Production and structure characterisation of recombinant chromogranin A N-terminal fragments (vasostatins) -- evidence of dimer-monomer equilibria.
Vasostatins (VS) are vasoinhibitory peptides derived from the N-terminal domain of chromogranin A, a secretory protein present in the electron-dense granules of many neuroendocrine cells. In this work we describe a method for the production in Escherichia coli of large amounts of recombinant vasostatins, corresponding to chromogranin A residues 1-78 (VS-1), and 1-115 (VS-2), and the use of these materials for structure characterisation. The masses of both products were close to the expected values, by SDS/PAGE and mass spectrometry analysis. However, their hydrodynamic behaviours in size-exclusion chromatography corresponded to that of proteins with a larger size. SDS/PAGE analysis of VS-1 and VS-2 after cross-linking with disuccinimidyl suberate indicated that both polypeptides form dimers. VS-2 was almost entirely dimeric at > 4 microM, but rapidly converted to monomer after dilution to 70 nM. The rapid dimer-monomer transition of VS-2 after dilution could be part of a mechanism for regulating its activity and localising its action. Immunological studies of VS-1 have shown that residues 37-70 constitute a highly antigenic region characterised by an abundance of linear epitopes efficiently mimicked by synthetic peptides. The recombinant products and the immunological reagents developed in this work could be valuable tools for further investigating the structure and the function of chromogranin A and its fragments.
Corti
A
A
DIBIT, Department of Biological and Technological Research, San Raffaele H. Scientific Institute, Milan, Italy. cortia@dibit.hsr.it
Sanchez
L P
LP
Gasparri
A
A
Curnis
F
F
Longhi
R
R
Brandazza
A
A
Siccardi
A G
AG
Sidoli
A
A
D000911:Antibodies, Monoclonal / Q000276:immunology; D015153:Blotting, Western; D002851:Chromatography, High Pressure Liquid; D053379:Chromogranin A; D002864:Chromogranins / Q000737:chemistry* / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification; D003001:Cloning, Molecular; D003432:Cross-Linking Reagents; D019281:Dimerization; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D018604:Epitope Mapping; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D013058:Mass Spectrometry; D010455:Peptides / Q000737:chemistry / Q000276:immunology; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification; D013388:Succinimides / Q000378:metabolism
D000911:Antibodies, Monoclonal; C506150:CHGA protein, human; D053379:Chromogranin A; D002864:Chromogranins; D003432:Cross-Linking Reagents; D010455:Peptides; D011994:Recombinant Proteins; D013388:Succinimides; C019358:disuccinimidyl suberate