Human placental Fc gamma receptor. Studies on affinity labeling of the receptor with IgG.
The aim of the presented work was to find out whether the Fc gamma receptor from human placental syncytiotrophoblast plasma membranes in its native membrane-bound state is composed of more subunit chains than previously found in our laboratory in the purified receptor. The chains might be lost during the purification procedure with the use of various chaotropic reagents, e.g. acidic or alkaline buffers, detergents. To study this problem, affinity labeling technique and bifunctional crosslinking reagents were used to covalently link IgG with the Fc gamma receptor. The reagents used were: noncleavable dimethylsuberimidate (DMS), cleavable dimethyl 3,3-dithiobispropionimidate (DTBP), and photoactivable sulfosuccinimidyl 6-/4' azido-2'nitrophenylamino/hexanoate (sulfo-SANPAH). Human 125I-IgG were crosslinked to the membrane-bound receptor or unlabeled IgG was crosslinked to 3H-labeled placental membranes. When 125I-IgG were used in the presence of an excess of unlabeled IgG, recovery of radioactivity after crosslinking was much lower, indicating that human IgG and the placental Fc gamma receptor were involved in the formation of crosslinking of ligand-receptor complexes. The products of crosslinking were analyzed in SDS-PAGE. In the absence of reducing agents, high molecular weight products were present which did not enter the gel or formed broad diffused bands at the top of the gel. Therefore, the products of crosslinking were analyzed under reducing conditions. Analysis by SDS-PAGE demonstrated a major polypeptide band Mr of 160,000 in soluble products of crosslinking of IgG to the placental Fc gamma receptor, regardless of which noncleavable crosslinker was used. The protein is built either of two molecules of the receptor subunit chain, Mr of 60,000 and one IgG heavy chain (Mr of 56,000) or of two IgG heavy chains and one molecule of the receptor chain. The presence of this protein in control samples was not observed at all or its content was markedly lower. The same effect was also observed when DTBP, a cleavable crosslinking reagent was used. In this case, 125I-IgG heavy and light chains or the receptor subunit chains were found after SDS-PAGE under reducing conditions. The results presented in this paper do not suggest a presence of additional chains in the placental Fc gamma receptor others than described in our previous paper concerning the subunit structure of this receptor.
Mikulska
J
J
Department of Immunochemistry, Polish Academy of Sciences, Wrocław.
Hałas
E
E
Lisowski
J
J
D003432:Cross-Linking Reagents; D005260:Female; D006801:Humans; D007143:Immunoglobulin Heavy Chains / Q000378:metabolism*; D007142:Immunoglobulin gamma-Chains / Q000378:metabolism*; D010920:Placenta / Q000276:immunology*; D011961:Receptors, Fc / Q000032:analysis* / Q000187:drug effects
D003432:Cross-Linking Reagents; D007143:Immunoglobulin Heavy Chains; D007142:Immunoglobulin gamma-Chains; D011961:Receptors, Fc