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Katherine Cianflone, Wei Cui, Marc Lapointe, David Kalant, Magdalena Maslowska, Characterization of C3adesArg/acylation stimulating protein (ASP) binding to C5L2 transfected cells and 3T3-L1 preadipocytes (94.17), The Journal of Immunology, Volume 178, Issue 1_Supplement, April 2007, Page S173, https://doi.org/10.4049/jimmunol.178.Supp.94.17
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Abstract
C5L2 is a recently identified receptor for C5a and C3a/C3adesArg (ASP). C5a/C5adesArg binds C5L2 with high affinity with no functional activation. By contrast, some studies demonstrate C3a/ASP binding/activation of C5L2; others do not. We evaluated the influence of diverse ASP-C5L2 binding methods used.
Orphan receptors (n>30) were screened with 125I-ASP (20oC & 4oC) or fluorescently labeled ASP (Fl-ASP, 37oC). Only C5L2 transfected cells were positive. Cell-associated Fl-ASP increased markedly from transiently-transfected < stably transfected < Fl-ASP sorted HEK-C5L2 for both hC5L2 and mC5L2. Similar results were obtained with transfected C5L2-CHO and C5L2-SW872 cells. Non-transfected cells ± Fl-ASP demonstrated background fluorescence only.
Recombinant ASP prepared under non-denaturing conditions demonstrated 10X greater bioactivity vs. proteolytically-derived plasma ASP (maximal TG synthetic activity 100–300 nM vs. 5 uM).
In adherent HEK-C5L2 (F-ASP sorted) and 3T3-L1 cells, blocking with 10% FCS, protamine sulfate or ovalbumin prevented 125I-ASP non-specific binding (NSB), while BSA blocking increased it. Optimal specific binding (Kd 66±14 nM) was obtained at 20oC (vs.4oC) in PBS or HAG-CM buffer. By contrast, cells in suspension demonstrated only non-specific binding. ASP-C5L2 binding has distinct characteristics that lead to phosphorylation, internalization and increased TG.
Funding: CIHR
