Abstract
The mitochondrial capsule is a selenium- and disulfide-rich structure enchasing the outer mitochondrial membrane of mammalian spermatozoa. Among the proteins solubilized from the sperm mitochondrial capsule, we confirmed, by using a proteomic approach, the presence of phospholipid hydroperoxide glutathione peroxidase (PHGPx) as a major component, and we also identified the sperm mitochondrion-associated cysteine-rich protein (SMCP) and fragments/aggregates of specific keratins that previously escaped detection (Ursini, F., Heim, S., Kiess, M., Maiorino, M., Roveri, A., Wissing, J., and Flohé, L. (1999) Science 285, 1393-1396). The evidence for a functional association between PHGPx, SMCP, and keratins is further supported by the identification of a sequence motif of regularly spaced Cys-Cys doublets common to SMCP and high sulfur keratin-associated proteins, involved in bundling hair shaft keratin by disulfide cross-linking. Following the oxidative polymerization of mitochondrial capsule proteins, catalyzed by PHGPx, two-dimensional redox electrophoresis analysis showed homo- and heteropolymers of SMCP and PHGPx, together with other minor components. Adjacent cysteine residues in SMCP peptides are oxidized to cystine by PHGPx. This unusual disulfide is known to drive, by reshuffling oxidative protein folding. On this basis we propose that oxidative polymerization of the mitochondrial capsule is primed by the formation of cystine on SMCP, followed by reshuffling. Occurrence of reshuffling is further supported by the calculated thermodynamic gain of the process. This study suggests a new mechanism where selenium catalysis drives the cross-linking of structural elements of the cytoskeleton via the oxidation of a keratin-associated protein.
Highlights
More than 3 decades ago, evidence that radioactive selenium is concentrated in rat sperm mid-piece [1] and that severe selenium deficiency leads to male infertility [2] brought into focus the crucial role of this oligo-element in spermatogenesis
Among the proteins solubilized from the sperm mitochondrial capsule, we confirmed, by using a proteomic approach, the presence of phospholipid hydroperoxide glutathione peroxidase (PHGPx) as a major component, and we identified the sperm mitochondrion-associated cysteine-rich protein (SMCP) and fragments/aggregates of specific keratins that previously escaped detection (Ursini, F., Heim, S., Kiess, M., Maiorino, M., Roveri, A., Wissing, J., and Flohe, L. (1999) Science 285, 1393–1396)
The identification of bands containing intact PHGPx and sperm mitochondrion-associated cysteinerich protein” (SMCP) was further corroborated by Western blotting and migration of recombinant SMCP.4
Summary
Preparation of Rat Spermatozoa MC—Rat spermatozoa, obtained by squeezing the cauda epididymis and the vas deferens into phosphatebuffered saline, were centrifuged at 1,500 ϫ g for 10 min and washed twice in phosphate-buffered saline. Two-dimensional Redox Electrophoresis—The protein pellet obtained from RSCP was solubilized in 0.01 M ammonium bicarbonate, 1 mM EDTA, 1% SDS, pH 8.1, and diluted 1:1 with Laemmli sample buffer, where 2 mM N-ethylmaleimide was substituted for 2-mercaptoethanol [24]. (www.genscript.com; Piscataway, NJ), HPLC-purified, and used as substrate for PHGPx. PHGPx Activity on SMCP-derived Peptides—Synthetic peptides were purchased from GenScript Corp. (www.genscript.com; Piscataway, NJ), HPLC-purified, and used as substrate for PHGPx They were designed based on the rat/mouse SMCP sequence and contained one adjacent cysteine motif and no other cysteine residue. In the second peptide, -PPPPCCPPPP-, the second most frequent amino acid of rat/mouse/human SMCP replaced corresponding positions for each Lys residue. Peptides were purchased and tested as donor substrates for PHGPx, as described above
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