Structure-Activity Relationships of Chromogranin A in Cell Adhesion

doi:10.1074/jbc.M003796200

Structure-Activity Relationships of Chromogranin A in Cell Adhesion

IDENTIFICATION OF AN ADHESION SITE FOR FIBROBLASTS AND SMOOTH MUSCLE CELLS*

From the Department of Biological and Technological Research, San Raffaele H Scientific Institute, via Olgettina 58, 20132 Milan, Italy,^‡Consiglio Nazionale delle Ricerche-IRBM, via M. Bianco 9, 20132 Milan, Italy, and ^§INSERM Unité 338, “Biologie de la Communication Cellulaire,” 5 rue Blaise Pascal, 67084 Strasbourg Cedex, France

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 (Cys¹⁷ and Cys³⁸) 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 nmor added to the liquid phase at 5–10 μm, 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 Arg⁵³, His⁵⁴, and Leu⁵⁷. Substitutions of residues His⁵⁴ _, Gln⁵⁵, and Asn⁵⁶ 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.

Footnotes

↵* This work was supported by a grant from the Associazione Italiana per la Ricerca sul Cancro (AIRC).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¶ To whom correspondence should be addressed: Dept. of Biological and Technological Research, San Raffaele H Scientific Institute, via Olgettina 58, 20132 Milan, Italy. Tel.: 39 02 26434802; Fax: 39 02 26434786; E-mail: corti.angelo@hsr.it.
Published, JBC Papers in Press, June 29, 2000, DOI 10.1074/jbc.M003796200
↵2 F. Sato, N. Ishida, T. Hasegawa, and H. Mukoyama, GenBank™ accession number AB025570.
Abbreviations:

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
- Received May 4, 2000.
- Revision received June 5, 2000.
The American Society for Biochemistry and Molecular Biology, Inc.