Recombinant C3adesArg/acylation stimulating protein (ASP) is highly bioactive: A critical evaluation of C5L2 binding and 3T3-L1 adipocyte activation

C5L2 is a recently identified receptor for C5a/C5adesArg, C3a and C3adesArg (ASP). C5a/C5adesArg bind with high affinity, with no identified activation. By contrast, some studies demonstrate C3a/ASP binding/activation to C5L2; others do not. Our aim is to critically evaluate ASP/C3adesArg-C5L2 binding and bioactivity. Cell-associated fluorescent-ASP (FI-ASP) binding to C5L2 increased from transiently transfected < stably transfected <FI-ASP-sorted C5L2-HEK for both human C5L2 and mouse C5L2. Transfected C5L2-CHO cells had similar results. Endogenous C5L2 expression increased from 3T3-L1 preadipocytes <3T3-L1 adipocytes < primary mouse adipocytes. Non-transfected cells FI-ASP demonstrated background fluorescence only. In adherent C5L2-HEK (FI-ASP sorted) and 3T3-L1 cells, blocking with 10% fetal calf serum, protamine sulfate or ovalbumin prevented I-125-ASP non-specific binding (NSB, no cells), while albumin increased NSB. Binding to non-transfected HEK was comparable to NSB. Optimal specific binding was obtained at 20 degrees C (vs. 4 degrees C) in PBS or serum-free medium with K-d 83.7 +/- 23.7 nM (C51-2-HEK), 66 +/-15 nM (C5L2-CHO)and 76 +/- 14.3 nM1 (3T3-L1 preadipocytes); I-125-C5a binding had greater affinity. FI-ASP-C5L2 binding was comparable and concentration dependent (K-d 31 nM (direct binding) and IC50 35 nM (competition binding) regardless of conditions). Recombinant ASP (rASP) produced in modified Escherichia coli Origami (DE3) (allowing folding and disulphicle bridge formation), purified under non-denaturing conditions demonstrated 10 x greaterbioactivity vs. proteolytically derived plasma ASP for triglycericle synthesis and fatty acid uptake in 3T3-L1 adipocytes and preadipocytes while adipose tissue from C5L2 KO mice was non-responsive. rASP stimulation of adipocyte BODIPY-fatty acid uptake demonstrated EC50 115 +/- 93 nM and maximal stimulation of 413 +/- 33%, p < 0.001. ASP binding has distinct characteristics that lead to C5L2 activation and increased bioactivity. (C) 2009 Elsevier Ltd. All rights reserved.