Characterization of a purified cell surface glycoprotein as a contact site in Polysphondylium pallidum

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Polyclonal and monoclonal antibodies were prepared against a glycoprotein (gp 64) of Polysphondyliurnpallidurn previously shown to act as a target site of adhesion-blocking Fab prepared from antisera against whole membranes of aggregation-competent cells. The purified glycoprotein, with a nominal M, of 64000, could be fractionated into two subspecies, gp 64, and gp 64,,, with apparent M , of 66000 and 60000, as determined in 7.5% sodium dodecyl sulfate/polyacrylamide gels.Rabbit antibodies against purified gp 64 reacted not only with the two subspecies but also with many other membrane proteins. Almost all the cross-reactivity could be abolished by absorption of the antibodies with extensively purified gp 64. All monoclonal antibodies obtained by screening with gp 64 showed similar crossreactivity. One monoclonal antibody specifically precipitating gp 64 was selected by screening with antigen that had been pretreated with anhydrous hydrogen fluoride for removal of carbohydrates.Fab from polyclonal anti-(gp 64) sera as well as one monoclonal Fab completely blocked cell adhesion of aggregation-competent P. pallidurn cells. A carbohydrate fraction prepared by treatment of gp 64 with proteases and hydrazine completely neutralized the adhesion-blocking Fab. The product of hydrazinolysis contained less than 3 % of the original peptide as based on the glucosamine recovered, but the specific neutralizing activity of the carbohydrate was essentially the same as that of the glycoprotein. In conclusion, monoclonal as well as polyclonal adhesion-blocking Fab reacted with carbohydrates; gp 64 shared the relevant carbohydrate moieties with other membrane proteins.Polysphondylium pallidurn is a cellular slime mold which, like Dictyostelium discoideum, exists in the form of single amoebae during growth. When nutrients are depleted, the amoebae develop within about 6 h into aggregation-competent cells. On a solid substratum these cells start within less than an hour to initiate aggregation centers and to assemble into streams which are radially oriented around the centers, the sources of an attractant identified as N-propionyl-y-rglutamyl-r -0rnithine-h-lactam ethyl ester [l]. If cells of P. pallidurn and D. discoideum are mixed, they sort themselves out, assembling into separate multicellular structures not only because of their different chemoattractants, but also because of the incompatibility of their adhesion systems [2 -41.Work on cell adhesion in P. pallidurn was largely stimulated by the prospect of elucidating the molecular basis for speciesspecific and unspecific adhesion. Neutralization of adhesionblocking Fab has been traced to a glycoprotein of molecular mass 71 kDa [5]. A glycoprotein with an apparent molecular mass of 64 kDa, probably identical with the 71-kDa one, has been purified and shown to absorb 60 -70 % of the adhesion-

“…The glycoprotein with an apparent molecular mass of 64 kDa, that has been called contact site 1 [2,6], was purified as shown in Fig. 1 by a modification of the previously used method.…”

Section: Improved Purification Of Glycoprotein 64 (Gp 64)mentioning

confidence: 99%

“…Hybridomas were cloned by selecting, under the microscope, droplets containing single cells. Solid-phase radioimmunoassays, IgG and Fab preparations were done by standard methods [6,121. Fab and IgG were prepared from hybridoma cultures in RPMI 1640 medium containing 10 % fetal calf serum.…”

mentioning

confidence: 99%

Monoclonal anti‐glycoprotein antibody that blocks cell adhesion in Polysphondylium pallidum

Toda

Bozzaro

Lottspeich

et al. 1984

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“…Hitherto, two purification methods of gp64 have been reported (Bozzaro et al, 1981 ;Toda et al, 1984a). The improved method (Toda et al 1984a), consisting of butanol extraction, ammonium sulfate precipitation, Triton X-114 extraction (Bordier, 1981) and elution from SDS/PAGE, gave a recovery of 4.3 mg protein from 3 x 10'' aggregation-competent cells in Triton X-114 extraction step.…”

Section: Purification Of Gp64mentioning

confidence: 99%

“…In both species, cell aggregation is established through a signal-relay response of the surrounding cells towards chemoattractants released by center cells and through cell-adhesion proteins that enable the cells to form multicellular aggregates (Bozzaro et al, 1981).…”

mentioning

confidence: 99%

A purification method and N‐glycosylation sites of a 36‐cysteine‐containing, putative cell/cell adhesion glycoprotein gp64 of the cellular slime mold, Polysphondylium pallidum

Saito

Kumazaki

Ochiai

1993

A 64-kDa membrane-bound glycoprotein (gp64) of the cellular slime mold Polysphondyliurn pallidum, is a putative cell/cell adhesion protein identified by adhesion-blocking antibody fragments (Fab). gp64 is expressed on the cell surface of growth-phase cells and seems to mediate cell/cell adhesion. This paper describes an improved purification method based on the lipophilic nature of this protein. A critical step in the purification method is to collect an insoluble top layer appearing during ammonium sulfate precipitation. The sequence of cDNA encoding gp64 and its deduced amino acid sequence have been determined previously. Based on cDNA sequence data, the structure of gp64 protein was analyzed: almost all amino acid compositions and partial amino acid sequences of lysylendopeptidase-digested peptides of gp64 were determined by protein analysis; all six asparaginelinked glycosylation sites (Am-Xaa-Ser/Thr) in fact contain carbohydrates, and all 36 cysteine residues were involved in forming disulfide bridges. From these data, gp64 seems to be a unique protein among cell/cell adhesion proteins.Cells of the cellular slime mold Polysphondylium pallidurn, like the related species Dictyosteliuni discoideum, exhibit chemotactic migration in response to a chemoattractant secreted from center cells and finally form multicellular aggregates called pseudoplasmodia (Gerisch et al., 1980). In both species, cell aggregation is established through a signal-relay response of the surrounding cells towards chemoattractants released by center cells and through cell-adhesion proteins that enable the cells to form multicellular aggregates (Bozzaro et al., 1981).D . discoideum has two types of contact site, A and B (Beug et al., 1973). Contact site A is EDTA resistant and was expressed in aggregation-stage cells. Contact site B is EDTAsensitive and was observed in both growth-phase and aggregation-phase cells. The cell adhesion protein at contact site A was identified as a glycoprotein with a molecular mass of 80 kDa (Mueller and Gerisch, 1978;Mueller et al., 1979). Its cDNA sequence (Noegel et al., 1986), adhesion site (Kamboj et al., 1989) and glycosyl-PtdIns anchor (Stadler et al., 1989) have been reported.Correspondence to H. Ochiai, Department of Botany, Faculty of Science, Hokkaido University, Kita-ku, Sapporo, Japan 060Abbreviations. DACM, N-(7-dimethylamino-4-methyl-coumariny1)maleimide; Fab, antigen-binding fragment; gp, glycoprotein.Enzymes. Chymotrypsin (EC 3.4.21.1); lysyl endopeptidase (EC 3.4.21.50).Note. The novel amino acid sequence data published here have been submitted to the EMBL sequence data bank@) and are available under accession number(s) PS0434.Aggregation cells of the related species P. pallidurn also exhibit two types of cell/cell adhesion protein (Bozzaro and Gerisch, 1978). One is a minor component detected in aggregation-stage cells, and the other is a major component expressed during growth phase and later stages. The major component was identified as a glycoprotein with a molecular mass of 64 kDa (gp6...

“…Gp64 is a putative cell-cell adhesion protein expressed during growth and aggregation-phases (Bozzar0 and Gerisch, 1978;Bozzaro et al, 1981 ;Toda et al, 1984). This membrane protein is modified by six asparagine-linked oligosaccharides; all 36 cysteine residues are involved in forming disulfide bridges (Saito et al, 1993).…”

mentioning

confidence: 99%

Evidence for a glycolipid anchor of gp64, a putative cell‐cell adhesion protein of Polysphondylium pallidum

Saito

Ochiai

1993

The membrane-bound glycoprotein (gp64) of the cellular slime mold Polysphondylium pallidum, is a putative cell-cell adhesion protein identified by adhesion-blocking antibody fragments. Since gp64 can be purified in a few days and in substantial yields, it is a good candidate for clarifying the structure of a cell-cell adhesion protein. This study reveals that gp64 possesses a glycolipid anchor which is sensitive to deamination but resistant to phosphatidylinositol-specific phospholipase C from Bacillus thuringiensis. Although the anchor resistance to phosphatidylinositol-specific phospholipase C can be ascribed to the presence of an additional acyl chain on the inositol ring in the glycosyl phosphatidylinositol anchors, this was not the case. After a mild-base treatment that released an additional acyl chain on the inositol ring, only a trace amount of fatty acid was detected but, after strong acid hydrolysis, we detected both amide-linked fatty acids and a long-chain base. The long-chain base was further analysed by gas-chromatography/mass spectrometry and was found to be phytosphingosine. Both fatty acids and myo-inositol were also analysed by gas-chromatography/mass spectrometry. These data suggest that gp64 possesses a glycolipid anchor which contains ceramide and myo-inositol.