Mutation analysis of five new patients affected by prolidase deficiency: the lack of enzyme activity causes necrosis-like cell death in cultured fibroblasts

Abstract

Prolidase, a ubiquitously distributed dipeptidase, is involved in the latter stage of degradation of endogenous and dietary proteins and is particularly important in collagen catabolism. It hydrolyzes dipeptides containing proline or hydroxyproline at the C-terminal position. Mutations in the gene encoding for prolidase cause prolidase deficiency (PD), an autosomal recessive disorder mainly characterized by skin lesions, mental retardation and recurrent infectious. In this work we reported the identification of the molecular defect in five PD patients. Direct sequencing of PCR amplified genomic DNA showed a homozygous G>A transversion in two siblings leading to a G448R substitution. A heterozygous IVS11+1G>C transition causing the skipping of exon 11 and a null allele were detected in a third proband. In two unrelated patients, a homozygous IVS7-1G>A transversion was identified and shown to cause multiple alternative spliced transcripts. All the mutations result in loss of prolidase activity. Long-term cultured fibroblasts from these PD patients were used to develop an in vitro model that allowed investigation of the affected cells. Light and electron microscopy revealed that PD cells were more round and branched out than controls with increased cytosolic vacuolization, interruptions of the plasma membrane, mitochondria swelling, mitochondrial matrix and cristae modifications. JC-1 labeling showed decreased mitochondrial membrane potential. A significant intracellular accumulation of the Gly-Pro dipeptide was detected by capillary electrophoresis analysis. Our results provide the first evidence that absence of prolidase activity causes the activation of a necrosis-like cellular death, which could be responsible for the typical skin lesions in PD.

Prolidase deficiency associated with systemic lupus erythematosus (SLE): single site experience and literature review

“…Cultured fibroblasts from patients with PD show changes that might be associated with a necrosis-like cellular death [13]. This change may be related to the intracellular accumulation of the Gly-Pro dipeptides which could be responsible for the typical skin lesions in PD, as well as for the association with SLE caused by exposure to intracellular protein.…”

Characterization of a new PEPD allele causing prolidase deficiency in two unrelated patients: natural-occurrent mutations as a tool to investigate structure–function relationship

“…4). In details, the in-frame deletions of exon 5 and exon 7 (Ohhashi et al 1990), located out of the ''pita-bread'' structure, respectively, cause the loss of 15 and 16 amino acids and the synthesis of either an unstable transcript or an unstable protein; the deletion of exons 8-9, falling in the a1 and a2 region of the ''pita-bread'' structure, compromised the stability of the transcript (Forlino et al 2002). Two different mutations at position 184 had been described: R184Z and R184X (Kikuchi et al 2000;Ledoux et al 1996).…”

“…The skipping of exon 11 causes the deletion of the b A and b B domains according to Bazan et al model and results in unstable transcript and inactive enzymes (Forlino et al 2002). The deletion of the sequence of a full exon in region two of the prolidase compromises enzyme stability.…”

“…cDNA synthesis was carried out with first strand cDNA synthesis kit for RT-PCR (Roche, Monza, Milan, Italy) according to the manufacturer's specifications. The entire prolidase transcript was amplified as seven overlapping fragments approximately 300-400 bp long using Taq polymerase (Clontech, San Jose, CA, USA) and the primer pairs and PCR conditions described by Forlino et al (2002). Both for patients and controls, all amplified fragments were first run on 1.8% agarose gel and then analyzed by single-strand conformation polymorphisms (SSCP).…”

Structural Analysis and Bioengineering of Thermostable Pyrococcus furiosus Prolidase for the Optimization of Organophosphorus Nerve Agent Detoxification

“…However, in humans, prolidase is involved in the final stage of the degradation of endogenous and dietary protein and is particularly critical for collagen catabolism (Endo, Hata et al 1987). Mutations in the gene coding for human prolidase can cause prolidase deficiency, an autosomal recessive disorder characterized by skin lesions, mental retardation, and recurrent infections (Scriver et al 1983;Endo et al 1989;Endo and Matsuda 1991;Forlino et al 2002;Lopes et al 2002;Perugini et al 2005). …”