Structure-Activity Relationships of Chromogranin A in Cell Adhesion: IDENTIFICATION OF AN ADHESION SITE FOR FIBROBLASTS AND SMOOTH MUSCLE CELLS

Sara Ratti,Flavio Curnis,Renato Longhi,Barbara Colombo,Anna Gasparri,Fulvio Magni,Ernesto Manera,Marie-Hélène Metz-Boutigue,Angelo Corti

doi:10.1074/jbc.m003796200

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 structure-activity 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 microm, 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.

Highlights

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
In the attempt to identify the molecular determinants of CgA cell adhesion activity, we have investigated the structure-activity relationships of vasostatin-1 using several recombinant and synthetic peptides and anti-CgA antibodies
We show that the region 47–57 of CgA N-terminal fragments contains an accessible site that induces adhesion and spreading of fibroblasts and smooth muscle cells on solid phases

Summary

The abbreviations used are

CgA, chromogranin A; mAb, monoclonal antibody; PBS, phosphate-buffered saline; BSA, bovine serum albumin; FBS, fetal bovine serum; DMEM, Dulbecco’s modified Eagle’s medium; ELISA, enzyme-linked immunosorbent assay; CASMC, coronary artery smooth muscle cell(s); HPLC, high pressure liquid chromatography. The intracellular and extracellular functions of CgA and its proteolytic fragments are still unclear. Fragments corresponding to amino acids 1–76 and 1–113, named vasostatin-1 and vasostatin-2, inhibit vascular tension (18 –20). These N-terminal fragments are released from the adrenal medulla [20] and from sympathetic nerve terminals in response to stimulation [21]. In the attempt to identify the molecular determinants of CgA cell adhesion activity, we have investigated the structure-activity relationships of vasostatin-1 using several recombinant and synthetic peptides and anti-CgA antibodies. We show that the region 47–57 of CgA N-terminal fragments contains an accessible site that induces adhesion and spreading of fibroblasts and smooth muscle cells on solid phases

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION