Histidine-rich glycoprotein and concanavalin A synergistically stimulate the phosphatidylinositol 3-kinase-independent signaling pathway in leukocytes leading to increased cell adhesion and changes in cell morphology.

Histidine-rich glycoprotein (HRG) promoted the adhesion and morphological changes of human T-cell line MOLT-4 in a Con A-dependent manner. This morphological change-promoting activity was specific for HRG and the Arg23-Lys66 glycopeptide from human HRG. The carbohydrate chain at Asn45 was essential for this activity. The morphological changes of MOLT-4 cells caused by HRG and Con A (HRG/Con A) were not inhibited by phosphatidylinositol 3-kinase inhibitor, wortmannin or LY294002, while the changes by Con A alone were completely inhibited by these reagents, suggesting that HRG/Con A cooperate to activate leukocytes via a signaling pathway distinct from that by Con A alone. The morphological changes by Con A were associated with pseudopodia like structure. On the other hand, the morphological changes caused by HRG/Con A were associated not only with pseudopodia like structure but also with an increase of the F-actin-rich surface protrusions. Wortmannin inhibited only the formation of pseudopodia like structure.

High affinity interaction between histidine-rich glycoprotein and the cell surface type ATP synthase on T-cells.

Histidine-rich glycoprotein (HRG) is a plasma protein implicated in the innate immune system. In recent studies, we showed that either HRG, or the Arg23-Lys66 glycopeptide derived from HRG, in concert with concanavalin A (Con A), promotes a morphological change and adhesion of the human leukemic T-cell line MOLT-4 to culture dishes, and that cell surface glycosaminoglycan or Fcgamma receptors do not participate in this cellular event. In the present study, we identified the alpha-subunit of ATP synthase as one of the HRG-binding proteins on the surface of T-cells by HRG-derived glycopeptide affinity chromatography and by a peptide mass finger printing method. HRG specifically interacted with mitochondrial ATP synthase with a dissociation constant of 66 nM. The presence of alpha- and beta-subunits of ATP synthase on the plasma membrane of MOLT-4 cell was demonstrated by immunofluorescent staining and FACS analysis. The HRG/Con A-induced morphological changes of MOLT-4 cells were specifically inhibited by a monoclonal antibody against the beta-subunit of ATP synthase. These results strongly suggest that the cell surface ATP synthase functions as a binding protein for HRG on MOLT-4 cells, which is required for the morphological changes observed in MOLT-4 cells following treatment with HRG/Con A.