less than 20 microM) and was greater than 100-fold more efficient (Vmax/Km) when using lysosomal enzymes as acceptors as compared to nonlysosomal glycoproteins that contain high mannose oligosaccharide units. Heat denaturation of the lysosomal enzymes resulted in the loss of acceptor activity. The model compounds alpha-methylmannoside and Man5--8GlcNAc were poor acceptors. We propose that this enzyme catalyzes the initial, determining step by which synthesized acid hydrolases are distinguished from other newly synthesized glycoproteins and thus are eventually targeted to lysosomes.

Biosynthesis and maturation of HLA-DR antigens in vivo.

The biosynthesis of the HLA-DR antigens was studied in the B lymphoblastoid cell line BRI 8. Three chains, of molecular weights 33,000 (alpha), 31,000 (p31), and 26,000 (beta) were detected intracellularly in pulse-labeled cells by immunoprecipitation with anti-(HLA-DR) sera. The alpha and beta chains were inserted asymmetrically into the rough endoplasmic reticulum as transmembrane polypeptides with the majority of the polypeptide chains oriented in the lumen. At this stage, both chains carried "high mannose" oligosaccharide units which were processed to the complex form during subsequent intracellular transport to the cell surface. The Mr = 31,000 polypeptide was also glycosylated but was structurally distinct from the alpha chain and was probably oriented differently in the lipid bilayer, with a much greater proportion of its polypeptide chain exposed in the cytoplasm. It ws not, therefore, a precursor of the alpha chain. The mature HLA-DR antigens at the plasma membrane comprised polypeptides of Mr = 34,000 and 28,000. These chains corresponded to the processed alpha and beta chains. Although the Mr = 31,000 component was only detected intracellularly, it was not ruled out that some or all of it may have been processed and exposed on the cell surface with an apparent molecular weight indistinguishable from that of the alpha chain.

UDP-N-acetylglucosamine:glycoprotein N-acetylglucosamine-1-phosphotransferase. Proposed enzyme for the phosphorylation of the high mannose oligosaccharide units of lysosomal enzymes.

The recognition marker for the targeting of lysosomal enzymes contains mannose 6-phosphate. The recent discovery of phosphate in diester linkage between N-acetylglucosamine (GlcNAc) and mannose in newly synthesized beta-glucuronidase led to the proposal that the phosphate might be acquired via N-acetylglucosamine-phosphate transfer from UDP-GlcNAc (Tabas, I., and Kornfeld, S. (1980) J. Biol. Chem. 255, 6633-6639). We describe the synthesis of [beta-32P]UDP-[3H]GlcNAc and the use of this compound to demonstrate a UDP-GlcNAc:glycoprotein N-acetylglucosamine-1-phosphotransferase. The basis of the enzyme assay is the incorporation of 32P and 3H into glycopeptides with a high affinity for Concanavalin A-Sepharose. This membrane-associated transferase is neither inhibited by tunicamycin nor stimulated by dolichol-phosphate, indicating that the reaction does not proceed via a dolichylpyrophosphoryl-N-acetylglucosamine intermediate. Characterization of the enzyme reaction products (derived from either endogenous or exogenous acceptors) demonstrated that alpha-linked N-acetylglucosamine 1-phosphate is transferred en bloc to the 6-hydroxyl of mannose in high mannose oligosaccharides of glycoproteins. We propose that the function of this enzyme is to donate N-acetylglucosamine 1-phosphate to mannose residues of newly synthesized lysosomal enzymes.

Identification of a rat liver alpha-N-acetylglucosaminyl phosphodiesterase capable of removing “blocking” alpha-N-acetylglucosamine residues from phosphorylated high mannose oligosaccharides of lysosomal enzymes.

We recently reported that the high mannose-type oligosaccharides of the biosynthetic intermediates of beta-glucuronidase contain phosphate groups in diester linkage between mannose residues and outer alpha-linked N-acetylglucosamine residues (Tabas, I., and Kornfeld, S. (1980) J. Biol. Chem. 255, 6633-6639). We now describe an alpha-N-acetylglucosaminyl phosphodiesterase from rat liver that is capable of removing the N-acetyl-glucosamine residues, leaving phosphomonoester groups on the high mannose oligosaccharide units. This activity is greatly enriched in smooth membrane preparations. It can be distinguished from a lysosomal alpha-N-acetylglucosaminidase by several criteria, including subcellular localization and differential inhibition by amino sugars. In addition, human fibroblasts with mutations which lead to a deficiency of the lysosomal activity have normal levels of the alpha-N-acetylglucosaminyl phosphodiesterase. This enzyme may be involved in the "unmasking" of the phosphomannosyl recognition marker on newly synthesized acid hydrolases which could then direct the targeting of these enzymes to lysosomes.

Structural studies of the major high mannose oligosaccharide units from Chinese hamster ovary cell glycoproteins.

The major high mannose-type glycopeptides present in Chinese hamster ovary cells have the compositions (Man)9(GlcNAc)2-Asn, (Man)8(GlcNAc)2-Asn, and (Man)6(GlcNAc)2-Asn. The structures of these glycopeptides were determined by the combination of methylation analysis, acetolysis, Smith periodate degradation, and alpha- and beta-mannosidase digestion. Their complete structures are: manalpha1 leads to 2Manaalpha1 leads to 6(Manalpha1 leads to 2Manalpha1 leads to 3)Manalpha1 leads to 6(Manalpha1 leads to 2Manalpha1 leads to 2Manalpha1 leads to 3)Manbeta1 leads to 4GlcNAcbeta1 leads to 4GlcNAc-Asn, Manalpha1 leads to 2Manalpha1 leads to 6(Manalpha1 leads to 3)Manalpha1 leads to 6(Manalpha1 leads to 2Manalpha1 leads to 2Manalpha1 leads to 3)Manbeta1 leads to 4GlcNAcbeta1 leads to 4GlcNAc-Asn, and Manalpha1 leads to 6(Manalpha1 leads to 3)Manalpha1 leads to 6(Manalpha1 leads to 2Manalpha1 leads to 3)Manbeta1 leads to 4GlcNAcbeta1 leads to 4GlcNAc-Asn. These structures are compared with the structures of the peptide-bound oligosaccharide intermediates that are processed to form complex-type oligosaccharides. From these results, it is proposed that the high mannose-type oligosaccharides are a product of incomplete processing of the protein-bound oligosaccharide along the same pathway which leads ultimately to the formation of a complex-type oligosaccharide.