Joachim Stadler

Complete sequence and transcript regulation of a cell adhesion protein from aggregating Dictyostelium cells.

Three cDNA clones coding for the contact site A (csA) protein, a cell adhesion molecule of Dictyostelium discoideum, were isolated by screening a cDNA library with monoclonal antibodies. Two of these clones contained the complete coding region for the csA protein of 1542 bp including a sequence of 57 bp coding for the leader. The N terminus of the mature protein, as it was published previously, was identified in the amino acid sequence derived from both full‐length cDNA clones. Southern blot analysis suggests the presence of only one csA gene in the haploid genome. Accumulation of the csA‐specific message of 1.9 kb begins during development on nitrocellulose filters at 9 h of starvation, and reaches a maximum at 12 h, the time of cell aggregation. Expression of the csA glycoprotein follows closely accumulation of the transcripts. In the multicellular slug stage following cell aggregation, the amount of csA transcripts rapidly declines to low levels.

The contact site A glycoprotein of Dictyostelium discoideum carries a phospholipid anchor of a novel type.

The contact site A glycoprotein, a cell adhesion protein of aggregating Dictyostelium cells, was labeled with fatty acid, myo‐inositol, phosphate and ethanolamine in vivo, indicating that the protein is anchored in the membrane by a lipid. This lipid was not susceptible to phosphatidyl inositol specific phospholipase C. When cleaved with nitrous acid or when subjected to acetolysis, the anchor released lipids which were different from those released from Trypanosoma variant cell surface glycoprotein, a protein with a known phosphatidyl inositol‐glycan anchor. Resistance to weak and sensitivity to strong alkali indicated that the fatty acid in the contact site A glycolipid anchor was in an amide bond. On incubation with sphingomyelinase, a lipid with the chromatographic behavior of ceramide was released. These results suggest that the contact site A glycoprotein is anchored by a ceramide based lipid glycan.

Probing an adhesion mutant of Dictyostelium discoideum with cDNA clones and monoclonal antibodies indicates a specific defect in the contact site A glycoprotein

Expression of developmentally regulated membrane proteins of aggregating cells of Dictyostelium discoideum is subject to several control mechanisms. One of them involves periodic cyclic‐AMP pulses as signals for gene expression. To increase the probability of selecting mutants specifically defective in the contact site A (csA) glycoprotein, one of the characteristic proteins of aggregating cells, we have bypassed the requirement for both cyclic‐AMP pulses and another control element by two runs of mutagenesis. A ‘double bypass’ mutant, HG592, was obtained which aggregated in nutrient medium where wild‐type did not develop. Mutants defective in expression of the csA‐glycoprotein were selected from HG592 by fluorescence‐activated cell sorting and colony immunoblotting using a monoclonal antibody specific for that protein. One among 51 csA‐negative mutants, HG693, specifically lacked the capability of forming EDTA‐stable intercellular contacts. It acquired chemotactic responsiveness and developed into fruiting bodies. Expression of the transcripts for eight developmentally regulated proteins was determined in HG693. Seven of the RNA species were normally expressed; they were recognized by cDNA clones which had been produced from poly(A)+ RNA isolated from membrane‐bound polysomes. The single RNA species which was not substantially expressed in HG693 was recognized by a cDNA clone that was obtained by screening a λgt11 library with an antibody specific for the csA‐glycoprotein. When probing RNA from wild‐type cells, this clone hybridized with a single developmentally regulated RNA species of 1.9 kb whose expression was strongly enhanced by cyclic‐AMP pulses. Appearance of this RNA coincided with the expression of the csA‐glycoprotein.

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 [35S]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 [35S]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 [35S]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 [35S]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.

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.

In vivo acylation of Dictyostelium actin with palmitic acid.

Cells of Dictyostelium discoideum were incubated with [3H]palmitic acid during development, and recovery of the fatty acid label in soluble and membrane‐associated proteins was investigated. One of the major labeled proteins was found exclusively in the soluble fraction. This protein, with an apparent mol. wt. of 44 kd, was identified as actin based on its labeling with a monoclonal anti‐actin antibody, its coincidence with the major [35S]methionine‐labeled protein after two‐dimensional electrophoresis and its binding to a DNase I affinity column. The 3H‐label was resistant to chloroform‐methanol extraction and boiling in SDS‐containing buffer. After partial purification by preparative SDS‐polyacrylamide gel electrophoresis, the 44‐kd protein was treated with KOH, the fatty acids released were derivatized to methyl esters and palmitic acid methylester was identified by gas‐liquid chromatography.

Acylation in vivo of the contact site A glycoprotein and of other membrane proteins in Dictyostelium discoideum

Cells of Dictyostelium discoideum were incubated during the preaggregation phase with [3H] palmitic acid, and association of the fatty acid label with proteins was investigated by SDS‐polyacrylamide gel electrophoresis and fluorography. Three proteins with apparent molecular masses of 44, 80 and 130kDa were preferentially labeled. The 80‐kDa protein proved to be identical with an integral membrane glycoprotein called contact site A. The protein retained the 3H label after chloroform‐methanol extraction and boiling in SDS‐containing buffer, but not after mild hydroxylamine treatment, indicating an ester bond. After treatment of the protein with KOH the 3H label was recovered in free palmitic acid, as demonstrated by gas‐liquid chromatography of the methyl ester.