Publications (14) View all
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Article: Oocyte specific oolemmal SAS1B involved in sperm binding through intra-acrosomal SLLP1 during fertilization.
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ABSTRACT: Molecular mechanisms by which fertilization competent acrosome-reacted sperm bind to the oolemma remain uncharacterized. To identify oolemmal binding partner(s) for sperm acrosomal ligands, affinity panning was performed with mouse oocyte lysates using sperm acrosomal protein, SLLP1 as a target. An oocyte specific membrane metalloproteinase, SAS1B (Sperm Acrosomal SLLP1 Binding), was identified as a SLLP1 binding partner. cDNA cloning revealed six SAS1B splice variants, each containing a zinc binding active site and a putative transmembrane domain, with signal peptides in three variants. SAS1B transcripts were ovary specific. SAS1B protein was first detected in early secondary follicles in day 3 ovaries. Immunofluorescence localized SAS1B to the microvillar oolemma of M2 oocytes. After fertilization, SAS1B decreased on the oolemma and became virtually undetectable in blastocysts. In transfected CHO-K1 cells SAS1B localized to the surface of unpermeabilized cells. Recombinant and native SLLP1 co-localized with SAS1B to the microvillar domain of ovulated M2 oocytes. Molecular interactions between mouse SLLP1 and SAS1B were demonstrated by surface plasmon resonance, far-western, yeast two-hybrid, recombinant- and native-co-IP analyses. SAS1B bound to SLLP1 with high affinity. SAS1B had protease activity, and SAS1B protein or antibody significantly inhibited fertilization. SAS1B knockout female mice showed a 34% reduction in fertility. The study identified SAS1B-SLLP1 as a pair of novel sperm-egg binding partners involving the oolemma and intra-acrosomal compartment during fertilization.Developmental Biology 12/2011; 363(1):40-51. · 4.07 Impact Factor -
Article: CABYR binds to AKAP3 and Ropporin in the human sperm fibrous sheath.
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ABSTRACT: Calcium-binding tyrosine phosphorylation-regulated protein (CABYR) is a highly polymorphic calcium-binding tyrosine- and serine-/threonine-phosphorylated fibrous sheath (FS) protein involved in capacitation. A putative domain (amino acids 12-48) homologous to the regulatory subunit of type II cAMP-dependent protein kinase A (RII) dimerisation and A kinase-anchoring protein (AKAP)-binding domains of protein kinase A at the N-terminus suggests that CABYR may self-assemble and bind to AKAPs. Moreover, there is evidence that CABYR has limited interaction with AKAPs. However, further evidence and new relationships between CABYR and other FS proteins, including AKAPs, will be helpful in understanding the basic physiology of FS. In this study, a new strategy for co-immunoprecipitation of insoluble proteins, as well as the standard co-immunoprecipitation method in combination with mass spectrometry and western blot, was employed to explore the relationship between CABYR, AKAP3 and Ropporin. The results showed that AKAP3 was co-immunoprecipitated with CABYR by the anti-CABYR-A polyclonal antibody, and, conversely, CABYR was also co-immunoprecipitated with AKAP3 by the anti-AKAP3 polyclonal antibody. Another RII-like domain containing protein, Ropporin, was also co-immunoprecipitated with CABYR, indicating that Ropporin is one of CABYR's binding partners. The interactions between CABYR, AKAP3 and Ropporin were confirmed by yeast two-hybrid assays. Further analysis showed that CABYR not only binds to AKAP3 by its RII domain but binds to Ropporin through other regions besides the RII-like domain. This is the first demonstration that CABYR variants form a complex not only with the scaffolding protein AKAP3 but also with another RII-like domain-containing protein in the human sperm FS.Asian Journal of Andrology 01/2011; 13(2):266-74. · 1.52 Impact Factor -
Article: CABYR isoforms expressed in late steps of spermiogenesis bind with AKAPs and ropporin in mouse sperm fibrous sheath.
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ABSTRACT: CABYR is a polymorphic calcium-binding protein of the sperm fibrous sheath (FS) which gene contains two coding regions (CR-A and CR-B) and is tyrosine as well as serine/threonine phosphorylated during in vitro sperm capacitation. Thus far, the detailed information on CABYR protein expression in mouse spermatogenesis is lacking. Moreover, because of the complexity of this polymorphic protein, there are no data on how CABYR isoforms associate and assemble into the FS. The capacity of mouse CABYR isoforms to associate into dimers and oligomers, and the relationships between CABYR and other FS proteins were studied by gel electrophoresis, Western blotting, immunofluorescence, immunoprecipitation and yeast two-hybrid analyses. The predominant form of mouse CABYR in the FS is an 80 kDa variant that contains only CABYR-A encoded by coding region A. CABYR isoforms form dimers by combining the 80 kDa CABYR-A-only variant with the 50 kDa variant that contains both CABYR-A and CABYR-B encoded by full length or truncated coding region A and B. It is proposed that this step is followed by the formation of larger oligomers, which then participate in the formation of the supramolecular structure of the FS in mouse sperm. The initial expression of CABYR occurs in the cytoplasm of spermatids at step 11 of spermiogenesis and increases progressively during steps 12-15. CABYR protein gradually migrates into the sperm flagellum and localizes to the FS of the principal piece during steps 15-16. Deletion of the CABYR RII domain abolished the interaction between CABYR and AKAP3/AKAP4 but did not abolish the interaction between CABYR and ropporin suggesting that CABYR binds to AKAP3/AKAP4 by its RII domain but binds to ropporin through another as yet undefined region. CABYR expresses at the late stage of spermiogenesis and its isoforms oligomerize and bind with AKAPs and ropporin. These interactions strongly suggest that CABYR participates in the assembly of complexes in the FS, which may be related to calcium signaling.Reproductive Biology and Endocrinology 01/2010; 8:101. · 2.05 Impact Factor -
Article: Immunogenicity of a multi-component recombinant human acrosomal protein vaccine in female Macaca fascicularis.
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ABSTRACT: A vaccine formula comprised of five recombinant human intra-acrosomal sperm proteins was inoculated into female monkeys to test whether specific antibodies to each component immunogen could be elicited in sera and whether antibodies elicited by the vaccine affected in vitro fertilization. Acrosomal proteins, ESP, SLLP-1, SAMP 32, SP-10 and SAMP 14, were expressed with his-tags, purified by nickel affinity chromatography and adsorbed to aluminum hydroxide. Five female cynomolgus monkeys were inoculated intramuscularly three times at monthly intervals. All five monkeys developed both IgG and IgA serum responses to each recombinant immunogen on Western blots. Each serum stained the acrosome of human sperm and bound to the cognate native protein on Western blots of human sperm extracts. By ELISA, all monkeys developed IgG to each immunogen, with the highest average absorbance values to ESP, SAMP 32 and SP-10, followed by lower values for SLLP-1 and SAMP 14. IgA was also generated to each component immunogen with the highest average absorbance values to SLLP-1 and SP-10. For antigens that induced an IgA response, the duration of the IgA response was longer than the IgG response to the same antigens. This study supports the concept that a multivalent contraceptive vaccine may be administered to female primates evoking both peripheral (IgG) and mucosal (IgA) responses to each component immunogen following an intramuscular route of inoculation with a mild adjuvant, aluminum hydroxide, approved for human use.Journal of Reproductive Immunology 05/2008; 77(2):126-41. · 2.97 Impact Factor -
Article: FSCB, a novel protein kinase A-phosphorylated calcium-binding protein, is a CABYR-binding partner involved in late steps of fibrous sheath biogenesis.
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ABSTRACT: We report characterization of a novel testis- and sperm-specific protein, FSCB (fibrous sheath CABYR binding), that is expressed post-meiotically and localized in mouse sperm flagella. FSCB was identified as a binding partner of CABYR, a calcium-binding protein that is tyrosine-phosphorylated during capacitation. Orthologous genes of FSCB are present in other mammals, including rat and human, and conserved motifs in FSCB include PXXP, proline-rich and extensin-like regions. FSCB is phosphorylated by protein kinase A as shown by in vitro phosphorylation assay and also by determining phosphorylation sites in native FSCB from mouse sperm. Calcium overlay assay showed that FSCB is a calcium-binding protein from sperm. FSCB is a post meiotic protein first expressed at step 11 of mouse spermatogenesis in the elongating spermatids, and it subsequently incorporates into the flagellar principal piece of the sperm. Ultrastructurally, FSCB localized to a cortical layer of intermediate electron density at the surface of the ribs and longitudinal columns of the fibrous sheath. Due to its temporal appearance during spermiogenesis and location at the cortex of the fibrous sheath, FSCB is postulated to be involved in the later stages of fibrous sheath assembly.Journal of Biological Chemistry 12/2007; 282(47):34104-19. · 4.77 Impact Factor
