Effects of missense mutations and deletions on membrane anchoring and enzyme function of human steroid 21-hydroxylase (P450c21)

We studied membrane binding and enzyme function of six variant forms of human steroid 21-hydroxylase (P450c21), a mutant (P30Q) from a patient with congenital adrenal hyperplasia, four artificial deletions in the amino terminal region (delS1 and del S2; the first and second hydrophobic segment, delS3; the region in between, delS4; the combination of these), and one naturally ocurring polymorphism in a region implicated to be critical for membrane integration (delL10). Enzyme function was assayed after transient expression in COS-1 cells, and membrane binding was studied by coupledin vitrotranscription-translation in the presence of microsomal membranes. P450c21(delS1) retained some enzyme activity but showed severely reduced membrane binding. P450c21(P30Q), P450c21 (delS2), P450c21(delS3), and P450c21(delS4) had abolished enzyme function. P450c21(P30Q) and P450c21 (delS2) did not affect membrane binding, P450c21 (delS3) had slightly reduced binding with a qualitative difference suggested by the absence of a glycosylated form of the protein, and P450c21(delS4) had abolished membrane integration. No significant differences could be identified for the delL10 variant. These data support that P450c21 spans the membrane through its first hydrophobic domain only, and that the protein lacking this segment retains sufficiently normal structure to enable catalysis. They also confirm that P30Q is responsible for the severe phenotype of the patient in which it was found, and indicate that the common delL10 polymorphism does not have a major effect on enzyme function.