Structure-Activity Relationships of Chromogranin A in Cell Adhesion

Abstract

Previous studies showed that chromogranin A (CgA), a glycoprotein stored and co-released with various hormones by neuroendocrine cells and neurons, can modulate cell adhesion. We have investigated the structureactivity relationships of CgA using fibroblasts and coronary artery smooth muscle cells in adhesion assays. A recombinant CgA fragment 1-78 and a peptide 7-57 containing reduced and alkylated cysteines (Cys 17 and Cys 38 ) induced cell adhesion after adsorption onto solid phases at 50 -100 nM. Peptides lacking the disulfide loop region, including residues 47-68, 39 -59, and 39 -68, induced cell adhesion, either bound to solid phases at 200 -400 nM or added to the liquid phase at 5-10 M, whereas peptide 60 -68 was inactive, suggesting that residues 47-57 are important for activity. The effect of CgA-(1-78) was blocked by anti-CgA antibodies against epitopes including residues Arg 53 , His 54 , and Leu 57 . Substitutions of residues His 54 , Gln 55 , and Asn 56 with alanine decreased the cell adhesion activity of peptide 47-68. These results suggest that the region 47-57 (RILSILRHQNL) contains a cell adhesion site and that the disulfide bridge is not necessary for the proadhesive activity. The ability of soluble peptides to elicit proadhesive effects suggests an indirect mechanism. The high sequence conservation and accessibility to antibodies suggest that this region is important for the physiological role of CgA.

Cleavage of Chromogranin A N-terminal Domain by Plasmin Provides a New Mechanism for Regulating Cell Adhesion

“…The cell line was routinely checked for mycoplasma contamination. Mouse antiCgA monoclonal antibodies (mAbs) 4D5, 7D1, 5A8, B4E11, and A11 were described previously (19,32,33).…”

“…Recombinant NH 2 -Ser-Thr-Ala-hCgA , NH 2 -Ser-Thr-Ala-hCgA , hereinafter termed (STA)1-78 and (STA)1-115, were obtained by expression in Escherichia coli cells (19). The products were purified by reverse phase HPLC using a SOURCE 15 RPC column (Pharmacia-Upjohn), followed by gel filtration chromatography on a Sephacryl S-200 HR column (Pharmacia-Upjohn), as described (32). The molecular masses of (STA)1-78 and (STA)1-115 were 9067.72 Ϯ 0.5 and 13,247.4 Ϯ 0.5, respectively, by electrospray mass spectrometry (ESI-MS) analysis.…”

“…Adhesion Assays-Solid phase and liquid phase adhesion assays were carried out using 96-well polystyrene cell culture plates as we described previously in detail (32). Briefly, serum-dependent adhesion assays with solid phase bound peptides were carried out by: (a) coating plates with peptide solutions in PBS (0.15 M sodium chloride, 0.05 M sodium phosphate buffer, pH 7.3; 50 l/well, 90 min at 37°C); (b) blocking with 3% bovine serum albumin (Sigma) in DMEM (200 l/well, 1 h at 37°C); and (c) seeding NIH3T3 cell suspension (50 l/well in DMEM-GPS) containing 0.1-0.6% FBS.…”

“…In previous work we showed that a recombinant CgA N-terminal fragment, called (STA)1-78, increases cell adhesion and spreading of fibroblasts to solid phases and that a pro-adhesive site is located within residues 47-57 (RILSILRHQNL) (32). To identify the antiadhesive sites of CgA, we tested the effect of several monoclonal antibodies on the anti/pro-adhesive activity of human CgA, (STA)1-115, and (STA)1-78, using the NIH 3T3 fibroblast adhesion assay.…”

“…Other studies showed that CgA, at nanomolar concentrations, may increase deposition of basement membrane components, such as collagen type IV, laminin, and perlecan by mammary epithelial cells, and alter ductal morphogenesis in vitro (31), reinforcing the hypothesis of a role of CgA in cell adhesion and tissue morphogenesis. Structural determinants of the pro-adhesive activity were found to be located within the conserved residues 47-57 (RIL-SILRHQNL) (32), whereas the location of the anti-adhesive site has not been identified yet. In this work we have studied the structural determinants of the anti-adhesive activity of CgA using natural, recombinant, and synthetic fragments and fibroblast adhesion assays.…”