G2Cdb::Gene report

Gene id
Gene symbol
Homo sapiens
G00000383 (Mus musculus)

Databases (5)

ENSG00000115275 (Ensembl human gene)
7841 (Entrez Gene)
751 (G2Cdb plasticity & disease)
601336 (OMIM)
Protein Sequence
Q13724 (UniProt)

Literature (38)

Pubmed - other

  • N-Glycans in the gp120 V1/V2 domain of the HIV-1 strain NL4-3 are indispensable for viral infectivity and resistance against antibody neutralization.

    Wolk T and Schreiber M

    Bernhard Nocht Institute for Tropical Medicine, Bernhard Nocht Str. 74, 20359 Hamburg, Germany.

    Here we report that N-glycans within the V1/V2 variable regions of the NL4-3 gp120 glycoprotein are indispensable to maintain viral functionality and are masking neutralizing epitopes. Fifteen variants of HIV-1 isolate NL4-3 with mutations of the six N-glycosylation sites g2-g7 within the V1 (g2-g4) and V2 loop (g5-g7) of gp120 were analyzed for viral infectivity and their sensitivity to neutralization. Presence of the N-glycans g4, g5, g6 and g7 was an important prerequisite to maintain viral infectivity, since virus mutants lacking these N-glycans were highly deficient in virus entry. Lack of g4 or g7 correlated to a reduction of infectivity to less than 3% of the infectivity observed for NL4-3 wild type. In contrast, mutants lacking N-glycans g2 and g3 showed a 50% increase in infectivity compared to NL4-3. Mutants lacking g2, g3, g5 and g6 with an infectivity of more than 10% of the NL4-3 wt virus were tested for neutralization and showed a high sensitivity against human serum antibody from HIV-1 infected individuals.

    Medical microbiology and immunology 2006;195;3;165-72

  • Diversification of transcriptional modulation: large-scale identification and characterization of putative alternative promoters of human genes.

    Kimura K, Wakamatsu A, Suzuki Y, Ota T, Nishikawa T, Yamashita R, Yamamoto J, Sekine M, Tsuritani K, Wakaguri H, Ishii S, Sugiyama T, Saito K, Isono Y, Irie R, Kushida N, Yoneyama T, Otsuka R, Kanda K, Yokoi T, Kondo H, Wagatsuma M, Murakawa K, Ishida S, Ishibashi T, Takahashi-Fujii A, Tanase T, Nagai K, Kikuchi H, Nakai K, Isogai T and Sugano S

    Life Science Research Laboratory, Central Research Laboratory, Hitachi, Ltd., Kokubunji, Tokyo, 185-8601, Japan.

    By analyzing 1,780,295 5'-end sequences of human full-length cDNAs derived from 164 kinds of oligo-cap cDNA libraries, we identified 269,774 independent positions of transcriptional start sites (TSSs) for 14,628 human RefSeq genes. These TSSs were clustered into 30,964 clusters that were separated from each other by more than 500 bp and thus are very likely to constitute mutually distinct alternative promoters. To our surprise, at least 7674 (52%) human RefSeq genes were subject to regulation by putative alternative promoters (PAPs). On average, there were 3.1 PAPs per gene, with the composition of one CpG-island-containing promoter per 2.6 CpG-less promoters. In 17% of the PAP-containing loci, tissue-specific use of the PAPs was observed. The richest tissue sources of the tissue-specific PAPs were testis and brain. It was also intriguing that the PAP-containing promoters were enriched in the genes encoding signal transduction-related proteins and were rarer in the genes encoding extracellular proteins, possibly reflecting the varied functional requirement for and the restricted expression of those categories of genes, respectively. The patterns of the first exons were highly diverse as well. On average, there were 7.7 different splicing types of first exons per locus partly produced by the PAPs, suggesting that a wide variety of transcripts can be achieved by this mechanism. Our findings suggest that use of alternate promoters and consequent alternative use of first exons should play a pivotal role in generating the complexity required for the highly elaborated molecular systems in humans.

    Genome research 2006;16;1;55-65

  • A human protein-protein interaction network: a resource for annotating the proteome.

    Stelzl U, Worm U, Lalowski M, Haenig C, Brembeck FH, Goehler H, Stroedicke M, Zenkner M, Schoenherr A, Koeppen S, Timm J, Mintzlaff S, Abraham C, Bock N, Kietzmann S, Goedde A, Toksöz E, Droege A, Krobitsch S, Korn B, Birchmeier W, Lehrach H and Wanker EE

    Max Delbrueck Center for Molecular Medicine, 13092 Berlin-Buch, Germany.

    Protein-protein interaction maps provide a valuable framework for a better understanding of the functional organization of the proteome. To detect interacting pairs of human proteins systematically, a protein matrix of 4456 baits and 5632 preys was screened by automated yeast two-hybrid (Y2H) interaction mating. We identified 3186 mostly novel interactions among 1705 proteins, resulting in a large, highly connected network. Independent pull-down and co-immunoprecipitation assays validated the overall quality of the Y2H interactions. Using topological and GO criteria, a scoring system was developed to define 911 high-confidence interactions among 401 proteins. Furthermore, the network was searched for interactions linking uncharacterized gene products and human disease proteins to regulatory cellular pathways. Two novel Axin-1 interactions were validated experimentally, characterizing ANP32A and CRMP1 as modulators of Wnt signaling. Systematic human protein interaction screens can lead to a more comprehensive understanding of protein function and cellular processes.

    Cell 2005;122;6;957-68

  • The status, quality, and expansion of the NIH full-length cDNA project: the Mammalian Gene Collection (MGC).

    Gerhard DS, Wagner L, Feingold EA, Shenmen CM, Grouse LH, Schuler G, Klein SL, Old S, Rasooly R, Good P, Guyer M, Peck AM, Derge JG, Lipman D, Collins FS, Jang W, Sherry S, Feolo M, Misquitta L, Lee E, Rotmistrovsky K, Greenhut SF, Schaefer CF, Buetow K, Bonner TI, Haussler D, Kent J, Kiekhaus M, Furey T, Brent M, Prange C, Schreiber K, Shapiro N, Bhat NK, Hopkins RF, Hsie F, Driscoll T, Soares MB, Casavant TL, Scheetz TE, Brown-stein MJ, Usdin TB, Toshiyuki S, Carninci P, Piao Y, Dudekula DB, Ko MS, Kawakami K, Suzuki Y, Sugano S, Gruber CE, Smith MR, Simmons B, Moore T, Waterman R, Johnson SL, Ruan Y, Wei CL, Mathavan S, Gunaratne PH, Wu J, Garcia AM, Hulyk SW, Fuh E, Yuan Y, Sneed A, Kowis C, Hodgson A, Muzny DM, McPherson J, Gibbs RA, Fahey J, Helton E, Ketteman M, Madan A, Rodrigues S, Sanchez A, Whiting M, Madari A, Young AC, Wetherby KD, Granite SJ, Kwong PN, Brinkley CP, Pearson RL, Bouffard GG, Blakesly RW, Green ED, Dickson MC, Rodriguez AC, Grimwood J, Schmutz J, Myers RM, Butterfield YS, Griffith M, Griffith OL, Krzywinski MI, Liao N, Morin R, Morrin R, Palmquist D, Petrescu AS, Skalska U, Smailus DE, Stott JM, Schnerch A, Schein JE, Jones SJ, Holt RA, Baross A, Marra MA, Clifton S, Makowski KA, Bosak S, Malek J and MGC Project Team

    The National Institutes of Health's Mammalian Gene Collection (MGC) project was designed to generate and sequence a publicly accessible cDNA resource containing a complete open reading frame (ORF) for every human and mouse gene. The project initially used a random strategy to select clones from a large number of cDNA libraries from diverse tissues. Candidate clones were chosen based on 5'-EST sequences, and then fully sequenced to high accuracy and analyzed by algorithms developed for this project. Currently, more than 11,000 human and 10,000 mouse genes are represented in MGC by at least one clone with a full ORF. The random selection approach is now reaching a saturation point, and a transition to protocols targeted at the missing transcripts is now required to complete the mouse and human collections. Comparison of the sequence of the MGC clones to reference genome sequences reveals that most cDNA clones are of very high sequence quality, although it is likely that some cDNAs may carry missense variants as a consequence of experimental artifact, such as PCR, cloning, or reverse transcriptase errors. Recently, a rat cDNA component was added to the project, and ongoing frog (Xenopus) and zebrafish (Danio) cDNA projects were expanded to take advantage of the high-throughput MGC pipeline.

    Funded by: PHS HHS: N01-C0-12400

    Genome research 2004;14;10B;2121-7

  • Hyperglycosylated mutants of human immunodeficiency virus (HIV) type 1 monomeric gp120 as novel antigens for HIV vaccine design.

    Pantophlet R, Wilson IA and Burton DR

    Department of Immunology, The Scripps Research Institute, La Jolla, California 92037, USA.

    The ability to induce broadly neutralizing antibodies should be a key component of any forthcoming vaccine against human immunodeficiency virus type 1. One potential vaccine candidate, monomeric gp120, has generally failed to elicit such antibodies. We postulated that gp120 might be a better immunogen if it could be engineered to preferentially bind known broadly neutralizing antibodies. In a first study, we found that four alanine substitutions on the perimeter of the so-called Phe-43 cavity of gp120 could reduce binding of weakly neutralizing CD4-binding site antibodies (R. Pantophlet, E. O. Saphire, P. Poignard, P. W. H. I. Parren, I. A. Wilson, and D. R. Burton, J. Virol. 77:642-658, 2003), while slightly enhancing binding of the potent, broadly neutralizing antibody b12. In the present study, we sought to reduce or abolish the binding of a wider range of nonneutralizing antibodies, by incorporating extra N-glycosylation motifs at select positions into the hypervariable loops and the gp120 core. A hyperglycosylated mutant containing seven extra glycosylation sequons (consensus sequences) and the four alanine substitutions described above did not bind an extensive panel of nonneutralizing and weakly neutralizing antibodies, including a polyclonal immunoglobulin preparation (HIVIG) of low neutralizing potency. Binding of b12, at lowered affinity, and of four antibodies to the C1 and C5 regions was maintained. Removal of N- and C-terminal residues in the C1 and C5 regions, respectively, reduced or abolished binding of the four antibodies, but this also adversely affected b12 binding. The hyperglycosylated mutant and its analogues described here are novel antigens that may provide a new approach to eliciting antibodies with b12-like neutralizing properties.

    Funded by: NIAID NIH HHS: AI33292, R01 AI033292, R37 AI033292; NIGMS NIH HHS: GM46192, R01 GM046192

    Journal of virology 2003;77;10;5889-901

  • (Arg)3 within the N-terminal domain of glucosidase I contains ER targeting information but is not required absolutely for ER localization.

    Hardt B, Kalz-Fuller B, Aparicio R, Volker C and Bause E

    Institut Für Physiologische Chemie, Universität Bonn Nussallee 11, 53115 Bonn, Germany.

    Glucosidase I is an endoplasmic reticulum (ER) type II membrane enzyme that cleaves the distal alpha1,2-glucose of the asparagine-linked GlcNAc2-Man9-Glc3 precursor. To identify sequence motifs responsible for ER localization, we prepared a protein chimera by transferring the cytosolic and transmembrane domain of glucosidase I to the luminal domain of Golgi-Man9-mannosidase. The GIM9 hybrid was overexpressed in COS 1 cells as an ER-resident protein that displayed alpha1,2-mannosidase activity, excluding the possibility that the glucosidase I-specific domains interfere with folding of the Man9-mannosidase catalytic domain. After substitution of the Args in position 7, 8, or 9 relative to the N-terminus by leucine, the GIM9 mutants were transported to the cell surface indicating that the (Arg)3 sequence functions as an ER-targeting motif. Cell surface expression was also observed after substitution of Arg-7 or Arg-8 but not Arg-9 in GIM9 by either lysine or histidine. Thus the side chain structure, including its positive charge, appears to be essential for signal function. Analysis of the N-linked glycans suggests that the (Arg)3 sequence mediates ER localization through Golgi-to-ER retrograde transport. Glucosidase I remained localized in the ER after truncation or mutation of the N-terminal (Arg)3 signal, in contrast to comparable GIM9 mutants. ER localization was also observed with an M9GI chimera consisting of the cytosolic and transmembrane domain of Man9-mannosidase and the glucosidase I catalytic domain. ER-specific targeting information must therefore be provided by sequence motifs contained within the glucosidase I luminal domain. This structural information appears to direct ER localization by retention rather than by retrieval, as concluded from N-linked Man9-GlcNAc2 being the major glycan released from the wild-type enzyme.

    Glycobiology 2003;13;3;159-68

  • Glycosylation inhibitors and neuraminidase enhance human immunodeficiency virus type 1 binding and neutralization by mannose-binding lectin.

    Hart ML, Saifuddin M and Spear GT

    Department of Immunology/Microbiology, Rush-Presbyterian-St Luke's Medical Center, 1563 W. Congress Parkway, Chicago, IL 60612, USA.

    Mannose-binding lectin (MBL), a C-type lectin component of the human innate immune system, binds to the gp120 envelope glycoprotein of human immunodeficiency virus type 1 (HIV-1). The objective of this study was to assess the effects of inhibitors of endoplasmic reticulum glucosidases and Golgi mannosidase as well as neuraminidase (NA) on the interaction between HIV and MBL. Production of HIV in the presence of the mannosidase I inhibitor deoxymannojirimycin (dMM) significantly enhanced binding of HIV to MBL and increased MBL neutralization of an M-tropic HIV primary isolate. In contrast, culturing HIV in the presence of alpha-glucosidase I and II inhibitors castanospermine and deoxynojirimycin only slightly affected virus binding and neutralization by MBL. Removal of sialic acid from HIV by NA also significantly enhanced virus binding and neutralization by MBL. Treatment of virus grown in the presence of dMM with endoglycosidase F1 substantially reduced binding to MBL, indicating that dMM increased MBL binding by increasing high-mannose carbohydrates on the virus. In contrast, endoglycosidase F1 did not decrease the MBL interaction with NA-treated virus, suggesting that NA exposed novel MBL binding sites. Treatment with dMM increased the immunocapture of HIV by monoclonal antibodies 2F5 and 2G12, indicating that altering the glycosylation of viral glycoproteins increases the accessibility or reactivity of some epitopes. This study shows that specific alterations of the N-linked carbohydrates on HIV gp120/gp41 can enhance MBL-mediated neutralization of virus by strengthening the interaction of HIV-1 with MBL.

    Funded by: NIAID NIH HHS: AI46963

    The Journal of general virology 2003;84;Pt 2;353-60

  • Cloning MafF by recognition site screening with the NFE2 tandem repeat of HS2: analysis of its role in globin and GCSl genes regulation.

    Marini MG, Asunis I, Chan K, Chan JY, Kan YW, Porcu L, Cao A and Moi P

    Istituto di Neurogenetica e Neurofarmacologia, CNR, Cagliari 09121, Italy.

    The erythroid-specific enhancer within hypersensitivity site 2 (HS2) of the human beta-globin locus control region is required for high level globin gene expression. We used an oligonucleotide of the NF-E2 tandem repeat, within HS2, as recognition site probe to screen a K562 cDNA library for interacting transcription factors. A 2.3 kb full length cDNA encoding the b-zip transcription factor MafF was isolated. MafF can form both homodimers and high affinity heterodimers with Nrf1, Nrf2 and Nf-E2, three members of the CNC-bZip family. Despite obvious structural similarities with the other small Maf proteins, MafF differs in its tissue distribution and its inability to repress transcription when overexpressed as homodimer. In fact, in different cell lines and on different promoters (gamma-globin, beta-globin and glutamylcysteine synthetase genes) the MafF homodimers do not appreciably affect transcription of target promoters, whereas MafF/CNC member heterodimers act as weak transcriptional activators. Even though MafF was cloned using probes derived from the globin LCR, it is in the context of the GCSl promoter and in combination with Jun that MafF shows a rather distinct and specific regulatory role. These observations suggest that a complex network of small Maf and CNC-AP1 protein interactions might be involved in regulating transcription in diverse tissues or developmental stages.

    Funded by: Telethon: A.121

    Blood cells, molecules & diseases 2002;29;2;145-58

  • Processing of N-linked carbohydrate chains in a patient with glucosidase I deficiency (CDG type IIb).

    Völker C, De Praeter CM, Hardt B, Breuer W, Kalz-Füller B, Van Coster RN and Bause E

    Institut für Physiologische Chemie, Universität Bonn, Bonn, Germany.

    Recently, we reported a novel congenital disorder of glycosylation (CDG-IIb) caused by severe deficiency of the glucosidase I. The enzyme cleaves the alpha1,2-glucose residue from the asparagine-linked Glc(3)-Man(9)-GlcNAc(2) precursor, which is crucial for oligosaccharide maturation. The patient suffering from this disease was compound-heterozygous for two mutations in the glucosidase I gene, a T-->C transition in the paternal allele and a G-->C transition in the maternal allele. This gives rise in the glucosidase I polypeptide to the substitution of Arg486 by Thr and Phe652 by Leu, respectively. Kinetic studies using detergent extracts from cultured fibroblasts showed that the glucosidase I activity in the patient's cells was < 1% of the control level, with intermediate values in the parental cells. No significant differences in the activities of other processing enzymes, including oligosaccharyltransferase, glucosidase II, and Man(9)-mannosidase, were observed. By contrast, the patient's fibroblasts displayed a two- to threefold higher endo-alpha1,2-mannosidase activity, associated with an increased level of enzyme-specific mRNA-transcripts. This points to the lack of glucosidase I activity being compensated for, to some extent, by increase in the activity of the pathway involving endo-alpha1,2-mannosidase; this would also explain the marked urinary excretion of Glc(3)-Man. Comparative analysis of [(3)H]mannose-labeled N-glycoproteins showed that, despite the dramatically reduced glucosidase I activity, the bulk of the N-linked carbohydrate chains (>80%) in the patient's fibroblasts appeared to have been processed correctly, with only approximately 16% of the N-glycans being arrested at the Glc(3)-Man(9-7)-GlcNAc(2) stage. These structural and enzymatic data provide a reasonable basis for the observation that the sialotransferrin pattern, which frequently depends on the type of glycosylation disorder, appears to be normal in the patient. The human glucosidase I gene contains four exons separated by three introns with exon-4 encoding for the large 64-kDa catalytic domain of the enzyme. The two base mutations giving rise to substitution of Arg486 by Thr and Phe652 by Leu both reside in exon-4, consistent with their deleterious effect on enzyme activity. Incorporation of either mutation into wild-type glucosidase I resulted in the overexpression of enzyme mutants in COS 1 cells displaying no measurable catalytic activity. The Phe652Leu but not the Arg486Thr protein mutant showed a weak binding to a glucosidase I-specific affinity resin, indicating that the two amino acids affect polypeptide folding and active site formation differently.

    Glycobiology 2002;12;8;473-83

  • Perturbation of free oligosaccharide trafficking in endoplasmic reticulum glucosidase I-deficient and castanospermine-treated cells.

    Durrant C and Moore SE

    Unité de Glycobiologie et Signalisation Cellulaire, U504, Bâtiment INSERM, 16 avenue Paul Vaillant-Couturier, 94807 Villejuif Cedex, France.

    Free oligosaccharides (FOS) are generated both in the endoplasmic reticulum (ER) and in the cytosol during glycoprotein biosynthesis. ER lumenal FOS possessing the di-N-acetylchitobiose moiety at their reducing termini (FOSGN2) are exported into the cytosol where they, along with their cytosolically generated counterparts possessing a single N-acetylglucosamine residue at their reducing termini (FOSGN1), are trimmed in order to be imported into lysosomes for final degradation. Both the ER and lysosomal FOS transport processes are unable to translocate triglucosylated FOS across membranes. In the present study, we have examined FOS trafficking in HepG2 cells treated with the glucosidase inhibitor castanospermine. We have shown that triglucosylated FOSGN2 generated in the ER are transported to the Golgi apparatus where they are deglucosylated by endomannosidase and acquire complex, sialic acid-containing structures before being secreted into the extracellular space by a Brefeldin A-sensitive pathway. FOSGN2 are also secreted from glucosidase I-deficient Lec23 cells and from the castanospermine-treated parental Chinese-hamster ovary cell line. Despite the secretion of FOSGN2 from Lec23 cells, we noted a transient intracellular accumulation (60 nmol/g cells) of triglucosylated FOSGN1 in these cells. Finally, in glucosidase I-compromised cells, FOS trafficking was severely perturbed leading to both the secretion of FOSGN2 into the extracellular space and a growth-dependent pile up of triglucosylated FOSGN1 in the cytosol. The possibility that these abnormalities contributed to the severe and rapidly progressive pathology in a patient with congenital disorders of glycosylation type IIb (glucosidase I deficiency) is discussed.

    The Biochemical journal 2002;365;Pt 1;239-47

  • Folding of the human immunodeficiency virus type 1 envelope glycoprotein in the endoplasmic reticulum.

    Land A and Braakman I

    Department of Bio-Organic Chemistry, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands.

    The lumen of the endoplasmic reticulum (ER) provides a unique folding environment that is distinct from other organelles supporting protein folding. The relatively oxidizing milieu allows the formation of disulfide bonds. N-linked oligosaccharides that are attached during synthesis play multiple roles in the folding process of glycoproteins. They stabilize folded domains and increase protein solubility, which prevents aggregation of folding intermediates. Glycans mediate the interaction of newly synthesized glycoproteins with some resident ER folding factors, such as calnexin and calreticulin. Here we present an overview of the present knowledge on the folding process of the heavily glycosylated human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein in the ER.

    Biochimie 2001;83;8;783-90

  • A novel disorder caused by defective biosynthesis of N-linked oligosaccharides due to glucosidase I deficiency.

    De Praeter CM, Gerwig GJ, Bause E, Nuytinck LK, Vliegenthart JF, Breuer W, Kamerling JP, Espeel MF, Martin JJ, De Paepe AM, Chan NW, Dacremont GA and Van Coster RN

    Department of Pediatrics, Division of Neonatal Intensive Care, Ghent University Hospital, B9000 Ghent, Belgium.

    Glucosidase I is an important enzyme in N-linked glycoprotein processing, removing specifically distal alpha-1,2-linked glucose from the Glc3Man9GlcNAc2 precursor after its en bloc transfer from dolichyl diphosphate to a nascent polypeptide chain in the endoplasmic reticulum. We have identified a glucosidase I defect in a neonate with severe generalized hypotonia and dysmorphic features. The clinical course was progressive and was characterized by the occurrence of hepatomegaly, hypoventilation, feeding problems, seizures, and fatal outcome at age 74 d. The accumulation of the tetrasaccharide Glc(alpha1-2)Glc(alpha1-3)Glc(alpha1-3)Man in the patient's urine indicated a glycosylation disorder. Enzymological studies on liver tissue and cultured skin fibroblasts revealed a severe glucosidase I deficiency. The residual activity was <3% of that of controls. Glucosidase I activities in cultured skin fibroblasts from both parents were found to be 50% of those of controls. Tissues from the patient subjected to SDS-PAGE followed by immunoblotting revealed strongly decreased amounts of glucosidase I protein in the homogenate of the liver, and a less-severe decrease in cultured skin fibroblasts. Molecular studies showed that the patient was a compound heterozygote for two missense mutations in the glucosidase I gene: (1) one allele harbored a G-->C transition at nucleotide (nt) 1587, resulting in the substitution of Arg at position 486 by Thr (R486T), and (2) on the other allele a T-->C transition at nt 2085 resulted in the substitution of Phe at position 652 by Leu (F652L). The mother was heterozygous for the G-->C transition, whereas the father was heterozygous for the T-->C transition. These base changes were not seen in 100 control DNA samples. A causal relationship between the alpha-glucosidase I deficiency and the disease is postulated.

    American journal of human genetics 2000;66;6;1744-56

  • Effect of various glycosidase treatments on the resistance of the HIV-1 envelope to degradation.

    Papandreou MJ and Fenouillet E

    CNRS, UMR 6560, IFR Jean Roche, Faculté de Médecine Nord, Marseille, France.

    Using a CD4-binding assay to assess the conformation of the human immunodeficiency virus envelope glycoprotein (CHO+ Env), we studied the effect of treatment with various glycosidases on the stability of Env in denaturing environments and in biological media: cleavage from Env of either high-mannose-type glycans (HMT- Env) by endoglycosidase H or sialic acid residues (Sial- Env) by sialidase did not alter Env stability whereas its complete deglycosylation (CHO- Env) by N-glycanase had a large effect. The influence of glycan removal on Env sensitivity to proteases was also studied. Thrombin cleavage within V3 was affected by N-glycanase treatment; both HMT- Env and CHO- Env displayed an increased sensitivity to other endoproteases. Thus, partial deglycosylation increases Env sensitivity to proteases but only its total deglycosylation alters its stability.

    FEBS letters 1997;406;1-2;191-5

  • N-butyldeoxynojirimycin-mediated inhibition of human immunodeficiency virus entry correlates with changes in antibody recognition of the V1/V2 region of gp120.

    Fischer PB, Karlsson GB, Butters TD, Dwek RA and Platt FM

    Department of Biochemistry, Glycobiology Institute, University of Oxford, United Kingdom.

    The alpha-glucosidase inhibitor N-butyldeoxynojirimycin (NB-DNJ) is an inhibitor of human immunodeficiency virus (HIV) replication and HIV-induced syncytium formation in vitro. Although NB-DNJ appears to inhibit HIV entry at the level of post-CD4 binding (P.B. Fischer, M. Collin, G.B. Karlsson, W. James, T.D. Butters, S.J. Davis, S. Gordon, R.A. Dwek, and F.M. Platt, J. Virol. 69:5791-5797, 1995), the exact mechanism of action remains to be established. In this study we have examined the effect of NB-DNJ on the structure of recombinant gp120 (rgpl20), expressed in CHO cells, by using a panel of 40 monoclonal antibodies. The levels of binding of antibodies to rgp120 produced in the presence [rgpl20(+)] and absence [rgpl20(-)] of NB-DNJ were compared by enzyme-linked immunosorbent assay and surface plasmon resonance (BIAcore; Pharmacia). The results showed an increase in the binding to rgp120(+) of antibodies directed against the C1 and C2 regions and a decrease in the binding of antibodies directed against the V1/V2 loops compared with antibody binding to rgpl20(-). A decrease in the binding to rgpl20(+) of antibodies directed against discontinuous epitopes was also observed. No differences were seen in the binding of antibodies directed against the crown of the V3 loop and the C4 region of gp120. Treatment of rgpl20 with alpha-glucosidases I and II had no effect on the differential binding observed, whereas treatment with sialidase abolished the differences seen in the binding of antibodies directed against the C1 and C2 regions of gp120. In addition to these findings, rgpl20(+) showed increased sensitivity to proteases released by CHO cells during expression, as well as to exogenous thrombin. Taken together, the data presented in this paper suggest that production of gp120 in the presence of NB-DNJ affects the conformation of the Vl/V2 loops of gpl20, as well as the overall charge of the C1 and C2 regions. These effects may play a role in the previously described NB-DNJ-mediated inhibition of HIV entry at the level of post-CD4 binding.

    Funded by: Wellcome Trust

    Journal of virology 1996;70;10;7143-52

  • N-butyldeoxynojirimycin-mediated inhibition of human immunodeficiency virus entry correlates with impaired gp120 shedding and gp41 exposure.

    Fischer PB, Karlsson GB, Dwek RA and Platt FM

    Department of Biochemistry, Glycobiology Institute, University of Oxford, United Kingdom.

    The alpha-glucosidase inhibitor N-butyldeoxynojirimycin (NB-DNJ) is an inhibitor of human immunodeficiency virus (HIV) replication and HIV-induced syncytium formation in vitro. Although an NB-DNJ-mediated change in viral envelope N-glycan composition inhibits HIV entry at the level of post-CD4 binding, the exact mechanism of inhibition remains to be established. In this study we have examined the effects of NB-DNJ on virion envelope composition and CD4-induced gp120 shedding and gp41 exposure. Virion composition analysis revealed an NB-DNJ-mediated reduction of 15% in overall virion envelope glycoprotein content and a reduction of 26% in the proteolytic maturation of virion gp160. Taken together, these two effects resulted in a reduction of approximately 40% in virion gp120 content. CD4-induced shedding of gp120 from the surfaces of envelope-transfected Cos cells was undetectable when gp120 was expressed in the presence of NB-DNJ. Similarly, the shedding of virion-associated gp120 was reduced 7.4-fold. CD4-induced exposure of cryptic gp41 epitopes on the surfaces of HIV-expressing ACH-2 cells was also greatly impaired, and the exposure of virion-associated gp41 epitopes was reduced 4.0-fold. Finally, CD4-induced increases in the binding of antibodies to the V3 loop of ACH-2-cell-expressed envelope glycoproteins were reduced 25-fold when the glycoproteins were expressed in the presence of NB-DNJ. These results suggest that the NB-DNJ-mediated retention of glycosylated N-glycans inhibits HIV entry by a combined effect of a reduction in virion gp120 content and a qualitative defect within the remaining gp120, preventing it from undergoing conformational changes after CD4 binding.

    Funded by: Wellcome Trust

    Journal of virology 1996;70;10;7153-60

  • Influence of N-linked glycans in V4-V5 region of human immunodeficiency virus type 1 glycoprotein gp160 on induction of a virus-neutralizing humoral response.

    Bolmstedt A, Sjölander S, Hansen JE, Akerblom L, Hemming A, Hu SL, Morein B and Olofsson S

    Department of Clinical Virology, University of Göteborg, Sweden.

    One of the functions of N-linked glycans of viral glycoproteins is protecting otherwise accessible neutralization epitopes of the viral envelope from neutralizing antibodies. The aim of the present study was to explore the possibility to obtain a more broadly neutralizing immune response by immunizing guinea pigs with gp160 depleted of three N-linked glycans in the CD4-binding domain by site-directed mutagenesis. Mutant and wild type gp160 were formulated into immunostimulating complexes and injected s.c. into guinea pigs. Both preparations induced high serum antibody response to native gp120 and V3 peptides. Both preparations also induced antibodies that bound equally well to the V3 loop or the CD4-binding region, as determined by a competitive enzyme-linked immunosorbent assay (ELISA). The sera from animals, immunized with mutated glycoprotein, did not neutralize nonrelated HIV strains better than did sera from animals, immunized with wild type glycoprotein. Instead, a pattern of preferred homologous neutralization was observed, i.e., sera from animals, immunized with mutant gp160, neutralized mutant virus better than wild type virus, and vice versa. These data indicated that elimination of the three N-linked glycans from gp160 resulted in an altered local antigenic conformation but did not uncover hidden neutralization epitopes, broadening the immune response.

    Journal of acquired immune deficiency syndromes and human retrovirology : official publication of the International Retrovirology Association 1996;12;3;213-20

  • Localization of the human glucosidase I gene to chromosome 2p12-p13 by fluorescence in situ hybridization and PCR analysis of somatic cell hybrids.

    Kalz-Füller B, Heidrich-Kaul C, Nöthen M, Bause E and Schwanitz G

    Institut für Physiologische Chemie, Universität Bonn, Nussallee 11, Bonn, 53115, USA.

    Genomics 1996;34;3;442-3

  • Cloning and expression of glucosidase I from human hippocampus.

    Kalz-Füller B, Bieberich E and Bause E

    Institut für Physiologische Chemie, Bonn, Germany.

    Glucosidase I, the first enzyme in the N-linked oligosaccharide processing pathway, cleaves the distal alpha 1,2-linked glucose residue from the Glc3-Man9-GlcNAc2 oligosaccharide precursor highly specifically. A human hippocampus cDNA library was screened against oligonucleotide probes, generated by PCR using primers derived from the amino acid sequences of tryptic peptides of pig liver glucosidase I. Two independent lambda clones were isolated which allowed the construction of a full-length glucosidase I cDNA of 2881 bp. This cDNA construct encodes, in a single open reading frame, a polypeptide of 834 amino acids corresponding to a molecular mass of 92 kDa. The 92-kDa protein contains a single N-glycosylation site of the Asn-Xaa-Thr/Ser type at Asn655, as well as a strongly hydrophobic sequence close to its N-terminus (amino acids 38-58) which, most likely, functions as a transmembrane anchor. The amino acid sequences of all tryptic peptides of the pig liver enzyme were found, with little deviation, within the coding sequence. This demonstrates the authenticity of the cDNA construct and the close evolutionary relationship between the enzymes from human hippocampus and pig liver. In contrast, the nucleotide and amino acid sequence revealed no homology with other processing enzymes cloned so far. Transfection of COS 1 cells with the glucosidase I cDNA construct resulted in overexpression (about fourfold) of enzymic activity, which was inhibited strongly by 1-deoxynojirimycin or N,N-dimethyl-deoxynojirimycin. The expressed enzyme, with a molecular mass of 95 kDa, is degraded by endoglycosidase H to a 93-kDa form, indicating that it carries a high-mannose oligosaccharide chain at Asn655. The presence of this glycan is in line with the localization of glucosidase I in the lumen of the endoplasmic reticulum, shown by immunofluorescence microscopy. The hydrophobicity profile as well as the removal by trypsin of an approximately 4-kDa polypeptide from the membrane-associated glucosidase I in intact microsomal structures, supports the view that the enzyme is a type-II transmembrane glycoprotein, which contains a short cytosolic tail of approximately 37 amino acids, followed by a single transmembrane domain and a large C-terminal catalytic domain located on the luminal side of the endoplasmic reticulum membrane.

    European journal of biochemistry 1995;231;2;344-51

  • Site-specific N-glycosylation and oligosaccharide structures of recombinant HIV-1 gp120 derived from a baculovirus expression system.

    Yeh JC, Seals JR, Murphy CI, van Halbeek H and Cummings RD

    Department of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, Oklahoma City 73190.

    We report the complete structures of the N-linked oligosaccharides and the site-specificity of the N-glycosylation of recombinant gp120 (rgp120) of the HIV-1 BH8 isolate produce by a baculovirus expression system. Glycopeptides derived from the tryptic digests of intact rgp120 or of cyanogen bromide-generated fragments of rgp120 were isolated by their binding to concanavalin A-Sepharose and were purified by reversed-phase HPLC. The isolated glycopeptides were treated with PNGase F, releasing the carbohydrate moiety while converting Asn to Asp, and identified by amino acid analysis and/or peptide sequencing. Our results indicate that all 22 potential N-glycosylation sites in the rgp120 sequence are utilized. We did not detect N-acetylgalactosamine in rgp120, indicating that the glycoprotein lacks typical O-linked oligosaccharides. To investigate the oligosaccharide structures at the sites of glycosylation, we determined the carbohydrate composition for each site and characterized the oligosaccharides by 1H-NMR spectroscopy and by oligosaccharide mapping using high pH anion-exchange chromatography. Mannose and N-acetylglucosamine were the only sugars observed in the intact rgp120 and likewise in individual glycopeptides. All glycopeptides derived from rgp120 contained high mannose-type N-linked oligosaccharides, ranging from GlcNAc2Man5 to GlcNAc2Man9. However, different glycosylation sites showed varied degrees of processing of the high mannose-type oligosaccharides, as characterized by the ratio of GlcNAc2Man8-9 to GlcNAc2Man5-7. These results demonstrate that N-glycosylation of rgp120 in the baculovirus expression system occurs at all potential sites and is site specific in terms of oligosaccharide structures.

    Funded by: NIAID NIH HHS: AI27135

    Biochemistry 1993;32;41;11087-99

  • Effects of the imino sugar N-butyldeoxynojirimycin on the N-glycosylation of recombinant gp120.

    Karlsson GB, Butters TD, Dwek RA and Platt FM

    Department of Biochemistry, University of Oxford, United Kingdom.

    The imino sugar N-butyldeoxynojirimycin (NB-DNJ) exhibits anti-HIV activity in vitro and inhibits the purified glycoprocessing enzyme alpha 1,2-glucosidase I. It has been speculated that the anti-viral activity of this compound may result from inhibition of HIV envelope glycoprotein processing. However, structural evidence that glucosidase inhibition takes place in intact cells at the anti-viral concentration (0.5 mM) is lacking. In this study, N-linked glycosylation of recombinant gp120 expressed in Chinese hamster ovary cells cultured in the presence or absence of NB-DNJ has been characterized. Immunoprecipitation, in conjunction with endoglycosidase H (endo H) digestion and SDS-polyacrylamide gel electrophoresis analysis, revealed that the glycosylation of gp120 was profoundly altered in the presence of NB-DNJ. The majority of the gp120 oligosaccharides from untreated cells were resistant to endo H. However, nearly complete endo H sensitivity was observed following treatment with 0.5 mM NB-DNJ indicating that gp120 expressed in treated cells carries immature, high mannose type oligosaccharides. In addition, using metabolic labeling with [3H]mannose, gel filtration chromatography, and digestion with highly purified glucosidases I and II, we provide the first definitive evidence that glucosidase I inhibition occurs at the anti-viral concentration of NB-DNJ. These data indicate that glucosidase inhibition is a candidate mechanism for the anti-viral activity of this compound.

    The Journal of biological chemistry 1993;268;1;570-6

  • Functional role of the glycan cluster of the human immunodeficiency virus type 1 transmembrane glycoprotein (gp41) ectodomain.

    Fenouillet E, Jones I, Powell B, Schmitt D, Kieny MP and Gluckman JC

    Laboratoire de Biologie et Génétique des Pathologies Immunitaires, CNRS URA 1463, France.

    To examine the role of the glycans of human immunodeficiency virus type 1 transmembrane glycoprotein gp41, conserved glycosylation sites within the env sequence (Asn-621, Asn-630, and Asn-642) were mutated to Gln. The mutated and control wild-type env genes were introduced into recombinant vaccinia virus and used to infect BHK-21 or CD4+ CEM cells. Mutated gp41 appeared as a 35-kDa band in a Western blot (immunoblot), and it comigrated with the deglycosylated form of wild-type gp41. Proteolytic cleavage of the recombinant wild-type and mutant forms of the gp160 envelope glycoprotein precursor was analyzed by pulse-chase experiments and enzyme-linked immunosorbent assay: gp160 synthesis was similar whether cells were infected with control or mutated env-expressing recombinant vaccinia virus, but about 10-fold less cleaved gp120 and gp41 was produced by the mutated construct than the control construct. The rates of gp120-gp41 cleavage at each of the two potential sites appeared to be comparable in the two constructs. By using a panel of antibodies specific for gp41 and gp120 epitopes, it was shown that the overall immunoreactivities of control and mutated gp41 proteins were similar but that reactivity to epitopes at the C and N termini of gp120, as present on gp160 produced by the mutated construct, was enhanced. This was no longer observed for cleaved gp120 in supernatants. Both gp120 proteins, from control and mutated env, were expressed on the cell surface under a cleaved form and could bind to membrane CD4, as determined by quantitative immunofluorescence assay. In contrast, and despite sufficient expression of env products at the cell membrane, gp41 produced by the mutated construct was unable to induce membrane fusion. Therefore, while contradictory results reported in the literature suggest that gp41 individual glycosylation sites are dispensable for the bioactivity and conformation of env products, it appears that such is not the case when the whole gp41 glycan cluster is removed.

    Journal of virology 1993;67;1;150-60

  • Role of asparagine-linked glycosylation in human immunodeficiency virus type 1 transmembrane envelope function.

    Dedera DA, Gu RL and Ratner L

    Department of Medicine, Washington University School of Medicine, St. Louis, Missouri 63110.

    Transmembrane envelope protein (TM) residues 100, 105, and 128 of human immunodeficiency virus type 1 (HIV-1) strain HXB2 are potential sites for asparagine-linked oligosaccharide additions which are conserved among HIV-1 isolates, and all other lentivirus TM proteins. Site-specific mutants of each of the asparagine residues did not eliminate the ability of the virus to infect and replicate in CD4+ cells, but infectivity was reduced with all of these mutants, and syncytia induction was attenuated with two of these mutants. Studies of envelope expression of the mutant with the most severe defect demonstrated no significant effects on envelope protein synthesis, conformation, processing, multimerization, or release into the culture medium, suggesting that N-linked oligosaccharides are important in the specific fusion activity of TM.

    Funded by: NIAID NIH HHS: AI25903, AI27475

    Virology 1992;187;1;377-82

  • Effect of a glucosidase inhibitor on the bioactivity and immunoreactivity of human immunodeficiency virus type 1 envelope glycoprotein.

    Fenouillet E and Gluckman JC

    Laboratoire de Biologie et Génétique des Pathologies Immunitaires, URA CNRS 1463, CERVI, Hôpital de la Pitié-Salpêtrière, Paris, France.

    Apparently conflicting results have been reported regarding the role of env glycoprotein glycans in human immunodeficiency virus type 1 (HIV-1) infectivity and cytopathogenicity. Whereas we have shown that enzymic removal of carbohydrates from mature envelope glycoproteins has only limited effect on the ability of HIV-1 to bind to CD4 and to infect target cells, sugar analogues that interfere with the glycosylation process of the nascent molecule markedly reduce virus infectivity. Here we have investigated the effect of a glucosidase inhibitor, 1-deoxynojirimycin (dNM), on the bioactivity and immunoreactivity of precursor gp160 produced by recombinant vaccinia virus-infected BHK-21 cells (rgp160). dNM (4 mM) did not affect the amount of rgp160 recovered nor its secretion from the cells. As described by other authors the effect of dNM was incomplete, resulting in the production of rgp160, the glycosylation of which was heterogeneous with respect to apparent Mr distribution and to sensitivity to endoglycosidase H and endoglycosidase F, all the species being susceptible to N-glycanase. A major reduction of the binding to CD4+ cells was noted with rgp 160 produced by dNM-treated cells using a quantitative indirect immunofluorescence assay and labelling with polyclonal human anti-HIV IgG. Similarly, dNM treatment altered the accessibility to murine monoclonal antibody 110-4 of the exposed V3 loop of HIV-1 gp120 by at least 10-fold, as determined by either ELISA capture assay or immunoaffinity purification. Such bioactivity and conformation modifications, which result from the abnormal folding of the nascent glycoprotein due to aberrant glycosylation, may account for the impaired HIV-1 infectivity elicited by dNM.

    The Journal of general virology 1991;72 ( Pt 8);1919-26

  • Inhibition of HIV and SIV infectivity by blockade of alpha-glucosidase activity.

    Ratner L, vander Heyden N and Dedera D

    Department of Medicine, Washington University, St. Louis, Missouri 63110.

    Processing of HIV and SIV envelope oligosaccharides is critical for proper intracellular trafficking and function. An inhibitor of alpha-glucosidases I and II, N-butyl deoxynojirimycin (N-BuDNJ), retards HIV-1 and SIVmac spread in lymphocytes and monocytes by diminishing virus infectivity, and also causes a reduction in syncytia formation between infected cells and uninfected lymphocytes. N-BuDNJ retards envelope processing from the precursor form to the mature surface (SU) and transmembrane proteins in HIV-1- and SIVmac-infected cells, as well as in cells infected with vaccinia-HIV-1 envelope recombinant virus. However, no significant reduction is seen in the amount of SU in released virus particles, though the virus particle-associated SU from N-BuDNJ-treated cells has an altered electrophoretic mobility. In contrast, N-BuDNJ had no effect on GAG protein synthesis and processing. These findings demonstrate a critical requirement for oligosaccharide processing by alpha-glucosidases I and II for HIV-1 and SIVmac envelope processing and fusogenicity.

    Funded by: NIAID NIH HHS: AI24745, AI25903, AI27302

    Virology 1991;181;1;180-92

  • Temporal expression of HIV-1 envelope proteins in baculovirus-infected insect cells: implications for glycosylation and CD4 binding.

    Murphy CI, Lennick M, Lehar SM, Beltz GA and Young E

    Cambridge Bioscience Corporation, Worcester, MA 01605.

    Three different human immunodeficiency virus type I (HIV-1) envelope derived recombinant proteins and the full length human CD4 polypeptide were expressed in Spodoptera frugiperda (Sf9) cells. DNA constructs encoding CD4, gp120, gp160, and gp160 delta (full length gp160 minus the transmembrane and cytoplasmic region of gp41) were cloned into the baculovirus expression vector pVL941 or a derivative and used to generate recombinant viruses in a cotransfection with DNA from Autographa californica nuclear polyhedrosis virus (AcMNPV). Western blotting of cell extracts of the recombinant HIV-1 proteins showed that for each construct two major bands specifically reacted with anti-HIV-1 envelope antiserum. These bands corresponded to glycosylated and nonglycosylated versions of the HIV proteins as determined by 3H-mannose labeling and tunicamycin treatment of infected cells. A time course of HIV envelope expression revealed that at early times post-infection (24 hours) the proteins were fully glycosylated and soluble in nonionic detergents. However, at later times postinfection (48 hours), expression levels of recombinant protein reached a maximum but most of the increase was due to a rise in the level of the nonglycosylated species, which was largely insoluble in nonionic detergents. Thus, it appears that Sf9 cells cannot process large amounts of glycosylated recombinant proteins efficiently. As a measure of biological activity, the CD4 binding ability of both glycosylated and nonglycosylated recombinant HIV envelope proteins was tested in a coimmunoprecipitation assay. The results showed that CD4 and the glycosylated versions of recombinant gp120 or gp160 delta specifically associated with one another in this analysis. Nonglycosylated gp120 or gp160 delta proteins from tunicamycin-treated cultures did immunoprecipitate with anti-HIV-1 antiserum but did not interact with CD4. We conclude that production of native HIV envelope proteins, as measured by addition of carbohydrate side chains and ability to bind CD4, peaks early after infection in baculovirus-infected insect cells.

    Funded by: NIAID NIH HHS: AI27135

    Genetic analysis, techniques and applications 1990;7;6;160-71

  • AIDS and glycosylation.

    Feizi T and Larkin M

    Glycoconjugates Section, MRC Clinical Research Centre, Harrow, Middlesex, UK.

    Glycobiology 1990;1;1;17-23

  • 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.

    Leonard CK, Spellman MW, Riddle L, Harris RJ, Thomas JN and Gregory TJ

    Department of Medicinal Chemistry, Genentech, Inc., South San Francisco, California 94080.

    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.

    The Journal of biological chemistry 1990;265;18;10373-82

  • Effect of N-(3-phenyl-2-propenyl)-1-deoxynojirimycin on the lectin binding to HIV-1 glycoproteins.

    Shimizu H, Tsuchie H, Honma H, Yoshida K, Tsuruoka T, Ushijima H and Kitamura T

    AIDS Research Center, National Institute of Health, Tokyo, Japan.

    The effect of N-(3-phenyl-2-propenyl)-1-deoxynojirimycin (ppDNM) on the lectin binding to HIV-1 glycoprotein was analyzed by using biotinylated lectins of various sugar specificities as probes. ppDNM potentially inhibited HIV-1-induced syncytium formation and viral infectivity of HIV-1 without cytotoxicity. The lectin binding assay showed that ppDNM treatment reduced Con A binding to gp120 of HIV-1.

    Japanese journal of medical science & biology 1990;43;3;75-87

  • Characterization of the secreted, native gp120 and gp160 of the human immunodeficiency virus type 1.

    Kalyanaraman VS, Rodriguez V, Veronese F, Rahman R, Lusso P, DeVico AL, Copeland T, Oroszlan S, Gallo RC and Sarngadharan MG

    Bionetics Research Inc., Kensington, MD 20895.

    We have previously shown that the cell line 6D5(451) chronically infected with the HIV-1 isolate HTLV-III(451), secretes the HIV-1 envelope glycoproteins gp120 and gp160 in the extracellular medium. The HTLV-III(451) gp120 and gp160 were purified by sequential affinity chromatographic steps using a monoclonal antibody to HIV-1 gp41 and an anti-HIV-1-positive human serum. Amino acid sequence analysis of gp120 and gp160 showed the loss of the signal peptide. Digestion of the purified gp120 and gp160 with endoglycosidases revealed that both proteins are heavily glycosylated and contain complex carbohydrates, in contrast to the intracellular form of gp160 which has been shown to contain mannose-rich immature sugars. Competitive binding analysis showed that while both gp120 and gp160 bind CD4, the affinity of gp160 was five times lower than that of gp120. Both gp120 and gp160 inhibited syncytia formation by HIV-1-infected cells when mixed with CD4+ cells. Furthermore, both gp120 and gp160 had strong mitogenic effects on the T cells from HIV-1-infected gibbons but not on cells from uninfected gibbons.

    AIDS research and human retroviruses 1990;6;3;371-80

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

    Dewar RL, Vasudevachari MB, Natarajan V and Salzman NP

    Division of Molecular Virology and Immunology, Georgetown University School of Medicine, D.C. 20007.

    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.

    Funded by: NIAID NIH HHS: N01-AI-72623

    Journal of virology 1989;63;6;2452-6

  • Role of oligosaccharides in the processing and maturation of envelope glycoproteins of human immunodeficiency virus type 1.

    Pal R, Hoke GM and Sarngadharan MG

    Department of Cell Biology, Bionetics Research, Inc., Kensington, MD 20895.

    The processing and maturation of envelope glycoproteins of human immunodeficiency virus type 1 (HIV-1) were studied in infected cells treated with inhibitors of oligosaccharide processing. In MOLT-3 cells chronically infected with HIV-1 (strain HTLV-IIIB), tunicamycin severely inhibited the glycosylation of envelope proteins. Deoxynojirimycin, an inhibitor of glucosidase I in the rough endoplasmic reticulum, inhibited the proteolytic processing of gp160, whereas no such effect was noted with either deoxymannojirimycin or swainsonine, inhibitors of mannosidase I and II, respectively, in the Golgi complex. The processed gp120 and gp41 synthesized in the presence of deoxymannojirimycin were found to contain mannose-rich oligosaccharide cores as evidenced by their susceptibility to endoglycosidase H digestion. The formation of syncytia normally observed when CEM cells are cocultured with HIV-1-infected cells was markedly inhibited in the presence of deoxynojirimycin, but such inhibition was not observed in cells treated with deoxymannojirimycin or swainsonine. The infectivity of virions released from MOLT-3/HTLV-IIIB cells treated with deoxynojirimycin or deoxymannojirimycin was significantly lower than the infectivity of virions released from untreated cells. On the other hand, treatment with swainsonine did not affect the infectivity of the progeny virus. These results suggest that the proteolytic processing of gp160 takes place in infected cells when the glycoprotein has mannose-rich oligosaccharide structures. Trimming of glucose residues and the primary trimming of mannose residues are necessary for the release of infectious virus.

    Funded by: NCI NIH HHS: N0L-CP-73725

    Proceedings of the National Academy of Sciences of the United States of America 1989;86;9;3384-8

  • Glycosylation and processing of the human immunodeficiency virus type 1 envelope protein.

    Kozarsky K, Penman M, Basiripour L, Haseltine W, Sodroski J and Krieger M

    Department of Biology, Massachusetts Institute of Technology, Cambridge 02139.

    The human immunodeficiency virus type 1 (HIV-1) envelope protein is synthesized as a gp160 precursor that is cleaved to a 120 kDa exterior glycoprotein (gp120) and a 41 kDa transmembrane glycoprotein (gp41). The HIV-1 envelope protein was stably expressed under the control of the transactivator proteins tat and rev, in wild-type and mutant Chinese hamster ovary (CHO) cells. The mutant, ldlD, is conditionally defective for the addition of galactose and N-acetylgalactosamine to oligosaccharide chains. The effects of glycosylation modification on the HIV-1 envelope's structure and function were examined. The effects of galactosylation on the structure of the envelope proteins suggest that cleavage of the gp160 precursor into gp120 and gp41 occurs intracellularly, apparently concurrent with the addition of galactose to N-linked oligosaccharides of the envelope proteins. No evidence for O-linked glycosylation of the envelope proteins in CHO cells was observed. The envelope protein in the transfected hamster cells mediated the fusion of these cells with CD4-positive lymphocytes, and this fusogenic activity was independent of the addition of either galactose or N-acetylgalactosamine to oligosaccharides in the transfected cells.

    Funded by: NIAID NIH HHS: AI 24755

    Journal of acquired immune deficiency syndromes 1989;2;2;163-9

  • Role of protein N-glycosylation in pathogenesis of human immunodeficiency virus type 1.

    Montefiori DC, Robinson WE and Mitchell WM

    Department of Pathology, Vanderbilt University, School of Medicine, Nashville, TN 37232.

    Human immunodeficiency virus type 1 (HIV-1), the retrovirus responsible for acquired immunodeficiency syndrome (AIDS), contains two heavily glycosylated envelope proteins, gp120 and gp41, which mediate attachment of virions to glycosylated cell surface receptor molecules (CD4 antigens) and appear to be responsible for syncytium formation and associated cytopathic effects of this virus. A comprehensive study of the effects of N-linked glycoprotein processing inhibitors on HIV-1 replication, infectivity, cytopathicity, target-cell infectibility, syncytium formation, and gp120 electrophoretic mobility was conducted to assess the importance of protein glycosylation in the pathogenesis of HIV-1 in vitro. The electrophoretic mobility of gp120 was decreased when gp120 was synthesized in the presence of castanospermine or 1-deoxynojirimycin (inhibitors of glucosidase I), increased when gp120 was synthesized in the presence of 1-deoxymannojirimycin (mannosidase I) or swainsonine (mannosidase II), and unaffected when gp120 was synthesized in the presence of bromoconduritol (glucosidase II). Inhibition by tunicamycin (lipid-linked oligosaccharide precursor synthesis), castanospermine, 1-deoxynojirimycin, and 1-deoxymannojirimycin attenuated HIV-1 infectivity and blocked HIV-1-induced syncytium formation and cytopathicity, whereas bromoconduritol and swainsonine failed to have such effects. None of the inhibitors interfered with virus replication in acutely infected cells or affected the ability of target cells to form syncytia with untreated HIV-1-infected cells. These results demonstrate that protein N-glycosylation is critical to the pathogenesis of HIV-1 at the levels of viral infectivity and cytopathicity but not at the level of virus replication or of host-cell infectibility.

    Funded by: NIAID NIH HHS: AI25272

    Proceedings of the National Academy of Sciences of the United States of America 1988;85;23;9248-52

  • Interference with HIV-induced syncytium formation and viral infectivity by inhibitors of trimming glucosidase.

    Gruters RA, Neefjes JJ, Tersmette M, de Goede RE, Tulp A, Huisman HG, Miedema F and Ploegh HL

    Central Laboratory, Netherlands Red Cross Blood Transfusion Service, University of Amsterdam.

    Human immunodeficiency virus (HIV), the causative agent of AIDS, infects human lymphocytes and monocytes. An interaction between the viral envelope gp 120 and CD4 protein is required to initiate an infectious cycle. HIV infection in vitro induces syncytium formation by cell-to-cell fusion; this aspect of viral cytopathogenicity is even more dependent on gp120-CD4 interactions. That gp120 is extremely heavily glycosylated (31-36 N-linked glycans per molecule), suggests involvement of N-linked glycans in the gp120-CD4 interaction. We therefore investigated the effects of castanospermine, 1-deoxynojirimycin (dNM) and 1-deoxymannojirimycin (dMM), three trimming glycosidase inhibitors which perturb N-linked glycan structure, on induction of the formation of syncytium between HIV-infected and CD4-expressing cells. The glucosidase inhibitors castanospermine and dNM, but not the mannosidase inhibitor dMM, inhibited syncytium formation and interfered with infectivity. The potential of glucosidase inhibitors as anti-HIV therapeutic agents deserves further investigation, especially because dNM and related compounds show little toxicity in vitro and in vivo.

    Nature 1987;330;6143;74-7

  • Inhibition of human immunodeficiency virus syncytium formation and virus replication by castanospermine.

    Walker BD, Kowalski M, Goh WC, Kozarsky K, Krieger M, Rosen C, Rohrschneider L, Haseltine WA and Sodroski J

    Infectious Disease Unit, Massachusetts General Hospital, Boston.

    Castanospermine (1,6,7,8-tetrahydroxyoctahydroindolizine) is a plant alkaloid that modifies glycosylation by inhibiting alpha-glucosidase I. Castanospermine is shown to inhibit syncytium formation induced by the envelope glycoprotein of the human immunodeficiency virus and to inhibit viral replication. The decrease in syncytium formation in the presence of castanospermine can be attributed to inhibition of processing of the envelope precursor protein gp160, with resultant decreased cell surface expression of the mature envelope glycoprotein gp120. In addition, castanospermine may cause defects in steps involved in membrane fusion after binding of CD4 antigen. The antiviral effects of castanospermine may be due to modifications of the envelope glycoprotein that affect the ability of the virus to enter cells after attachment to the CD4 cell receptor.

    Funded by: NCI NIH HHS: CA28151, CA40658, CA40987

    Proceedings of the National Academy of Sciences of the United States of America 1987;84;22;8120-4

  • Evidence that mannosyl residues are involved in human immunodeficiency virus type 1 (HIV-1) pathogenesis.

    Robinson WE, Montefiori DC and Mitchell WM

    Department of Pathology, Vanderbilt University School of Medicine, Nashville, TN 37232.

    The Human Immunodeficiency Virus (HIV), the causative agent of AIDS, is thought to bind to T4+ (CD4+) target cells through the heavily glycosylated gp120 envelope glycoprotein. Plant lectins bind glycoproteins through noncovalent interaction with specific hexose residues; therefore, lectins were evaluated for their ability to inactivate HIV in vitro. The mannose-specific lectins concanavalin-A and succinyl concanavalin-A completely inactivated HIV while lentil lectin, wheat germ agglutinin, and phytohemagglutinin-P substantially inactivated HIV. BS-II, Vicia villosa (hairy vetch), and Ptilota plumosa (red marine algae) failed to alter the infectibility of HIV. Neither simple stearic hindrance, viral aggregation, nor lectin-cell interactions served to explain this phenomenon. Glycoprotein glycosylation was evaluated by differential lectin binding as well as molecular weight changes in gp120 when virus was produced in the presence of swainsonine, a glycosylation inhibitor. Lentil lectin bound gp120 better than concanavalin-A, suggesting the majority of glycosylation sites are fucosylated. The apparent molecular weight of gp120 was reduced by swainsonine, although HIV infectivity and concanavalin-A inactivation were retained. Thus, at least some N-glycosylation sites are complex-type glycoproteins but regions external to the (GlcNAc)2(Man)3 "core" pentasaccharide region are not required for HIV infectivity. It appears that the site or sites involved are nonfucosylated, high mannose and/or biantennary, nonsialylated, N-glycosylated regions of gp120 or gp41. Alternatively, they may be in close approximation to such carbohydrate regions.

    Funded by: NIAID NIH HHS: AI25272

    AIDS research and human retroviruses 1987;3;3;265-82

  • Glycosylation inhibitors block the expression of LAV/HTLV-III (HIV) glycoproteins.

    Blough HA, Pauwels R, De Clercq E, Cogniaux J, Sprecher-Goldberger S and Thiry L

    The glycosylation inhibitors 2-deoxy-D-glucose (2-dGlc) and, to a lesser extent, beta-hydroxynorvaline blocked the formation of syncytia in HIV (LAV/HTLV-III)-infected cells. Using monospecific polyclonal antibodies against recombinant envelope proteins gp110 and gp41 or monoclonal antibodies against env gp110, we could demonstrate a marked reduction in the immunoreactivity of these antigens in HIV-infected cells exposed to the glycosylation inhibitors. There was concomitant accumulation of core proteins p15 and p24, as shown by a solid phase radio-immunoassay, and a decreased oligosaccharide synthesis of env proteins, as monitored by the incorporation of [6-3H]GlcNAc. The reverse transcriptase was not affected by the compounds. Glycosylation inhibitors may be considered for the chemotherapy of AIDS or AIDS-related complex, or chemoprophylaxis of HIV-positive individuals.

    Biochemical and biophysical research communications 1986;141;1;33-8

Gene lists (5)

Gene List Source Species Name Description Gene count
L00000009 G2C Homo sapiens Human PSD Human orthologues of mouse PSD adapted from Collins et al (2006) 1080
L00000016 G2C Homo sapiens Human PSP Human orthologues of mouse PSP adapted from Collins et al (2006) 1121
L00000061 G2C Homo sapiens BAYES-COLLINS-MOUSE-PSD-CONSENSUS Mouse cortex PSD consensus (ortho) 984
L00000069 G2C Homo sapiens BAYES-COLLINS-HUMAN-PSD-FULL Human cortex biopsy PSD full list 1461
L00000071 G2C Homo sapiens BAYES-COLLINS-MOUSE-PSD-FULL Mouse cortex PSD full list (ortho) 1556
© G2C 2014. The Genes to Cognition Programme received funding from The Wellcome Trust and the EU FP7 Framework Programmes:
EUROSPIN (FP7-HEALTH-241498), SynSys (FP7-HEALTH-242167) and GENCODYS (FP7-HEALTH-241995).

Cookies Policy | Terms and Conditions. This site is hosted by Edinburgh University and the Genes to Cognition Programme.