Glycoprotein gp130 ofDictyostelium discoideumInfluences Macropinocytosis and Adhesion

Abstract

Glycoprotein gp130, found on the plasma membrane of Dictyostelium discoideum amoebae, was postulated previously to play a role in phagocytosis. The gene for gp130 was cloned and when translated, yielded a 768 amino acid preproprotein of 85.3 kDa. It had nearly 40% similarity to the 138 kDa family of glycoproteins implicated in sexual cell fusion during macrocyst formation in D. discoideum. The difference between the calculated size and observed M r of 130 kDa on protein gels likely was due to N-glycosylation that was confirmed by lectin blots. Consistent with its surface-exposure, an antibody raised against recombinant protein stained the plasma membrane of D. discoideum amoebae. Gp130 and its transcripts were high during axenic growth of cells, but relatively low during growth on bacteria. The gene for gp130 was disrupted and cell lines lacking the glycoprotein were efficient phagocytes, indicating that gp130 was dispensable for phagocytosis. Gp130-null cells were similar in size to parent DH1 cells, had enhanced macropinocytosis and grew faster to higher densities. They also exhibited weaker cell-substrate adhesion but displayed greater cell-cell cohesion. Collectively, the data indicated that gp130 influenced macropinocytosis and played a role in adhesion during vegetative growth. INTRODUCTIONThe processes of phagocytosis, cell-cell and cell-substrate adhesion share a common initial event of recognition, whether it is of specific ligands or the chemical properties of the partner(s) in the interaction. With the long-term aim of understanding the mechanism of phagocytosis at the molecular level, particularly the early steps of recognition and binding, we have focused on surface-exposed molecules of the phagocytically active amoebae of Dictyostelium discoideum. D. discoideum cells are studied also for the related processes of motility and aggregation. When starved, individual but clonal cells coalesce into multicellular structures that, within a day, differentiate into fruiting bodies called sorocarps. Cell-cell recognition and adhesion are vital aspects of this developmental progression, and researchers have established that cell-surface molecules, including gp24 (Knecht et al., 1987;Loomis, 1988;Brar and Siu, 1993), gp80 (Muller and Gerisch, 1978;Noegel et al., 1986), and gp150 (Gao et al., 1992;Wang et al., 2000), are mediating these interactions (reviewed in Kessin, 2001 andSiu et al., 2004). An understanding of the plasma membrane molecules involved in adhesive events during phagocytosis and motility of vegetative amoebae is emerging with the recent identification of cell-substrate adhesion molecule, sadA (Fey et al., 2002) and the phg1 family of transmembrane 9 proteins (Cornillon et al., 2000;Benghezal et al., 2003).In this study, we present evidence that a plasma membrane glycoprotein gp130 also played a role in cell-substrate adhesion during vegetative growth. Postulated to be a phagocytosis receptor (Chia, 1996), gp130 is possibly the same molecule as gp126, a surface-exposed glycoprotein suggested to ...

Bacterial Discrimination by Dictyostelid Amoebae Reveals the Complexity of Ancient Interspecies Interactions

“…Dictyostelium strains carrying mutations in clkB , spc3 , alyL , or the insertion at Ω1334 grew poorly on the seven Gram(−) bacterial species, but grew well on the five Gram(+) bacterial species tested (Figure 2, and supplemental Figure S2a). We also examined a previously characterized mutant of gp130 which we hypothesized might be a receptor for Gram(+) bacteria[29, 30]. Interestingly, the gp130 -null mutant exhibited a growth defect on lawns of Gram(+) but not on Gram(−) bacteria (Figure 2a, and supplemental Figure S2a).…”

“…Amoebal growth on Gram(+) bacteria appears to be critically dependent on one or more glycoproteins and Gp130 appears to be one of them, so the swp1 mutant growth phenotype might be entirely attributable to reduced Gp130 function or levels [29, 30]. Pharmacological interruption of the N-linked glycosylation pathway of the amoebae would allow Gram(+) bacteria to evade predation by amoebae, and this appears to be a weakness that the Gram(+) Streptomyces bacteria seem to exploit for their survival by secreting tunicamycin [33, 36].…”

Genome-wide transcriptional changes induced by phagocytosis or growth on bacteria in Dictyostelium

“…The V-H+ ATPase, Rab7 and calreticulin are typical markers of phagosomal traffic [ 16 , 81 - 83 ], whereas gp24, discoidin II and gp130 are extracellular or membrane proteins that could be entrapped in the phagosome. However, knockout mutants for both gp24 and gp130 are not defective in phagocytosis [ 84 ]. Discoidins have not been involved in phagocytosis sofar, but their role could be re-evaluated, considering this result, their lectin activity and the finding that both discoidin I and discoidin II genes are strongly up-regulated in V12M2.…”

Glycopeptidome of a Heavily N-Glycosylated Cell Surface Glycoprotein of Dictyostelium Implicated in Cell Adhesion

“…The BamH1-digested gel-purified PCR product was directionally cloned into the BglII site of pVS, 23 and pVS-CD11 was electroporated into the gp130-null strain DBS0235414. 4 Producers were selected in 20 µg/mL G418 and clones were screened using silver-stained SDS-PAGE gels. Clone CC11 was used for the present study.…”