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Biomedical subjects

G Gerisch

Publications and source records attributed to G Gerisch.

At least 109 records · Page 6Linked to original sources
PubMed1985

Incomplete contact site A glycoprotein in HL220, a modB mutant of Dictyostelium discoideum.

HL220, a modB mutant that lacks a modification of certain membrane proteins of Dictyostelium discoideum, has been shown to aggregate and to form EDTA-stable intercellular contacts typical of aggregating wild-type cells. A developmentally regulated glycoprotein of 80 X 10(3) apparent molecular weight has been identified as a target site of adhesion-blocking Fab and thought to be involved in EDTA-stable cell contact formation (Müller & Gerisch, 1978). In the HL220 mutant this glycoprotein is no longer recognized by a modB-specific antibody. Therefore, it has been suggested that the 80 X 10(3) Mr glycoprotein, or a modification on it, is not required for the EDTA-stable cell contact of aggregating cells. We show that HL220 synthesizes an equivalent of the 80 X 10(3) Mr glycoprotein with an apparent molecular weight of 68 X 10(3). The mutant product reacted with certain monoclonal antibodies highly specific for the 80 X 10(3) Mr glycoprotein in the wild type, and was developmentally regulated like the 80 X 10(3) Mr glycoprotein. These results indicate that the 68 X 10(3) Mr protein of the mutant lacks a modification, most likely an oligosaccharide residue, the absence of which causes the substantial shift of the apparent molecular weight from 80 X 10(3) to 68 X 10(3). Monoclonal antibodies that did not react with proteins of the mutant could be classified according to their reactions with different sub-sets of wild-type proteins. These results indicate that the proteins that reacted with either one or the other antibody were not modified by a uniform structure. The modification rather varies from one sub-set of cross-reacting proteins to another, suggesting differences between the glycosyl residues of the partially cross-reacting proteins. HL220 cells showed strongly reduced EDTA-stable contact formation under our conditions. EDTA-sensitive intercellular adhesion was undetectable in the mutant, whereas adhesion of the cells to the substratum appeared to be strengthened. The rear ends of the cells, in particular, were tightly attached to glass or Teflon surfaces. The mutant cells were capable of responding chemotactically. Propagated excitation waves like those known to be based on periodic cyclic AMP production and relay were clearly seen. Extracellular phosphodiesterase induction by cyclic AMP and phosphodiesterase inhibitor production were normal. These results indicate that the generation of chemotactic signals and the cellular responses to cyclic AMP are not severely affected by the mutation.

Antibodies, Fungal↗
PubMed1984

Localization of two phosphorylation sites adjacent to a region important for polymerization on the tail of Dictyostelium myosin.

Phosphorylation of the myosin heavy chains of Dictyostelium discoideum is known to be inhibited following chemotactic stimulation of the cells. Effects of dephosphorylation on the assembly of myosin and on its actin-activated ATPase activity raised the question of where the phosphorylated sites are located with respect to sites responsible for polymerization and actin binding. Using seven monoclonal antibodies the binding sites of which were mapped in the electron microscope, two phosphorylation sites, i.e., threonine residues that were phosphorylated by a kinase from D. discoideum, were localized by immunoblotting of chymotryptic fragments. Two of the antibodies bound to the terminal one fifth of the tail and recognized a phosphorylated chymotryptic fragment of 38 kd. The non-phosphorylated form and single and double phosphorylated forms of this fragment were separated by two-dimensional electrophoresis. Antibody labeling of lower mol. wt. polypeptides indicated that both phosphorylation sites were located at least 32 kd from the end of the tail. A non-phosphorylated fragment, that was insoluble at low ionic strength due to polymerization, proved to be an internal cleavage product of the tail. A segment of this fragment necessary for polymerization was mapped adjacent to the phosphorylation sites.

Journal Article↗
PubMed1984

Monoclonal antibodies that block cell adhesion in Polysphondylium pallidum: reaction with L-fucose, a terminal sugar in cell-surface glycoproteins.

By permethylation analysis the linkages of L-fucose and D-mannose in the oligosaccharide residues of cell surface glycoproteins of Polysphondylium pallidum were determined. Mannose was found in terminal positions, in 1,2-, 1,3- and 1,6-intra-chain linkages, and at branch points. Fucose was exclusively located at nonreducing ends. Fab of a monoclonal antibody, mAb 293, has been previously shown to inhibit cell adhesion in P. pallidum completely. Binding of this antibody to glycoprotein was blocked by L-fucose, and at very high concentrations also by D-mannose. The dissociation constant for the antibody-fucose complex was Kd = 70 microM, which was two orders of magnitude higher than estimated for the natural oligosaccharide. Antibody-glycoprotein complexes dissociated in the presence of 100 mM L-fucose with a half-time of about 56 s. The blockage by L-fucose is taken as evidence that the adhesion-blocking antibody binds to oligosaccharide end groups containing L-fucose as the terminal sugar.

Adhesiveness↗
PubMed1984

Monoclonal anti-glycoprotein antibody that blocks cell adhesion in Polysphondylium pallidum.

Polyclonal and monoclonal antibodies were prepared against a glycoprotein (gp 64) of Polysphondylium pallidum 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 Mr of 64000, could be fractionated into two subspecies, gp 64I and gp 64II, with apparent Mr 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 cross-reactivity. 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. pallidum 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.

Adhesiveness↗
PubMed1984

New actin-binding proteins from Dictyostelium discoideum.

Dictyostelium discoideum contains a soluble actin-binding protein that caps actin filaments at their fast growing ends. The purified protein consists of two subunits with 34 kd and 32 kd apparent mol. wts. Like similar proteins from Acanthamoeba and bovine brain the capping protein from D. discoideum acts in a Ca -independent manner. It lacks severing activity as indicated by its inability to disrupt the stress fibers and the microfilament network in detergent-extracted cells. Two actin-binding proteins from a plasma membrane-enriched fraction were labeled with [I]actin using a gel overlay technique. One of these proteins, with an apparent mol. wt. of 17 kd in SDS-polyacrylamide gels, has been purified from high-salt extracts, the other protein with an apparent mol. wt. of 31 kd has been purified from Triton X-100 extracted membranes. Monoclonal antibodies were raised against D. discoideum severin, alpha-actinin, the larger subunit of the capping protein, and the 17-kd membrane-associated protein. Immunoblotting of proteins from whole cell lysates showed that all these actin-binding proteins were present in both growth phase and aggregation-competent cells.

Journal Article↗
PubMed1984

Wheat germ agglutinin binds to the contact site A glycoprotein of Dictyostelium discoideum and inhibits EDTA-stable cell adhesion.

Wheat germ agglutinin (WGA), a lectin that primarily reacts with N-acetylglucosamine residues, specifically inhibits the EDTA-stable type of intercellular adhesion of aggregation competent Dictyostelium discoideum cells. The major WGA-binding protein of these cells is a developmentally-regulated glycolipoprotein of 80 kd apparent mol. wt., designated as contact site A. This glycoprotein is a target site of antibody fragments that block the EDTA-stable cell adhesion, and is characterized by sulfated carbohydrate residues. WGA does not significantly bind to glycoproteins of a mutant, HL220, which produces a 68-kd component in place of the 80-kd glycoprotein. Inhibition of N-glycosylation by tunicamycin causes wild-type cells to produce a WGA-binding but unsulfated 66-kd component and a non-binding 53-kd component. These results indicate that the 80-kd glycoprotein contains two classes of carbohydrate residues, a WGA-binding one that is defective in HL220, and another, sulfated, one that is absent from the 66-kd wild-type product; both are missing in the 53-kd protein. WGA and a monoclonal antibody that is blocked by N-acetylglucosamine were further used to probe for glycoproteins in the multicellular slug stage that share carbohydrate structures - and possibly functions - with the contact site A glycoprotein. Glycoproteins in the 95-kd range have previously been implicated in cell-to-cell adhesion during the slug stage. We distinguished a 95-kd glycoprotein that binds WGA from another one that binds antibody.

Journal Article↗
PubMed1984

Monoclonal antibody against cytoplasmic lectins of Dictyostelium discoideum: cross-reactivity with a membrane glycoprotein, contact site A, and with E. coli beta-galactosidase and lac repressor.

Monoclonal antibodies were raised against two soluble, galactose-binding lectins from cells of Dictyostelium discoideum, discoidin I and II. These antibodies reacted not only with both discoidins, but also with a plasma membrane glycoprotein of aggregation competent cells, called contact site A, and with two carbohydrate-binding proteins of E. coli, beta-galactosidase and lac repressor. The possibility that the antibody recognizes a structure common to different carbohydrate-binding proteins is discussed. The two carbohydrate-binding proteins of E. coli share with discoidin I the sequence -Ser-X-X-Ile-His(Pro)-Pro(His)-Leu-Thr- which might be responsible for the cross-reactivity.

Amino Acid Sequence↗
PubMed1983

Myosin heavy chain kinase inactivated by Ca2+/calmodulin from aggregating cells of Dictyostelium discoideum.

Soluble myosin heavy chain kinases (MHC kinases) were partially purified from growth phase and aggregation-competent cells of Dictyostelium discoideum. In the aggregation-competent cells, two MHC kinases were distinguishable. One of these enzymes, called MHC kinase II, was inactivated by Ca2+ and calmodulin in a highly temperature-dependent reaction. A MHC kinase found in growth phase cells did not have these regulatory properties. Substrate specificities were analysed for MHC kinase II and for the MHC kinase from growth phase cells. Both enzymes phosphorylated threonine residues of the myosin heavy chains of D. discoideum and Physarum polycephalum. Phosphopeptide mapping of D. discoideum myosin and determination of the stoichiometry of its phosphorylation suggested the presence of two phosphorylation sites per heavy chain. Both sites were contained within a 38-kd chymotryptic fragment. The inactivation of MHC kinase II by Ca2+ plus calmodulin suggests this enzyme has a role in the regulation of myosin functions during the chemotactic response of a cell. The phosphorylated myosin had about one third the actin-activated Mg2+-ATPase activity of the non-phosphorylated myosin. Previous findings indicated that stimulation of D. discoideum cells with the chemo-attractant cAMP increases the cytoplasmic Ca2+ concentration. Under these conditions MHC kinase II might be inhibited and the dephosphorylated, more active form of myosin would accumulate.

Calcium↗
PubMed1983

In vivo sulfation of the contact site A glycoprotein of Dictyostelium discoideum.

During the development of Dictyostelium discoideum from the growth phase to the aggregation stage, a glycoprotein with an apparent mol. wt. of 80 kd is known to be expressed on the cell surface. This glycoprotein, referred to as contact site A, has been implicated in the formation of species-specific, EDTA-stable contacts of aggregating cells. When developing cells were labeled in vivo with [S]sulfate, the 80-kd glycoprotein was found to be the most prominently sulfated protein. Another strongly sulfated protein had an apparent mol. wt. of 130 kd and was, like the 80-kd glycoprotein, developmentally regulated and associated with the particulate fraction of the cells. The [S]sulfate incorporated into the 80-kd and 130-kd proteins was not present as tyrosine-O-sulfate, a modified amino acid found in many proteins of mammalian cells. D. discoideum cells incubated with [S]sulfate in the presence of tunicamycin, an inhibitor of N-glycosylation, produced a 66-kd protein that reacted with monoclonal antibodies raised against the 80-kd glycoprotein, but no longer contained [S]sulfate. These results suggest that sulfation of the 80-kd glycoprotein occurred on carbohydrate residues. The possible importance of sulfation for a role of the 80-kd glycoprotein in cell adhesion is discussed.

Journal Article↗
PubMed1983

Responses of Dictyostelium discoideum amoebae to local stimulation by light.

Single amoebae of Dictyostelium discoideum were locally stimulated with microbeams of white and monochromatic light. Low illuminance stimulation favored formation of pseudopodia at the irradiated parts of the cells, high illuminance stimulation locally suppressed the extension of pseudopodia. When the high illuminance light spot was placed on any portion of the cell other than the moving front, no response could be observed. The results are compatible with the assumption that, during their phototactic response, single amoebae detect the direction of light by a shadowing effect caused by pigments like cytochromes, and/or by light scattering of particles in the cytoplasm.

Dictyostelium↗
PubMed1982

Monoclonal antibodies against contact sites A of Dictyostelium discoideum: detection of modifications of the glycoprotein in tunicamycin-treated cells.

Tunicamycin acts on cell aggregation in Dictyostelium discoideum by changing cell movement and by inhibiting the EDTA-stable type of intercellular adhesion. Tunicamycin-treated cells show unco-ordinated pseudopodial activity such that pseudopods are simultaneously extended from all parts of the cell surface, and the cells are unable to move in straight paths. Concurrent with the inhibition of formation of EDTA-stable contacts, N-glycosylation of a glycoprotein specific for aggregation-competent cells is inhibited. This glycoprotein, previously called contact site A, has an apparent mol. wt. of 80 kilodaltons (kd). In membranes of tunicamycin-treated cells, two components are detected that react with certain monoclonal antibodies against contact sites A: one component of 66 kd, the other of 53 kd apparent mol. wt. Another group of monoclonal antibodies reacts only with the 80-kd glycoprotein and the 66-kd component. These results are in accord with the assumption that the glycoprotein carries two carbohydrate chains, and that the antibodies differ in their requirement for glycosylation of the antigen. Despite the coincidence between blockage of EDTA-stable cell adhesion and inhibited glycosylation of contact sites A, direct involvement of the carbohydrate moieties of this glycoprotein in intercellular adhesion seems questionable. EDTA-stable cell adhesion has not been blocked by Fab fragments from antibodies that specifically react with the glycosylated protein.

Journal Article↗
PubMed1982

Electron microscopic mapping of monoclonal antibodies on the tail region of Dictyostelium myosin.

The binding sites of five monoclonal antibodies against myosin of Dictyostelium discoideum have been mapped. These antibodies bind to the tail region of the myosin molecule. By rotary shadowing, images of myosin-antibody complexes were obtained in which the mean distance of the midpoint of an antibody molecule from the myosin heads was localized with a precision better than 2 nm (90% confidence limit). Other quantitative data extracted from electron micrographs provided information on the stoichiometry of antibody-myosin interaction. Certain antibodies interacted with myosin molecules only at a ratio of 1:1. Other antibodies formed complexes of two molecules bound to homologous sites on a double-stranded myosin tail. Affinities were estimated and the abilities of different antibodies to cross-connect two myosin molecules were evaluated.

Journal Article↗
PubMed1982

Stage-specific antigens reacting with monoclonal antibodies against contact site A, a cell-surface glycoprotein of Dictyostelium discoideum.

Monoclonal antibodies against a glycoprotein presumably involved in adhesion of aggregating Dictyostelium discoideum cells have been used for labeling of the antigen at the cell surface. The antigen is distributed over the whole surface of the cells, apparently in form of small clusters. The antigen appears concomitantly with the acquisition of EDTA-stable adhesiveness typical of aggregation competent cells. In contrast, discoidin I, a lectin whose accumulation during development parallels EDTA-stable adhesiveness in another strain (NC-4), is present in nearly the same amounts of growth phase and aggregating cells of AX2-214, the strain used by use. Thus, no correlation exists in this strain between the expression of discoidin I and the development of cell adhesiveness. The 80 kilodalton glycoprotein typical of aggregation competent cells has been purified by affinity chromatography on a monoclonal antibody column. The purified antigen absorbs adhesion-blocking Fab from rabbits. Another antigen strongly reacting with the same monoclonal antibodies has an apparent molecular weight of 106 000 and is not detectable before slugs are formed.

Animals↗
PubMed1982

Improved purification and N-terminal amino acid sequence determination of the contact site A glycoprotein of Dictyostelium discoideum.

On aggregating cells of Dictyostelium discoideum a specific glycoprotein is expressed which is absent from growth phase cells of this organism. The glycoprotein has been related to cell surface antigens, called contact sites A, that are defined by their reaction with aggregation blocking antibody fragments. In the present paper an improved purification scheme for the glycoprotein is described and its N-terminal sequence is given. The purification has been monitored by use of a monoclonal antibody. The contact site A glycoprotein has the properties of an integral membrane protein.

Amino Acid Sequence↗