Individuals with mutations in XPNPEP3, which encodes a mitochondrial protein, develop a nephronophthisis-like nephropathy

Abstract

The autosomal recessive kidney disease nephronophthisis (NPHP) constitutes the most frequent genetic cause of terminal renal failure in the first 3 decades of life. Ten causative genes (NPHP1-NPHP9 and NPHP11), whose products localize to the primary cilia-centrosome complex, support the unifying concept that cystic kidney diseases are "ciliopathies". Using genome-wide homozygosity mapping, we report here what we believe to be a new locus (NPHP-like 1 [NPHPL1]) for an NPHP-like nephropathy. In 2 families with an NPHP-like phenotype, we detected homozygous frameshift and splice-site mutations, respectively, in the X-prolyl aminopeptidase 3 (XPNPEP3) gene. In contrast to all known NPHP proteins, XPNPEP3 localizes to mitochondria of renal cells. However, in vivo analyses also revealed a likely cilia-related function; suppression of zebrafish xpnpep3 phenocopied the developmental phenotypes of ciliopathy morphants, and this effect was rescued by human XPNPEP3 that was devoid of a mitochondrial localization signal. Consistent with a role for XPNPEP3 in ciliary function, several ciliary cystogenic proteins were found to be XPNPEP3 substrates, for which resistance to N-terminal proline cleavage resulted in attenuated protein function in vivo in zebrafish. Our data highlight an emerging link between mitochondria and ciliary dysfunction, and suggest that further understanding the enzymatic activity and substrates of XPNPEP3 will illuminate novel cystogenic pathways.

Figure 1. Positional cloning of the XPNPEP3 gene, as mutated in NPHPL1.

(A) Parametric multipoint LOD score profile across the human genome of consanguineous kindred A131. Human chromosomes (numbered on top) are concatenated from pter (left) to qter (right) on the x-axis. Genetic distance is given in cM. Note the presence of a significant maximum LOD score of 3.6 on human chromosome 22q13.2 (arrowhead), defining a new gene locus (NPHPL1) for an NPHP-like kidney disease. (B) In kindred A131, the NPHPL1 locus, which is homozygous by descent, is delimited by heterozygous markers SNP_A-1516630 and SNP_A-1649765 to a 4.3-Mb interval, which contains 101 positional candidate genes (per the UCSC sequence; http://genome.ucsc.edu/). Mutations were detected in XPNPEP3 (encircled red). (C) The XPNPEP3 gene extends over 70.8 kb and contains 10 exons (vertical hatches). (D) Exon structure of human full-length XPNPEP3 cDNA (3,056 bp). Positions of start codon (ATG) at nt +1 and of stop codon (TGA) are indicated. Exon sizes, ranging from 63 bp to 997 bp, are approximated. (E) Positions of the mitochondrial localization signal (Mito sign). The SMART program (http://smart.embl-heidelberg.de) predicts a putative N-terminal aminopeptidase P domain (AMP_N; amino acid 67–213), and prolidase domain (amino acid 253–490), which are drawn in relation to the encoding exon positions in D. (F) Two homozygous mutations of XPNPEP3 detected in families A131 and F543 with NPHPL1 (see Table 1).

Figure 2. Renal histology of patient A131 II-4.

Morphologic changes are characteristic for NPHP and include (a) thickening, splitting, and attenuation of tubular basement membranes in tubules with disorganized epithelium (thick arrows); (b) atrophic tubules that contain protein casts (thin arrows); (c) dilated collecting ducts (cd), which are lined by exceptionally tall epithelium; and (d) a mild degree of interstitial fibrosis. Most glomeruli appeared normal, but there were a few scattered obsolescent glomeruli (not shown). Scale bar: 30 μm.

Figure 3. Human XPNPEP3 is targeted to mitochondria by an amino-terminal mitochondrial signal sequence.

(A) Whole kidney homogenates from mice were fractionated into mitochondrial fractions (lanes M1 and M3) and cytosolic fractions (lanes C2 and C4). Immunoblotting with α-XPNPEP3 yielded a single band in the mitochondrial fraction at approximately 51 kDa (lane M1), consistent with the product of cleavage of the mitochondrial signal peptide (after amino acid 53) predicted to occur after mitochondrial import. A doublet was seen in the cytosolic fraction (lane C2) at approximately 57 kDa, which is compatible with the full-length isoform 1 of XPNPEP3. Reblotting with a monoclonal antibody against the mitochondrial protein cytochrome c (Cyt C) demonstrates that cytochrome c is enriched in the mitochondrial fraction (lane M3), yielding a band at approximately 15 kDa (arrowhead), which was absent from the cytosolic fraction (lane C4). (B) Immunofluorescent microscopy was performed on IMCD3 cells that stably express the human full-length XPNPEP3-GFP (in 90% of cells) (left panel), which contains the mitochondrial leader sequence. Upon costaining with the mitochondrial marker, Mitotracker Red, there was colocalization (right panel) in mitochondria. Scale bar: 10 μm. Iso 1, isoform 1. (C) By contrast, in IMCD3 cells that stably express an amino-terminal deletion construct of XPNPEP3-GFP, lacking the 53–amino acid N-terminal mitochondrial signal sequence (ΔN-XPNPEP3-GFP) (left panel), XPNPEP3 expression occurred diffusely in the cytoplasm rather than in mitochondria (right panel). Mitochondria were again labeled with the marker Mitotracker Red. Scale bar: 10 μm. (D) To detect presence of endogenous Xpnpep3 in mitochondria of distal tubular segments in rat kidney, α-XPNPEP3 was labeled with 10-nm gold particles. Endogenous Xpnpep3 labeling was detected in mitochondria of distal tubular segments. Scale bar: 1 μm.

Figure 4. Full-length human XPNPEP3-GFP transiently expressed in IMCD3 cells localizes to mitochondria and not to the cilium/basal body/centrosome complex in IMCD3 cells.

Immunofluorescent microscopy was performed in IMCD3 cells that stably express human full-length XPNPEP3-GFP, which contains the mitochondrial signal sequence, demonstrating expression of XPNPEP3 in mitochondria. XPNPEP3-GFP was not detected in primary cilia (A), basal bodies (B), or centrosomes (C), when counterstaining with acetylated α-tubulin (Ac-α-tublin) (A), γ-tubulin (B), or pericentrin (C), respectively (middle and right panels). Scale bar: 10 μm.

Figure 5. Peptide cleavage by the XPNPEP3 ortholog, ecAPP.

(A) Expression and purification of ecAPP. Coomassie staining of purified protein (lane 1) and immunoblot with anti-His tag antibody (lane 2) are shown. (B) Peptide substrates designed for enzymatic assay. Arrows indicate the cleavage site(s). Proline residue recognized by ecAPP is highlighted in red. Molecular weights are listed in Da. BN, bradykinin; N6, CEP290/NPHP6; AL, ALMS1; LR, LRRC50; DN, dynein. (C) Peptide cleavage detected by electrospray ionization–liquid chromatography–mass spectrometry. Mass spectrometry of peptides before and after digestion is shown. M+nH indicates the addition of n protons to the mass after ionization. intens, intensity.

Figure 6. N-terminal cleavage of LRRC50 is required to rescue lrrc50 morphant phenotypes in zebrafish.

(A) Quantitative representation of the effect of lrrc50 MO on gastrulation development and rescue efficiency of different human lrrc50 RNA isoforms. The developmental phenotype of embryos was scored as Class I–II as described previously (22). Normal, indistinguishable from WT; Class I, mildly affected with a shortened body axis, small anterior structures, mild somite defects; Class II, severely affected with a short body axis, poorly defined head and eyes, broadening and kinking of the notochord, broad, thin somites, and tail extension defects. (B) Representative examples of embryos showing the gastrulation defect caused by lrrc50 MO, which could be rescued by WT and proline-to-alanine human LRRC50 but not other mutants. PA, proline to alanine; PV, proline to valine; PD, proline to aspartic acid; PR, proline to arginine.