Tysnd1 Deficiency in Mice Interferes with the Peroxisomal Localization of PTS2 Enzymes, Causing Lipid Metabolic Abnormalities and Male Infertility

<div><p>Peroxisomes are subcellular organelles involved in lipid metabolic processes, including those of very-long-chain fatty acids and branched-chain fatty acids, among others. Peroxisome matrix proteins are synthesized in the cytoplasm. Targeting signals (PTS or peroxisomal targeting signal) at the C-terminus (PTS1) or N-terminus (PTS2) of peroxisomal matrix proteins mediate their import into the organelle. In the case of PTS2-containing proteins, the PTS2 signal is cleaved from the protein when transported into peroxisomes. The functional mechanism of PTS2 processing, however, is poorly understood. Previously we identified Tysnd1 (Trypsin domain containing 1) and biochemically characterized it as a peroxisomal cysteine endopeptidase that directly processes PTS2-containing prethiolase Acaa1 and PTS1-containing Acox1, Hsd17b4, and ScpX. The latter three enzymes are crucial components of the very-long-chain fatty acids β-oxidation pathway. To clarify the <em>in vivo</em> functions and physiological role of Tysnd1, we analyzed the phenotype of <em>Tysnd1^−/−</em> mice. Male <em>Tysnd1^−/−</em> mice are infertile, and the epididymal sperms lack the acrosomal cap. These phenotypic features are most likely the result of changes in the molecular species composition of choline and ethanolamine plasmalogens. <em>Tysnd1^−/−</em> mice also developed liver dysfunctions when the phytanic acid precursor phytol was orally administered. Phyh and Agps are known PTS2-containing proteins, but were identified as novel Tysnd1 substrates. Loss of <em>Tysnd1</em> interferes with the peroxisomal localization of Acaa1, Phyh, and Agps, which might cause the mild Zellweger syndrome spectrum-resembling phenotypes. Our data established that peroxisomal processing protease Tysnd1 is necessary to mediate the physiological functions of PTS2-containing substrates.</p> </div

Male <i>Tysnd1</i>^−/− mice are infertile.

<p>A. Semi-thin (8–10 micron) testes sections of 20 weeks old <i>Tysnd1</i>^+/+ and <i>Tysnd1^−/−</i> mice were stained with hematoxylin-eosin. Abnormal, round sperm heads are visible in the seminiferous tubules of <i>Tysnd1</i>^−/− mice. B. Abnormal morphology of <i>Tysnd1</i>^−/− sperms. Epididymal sperms of 15 weeks-old <i>Tysnd1</i>^+/+ and <i>Tysnd1</i>^−/− mice were stained with mitochondrial stains (MitoFluor Red) and nuclear staining (DAPI). Scale bar = 20 µm. C. Percentage of sperms showing normal morphology in <i>Tysnd1</i>^+/+ (n = 4), <i>Tysnd1</i>^+/− (n = 4) and <i>Tysnd1</i>^−/− (n = 5) mice. Each error bar represents the mean ± SE. D. Anti-MN9 antibody immunostaining and Hoechst nuclear staining of epididymal sperms isolated from a 20 weeks old <i>Tysnd1</i>^−/− mouse (red: acrosome and blue: nucleus). Arrow heads and arrows indicate abnormal round-headed sperms and normal sperms, respectively. Scale bar = 5 µm. E. Acrosomes of a semi-thin testis section from a 10 weeks old <i>Tysnd1</i>^−/− and a heterozygous control mouse were stained with PNA-FITC (green) and Hoechst nuclear stain (blue). F. EM image of a <i>Tysnd1</i>^−/− caudal epididymal sperm. The round-headed sperm lacks the acrosome and shows an abnormal mitochondrial sheath (M) around the nucleus (N). Scale bar = 1 µm. G. EM image showing normal spermatogenesis in <i>Tysnd1</i>^+/− male mice. S7: step 7 round spermatid; S16: step 16 elongated spermatid. Acrosomes (A) are normally formed. Scale bar = 2 µm. H. EM image of <i>Tysnd1</i>^−/− elongated spermatid. S16: step 16 spermatid. In some spermatids the acrosome (*) is detached from the nucleus (N). Scale bar = 2 µm.</p

Tysnd1 processes Agps and Phyh <i>in vitro</i>.

<p>A. COS7 cells were transiently co-transfected with Agps-V5 and Tysnd1 expression plasmids. With increasing amounts of Tysnd1 unprocessed Agps-V5 decreased (arrow). Processed Agps-V5 is indicated by an arrowhead B. Agps processing by Tysnd1 is specific and was affected by MG132 proteasome inhibitor. C. Western blot of testes extract using anti-Agps antibody shows unprocessed (arrow) and processed (arrowhead) forms of Agps. D. COS7 cells were transiently co-transfected with Phyh-V5 and Tysnd1 expression plasmids. With increasing amounts of Tysnd1, unprocessed Phyh-V5 decreased (arrow) and processed Phyh-V5 increased (arrowhead). E. Western blot of liver extract using anti-Phyh antibody shows unprocessed (arrow) and processed (arrowhead) forms of Phyh.</p

Generation of <i>Tysnd1</i>^−/− mice.

<p>A. Map of <i>Tysnd1</i>^−/− targeting constructs. P1, P2 and P3 indicate primers used for genotyping by PCR. B. Identification of genotyping by PCR. Wild type and <i>Tysnd1</i>^−/− genotype were identified by 237 bp and 339 bp PCR products, respectively. C. Relative expression level of <i>Tysnd1</i> mRNA measured by quantitative real-time PCR in liver. D. Tysnd1 protein was absent in the liver of <i>Tysnd1</i>^−/− mice as shown by Western blotting using anti-Tysnd1 antibody. E. The expression of known Tysnd1 substrates in the liver homogenate was detected by Western blotting using anti-Acox1, -ScpX/Scp2, -Hsd17b4 and -Acaa1 antibodies. Processed forms of Tysnd1 substrates were not detected in <i>Tysnd1</i>^−/− mice. Arrows indicate the processed form of each enzyme. F. Peroxisomal β-oxidation activity was measured by [1-C¹⁴]lignoceric acids in 15 weeks old control diet-fed (CE2, Clea Japan) male mice liver homogenate. ***<i>p</i><0.001. Each error bar represents the mean ± SE in <i>n</i> = 3.</p

Ten-week-old male <i>Tysnd1</i>^−/− mice accumulate phytanic acid.

<p>A. Livers after 13 days of phytol feeding. Left: enlarged, beige-coloured liver of phytol-fed <i>Tysnd1</i>^−/− mouse. Right: liver of phytol-fed <i>Tysnd1</i>^+/+ mouse. B. Hematoxylin-eosin stained semi-thin liver sections. Lipid droplets are indicated by yellow arrow heads. Green arrow heads indicate giant cells. C. Blood serum-derived phytanic acid measurement by GC/MS normalized for C16∶0 (hexadecanoic acid) content. Each error bar represents the mean ± SE in n = 4–9. ***<i>p</i><0.001. D. Measurement of blood serum C24∶0 (tetracosanoic acid), C25∶0 (pentacosanoic acid) and C26∶0 (hexacosanoic acid) VLCFAs by GC/MS normalized by C22∶0 (docosanoic acid) content. Each error bars: mean ± SE in n = 4–9. ***<i>p</i><0.001. E. Measurement of liver peroxisomal β-oxidation activity with or without orally administered 0.5% phytol in carboxyl methyl cellulose (CMC). Each error bars: mean ± SE in <i>n</i> = 3–6. NS: not significant. ***<i>p</i><0.001. F. EM image of liver sections with or without orally administered phytol in CMC. The blue and purple arrow heads indicate peroxisomes and autophagosomes, respectively.Bar: 2 µm. G. Numbers of peroxisomes and autophagosomes counted within same field areas of EM images. Error bars: mean ± SE in n = 5–8. ***<i>p</i><0.001.</p

Proposed model of PTS2-protein import into peroxisomes in presence or absence of Tysnd1.

<p>A. Unprocessed PTS2 protein is imported from the cytoplasm into the peroxisome by Pex7 in association with the long isoform of Pex5 (Pex5pL). Tysnd1 processes the imported protein after the PTS2 signal and Pex7 and Pex5pL are recycled to the cytoplasm. The PTS2-containing fragment is exported from the peroxisome to the cytoplasm. Subsequent Pex5 and Pex7 docking components Pex13 and Pex14 are displayed as gray circles, but not individually labelled for reasons of simplicity. B. Tysnd1-deficient mouse peroxisome. Unprocessed PTS2-containing proteins remain bound as Pex7-Pex5pL cargo complex. Saturation of Pex7 and Pex5pL transport with relative shortage of free Pex5pL and Pex7 causes partial peroxisomal location appearance and accumulation of peroxisomal proteins outside the peroxisome.</p