Characterization of a new PEPD allele causing prolidase deficiency in two unrelated patients: natural-occurrent mutations as a tool to investigate structure-function relationship.
Prolidase deficiency (PD) is a rare autosomal recessive disorder characterized mainly by skin lesions of the legs and feet, mental retardation, and respiratory infections. Mutations at the PEPD locus, located on chromosome 19, are responsible for this disease. We identified a new PEPD allele in two unrelated Portuguese PD patients by analyses of reverse transcribed PCR-amplified cDNA. We used SSCP analysis of seven overlapping fragments spanning the entire coding region of the gene and detected abnormal SSCP bands in two of them: PD3 (nt 425-743) and PD4 (nt 661-973). Direct sequencing of the mutant cDNA and genomic DNA revealed a new homozygous 3-bp deletion (Y231del) in both cases. Transient expression in PD fibroblasts of wild-type and mutant prolidase cDNA confirmed reduced activity of the construct carrying the 3-bp deletion. The mutation results in a loss of prolidase activity in skin fibroblasts. Intracellular accumulation of Gly-Pro dipeptide in long-term cultured fibroblasts was detected by capillary electrophoresis. The mutation falls in the alpha2 domain of the "pita bread" structure proposed for E. coli and human prolidase by Bazan et al. on the bases of their sequence homology with E. coli methionine aminopeptidase. Taking into account the effects of the described mutations on stability and activity of the enzyme, we propose the identification of three different functional regions.
Department of Biochemistry, Section of Medicine and Pharmacology, University of Pavia, Pavia, Italy.
Anna
A
Lupi
Department of Biochemistry, Section of Medicine and Pharmacology, University of Pavia, Pavia, Italy.|Georgia Tech-IBB, Atlanta, GA, USA.
Antonio
A
De Riso
Department of Biochemistry, Section of Medicine and Pharmacology, University of Pavia, Pavia, Italy.
Sara Della
SD
Torre
Department of Biochemistry, Section of Medicine and Pharmacology, University of Pavia, Pavia, Italy.
Antonio
A
Rossi
Department of Biochemistry, Section of Medicine and Pharmacology, University of Pavia, Pavia, Italy.
Elena
E
Campari
Department of Clinical Biology, Medical Genetics Institute, Praca Pedro Nunes, Porto, Portugal.
Laura
L
Vilarinho
Department of Biochemistry, Section of Medicine and Pharmacology, University of Pavia, Pavia, Italy.
Giuseppe
G
Cetta
Department of Biochemistry, Section of Medicine and Pharmacology, University of Pavia, Pavia, Italy. aforlino@unipv.it.|Department of Biochemistry, University of Pavia, Via Taramelli 3/B, 27100, Pavia, Italy. aforlino@unipv.it.
Antonella
A
Forlino
D000483:Alleles; D001483:Base Sequence; D015152:Blotting, Northern; D002888:Chromosomes, Human, Pair 19 / Q000235:genetics*; D017931:DNA Primers; D018076:DNA, Complementary / Q000235:genetics; D004150:Dipeptidases / Q000172:deficiency* / Q000235:genetics*; D019075:Electrophoresis, Capillary; D005347:Fibroblasts / Q000378:metabolism; D005808:Genes, Recessive / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics*; D010455:Peptides / Q000378:metabolism; D010957:Plasmids / Q000235:genetics; D018807:Polymorphism, Single-Stranded Conformational; D011174:Portugal; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D013329:Structure-Activity Relationship
D017931:DNA Primers; D018076:DNA, Complementary; D010455:Peptides; D004150:Dipeptidases; C022449:proline dipeptidase