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 hum an steroid 21-hydroxylase (P450c21), a mutant (P30Q) from a patient with co ngenital adrenal hyperplasia, four artificial deletions in the amino termin al region (delS1 and del S2; the first and second hydrophobic segment, delS 3; the region in between, delS4; the combination of these), and one natural ly ocurring polymorphism in a region implicated to be critical for membrane integration (delL10). Enzyme function was assayed after transient expressi on in COS-1 cells, and membrane binding was studied by coupled in vitro tra nscription-translation in the presence of microsomal membranes. P450c21(del S1) retained some enzyme activity but showed severely reduced membrane bind ing. P450c21(P30Q), P450c21 (delS2), P450c21(delS3), and P450c21 (delS4) ha d abolished enzyme function. P450c21(P30Q) and P450c21 (delS2) did not affe ct membrane binding, P450c21 (delS3) had slightly reduced binding with a qu alitative difference suggested by the absence of a glycosylated form of the protein, and P450c21(delS4) had abolished membrane integration. No signifi cant differences could be identified for the delL10 variant. These data sup port that P450c21 spans the membrane through its first hydrophobic domain o nly, and that the protein lacking this segment retains sufficiently normal structure to enable catalysis, They also confirm that P30Q is responsible f or 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. (C) 1999 Academic Press.