FUNCTIONAL BIOACTIVE RECOMBINANT ACYLATION STIMULATING PROTEIN IS DISTINCT FROM C3A ANAPHYLATOXIN

Acylation stimulating protein (ASP) acts upon adipose tissue to stimul ate triglyceride synthesis and glucose transport. The aim of the prese nt study was to produce recombinant ASP and to measure its bioactivity . The cDNA region of the parent complement C3 sequence coding for ASP (C3adesArg) was cloned and expressed in E. coli. Bioactivity of the pu rified recombinant material was tested by determining its effect on tr iglyceride synthesis, glucose transport, and competition binding assay s. In standard assays, concentrations of 5.5 mu M recombinant ASP (rAS P) stimulated triglyceride synthesis comparably to plasma ASP (pASP); 228% versus 237%, respectively, in 3T3 preadipocytes and 568% versus 4 40% in human differentiated adipocytes. rASP also increased glucose tr ansport in L6 myocytes (163% at 10 mu M rASP) and in human differentia ted adipocytes (334% rASP vs. 329% pASP at 5 mu M). rASP competitively displaced radiolabeled plasma ASP from high affinity association with the cell surface in both human differentiated adipocytes and 3T3 prea dipocyte fibroblasts. Furthermore, immunoprecipitation of rASP and pAS P with a specific monoclonal antibody abolished stimulation of cellula r triglyceride synthesis. Lastly, we contrasted the structure: functio n activities of the arginated (C3a) and desarginated (ASP) proteins. T he lipogenic activity and the anaphylatoxic activity results from dist inct structural domains of the polypeptides. Thus rASP retains full bi ologic ASP activity and may provide a tool to study structure-function relationships in this physiologic system.