Direct implication of surface mannosyl residues in cell adhesion of Dictyostelium discoideum

In the cell adhesion of aggregation-competent Dictyostelium cells, the requirement for the carbohydrate moiety of the glycoprotein appeared to be indirect in that it acts to protect the protein moiety from proteolytic degradation; however, the effect was limited to the tunicamycin (TM)-sensitive carbohydrate moiety (Hirano, T., et al. (1983) J. Biochem. 93, 1249-1257). In the present study, we showed that the EDTA-stable adhesion of aggregation-competent Dictyostelium cells was abolished by the treatment of intact cells with jack bean alpha-mannosidase, whereas neuraminidase, beta-galactosidase, beta-N-acetylhexosaminidase, or alpha-L-fucosidase had no effect. The EDTA-stable cohesiveness of TM-treated cells in the presence of leupeptin (TM/LP cells) was also abolished by the treatment of the cells with alpha-mannosidase. The effect of alpha-mannosidase was not prevented in the presence of LP. The N-glycoside-deficient contact site A (an adhesion-mediating glycoprotein) was obtained from TM/LP cells and was shown to have a molecular weight of 70,000. This protein (p 70) was shown to still have carbohydrates as detected by polyacrylamide gel electrophoresis (PAGE) in the presence of sodium dodecyl sulfate (SDS) and subsequent staining of the gel with periodic acid-silver stain. Moreover, p 70 reacted with anti-gp 68, which has a specificity against alpha-mannosyl residues of carbohydrate chains. However, p 70 treated with alpha-mannosidase showed decreased reactivity with anti-gp 68. The monovalent antibody fragment of anti-contact site A or anti-p 70 inhibited EDTA-stable cell adhesion of both control and TM/LP cells. These results indicated that TM-resistant mannosyl residues of contact site A are directly involved in EDTA-stable adhesion of aggregation-competent cells. This is the first report of the direct involvement of the carbohydrate moiety in cell adhesion of aggregation-competent Dictyostelium cells. A schematic model is presented of the role of the carbohydrate moiety in EDTA-stable cell adhesion, including the direct effect of carbohydrates.     

Cell-free sulfation of the contact site A glycoprotein of Dictyostelium discoideum and of a partially glycosylated precursor

An 80-kDa glycoprotein of Dictyostelium discoideum, designated contact site A, has been implicated in EDTA-stable cell adhesion. This protein is known to be the major sulfated protein of aggregation-competent cells and has been shown to contain two types of carbohydrate, sulfated type 1 and unsulfated type 2 carbohydrate moieties. Here we investigate the cell-free sulfation of this protein. In the homogenate of developing cells, [35S]sulfate was transferred by endogenous sulfotransferase from [35S]3'-phosphoadenosine-5'-phosphosulfate to the contact site A glycoprotein and to various other endogenous proteins. The sulfate was transferred to carbohydrate rather than to tyrosine residues. After differential centrifugation of the homogenate, the capacity for sulfation of the contact site A glycoprotein was barely detected in the plasma membrane-enriched 10,000 X g pellet fraction which contained the bulk of this glycoprotein, but was largely recovered in the 100,000 X g pellet fraction which contained only a small portion of this glycoprotein. After sucrose gradient centrifugation, the membranes containing the sulfation capacity were found to have a density characteristic for Golgi membranes. In immunoblots, monoclonal antibodies raised against the contact site A glycoprotein recognized not only this 80-kDa protein, but also a sulfatable 68-kDa protein found in the 100,000 X g pellet fraction. The 68-kDa protein did not react with monoclonal antibodies against type 2 carbohydrate but was converted by endoglycosidases F and H into a 53-kDa protein, indicating that it was a partially glycosylated form of the 80-kDa glycoprotein containing only type 1 carbohydrate. Isoelectric focusing showed that a substantial portion of the 68-kDa glycoprotein was unsulfated, even after cell-free sulfation. The 68-kDa glycoprotein was not found in the plasma membrane-enriched 10,000 X g pellet fraction and did not accumulate in parallel with the 80-kDa contact site A glycoprotein during cell development. We conclude that the 68-kDa glycoprotein is a precursor that is converted by attachment of type 2 carbohydrate and sulfation of type 1 carbohydrate into the mature 80-kDa glycoprotein. The precursor nature of the 68-kDa glycoprotein was supported by results obtained with mutant HL220 which is defective in glycosylation (Murray, B. A., Wheeler, S., Jongens, T., and Loomis, W. F. (1984) Mol. Cell. Biol. 4, 514-519). This mutant specifically lacks type 2 carbohydrate and produces a 68-Kda glycoprotein instead of the 80-kDa contact site A glycoprotein (Yoshida, M., Stadler, J., Bertholdt, G., and Gerisch, G. (1984) EMBO J. 3, 2663-2670).(ABSTRACT TRUNCATED AT 400 WORDS)     

Immunological analysis of a glycoprotein (contact sites A) involved in intercellular adhesion of dictyostelium discoideum

We have prepared antisera in rabbits to the “contact sites A” glycoprotein (gp80) purified from Dictyostelium discoideum. IgG isolated from these anti-sera reacts with a number of different proteins in D discoideum lysates, as analyzed by immune precipitation and by antibody staining of gel electropherograms transferred to nitrocellulose. Blocking experiments indicate that this cross-reactivity reflects the presence of common antigeneic determinants on gp80 and other cellular proteins, rather than the presence of extraneous antibodies in the antisera. The spectrum of reactive proteins is different a: different stages of development. In particular, gp80 itself is synthesized only for a restricted period during the cell aggregation phase. The protein persists throughout development and can be detected in spores. Anti-gp80 Fab fragments bind to the surface of developing D discoideum cells and specifically block their developmentally regulated adhesion. After absorption with vegetative cells, the IgG stains only gp80 and (to a lesser extent) one other band in lysates of aggregation-competent cells. The absorbed antibodies also can block adhesion. Several proteins that appear late in development also arc stained by the absorbed IgG.     

Determination of the protective epitopes on the glycoproteins of Venezuelan equine encephalomyelitis virus by passive transfer of monoclonal antibodies

We have previously characterized seven unique antigenic epitopes on the two envelope glycoproteins of the Venezuelan equine encephalomyelitis (VEE) virus vaccine strain, TC-83, by using monoclonal antibodies. The in vitro function of virus neutralization was primarily associated with one epitope on the gp56 (gp56c). To determine which epitopes were important in protecting animals from VEE infection, purified monoclonal antibodies were inoculated i.v. into 3-wk-old Swiss mice. Twenty-four hours later these animals were challenged i.p. with 100 IPLD50 of virulent VEE virus (Trinidad donkey). High-avidity anti-gp56c, anti-gp50b, anti-gp50c, and anti-gp50d monoclonal antibodies protected animals from virus challenge. Rabbit antisera to the gp56 and the gp50 glycoproteins were also effective in protecting mice from challenge with virulent VEE virus. Less antibody was needed to protect animals if the antibody was directed against the critical neutralization site. Less avid antibodies to the gp56c and gp50b epitopes demonstrated little or no protection in vivo. Protection, therefore, appeared to be a function of the passive antibody's specificity, avidity, and ability to bind to virion antigenic determinants topologically proximal to the critical neutralization site.     

Distribution of linear antigenic sites on glycoprotein gp55 of human cytomegalovirus.

Human convalescent serum and bacterial fusion proteins constructed from overlapping open reading frames of the nucleotide sequence encoding the human cytomegalovirus gp55 component of the major envelope glycoprotein complex, gp55-116 (gB), were used to localize antigenic regions recognized by human antibodies. All donor serum analyzed contained antibody reactivity for an antigenic site(s) located between amino acids (AA) 589 and 645, a region containing a previously defined linear site recognized by neutralizing monoclonal antibodies (U. Utz, B. Britt, L. Vugler, and M. Mach, J. Virol. 63:1995-2001, 1989). Furthermore, in-frame insertion of two different synthetic oligonucleotides encoding four amino acids into the sequence at nucleotide 1847 (AA 616) eliminated antibody recognition of the fusion protein. A second antibody binding site was located within the carboxyl terminus of the protein (AA 703 through 906). A competitive binding inhibition assay in which monoclonal antibodies were used to inhibit human antibody reactivity with recombinant gp55-116 (gB) suggested that the majority of human anti-gp55-116 (gB) antibodies were directed against a single antigenic region located between AA 589 and 645. Furthermore, inoculation of mice with fusion proteins containing this antigenic site led to a boostable antibody response. These results indicated that the antigenic site(s) located between AA 589 and 645 was an immunodominant antibody recognition site on gp55 and likely the whole gp55-116 (gB) molecule. The enhanced immunogenicity of this region in vivo may account for its immunodominance.     

Molecular mechanisms of cell-cell interaction in Dictyostelium discoideum

During development of the cellular slime mold Dictyostelium discoideum, cells migrate in response to cAMP to form aggregates, which give rise to fruiting bodies consisting of two major cell types: spores and stalk cells. Multicellularity is achieved by the expression of two types of cell-cell adhesion sites. The EDTA-sensitive binding sites are expressed at the initial stage of development. At the aggregation stage, cells acquire EDTA-resistant binding sites, which are mediated by a cell-surface glycoprotein of Mr80,000 (gp80). gp80 is preferentially associated with cell surface filopodia, which are probably involved in the initiation of contact formation between cells. Covaspheres conjugated with gp80 bind specifically to aggregation-stage cells. The binding can be inhibited by precoating cells with an anti-gp80 monoclonal antibody, thus suggesting that gp80 mediates cell-cell binding via homophilic interaction. The structure of gp80 predicted from its cDNA sequence can be divided into three major domains: a membrane anchor, a hinge, and a globular region. An analysis of fusion proteins containing different gp80 segments shows that the cell-binding activity resides in the globular region. In the postaggregation stages, gp80 is replaced by other surface glycoproteins in maintaining cell-cell adhesion. One of them has a Mr of 150,000 (gp150). Anti-gp150 antibodies have no effect on aggregation-stage cells, but they disrupt cell-cell adhesion at subsequent stages. It becomes evident that the complex phenomena of cell adhesion and tissue organization involve the participation of a number of surface glycoproteins.     

Monoclonal antibody recognizing gp80, a membrane glycoprotein implicated in intercellular adhesion of Dictyostelium discoideum.

WE have raised a monoclonal antibody, designated E28D8, which reacts with an 80,000-dalton membrane glycoprotein (gp80) of Dictyostelium discoideum. gp80 has been implicated in the formation of the EDTA-resistant adhesions ("contact sites A") which appear during development. The monoclonal antibody reacted with other developmentally regulated proteins of D. discoideum, confirming previous results indicating the presence of common antigenic determinants recognized by polyclonal rabbit antibodies directed to gp80. Periodate sensitivity of the determinants suggests that carbohydrate may be necessary for reactivity. Thus, the determinant recognized by E28D8 may result from a posttranslational modification common to a number of proteins. Some of the proteins that carry the determinant were preferentially localized to posterior cells in slugs. Monoclonal antibody E28D8 did not inhibit contact-sites-A-mediated intercellular adhesion. However, gp80 affinity purified on immobilized monoclonal antibody was able to neutralize the adhesion-blocking effect of rabbit antiserum to gp80. Although gp80 itself may not be essential for cell-cell adhesion, it appears to carry the determinants associated with adhesion.     

Identification of a cell surface glycoprotein involved in cell aggregation in D. discoideum.

Analysis of the composition of cell surface-associated glycoproteins of D. discoideum by lactoper-oxidase-catalyzed radioiodination, followed by isolation by Con A-Sepharose chromatography, revealed that the developmentally regulated cell surface expression of a certain glycoprotein (gp150) parallels the onset of mutual cellular cohesiveness (Geltosky, Siu and Lerner, 1976). We have purified gp150 and raised specific antibodies to it. Through utilization of the specific antibody and a fluorescence-activated cell sorter, the expression of gp150 on the cell surface has been studied. Starting from a low level in noncohesive (vegetative) cells, there is a rapid accumulation of gp150 on the surfaces of aggregating cells. A peak level of expression is achieved by 10 hr and maintained at least until the steps of terminal differentiation. Most significantly, monovalent Fa'b derived from anti-gp150, when added to aggregation-competent cells, blocks the cells' ability to reaggregate. Fab's derived from antisera with different specificities were ineffective inhibitors of cell aggregation. These results suggest that gp150 serves an intimate role in cell adhesion.     

Glycoprotein gp118 of varicella-zoster virus: purification by serial affinity chromatography.

Glycoprotein gp118, one of the major glycosylated proteins specified by varicella-zoster virus, is biologically of great importance since it possesses an epitope which elicits a complement-independent neutralizing antibody response. To purify this glycoprotein from a Nonidet-solubilized extract of varicella-zoster virus-infected cells, we examined its affinity to a variety of ligands, including two lectins--concanavalin A and Lens culinaris, Cibacron blue and heparin, and finally an immunoadsorbent anti-gp118 monoclonal antibody. By serial affinity chromatography on three different columns consisting of, respectively (i) Cibacron blue dye-Sepharose, (ii) L. culinaris-Sepharose, and (iii) anti-gp118 murine monoclonal antibody bound to CNBr-activated Sepharose, we isolated varicella-zoster virus-specific gp118 essentially free of contamination by any other radiolabeled viral or cellular polypeptide. The fold purification was estimated at 1,025 and the percent recovery at 13.6. On the basis of its chromatographic properties, gp118 appeared to contain mainly asparagine-linked, biantennary, complex-type, and hybrid-type oligosaccharides.     

Two major groups of neutralizing anti-gp120 antibodies exist in HIV-infected individuals. Evidence for epitope diversity around the CD4 attachment site.

The aim of this study was to dissect neutralizing anti-gp120 antibody populations in seropositive asymptomatic individuals. Murine anti-Id mAb were raised against polyclonal affinity-purified human anti-gp120 antibodies. These anti-Id mAb were used to fractionate anti-gp120 antibodies from a pool of HIV-positive sera into idiotypically distinct anti-gp120 antibody (Id+Ab) preparations. Immunochemical and neutralization studies indicated that all Id+Ab that neutralized HIV-1 in vitro interacted with either the V3 loop or the CD4 attachment site of gp120. The V3-specific Id+Ab neutralized HIV-1 in a strain-restricted manner. Id+Ab specific for the CD4 attachment site exhibited different spectra of neutralizing activities against multiple strains of HIV-1. This finding indicates that multiple, antigenically diverse epitopes reside around the CD4 attachment site of gp120. Significantly, depletion of the Id+Ab from affinity-purified total anti-gp120 antibodies abrogated most of the neutralizing activities of these antibodies, suggesting that neutralizing anti-gp120 antibodies consist of two major specificities, either to the V3 region or to the CD4 attachment site. The understanding of specificities and neutralizing activities of different anti-gp120 antibodies in seropositive healthy individuals will be helpful for designing effective vaccines and immunotherapeutic strategies for AIDS.     

Oligomeric and conformational properties of a proteolytically mature,disulfide-stabilized human immunodeficiency virus type 1 gp140 envelope glycoprotein

We describe the further properties of a protein, designated SOS gp140, wherein the association of the gp120 and gp41 subunits of the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein is stabilized by an intersubunit disulfide bond. HIV-1(JR-FL) SOS gp140, proteolytically uncleaved gp140 (gp140(UNC)), and gp120 were expressed in stably transfected Chinese hamster ovary cells and analyzed for antigenic and structural properties before and after purification. Compared with gp140(UNC), SOS gp140 reacted more strongly in surface plasmon resonance and radioimmunoprecipitation assays with the neutralizing monoclonal antibodies (MAbs) 2G12 (anti-gp120), 2F5 (anti-gp41), and 17b (to a CD4-induced epitope that overlaps the CCR5-binding site). In contrast, gp140(UNC) displayed the greater reactivity with nonneutralizing anti-gp120 and anti-gp41 MAbs. Immunoelectron microscopy studies suggested a model for SOS gp140 wherein the gp41 ectodomain (gp41(ECTO)) occludes the "nonneutralizing" face of gp120, consistent with the antigenic properties of this protein. We also report the application of Blue Native polyacrylamide gel electrophoresis (BN-PAGE), a high-resolution molecular sizing method, to the study of viral envelope proteins. BN-PAGE and other biophysical studies demonstrated that SOS gp140 was monomeric, whereas gp140(UNC) comprised a mixture of noncovalently associated and disulfide-linked dimers, trimers, and tetramers. The oligomeric and conformational properties of SOS gp140 and gp140(UNC) were largely unaffected by purification. An uncleaved gp140 protein containing the SOS cysteine mutations (SOS gp140(UNC)) was also oligomeric. Surprisingly, variable-loop-deleted SOS gp140 proteins were expressed (although not yet purified) as cleaved, noncovalently associated oligomers that were significantly more stable than the full-length protein. Overall, our findings have relevance for rational vaccine design.     

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.     

The contact site A glycoprotein mediates cell-cell adhesion by homophilic binding in Dictyostelium discoideum

Dictyostelium discoideum expresses a developmentally regulated cell surface glycoprotein of Mr 80,000 (gp80), which has been implicated in the formation of the EDTA-resistant contact sites A at the cell aggregation stage. To determine whether gp80 participates directly in cell binding and, if so, its mode of action, we conjugated purified gp80 to Covaspheres (Covalent Technology Corp., Ann Arbor, MI) and investigated their ability to bind to cells. The binding of gp80- Covaspheres was dependent on the developmental stage of the cells, with maximal interaction at the late aggregation stage. Scanning electron microscopic studies revealed the clustering of gp80-Covaspheres at the polar ends of these cells, similar to the pattern of gp80 distribution on the cell surface as reported earlier (Choi, A. H. C., and Siu, C.- H., 1987, J. Cell Biol., 104:1375-1387). Precoating cells with an adhesion-blocking anti-gp80 monoclonal antibody inhibited the binding of gp80-Covaspheres, suggesting that Covasphere-associated gp80 might undergo homophilic interaction with gp80 on the cell surface. Quantitative binding of 125I-labeled gp80 to intact cells gave an estimate of 1.5 X 10(5) binding sites per cell at the aggregation stage. Binding of soluble gp80 to cells was blocked by precoating cells with the anti-gp80 monoclonal antibody. The ability of gp80 to undergo homophilic interaction was further tested in a filter-binding assay, which showed that 125I-labeled gp80 was able to interact with gp80 bound on nitrocellulose in a dosage-dependent manner. In addition, reassociation of cells was significantly inhibited in the presence of soluble gp80, suggesting that gp80 has a single cell-binding site. These results are consistent with the notion that gp80 mediates cell- cell binding at the aggregation stage of development via homophilic interaction.     

Analysis of the cross-reactive anti-gp120 antibody population in human immunodeficiency virus-infected asymptomatic individuals.

This study was undertaken to analyze the specificity and neutralizing properties of cross-reactive anti-gp120 antibodies (Abs) in the sera of two human immunodeficiency virus (HIV)-infected asymptomatic individuals. Two panels of murine monoclonal anti-idiotype Abs (anti-id MAbs) were established against cross-reactive polyclonal anti-gp120 Abs purified from HIV+ sera by sequential affinity chromatography using gp120SF2- and gp120IIIB-Sepharose columns. These panels of anti-id MAbs were then used to affinity purify idiotype-positive (Id+) anti-gp120 Abs from HIV+ sera. The recovery of each of these Id+ Abs by purification indicated that several idiotypically distinct cross-reactive anti-gp120 Abs are present in sera over a wide range of concentrations. Immunological and biological studies showed that although all of the Id+ Abs were reactive against gp120SF2 and gp120IIIB, they exhibited unique epitope specificities and distinct neutralizing activities. Most of the Id+ Abs were directed against epitopes in the CD4 attachment site (CD4 site epitopes) of gp120 and exhibited a spectrum of broadly neutralizing activities. On the other hand, a minor population of Id+ Abs showed specificity for the V3 region of gp120 and exhibited limited cross-neutralizing activities. Together, these studies indicate that the CD4 site epitope-specific Abs are heterogeneous with respect to their clonality, neutralizing activity, and concentration in sera. This heterogeneity suggests that anti-gp120 Abs to the CD4 attachment site are developed in response to multiple overlapping epitopes present on the original virus isolate and/or epitopes on mutated variants which emerged over time.     

Cell-cell adhesion molecules in Dictyostelium

Multicellularity in the cellular slime mold Dictyostelium discoideum is achieved by the expression of two types of cell-cell adhesion sites. The EDTA-sensitive adhesion sites are expressed very early in the development cycle and a surface glycoprotein of 24,000 Da is known to be responsible for these sites. The EDTA-resistant contact sites begin to accumulate on the cell surface at the aggregation stage of development. Several glycoproteins have been implicated in the EDTA-resistant type of cell-cell binding and the best characterized one has an Mr of 80,000 (gp80). gp80 mediates cell-cell binding via homophilic interaction and its cell binding site has been mapped to an octapeptide sequence. The mechanism by which gp80 mediates cell-cell adhesion will be discussed.     

Involvement of a triton-insoluble floating fraction in Dictyostelium cell-cell adhesion

We have isolated and characterized a Triton-insoluble floating fraction (TIFF) from Dictyostelium. Ten major proteins were consistently detected in TIFF, and six species were identified by mass spectrometry as actin, porin, comitin, regulatory myosin light chain, a novel member of the CD36 family, and the phospholipid-anchored cell adhesion molecule gp80. TIFF was enriched with many acylated proteins. Also, the sterol/phospholipid ratio of TIFF was 10-fold higher than that of the bulk plasma membrane. Immunoelectron microscopy showed that TIFF has vesicular morphology and confirmed the association of gp80 and comitin with TIFF membranes. Several TIFF properties were similar to those of Dictyostelium contact regions, which were isolated as a cytoskeleton-associated membrane fraction. Mass spectrometry demonstrated that TIFF and contact regions shared the same major proteins. During development, gp80 colocalized with F-actin, porin, and comitin at cell-cell contacts. These proteins were also recruited to gp80 caps induced by antibody cross-linking. Filipin staining revealed high sterol levels in both gp80-enriched cell-cell contacts and gp80 caps. Moreover, sterol sequestration by filipin and digitonin inhibited gp80-mediated cell-cell adhesion. This study reveals that Dictyostelium TIFF has structural properties previously attributed to vertebrate TIFF and establishes a role for Dictyostelium TIFF in cell-cell adhesion during development.     

Conducting polymer based fluorescence quenching as a new approach to increase the selectivity of immunosensors

Polypyrrole (Ppy) has been shown to be a superior matrix for fluorescence detection based immunosensors: (i) the fluorescence of polypyrrole and polypyrrole modified by entrapped proteins was almost not detectable when this polymer was excited by near UV 325 nm light; (ii) polypyrrole quenched the fluorescence of such fluorescence agents as fluoresceine 5(6)-isothiocyanate, rhodamine B and enzyme-horseradish peroxidase (HRP) by almost 100% if they were deposited in the solution as a drop at the Ppy surface followed by evaporation of the solvent. According to our knowledge, this work is first application of Ppy in the design of a fluorescence-based immunosensor, where low Ppy fluorescence background and Ppy induced fluorescence quenching were exploited. These sensors were devoted to the detection of antibodies against bovine leukemia virus (BLV) protein gp51 (anti-gp51-Ab). A biological recognition system of this fluorescence immunosensor model was based on polypyrrole with entrapped BLV proteins gp51 (gp51/Ppy). This gp51/Ppy layer was applied for the detection of anti-gp51-Ab. Secondary antibodies against anti-gp51-Ab labeled with HRP (Ab*) were applied as fluorescence-detectable labels that are able to recognize specifically and interact with the complex of gp51 proteins and anti-gp51-Ab antibodies (gp51/anti-gp51-Ab). It was demonstrated that fluorescence of non-specifically adsorbed Ab* was almost completely quenched by the Ppy substrate. In addition, enzymatic activity of HRP was exploited as a traditional reference method for verification of the formation of the immune complex gp51/anti-gp51-Ab/Ab*.     

Inhibition of cell-cell binding at the aggregation stage of Dictyostelium discoideum development by monoclonal antibodies directed against an 80,000-dalton surface glycoprotein

Monoclonal antibodies were prepared against a putative cell-cell adhesion molecule, a surface glycoprotein with an apparent Mr of 80,000 (gp80), from Dictyostelium discoideum. Seven monoclonal antibodies directed against gp80 were characterized and found to fall into three distinct classes. Class I consisted of one monoclonal antibody, is monospecific for gp80, and probably recognizes the peptide portion of the molecule. This class was capable of blocking the EDTA-resistant contact sites effectively. Class II recognized the carbohydrate moiety of gp80 and cross-reacted with a large number of glycoproteins. These monoclonal antibodies partially inhibited cell reassociation. Class III recognized gp80 and one other glycoprotein of Mr 95,000. This class had no effect on cell-cell binding. The class I monoclonal antibody was most potent in inhibiting cell reassociation at the aggregation stage of development. Its effect decreased drastically as development progressed and became negligible by the culmination stage. These observations are consistent with a direct role of gp80 in cell-cell binding and suggest a transient function for gp80 at the aggregation stage.     

Inhibition of cell adhesion in Dictyostelium discoideum by tunicamycin is prevented by leupeptin

The carbohydrate requirement for cell adhesion of aggregation-competent cells of Dictyostelium discoideum has been examined by use of a selective glycosylation inhibitor of N-glycosyl protein, tunicamycin (TM). TM completely inhibited EDTA-stable cell adhesion and glycosylation of some membrane glycoproteins in aggregation-competent cells of D. discoideum (Yamada, H., et al. (1982) J. Biochem. 92, 399-406). The present study showed that the inhibition of EDTA-stable cell adhesion by TM was prevented significantly when the cells were treated with TM in the presence of a protease inhibitor, leupeptin (LP), whereas the inhibition of glycosylation by TM was not prevented. The cell extract of aggregation-competent cells contained acid proteases, and LP strongly inhibited acid protease from D. discoideum in vitro. On analysis by SDS-polyacrylamide gel electrophoresis (PAGE), many protein bands present in the membrane fraction of control cells disappeared or decreased on TM treatment of the cells in the absence of LP, however, some of these proteins were restored when the cells were treated with TM in the presence of LP. These results strongly support an idea that EDTA-stable cell adhesion characteristic to aggregation-competent cells is mediated by glycoproteins with asparagine-linked carbohydrate. However, the requirement for the carbohydrate moiety of the glycoprotein in cell adhesion appears to be indirect in that it acts to protect the protein moiety from proteolytic degradation.     

Topographical rearrangements of visna virus envelope glycoprotein during antigenic drift.

Visna virus undergoes antigenic drift during persistent infection in sheep and thus eludes neutralizing antibodies directed against its major envelope glycoprotein, gp135. Antigenic variants contain point mutations in the 3' end of the genome, presumably within the envelope glycoprotein gene. To localize the changes in the viral proteins of antigenic mutants, we isolated 35 monoclonal antibodies (MAbs) against the envelope glycoprotein gp135 or the major core protein p27 of visna virus. The MAbs defined five partially overlapping epitopes on gp135. We used the MAbs and polyclonal immune sera directed against visna virus, gp135, or p27 in enzyme-linked immunosorbent assays to compare visna virus (strain 1514) with antigenic mutants (LV1-1 to LV1-6) previously isolated from a single sheep persistently infected with plaque-purified strain 1514. Polyclonal immune sera and anti-core p27 MAbs failed to distinguish antigenic differences among the viruses. By contrast, the anti-gp135 MAbs detected changes in all five epitopes of the envelope glycoprotein. Three gp135 epitopes, prominently exposed on strain 1514, were lost or obscured on the mutants; two covert gp135 epitopes, poorly exposed on strain 1514, were reciprocally revealed on the mutants. Even virus LV1-2, which is indistinguishable from parental strain 1514 by serum neutralization tests and which differs from it by only two unique oligonucleotides on RNase-T1 fingerprinting, displayed global changes in gp135. Our data suggest that visna virus variants may emerge more frequently during persistent infection than can be detected by serological tests involving the use of polyclonal immune sera, and the extent of phenotypic changes in their envelope glycoproteins may be greater than predicted by the small number of genetic changes previously observed. We suggest that topographical rearrangements in the three-dimensional structure of gp135 may magnify the primary amino acid sequence changes caused by point mutations in the env gene. This may complicate strategies to construct lentiviral vaccines by using the envelope glycoprotein.