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 (Fl-ASP) binding to C5L2 increased from transiently transfected < stably transfected < Fl-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 ± Fl-ASP demonstrated background fluorescence only.In adherent C5L2-HEK (Fl-ASP sorted) and 3T3-L1 cells, blocking with 10% fetal calf serum, protamine sulfate or ovalbumin prevented 125I-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 °C (vs. 4 °C) in PBS or serum-free medium with Kd 83.7 ± 23.7 nM (C5L2-HEK), 66 ± 15 nM (C5L2-CHO) and 76 ± 14.3 nM (3T3-L1 preadipocytes); 125I-C5a binding had greater affinity. Fl-ASP-C5L2 binding was comparable and concentration dependent (Kd 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 disulphide bridge formation), purified under non-denaturing conditions demonstrated 10× greater bioactivity vs. proteolytically derived plasma ASP for triglyceride 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.