Acylation-stimulating protein (ASP): structure-function determinants of cell surface binding and triacylglycerol synthetic activity

Acylation-stimulating protein (ASP or C3adesArg) is a potent lipogenic fact or in human and murine adipocytes and fibroblasts, The arginated form of AS P, i.e. complement C3a (C3a), stimulates immunological responses in human g ranulocytes, mast cells, guinea pig platelets and guinea pig macrophages; h owever, ASP is inactive in stimulating these responses. Thus both ASP and C 3a are bioactive across species but are not functionally interchangeable. T ertiary structure of both proteins by X-ray crystallography and NMR spectro scopy predicts a tightly linked core region consisting of three cc-helices linked via three disulphide bonds, with one of the a-helices extending out from the core and terminating in a flexible conformationally irregular carb oxy-tail region. The present studies were undertaken in order to define the functionally active domains of ASP, distinctive from those of C3a, using c hemical modifications, enzymic cleavage and synthetic peptide fragments. Th e results indicate that: (i) the N-terminal region (< 10 amino acids) plays little role in ASP receptor binding and triacylglycerol synthesis stimulat ion; (ii) the native C-terminal region had no activity, but modifications w hich increased hydrophobicity increased receptor binding, and led to some a ctivation of triacylglycerol synthesis stimulation; (iii) an intact disulph ide-linked core region is essential for triacylglycerol synthesis stimulati on activity but not for receptor interaction. Finally, basic charges in the carboxy region (His) are essential for ASP triacylglycerol synthesis stimu lation but not for receptor binding, whereas both functions are eliminated by the modification of Lys in the disulphide-linked core region. The presen t results suggest that there are two functional domains in ASP, one that is responsible for the initial binding to the cell surface receptor, and a se cond domain that activates and increases triacylglycerol synthesis stimulat ion. This contrasts markedly with the structure-function studies of C3a whe re both binding competency and function were dependent on the C-terminal Ar g. Thus ASP demonstrates distinct bioactivity.