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At least 19 recordsLinked to original sources
PubMed1992

The lymphoma transmembrane glycoprotein GP85 (CD44) is a novel guanine nucleotide-binding protein which regulates GP85 (CD44)-ankyrin interaction.

In this study, we have used photoaffinity labeling by [32P]azido-GTP as well as [32P]ADP-ribosylation by pertussis toxin (PT) and cholera toxin (CT) to identify GTP-binding proteins associated with mouse T-lymphoma plasma membranes. Our results indicate that GP85 (CD44) can be photoaffinity labeled by [32P] azido-GTP and [32P]ADP-ribosylated by both PT and CT. Using purified GP85 (CD44) obtained by Triton X-100 extraction, wheat germ agglutinin-Sepharose, and anti-GP85 (CD44) antibody affinity chromatographies, we have further characterized GP85 (CD44) as a GTP-binding protein. GP85 (CD44) is found to bind guanosine 5'-3-O-(thio)triphosphate (GTP gamma S) in a time- and dose-dependent manner with a dissociation constant of 0.83 nM. Importantly, GP85 (CD44) appears to display a GTPase activity which hydrolyzes [gamma-32P]GTP at a rate of 0.011 mol of Pi released/mol of GP85 (CD44)/min. This GTPase activity can be readily inhibited by PT- or CT-mediated ribosylation of GP85 (CD44). Most interestingly, GTP binding significantly enhances the interaction of purified GP85 (CD44) with ankyrin, whereas ADP-ribosylation of GP85 (CD44) by PT or CT inhibits the GTP-induced increase in ankyrin binding to GP85 (CD44). In addition to GP85 (CD44) being the first reported transmembrane GTP-binding protein, these results suggest that GTP plays an important role in promoting the interaction between GP85 (CD44) and its underlying membrane cytoskeleton through ankyrin.

Adenosine Diphosphate Ribose↗
PubMed1991

Acylation of the lymphoma transmembrane glycoprotein, GP85, may be required for GP85-ankyrin interaction.

The lymphoma plasma membrane glycoprotein, GP85, is a transmembrane glycoprotein that binds directly to ankyrin, a molecule known to link the plasma membrane with the underlying cytoskeleton. In this study, we have demonstrated that palmitic acid is incorporated into GP85 in vivo and that the amount of palmitic acid incorporated is greatly stimulated during lymphoma cap formation. The majority of the incorporated palmitic acid appears to be strongly linked to GP85 since it is not dissociated by strong detergents (e.g. sodium dodecyl sulfate) or by chloroform/methanol extraction, but is labile to alkaline or acid hydrolysis. Furthermore, we have established that deacylation of GP85 (i.e. removal of the palmitic acid moiety from GP85 by 1 M hydroxylamine treatment) significantly reduces the binding affinity between GP85 and ankyrin, and reacylation of GP85 restores the binding affinity. These findings suggest that fatty acid acylation of GP85 by palmitic acid may be required for the stable attachment of the cytoskeleton to the lymphoma plasma membrane.

Animals↗
PubMed1990

Expression of avian leukaemia virus env-gp85 in Spodoptera frugiperda cells by use of a baculovirus expression vector.

We studied the genetic expression of gp85 of avian leukaemia virus (ALV) subgroup A in a baculovirus/insect cell system. 5'terminal sequences of the gag gene were added to precede the ALV gp85 sequence and a stop codon was introduced at the boundary of gp85 and gp37. The resulting construct was then cloned into the baculovirus transfer vector pAcYM1, which contains the polyhedrin promoter of Autographa californica nuclear polyhedrosis virus (AcNPV). Cells of the insect Spodoptera frugiperda (Sf9) were cotransfected with the resulting recombinant transfer vector pAc85 and infectious AcNPV/E2 DNA. After cotransfection, recombinant baculovirus that lacked the polyhedrin gene and expressed gp85 was selected from the supernatant and used to infect Sf9 cells. The expression of the gp85 gene peaked 3 days after infection, but expression products were not released into the culture medium even though the signal peptide had been cleaved. Owing to incomplete N-glycosylation in the insect cells the largest gp85 product had an Mr of only 65,000. In immunofluorescence tests and immunoblots the recombinant gp85 products reacted with polyclonal and monoclonal antibodies directed against ALV gp85 of subgroup A. Chickens inoculated with crude lysates of Sf9 cells infected with gp85-expressing recombinant baculovirus developed antibodies directed against ALV gp85. These antibodies were not capable of neutralizing ALV.

Animals↗
PubMed1991

Post-translational protein modification and expression of ankyrin-binding site(s) in GP85 (Pgp-1/CD44) and its biosynthetic precursors during T-lymphoma membrane biosynthesis.

In this study, we have investigated the biosynthesis and processing of GP85 (Pgp-1/CD44), a lymphoma transmembrane glycoprotein known to contain ankyrin-binding site(s). Using a standard pulse-chase protocol, we have detected a 52-kDa polypeptide precursor (p52) within the first 5 min of pulse labeling which contains a high mannose-type N-linked oligosaccharide chains. The conversion of p52 to GP85 requires further glycosylation (both complex type N-linked and O-linked) which takes place in the Golgi complex within 10-20 min after p52 is synthesized. GP85 is then incorporated into the plasma membrane where its turnover rate is relatively slow, a t1/2 of approximately 8 h. Following tunicamycin treatment, we have detected two other precursor proteins: p42 which is unglycosylated and p58 which is O-glycosylated. p42 appears to be an immediate precursor of p52 because p52 is converted to p42 upon deglycosylation. Therefore, the biosynthesis of GP85 appears to occur in the following sequence: p42 in equilibrium to p52 in equilibrium to GP85. Further analysis reveals that all of the GP85 precursors (i.e. p42, p52, and p58) contain ankyrin-binding site(s). Chemical composition analysis of GP85 indicates that this molecule contains approximately 3 N-linked and 4-5 O-linked oligosaccharide chains. Although neither N-glycosylation nor O-glycosylation appears to play an important role in the formation of ankyrin-binding site(s), O-glycosylation (and to a lesser extent N-glycosylation) of GP85 is required for T-lymphoma cell surface interaction with both collagen and hyaluronic acid. These findings suggest that GP85 (Pgp-1/CD44) and its biosynthetic precursors play a pivotal role in regulating adhesion functions such as lymphocyte homing and binding to the extracellular matrix.

Animals↗
PubMed1991

Genetic analysis of the Rous sarcoma virus subgroup D env gene: mammal tropism correlates with temperature sensitivity of gp85.

Subgroup D avian sarcoma and leukosis viruses can penetrate a variety of mammalian cells in addition to cells from their natural host, chickens. Sequences derived from the gp85-coding domain within the env gene of a mammal-tropic subgroup D virus (Schmidt-Ruppin D strain of Rous sarcoma virus [SR-D RSV]) and a non-mammal-tropic subgroup B virus (Rous-associated virus type 2) were recombined to map genetic determinants that allow penetration of mammalian cells. The following conclusions were based on host range analysis of the recombinant viruses. (i) The determinants of gp85 that result in the mammal tropism phenotype of SR-D RSV are encoded within the 160 codons that lie 3' of codon 121 from the corresponding amino terminus of the gp85 protein. (ii) Small linear domains of the SR-D RSV gp85-coding domain placed in the subgroup B background did not yield viruses with titers equal to that of the subgroup D virus in a human cell line. (iii) Recombinant viruses that contained subgroup D sequences within the hr1 variable domain of gp85 showed modest-to-significant increases in infectivity on human cells relative to chicken cells. A recombinant virus that contained three fortuitous amino acid substitutions in the gp85-coding domain was found to penetrate the human cell line and give a titer similar to that of the subgroup D virus. In addition, we found that the subgroup D virus, the mutant virus, and recombinant viruses with an increased mammal tropism phenotype were unstable at 42 degrees C. These results suggest that the mammal tropism of the SR-D strain is not related to altered receptor specificity but rather to an unstable and fusogenic viral glycoprotein. A temperature sensitivity phenotype for infectivity of mammalian cells was also observed for another mammal-tropic avian retrovirus, the Bratislava 77 strain of RSV, a subgroup C virus, but was not seen for any other avian retrovirus tested, strengthening the correlation between mammal tropism and temperature sensitivity.

Animals↗
PubMed2026

Molecular characterization and biological characteristics of a highly pathogenic recombinant ALV-J strain (HUE2023) with cross-clade gp85 recombination.

Avian leukosis virus subgroup J (ALV-J) has undergone extensive diversification into phylogenetically distinct clades, yet whether recombination between these clades within the gp85 envelope glycoprotein generates variants with altered pathogenicity has received little direct investigation. A field strain (HUE2023) was recovered from breeding roosters displaying vascular tumors. The viral genome was sequenced and subjected to phylogenetic and recombination analyses. The three-dimensional structure of gp85 was predicted with AlphaFold3; electrostatic surface potentials and surface hydrophobicity were computed using the Adaptive Poisson-Boltzmann Solver and the Eisenberg hydrophobicity scale, respectively. Pathogenicity and immunosuppressive effects were assessed in Hy-Line Brown chickens. Recombination analysis revealed that HUE2023 is an inter-clade recombinant derived from Clade 1.1 (major parent: JS14NT01) and Clade 1.2 (minor parent: JS09GY3). A single-residue deletion at position 61 within receptor-binding domain 1 (RBD-1), unique to the recombinant, induced a localized conformational rearrangement that generated a concentrated electronegative surface patch and a contiguous hydrophobic pocket not observed in either parental gp85. Animal challenge showed that HUE2023 is highly pathogenic: female chickens in the high-dose group reached only 61% survival and displayed significant growth retardation (P&#x202f;<&#x202f;0.05) together with marked immunosuppression. The recombination in the RBD-1 led to local conformational rearrangement, resulting in a concentrated and negatively charged surface area as well as a continuous hydrophobic pocket, which were never present in any of the parental gp85 sequences. These results indicate that gp85 recombination across clades can yield variants with fundamentally altered receptor-binding surfaces and argue for integrating structural surveillance into ALV-J monitoring programmes.

Animals↗
PubMed1992

Expression of feline leukaemia virus gp85 and gag proteins and assembly into virus-like particles using the baculovirus expression vector system.

In order to test components of feline leukaemia virus (FeLV) as subunit vaccines, we have constructed recombinant baculoviruses that express the FeLV envelope glycoprotein gp85 [Autographa californica nuclear polyhedrosis virus (AcNPV)-gp85] and the structural protein, gag (AcNPVgag). The gag protein is expressed and shed into the medium of infected cells as particles which have a buoyant density on sucrose gradients and appearance by electron microscopy similar to those of authentic FeLV virions. The gag precursor protein within the particles is not fully processed and appears to be a result of partial cleavage of the gag polypeptide. Insect cells that are coinfected with AcNPVgag and AcNPVgp85 shed particles that contain both the gag protein and the gp85 glycoprotein.

Baculoviridae↗
PubMed1977

Proteins of Rous-associated virus type 61: polypeptide stoichiometry and evidence that glycoprotein gp35 is not a cleavage product of gp85.

The two glycoproteins, gp85 and gp35, of Rous-associated virus type 61 (RAV-61), were isolated from radiolabeled virions by gel electrophoresis and digested with trypsin. The chromatographic profile of the gp35 digest revealed no peaks in common with that of gp85; therefore, the smaller glycoprotein is not a cleavage product of gp85. The stoichiometry of radiolabeled RAV-61 proteins was studied by quantitative gel filtration and gel electrophoresis. Among the 11 polypeptides identified were 4 minor ones, including the beta(p91) and alpha(p64) chains of reverse transcriptase and two unidentified chains, p76 and p35; the latter two were unmasked by removing the virions' surface glycoproteins with a protease, bromelain. Virions contained some 15 to 30 molecules of reverse transcriptase.

Animals↗
PubMed1978

Proteins of Rous-associated virus 61, an avian retrovirus: common precursor for glycoproteins gp85 and gp35 and use of pactamycin to map translational order of proteins in the gag, pol, and env genes.

Cells infected by Rous-associated virus 61 (RAV-61) contained a precursor-like protein, pr90, that was specifically precipitated by antiserum directed against envelope glycoproteins, gp85 and gp35. Tryptic peptide mapping showed that pr90 contained tryptic sequences of both gp85 and gp35. Pactamycin mapping experiments indicated that the two glycoproteins are translated from the env-mRNA in the order (5') gp85--gp35. The pactamycin mapping experiments also indicated a translational order of p10--(p27, p12)--p15 for the gag proteins; this agreement with the order previously reported from tryptic mapping studies on precursor pr76 of avian myeloblastosis virus implied that the stoichiometry of the core proteins was unchanged when virions were assembled in the presence of pactamycin. The reverse transcriptase proteins, unlike those of the env and gag genes, fell on the right side of the pactamycin map. This result is in accord with the idea that most, if not all, of the reverse transcriptase protein is translated by read-through of the gag(pol) message rather than by translation of a hypothetical pol-mRNA devoted solely to synthesis of that protein.

Avian Leukosis Virus↗
PubMed1976

Expression of viral proteins in mammalian cells transformed by avian sarcoma viruses.

The expression of viral proteins in nine lines of hamster and rat cells transformed by avian sarcoma viruses (ASV) was studied by indirect immunofluorescence with monospecific antisera to purified gp85 and p27 of AMV-B and a polyvalent antiserum to all the p proteins of this same virus. The lines of ASV-transformed cells were either low virus producers (VP) or inducible or non-inducible non producers (NP). Cytoplasmic expression of p proteins was observed in all the cell lines except the least inducible NP cell line, and cytoplasmic expression of gp85 in all the cell lines. The degree of expression varied widely with the lines and was not related to the class of permissiveness or inducibility. However, in the inducible NP class, the expression of p proteins and gp85 was higher in the most inducible cell lines. The data also suggest that the expression of the p proteins must be uncoordinate in at least some cell lines and must also be uncoordinate with the expression of gp85. In the VP cell lines and the most inducible NP lines, g85 and some p proteins other than p27 were also expressed on the cell membrane. The membrane expression of gp85 and the p proteins which were expressed appeared to be coordinate and to parallel the degree of cytoplasmic expression. In contrast, no, or a negligible expression of viral proteins was observed on the membrane of the least inducible and the non-inducible cell lines. These results suggest that there may exist translational and/or post-translational controls of the expression of viral proteins in the ASV-transformed mammalian cells and that the permissiveness and the inducibility of the cells may depend on the insertion of viral proteins in the cell membrane. The failure of p27 to insert in the cell membrane could account for the low permissiveness or the non-permissiveness of the cells.

Alpharetrovirus↗
PubMed1991

An 85-kilodalton surface antigen gene family of Trypanosoma cruzi encodes polypeptides homologous to bacterial neuraminidases.

We have determined the sequence of a cDNA (Tt34c1) encoding a Trypanosoma cruzi trypomastigote stage-specific 85-kDa surface glycoprotein (gp85). Within the peptide sequence of Tt34c1 are two 8-amino acid motifs, Ser-X-Asp-X-Gly-X-Thr-Trp, that are characteristic of bacterial neuraminidases. Analysis of the Tt34c1 sequence predicts the presence of an amino-terminal signal sequence and a hydrophobic carboxy-terminus that is probably replaced by a glycosyl phosphatidylinositol membrane anchor. Gp85 is encoded by an extensive multigene family that is distributed throughout the genome and can be divided into subsets on the basis of oligonucleotide hybridisation patterns. By sequencing products of polymerase chain reaction (PCR) amplification of the 5' end of trypomastigote gp85 mRNA we show that multiple copies of the gene family are transcribed simultaneously in a trypanosome population. Comparison of the sequence of the PCR clones and another gp85 cDNA showed a highly conserved region 5' of the first methionine extending 180 nt into the coding sequence. Insertions and point mutations were observable outside these homologous sequences demonstrating the variant nature of the gp85 mRNAs.

Amino Acid Sequence↗
PubMed1977

Analysis of precursors to the envelope glycoproteins of avian RNA tumor viruses in chicken and quail cells.

Immune precipitation with monospecific antiserum was employed to study the intracellular synthesis of viral glycoproteins gp85 and gp37. Labeled gp85 and gp37 were detected from lysates of cells transformed with Rous sacroma virus, strain B77, after long-term labeling with radioactive glucosamine or phenylalanine. Immune precipitates prepared from lysates of cells pulse-labeled for a short time resulted in a glycoprotein of 92,000 molecular weight (gp92). This precursor was stable in B77-transformed Japanese quail cells for several hours, whereas in chicken cells it could be chased within a few hours into virion glycoproteins gp85 and gp37. Similarly, the precursor for the structural viral proteins, pr76, persisted in quail cells much longer than in chicken cells. During very short pulses or in the presence of a glucosamine block (25 mM glucosamine), the antiserum against the viral envelope glycoproteins detected a precursor of higher electrophoretic mobility of approximately 70,000 molecular weight, "p70." Fucose label entered gp92 and gp85 as well as "p70." Proteolytic treatment of virion-bound gp85 in vitro generated two discrete glycoproteins of 62,000 and 45,000 molecular weight, but did not result in an increase in the amount of gp37.

Animals↗
PubMed1976

Formation of an infectious virus-antibody complex with Rous sarcoma virus and antibodies directed against the major virus glycoprotein.

Preparations of Rous sarcoma virus (RSV) can form an infectious viral-antibody complex with antibodies raised against the major glycoprotein, gp85, isolated from avian myeloblastosis virus and Prague-RSV subgroup C. Binding of anti-gp85 antibodies to RSV can be demonstrated by the inhibition of focus-forming activity after addition of goat anti-rabbit immunoglobulin and by a shift in density of virions treated with anti-gp85 serum. Group- rather than subgroup- specific regions of viral gp85 appear to be the site of binding for infectious complex.

Animals↗
PubMed1978

Rous sarcoma virus-transformed avian cells express four different cell surface antigens that are distinguishable by a cell-mediated cytotoxicity-blocking test.

Japanese quails bearing avian sarcoma virus-induced tumors develop immune spleen cells that are cytotoxic in vitro against virally and chemically transformed cells, as well as against embryonic cells. The cell-mediated cytotoxicity can be blocked by soluble antigens extracted from in vitro cultured cells. The existence of partial as well as total blocking effects in tests with extracts from various transformed and untransformed virus-producing cells makes it possible to distinguish up to four different kinds of antigens expressed on sarcoma virus transformed cells: a) a subgroup-specific determinant of the virus-envelope glycoprotein gp85 (s-gp85) is expressed at the surface of productively infected, tranformed as well as untransformed cells; b) a group-specific determinant of gp85 (g-gp85) that is only expressed on the surface of virus-transformed cells; c) embryonic antigens, also detectable on chemically transformed as well as on primary normal embryonic cells, and finally; d) a sarcoma virus transformation-specific antigen (TSSA) that is not a structural constituent of the virus.

Animals↗
PubMed1978

Surface expression of murine leukemia virus structural polypeptides on host cells and the virion.

The expression of murine leukemia virus structural polypeptides on the surface of cells producing exogenous Friend leukemia virus, endogenous ecotropic AKR and xenotropic BALB/c virus was investigated. Antisera to Friend virus gp71, p30, p15E, p12 and p10 were employed in a complement-dependent chromium release assay and to immunoprecipitate lactoperoxidase iodinated surface polypeptides prior to analysis in polyacrylamide gel electrophoresis. With the latter technique gag-gene encoded proteins and their precursors were not discovered on the viral and cellular surface membranes. Only env-gene encoded polypeptides gp85, gp71, and p15E were detectable. p15E is embedded into the lipid membrane. gp85 is formed by disulfide linkage of p15E to surface-exposed gp71. The ratio of gp71 to gp85 is variable and apparently determined by the host cell. Antibodies of strong cytotoxicity are those against type- and group-specific epitopes of gp71 as well as type-specific epitopes of p12.

Antibodies, Viral↗