Probing the 121-136 domain of lecithin : cholesterol acyltransferase usingantibodies

Lecithin:cholesterol acyltransferase (LGAT) catalyzes the esterification of plasma lipoprotein cholesterol in mammals as part of the reverse cholester ol transport pathway. Studies of the natural mutations of LCAT revealed a r egion that is highly sensitive to mutations (residues 121-136) and it is hi ghly conserved in six animal species. The purpose of these studies was to i nvestigate the reactivity of wild type and several mutated forms of LCAT, w ith a series polyclonal antibodies to further characterize this specific do main (residues 121-136), Two polyclonal antibodies directed against the who le enzyme, one against human plasma LCAT and the other against purified rec ombinant LCAT, and one site specific polyclonal antibody, directed against the 121-136 region of LCAT, were employed. All three antibodies reacted wit h a recombinant form of purified LCAT; however, only the polyclonal antibod ies directed against the whole enzyme were able to recognize the LCAT when it was adsorbed to a hydrophobic surface in a solid phase immunoassay, or w hen bound to HDL in a sink immunoassay, These findings indicate that the ep itope(s) of the 121-136 region are not accessible to antibodies under these conditions, Three mutant forms of LCAT, representing alterations in the 12 1-136 region, were also examined for their immunoreactivity with the same p anel of antibodies and compared to the wildtype enzyme. These studies demon strate that in its native configuration the 121-136 region of LCAT is Likel y to reside on a surface of LCAT, Furthermore, mutations within this region appear to markedly impact the exposure of epitopes at additional sites. Th ese findings suggest that the 121-136 region could play an important role i n enzyme interaction with its hydrophobic lipoprotein substrates as mutatio ns within this region appear to alter enzyme conformation, catalytic activi ty, and the specificity of LCAT, (C) 2001 Academic Press.