Mitochondrial Capsule Selenoprotein Research Articles

PHGPx is the mitochondrial capsule selenoprotein of mammalian sperm

PHGPx is the mitochondrial capsule selenoprotein of mammalian sperm

Impairment of NF-kappaB activation and modulation of gene expression by calpastatin.

To address the involvement of the calpain system in both basal and silica-induced nuclear factor (NF)-kappaB activation, several human bronchial epithelial cell lines were established in which an intracellular inhibitor of calpain, calpastatin, was stably expressed. Reduced basal and silica-induced inhibitor (IkappaBalpha) degradation and NF-kappaB activation were observed in cells stably overexpressing calpastatin. In addition, the cells in which calpain was constitutively inhibited by the overexpression of calpastatin exhibited a notable morphological change. Whereas empty vector-transfected cells displayed a morphology indistinguishable from that of parental cells, cells overexpressing calpastatin exhibited a mosaic morphological change with reduced formation of lamella 30 min after the cells were seeded. Genefilter microarray experiments, in which 3,965 human genes can be evaluated for their expression at the same time, showed that calpastatin downregulated genes encoding several membrane-associated proteins or nuclear proteins and upregulated genes of collagen alpha2, DAZ, and mitochondrial capsule selenoprotein. These results suggest that, in addition to their proteolytic activities on cytoskeletal proteins and other cellular regulatory proteins, calpain-calpastatin systems can also affect the expression levels of genes encoding structural or regulatory proteins.

Cloning and expression of mitochondrial capsule selenoprotein gene in the golden hamster.

Mitochondrial capsule selenoprotein (MCS) has been known as a structural protein of the mitochondrial sheath in spermatoza. In the present study, a full-length cDNA encoding the MCS was first isolated from the testes of 10-week-old golden hamsters using a RACE (rapid amplification of cDNA ends) technique and its mRNA expression pattern was investigated from the hamster tissues by reverse transcription-polymerase chain reaction (RT-PCR) analysis. Hamster MCS cDNA was 820 bp long, including 24 bp of the 5'-untranslated region (UTR) and 243 bp of the 3'-UTR, and showed identity of 75.6% and 73.9% with mouse and rat MCS. According to the deduced amino acid (aa) sequence analysis, hamster MCS encoded a polypeptide of 184 aa, including a cysteine- and proline-rich domain which is the characteristic sequences of MCS, and contained 2 in-frame UGA codons for selenocysteine. Hamster MCS also shared aa identity of 64.4% with mouse MCS and contained an Arg-Lys-Ser-Thr-rich region in the N-terminus similar to the mitochondrial targeting signal. On the other hand, according to the RT-PCR analysis using the specific primers for hamster MCS, hamster MCS mRNA was expressed in various tissues as well as the testes. This finding indicates that MCS in hamster may have more than just a function of mitochondrial sheath formation of spermatozoa.

Expression pattern of mitochondrial capsule selenoprotein mRNA in the hamster testis.

Mitochondrial capsule selenoprotein (MCS) has been known as a structural protein of the mitochondrial sheath in spermatozoa. In the present study, to determine the expression pattern of MCS mRNA in the hamster testis, northern blot and in situ hybridization analyses using digoxigenin-labeled riboprobes for the hamster MCS were performed in the testes of 10-week-old golden hamsters. According to the northern blot analysis, hamster MCS was detected as a single transcript of about 1 kb in the testis. During spermatogenesis, the hamster MCS mRNA first appeared in step 6 spermatids, gradually increased in round spermatids during spermiogenesis, reached a peak in step 8 spermatids, and persisted a high level until step 13 spermatids. After step 14, the signal began to show a progressive decline in the spermatids and was weakly detected in the tails of step 17 spermatids. However, the signal was not observed in spermatogonia, spermatocytes, Sertoli cells, peritubular myoid cells, and interstitial cells. These findings indicate that hamster MCS is mainly related to the spermiogenesis during spermatogenesis.

Expression of Mitochondrial Capsule Selenoprotein mRNA Increases with Aging, but Decreases by Selenium Deficiency in the Mouse Testis.

Mitochondrial capsule selenoprotein (MCS) has been known as a structural protein of the mitochondrial sheath in spermatozoa. In this study, to investigate whether or not the expression of MCS mRNA in testes is affected by aging or selenium (Se) deficiency, the cDNA clone of MCS was isolated from the testes of 8-week-old ICR mice by reverse transcription-polymerase chain reaction. The nucleic acid sequence analysis confirmed that the cloned cDNA is an open reading frame of the MCS and contains 3 in-phase TGA codons that recognize specifically the selenocysteine in selenoprotein. According to Northern blot analysis, the MCS mRNA in testes greatly increased at 8 weeks old and then began to increase with aging. As compared with that of 8-week-old mice, the signal increased about 2.4-fold in the testes of 80-week-old mice. On the other hand, we generated Se-deficient and Se-sufficient mice by feeding weanling male mice (3 weeks old) with either a Se-deficient or a Se-sufficient diet for 11 weeks. MCS mRNA decreased about 21-fold in the testes of Se-deficient animals as compared with that of Se-sufficient animals. The present study provides a novel evidence that MCS mRNA level in testes increases with aging, but remarkably decreases due to Se deficiency.

Expression Pattern of the Mitochondrial Capsule Selenoprotein mRNA in the Mouse Testis after Puberty; in situ Hybridization Study.

Mitochondrial capsule selenoprotein (MCS) has been known as a structural protein of the mitochondrial sheath in spermatozoa. In this study, to determine the expression pattern of MCS mRNA in the mouse testis after puberty, in situ hybridization using digoxigenin-labeled RNA probes for MCS was performed in the testes of 8- and 20-week-old ICR mice. In the testes of both ages, MCS mRNA first appeared in step 3 round spermatids, gradually increased during early spermiogenesis, and persisted a high level until step 14 spermatids. After the step 14 spermatids, the signal began to decline and was weakly detected in steps 15-16 spermatids. On the other hand, compared with that in the testes of 8-week-old mice, MCS mRNA level in the testes of 20-week-old mice increased over 2-fold at stages VI-III, while it slightly increased at stages IV-V. These findings suggest that MCS gene transcription may be up-regulated after puberty in the mouse testis.

Isolation, Expression, and Chromosomal Localization of the Human Mitochondrial Capsule Selenoprotein Gene (MCSP)

The mitochondrial capsule selenoprotein (MCS) (HGMW-approved symbol MCSP) is one of three proteins that are important for the maintenance and stabilization of the crescent structure of the sperm mitochondria. We describe here the isolation of a cDNA, the exon–intron organization, the expression, and the chromosomal localization of the human MCS gene. Nucleotide sequence analysis of the human and mouse MCS cDNAs reveals that the 5′- and 3′-untranslated sequences are more conserved (71%) than the coding sequences (59%). The open reading frame encodes a 116-amino-acid protein and lacks the UGA codons, which have been reported to encode the selenocysteines in the N-terminal of the deduced mouse protein. The deduced human protein shows a low degree of amino acid sequence identity to the mouse protein (39%). The most striking homology lies in the dicysteine motifs. Northern and Southern zooblot analyses reveal that the MCS gene in human, baboon, and bovine is more conserved than its counterparts in mouse and rat. The single intron in the human MCS gene is approximately 6 kb and interrupts the 5′-untranslated region at a position equivalent to that in the mouse and rat genes. Northern blot andin situhybridization experiments demonstrate that the expression of the human MCS gene is restricted to haploid spermatids. The human gene was assigned to q21 of chromosome 1.

Cloning, Expression, and Chromosomal Localization of the Rat Mitochondrial Capsule Selenoprotein Gene (MCS): The Reading Frame Does Not Contain Potential UGA Selenocysteine Codons

The mitochondrial capsule selenoprotein (MCS) is a selenium-containing polypeptide. It is one of three proteins that are important for the maintenance and stabilization of the crescent structure of the sperm mitochondria. In this paper, we report the isolation and characterization of the rat MCS cDNA and gene. The cDNA contains a reading frame for a 145-amino-acid protein and it lacks the UGA codons, which have been found in the reading frame of the mouse MCS cDNA and have been presumed to encode the selenocysteine in the amino terminal of the deduced mouse amino acid sequence. The deduced amino acid sequence of the rat and mouse MCS shows a high level of homology (79%). The rat MCS gene contains two exons; the intron sequence interrupts the 5' untranslated sequence at the same position as in the mouse MCS gene. The transcription start site is located 184 bp upstream of the translation start site. Alignment of the 5'-flanking regions of the mouse and rat genes reveals that the first 400 nucleotides upstream of the transcription start site exhibit an overall sequence similarity of 73%. This conserved region contains no TATA or CAAT box motifs. Northern blot analysis indicates that the MCS mRNA is detectable only in the testis after day 30 of postnatal development. Moreover, in situ hybridization revealed that the rat MCS gene is mainly expressed in round spermatids. From the analysis of mouse-rat cell hybrids that segregate rat chromosomes, the MCS gene was assigned to rat chromosome 2.

Multiple Controls over the Efficiency of Translation of the mRNAs Encoding Transition Proteins, Protamines, and the Mitochondrial Capsule Selenoprotein in Late Spermatids in Mice

The mRNAs encoding protamines 1 and 2, transition proteins 1 and 2, and the mitochondrial capsule selenoprotein are translationally repressed with long poly(A) tracts in early spermatids and translationally active with heterogenous shortened poly(A) tracts in late spermatids (Kleene, 1989). In the present study, the spacing of ribosomes on the translationally active forms of each mRNA was calculated from the length of the coding region and the polysome size determined by sucrose gradient and Northern blot analysis. In addition, the rate of initiation of these five mRNAs was compared in the reticulocyte cell-free translation lysate. Our results reveal at least four additional forms of translational control over these mRNAs: (1) The vast majority of the active forms of the transition protein 1 mRNA and both protamine mRNAs sediment with polysomes in which the ribosomes are spaced closer than is typical of mammalian mRNAs (31-38 vs 80-100 bases apart). This implies that the rate of initiation is unusually fast and/or that the rate of elongation is unusually slow. (2) The mitochondrial capsule selenoprotein mRNA also initiates efficiently in vivo and in vitro, but sediments with polysomes in which the ribosomes are spaced wider than on the protamine mRNAs. The small size of these polysomes can be explained by inefficient insertion of selenocysteine residues at UGA codons. (3) The transition protein 2 mRNA is translated on small polysomes and a relatively large fraction sediments as free mRNPs in vivo. In addition, the transition protein 2 mRNA initiates translation inefficiently at high mRNA concentration and efficiently at low mRNA concentration in vitro. These observations suggest that the transition protein 2 mRNA may be translated inefficiently because it is a weak competitor for a limiting initiation factor. (4) Since low levels of cycloheximide fail to increase the polysome loading of transition protein 2 mRNA in culture, active single ribosomes may also be limited in late spermatids.

Sequence of the gene encoding the mitochondrial capsule selenoprotein of mouse sperm: identification of three in-phase TGA selenocysteine codons.

The mitochondrial selenoprotein is a major structural protein of the keratinous mitochondrial capsule in mammalian sperm, a structure that functions in shaping mitochondria into the helical sheath surrounding the flagellum. A cDNA clone (Kleene et al., 1990) was isolated previously encoding a protein whose predicted size and amino acid content of > 20% cysteine and proline closely resembled a selenoprotein in the bull mitochondrial capsule. The sequences of additional cDNAs and genomic DNA reported here reveal that the mouse mitochondrial capsule selenoprotein reading frame begins 54 codons further upstream than previously reported. Significantly, these 54 codons contain three in-phase UGA codons, which normally signify stop but encode selenocysteine in bacterial and mammalian selenoproteins. The coding region of the mitochondrial capsule selenoprotein gene is interrupted by a single intron. S1 mapping and primer extension demonstrate that the vast majority of MCS mRNAs are spliced using consensus 5' and 3' slice junctions in mammalian cells. However, two cDNAs have been identified that apparently represent rare mRNA variants produced by use of cryptic splice sites.

A study by in situ hybridization of the stage of appearance and disappearance of the transition protein 2 and the mitochondrial capsule seleno-protein mRNAs during spermatogenesis in the mouse.

Transition protein 2 is a basic chromosomal protein which functions as an intermediate in the replacement of histones by protamines, and the mitochondrial capsule seleno-protein is a constituent of the outer membrane of mitochondria which functions in constructing the mitochondrial sheath surrounding the flagellum. To determine precisely the stages in spermatogenesis when these mRNAs are present, paraffin sections of sexually mature testes were hybridized to 35S- and 3H-labeled antisense RNAs and exposed to autoradiographic emulsion. The cell types hybridizing to probes in situ were determined by staining with hematoxylin and periodic acid Schiff. The in situ hybridizations reveal that the transition protein 2 mRNA is first detectable in step 7 round spermatids, persists at high levels through step 13, and is degraded before step 14. By contrast, the mitochondrial capsule seleno-protein mRNA is first detected in step 3 round spermatids and persists at high levels until step 16, the end of spermiogenesis. The mitochondrial capsule seleno-protein mRNA appears to be expressed only in haploid cells since low levels could not be detected in Northern blots of RNA from pachytene primary spermatocytes from 18 day prepubertal mice. These results demonstrate that the transition protein 2 and mitochondrial capsule seleno-protein mRNAs are transcribed and degraded at different times during the haploid phase of spermatogenesis.

Sequence and developmental expression of the mRNA encoding the seleno-protein of the sperm mitochondrial capsule in the mouse

We have characterized cDNA clones encoding the selenium-containing polypeptide of the keratinous mitochondrial capsule in mouse sperm. The longest open reading frame encodes a polypeptide 143 amino acids long which contains 21% cysteine and 27% proline and closely resembles the size and amino acid composition of bull mitochondrial capsule seleno-protein (V. Pallini, B. Baccetti, and A. G. Burrini, 1979, in “The Spermatozoon,” D. W. Fawcett and J. M. Bedford, Eds., pp. 141–151, Urban & Schwartzenberg, Baltimore/Munich). The reading frame encoding the mitochondrial capsule seleno-protein ends with an amber stop codon suggesting that selenium is not incorporated cotranslationally into the protein by an opal supressor selenocysteyl-tRNA as has been found for several eukaryotic and bacterial proteins. Northern blots using RNA extracted from purified spermatogenic cells and staged prepuberal mice suggest that the mitochondrial capsule seleno-protein mRNA is first transcribed in late meiotic cells and that the levels of the mRNA increase after meiosis in early haploid cells. Southern blots demonstrate that there is one copy of the gene in the mouse genome. The identification of this cDNA clone, in combination with previous work (K. C. Kleene, 1989, Development 106, 367–373) demonstrates that the mRNA for the mitochondrial capsule seleno-protein is translationally repressed with long homogenous poly(A) tracts in round spermatids and translationally active with shortened heterogenous poly(A) tracts in elongating spermatids.