EXPRESSION AND MOLECULAR ANALYSIS OF MUTATIONS IN PROLIDASE DEFICIENCY

Prolidase (E.C.3.4.13.3) cleaves iminodipeptides. Prolidase deficiency (PD; McKusick 170100) is an autosomal recessive disorder with highly variable penetrance. We have identified two novel alleles in the proli dase gene (PEPD) by direct sequencing of PCR-amplified cDNA from a PD individual asymptomatic at age 11 years: a 551G-->A transition in exon 8 (R184Q) and a 833G-->A transition in exon 12 (G278D). To assess the biochemical phenotypes of these and two previously identified PEPD mu tations (G448R and delE452), we have designed a transient-expression s ystem for prolidase in COS-1 cells. The enzyme was expressed as a fusi on protein carrying an N-terminal tag, the HA1 epitope of influenza he magglutinin, allowing its immunological discrimination from the endoge nous enzyme with a monoclonal antibody. Expression of the R184Q mutati on produced 7.4% of control enzymatic activity whereas the expression of the G278D, G448R, and delE452 mutations produced inactive enzymes. Western analysis of the R184Q, G278D, and G448R prolidases revealed st able immunoreactive material whereas the delE452 prolidase was not det ectable. Pulse-chase metabolic labeling of cells followed by immunopre cipitation revealed that the delE452 mutant protein was synthesized bu t had an increased rate of degradation.