| Summary: | Prolidase (E.C.3.4.13.9) hydrolyzes imidodipeptides. Prolidase deficiency (PD) (McKusick no. 170100) is an autosomal recessive disorder characterized by a highly variable clinical phenotype. Mutation analysis was performed on a panel of 10 PD cell lines. Single-stranded conformation polymorphism analysis (SSCP) analysis on four overlapping cDNA-PCR products covering the entire coding region of the prolidase gene revealed seven novel mutations: G $ to$ A,551 (R184Q); G $ to$ A,833 (G278D); G $ to$ A, 1342 (G448R); G $ to$ A, 1354 (E452K); delGAG, 1354-1356 (delE452); a deletion of exon 5; and a deletion of exon 7. We used inverse PCR to clone intronic regions flanking exons 5 and 7 and designed primers for conventional PCR of these regions of the genome in patients expressing mRNAs with deleted exons. Two splice acceptor site mutations were identified: a G $ to$ C, nt $-$1 of intron 4 and an A $ to$ G, nt $-$2 of intron 6. To assess the biochemical phenotypes of four of these mutations (R184Q, G278D, G448R, and delE452), we have designed a transient expression system for prolidase in COS-1 cells. The enzyme was expressed as a fusion protein carrying the HA1 epitope of influenza hemagglutinin, allowing its immunological discrimination from the endogenous enzyme. Expression of the R184Q mutation produced 7.4% of control enzymatic activity while the expression of the G278D, G448R and delE452 produced inactive enzymes. Western analysis of the R184Q, G278D and G448R prolidases revealed stable immunoreactive material whereas the delE452 prolidase was not detectable. Pulse-chase experiments revealed that the delE452 mutant protein was synthesized but unstable. === The R184Q allele is carried by an asymptomatic individual, suggesting that its residual activity may be sufficient to prevent the development of symptoms. The other alleles, G278D, G448R, and delE452 which completely abolish enzyme activity associate with the symptomatic form of the disorder. Interestingly, these substitutions are all located at or very close to the putative metal binding residues of prolidase. === We have cloned and sequenced the mouse prolidase cDNA. We have cloned a genomic DNA fragment which carries exons 2, 3, and 4 of the mouse prolidase gene. We constructed a vector for the targeted disruption of the prolidase gene in mouse embryonic stem cells, to create a mouse model for prolidase deficiency.
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