Deletion of PREPL, a gene encoding a putative serine oligopeptidase, in patients with hypotonia-cystinuria syndrome

Abstract

In 11 patients with a recessive congenital disorder, which we refer to as "the hypotonia-cystinuria syndrome," microdeletion of part of the SLC3A1 and PREPL genes on chromosome 2p21 was found. Patients present with generalized hypotonia at birth, nephrolithiasis, growth hormone deficiency, minor facial dysmorphism, and failure to thrive, followed by hyperphagia and rapid weight gain in late childhood. Since loss-of-function mutations in SLC3A1 are known to cause isolated cystinuria type I, and since the expression of the flanking genes, C2orf34 and PPM1B, was normal, the extended phenotype can be attributed to the deletion of PREPL. PREPL is localized in the cytosol and shows homology with prolyl endopeptidase and oligopeptidase B. Substitution of the predicted catalytic residues (Ser470, Asp556, and His601) by alanines resulted in loss of reactivity with a serine hydrolase-specific probe. In sharp contrast to prolyl oligopeptidase and oligopeptidase B, which require both aminoterminal and carboxyterminal sequences for activity, PREPL activity appears to depend only on the carboxyterminal domain. Taken together, these results suggest that PREPL is a novel oligopeptidase, with unique structural and functional characteristics, involved in hypotonia-cystinuria syndrome.

Figure A1

Multiple sequence alignment of the 5′ UTR splice variants. The two alternative ATG-start codons are shaded. The aligment is stopped at the end of exon 2.

Figure 1

Clinical presentation of HCS. A, Patient 5-I during childhood (top) and adulthood (bottom). B, Growth curve of patient 3-I, illustrating the excellent response to growth hormone treatment. Vertical arrow indicates the age at which the treatment was started.

Figure 2

Genetics of HCS. A, Deletions present in the HCS families. All deletions disrupt the coding sequences of SLC3A1 and PREPL, located on chromosome 2p21. Sizes range from 23.8 kb to 75.5 kb. Sequences flanking the breakpoints are shown and are numbered in accordance with BAC clone 24i5. B, Junction fragments spanning the breakpoint. C, Haplotype analysis for deletion A, B, and C alleles. Dashed lines indicate the breakpoint. Conserved alleles are shaded in gray. For deletion B, the conserved subhaplotypes proximal to the breakpoint are shaded in light gray and dark gray. Families 5 (deletion B/deletion B) and 6 (deletion A/deletion B) are excluded because parents were not available. p= Paternal haplotype; m= maternal haplotype.

Figure 3

Expression analysis of PREPL. A, Genomic organization of PREPL. B, In silico analysis of the 5′ UTR revealed two transcriptional start sites. The first transcription start is present in isoforms PREPL₃ and PREPL₅, and the second is present in the remaining isoforms. Isoforms PREPL_1–4 use the start codon in exon 2, generating a 638-aa protein. PREPL_5–7 isoforms contain an additional start site in exon 1B, generating a 727-aa protein. C, Human multiple-tissue northern blot shows a broad tissue distribution of a 5.1-kb transcript, with highest expression in brain, kidney, and skeletal muscle. Tissue specific transcripts are present in brain (∼4.1 kb) and kidney (∼2.8 kb) tissue.

Figure 4

The PREPL homologues. A, Amino acid identity of different PREPL_S orthologues and amino acid similarity of different PREPL_S homologues. The light-gray and dark-gray bars represent the identity (left panel) or similarity (right panel) percentages of the complete sequence and catalytic domain, respectively. An asterisk indicates an incomplete amino acid sequence present in the database. B, Schematic representation of the domain structure of the PREPL isoforms. Identity/similarity percentages of the different domains with PREP and OpdB are shown.

Figure 5

Multiple sequence alignments of the PREPL variants with PREP and OpdB. Predicted secondary structure elements of PREPL are shown above the sequence, whereas the PREP secondary structure elements (based on Fulop et al. 1998) are shown below the sequence. Identical residues between PREPL and one homologue are shaded in light gray, whereas completely conserved residues are shaded in dark gray. Catalytic triad residues are indicated by an asterisk, whereas important residues of the S1 binding pocket are boxed.

Figure 6

Functional analysis of PREPL. A, Immunofluorescence analysis of PREPL_S and PREPL_L in CHO cells. Both isoforms show similar cytoplasmic staining, and similar results were obtained for PREPL_S when the FLAG epitope was linked to the carboxyterminus instead of to the aminoterminus. B, Enzymatic activity of immunopurified, recombinant proteins was determined using FP-biotin and was visualized with streptavidin-HRP. Expression of recombinant proteins was confirmed with biotinylated anti-FLAG M2 antibody. C, Wild-type PREPL_S (lane 2) and mutant His607Ala (lane 6) show reactivity with FP-biotin. In contrast, mutations of the predicted catalytic residues (lanes 3, 4, and 5) ablated activity. D, Full-length PREPL_A–B, OpdB, and PREP are all FP-biotin reactive. Reactivity was abolished in the truncated constructs of OpdB and PREP (lanes 4 and 6) but not in truncated PREPL (lane 2), confirming the fundamental difference in domain structure. ΔPREPL = truncated construct of PREPL (aa 342–638), ΔOpdB = truncated construct of OpdB (aa 405–687), and ΔPREP = truncated construct of PREP (aa 428–710).