齊藤 玉緒

The all-cis-5,9,12-heptadecatrienoic acid was identified in the cellular slime mold Polysphondylium pallidum. The structural elucidation was accomplished by capillary gas chromatography, argentation thin-layer chromatography, and gas chromatography/mass spectrometry. This fatty acid has not been reported previously.

The cellular slime mold, Polysphondylium pallidum, has two EDTA-resistant types of cell-cell adhesion, The major component of them has been identified as a glycoprotein with a molecular mass of 64 kDa on SDS-PAGE (referred to as gp64), We found that a substantial amount of the gp64 run as dimer, when gp64 was dissolved in SDS-sample buffer without 2-mercaptoethanol and then subjected to electrophoresis, The occurrence of a homophilic dimer was demonstrated by analyzing the dimer-like band on a gel for its amino acid sequence and amino acid composition, The dimer-like band also was analyzed by three sorts of monoclonal antibodies, two of which recognize respectively a conformational epitope and a denatured epitope of the protein moiety of gp64. The data indicate that the native conformation of gp64 is necessary for dimer formation.

The 64-kDa membrane-bound glycoprotein of the cellular slime mold Polysphondylium pallidum (referred to as gp64), seems to be implicated in cell-cell adhesion. Previously we have isolated a full-length gp64 cDNA, determined its nucleotide sequence, and found that all cysteine residues in the protein are involved in the formation of disulfide bonds. The disulfide arrangement of the 36 cysteines in gp64 was established by analysis of proteolytically cleaved protein and sequence analysis of cystine-containing fragments. Since gp64 has 36 Cys residues, 18 disulfide bonds must exist and the positions of 15 of them were determined. The 15 disulfide bonds in gp64 constitute five characteristic, so called Sushi do mains. In a Sushi domain, the first Cys in a sequence is connected to the third one and the second Cys to the fourth one. This is the first report describing the presence of Sushi domains in a cellular slime mold protein. From these data, gp64 appears to be distinct from all other previously described cell-adhesion proteins.

The cellular slime mold Polysphondylium pallidum expresses a cell surface glycoprotein (referred to as gp64), which seems to be implicated in cell-cell adhesion. We identified a near full-length gp64 cDNA (1,104 base) upon screening a P. pallidum lambdagt11 library with a monoclonal antibody. The open reading frame encodes a protein of 320 amino acids with a molecular mass of 32,752 Da; the protein includes hydrophobic segments at both a NH2- and a COOH-terminal ends. By an Edman degradation analysis of S-pyridylethylated gp64 and its COOH-terminal peptide, it was found that the NH2- and COOH-terminal segments are both removed from the precursor protein of gp64. The COOH-terminal segment was isolated from a lysyl endopeptidase digest of gp64 by an affinity method. The COOH-terminal segment was identified at positions 266-279 in the primary sequence deduced from the cDNA sequence. The mature gp64 consisted of 279 amino acid residues and extremely rich in Cys residues (36 Cys/279 amino acids = 12.9%). Although there was already maximal accumulation of gp64 mRNA in vegetative cells, the protein reached a maximal level during aggregation stage, decreased, and then leveled off through the developmental cycle.

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.

A 64-kDa membrane-bound glycoprotein (gp64) of the cellular slime mold Polysphondylium 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 asparagine-linked glycosylation sites (Asn-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.

Monoclonal antibodies were produced against a cell‐cell adhesion (contact site A) glycoprotein of Dictyostelium discoideum, isolated by preparative gel electrophoresis. The glycoprotein was recovered by electroelution from a polyacrylamide gel strip and used for the production of monoclonal antibodies. Four of the five antibodies obtained bound specifically to the protein moiety of the contact site A glycoprotein. The specificities of the antibodies were in striking contrast to those of antibodies raised against the contact site A glycoprotein purified by Triton X‐114 phase separation and DEAE chromatography. The majority of the latter antibodies recognized the carbohydrate moiety of the contact site A glycoprotein and cross‐reacted heavily with other membrane glycoproteins. Copyright © 1990 VCH Verlagsgesellschaft mbH