Recombinant production of enzymatically active male contraceptive drug target hTSSK2 - Localization of the TSKS domain phosphorylated by TSSK2.

The testis-specific serine/threonine kinase 2 (TSSK2) has been proposed as a candidate male contraceptive target. Development of a selective inhibitor for this kinase first necessitates the production of highly purified, soluble human TSSK2 and its substrate, TSKS, with high yields and retention of biological activity for crystallography and compound screening. Strategies to produce full-length, soluble, biologically active hTSSK2 in baculovirus expression systems were tested and refined. Soluble preparations of TSSK2 were purified by immobilized-metal affinity chromatography (IMAC) followed by gel filtration chromatography. The biological activities of rec.hTSSK2 were verified by in vitro kinase and mobility shift assays using bacterially produced hTSKS (isoform 2), casein, glycogen synthase peptide (GS peptide) and various TSKS peptides as target substrates. Purified recombinant hTSSK2 showed robust kinase activity in the in vitro kinase assay by phosphorylating hTSKS isoform 2 and casein. The ATP Km values were similar for highly and partially purified fractions of hTSSK2 (2.2 and 2.7 µM, respectively). The broad spectrum kinase inhibitor staurosporine was a potent inhibitor of rec.hTSSK2 (IC50 = 20 nM). In vitro phosphorylation experiments carried out with TSKS (isoform 1) fragments revealed particularly strong phosphorylation of a recombinant N-terminal region representing aa 1-150 of TSKS, indicating that the N-terminus of human TSKS is phosphorylated by human TSSK2. Production of full-length enzymatically active recombinant TSSK2 kinase represents the achievement of a key benchmark for future discovery of TSSK inhibitors as male contraceptive agents.

Dynamic Changes in Equatorial Segment Protein 1 (SPESP1) Glycosylation During Mouse Spermiogenesis.

ESP1/SPESP1 is a testis-specific, postmeiotic gene expressed in round spermatids that encodes equatorial segment protein 1, an intra-acrosomal protein found in the acrosomal matrix and on the luminal surface of the inner and outer acrosomal membranes within the equatorial segment domain of mature spermatozoa. A comparison of testicular protein extracts with caput, corpus, and caudal epididymal sperm proteins revealed striking differences in the apparent masses of SPESP1 isoforms. The predominant isoforms of SPESP1 in the testis were 77 and 67 kDa, with 47-kDa forms present to a minor degree. In contrast, SPESP1 isoforms of 47 and 43 kDa were found in caput, corpus, and caudal sperm, indicating that SPESP1 undergoes noticeable mass changes during spermiogenesis and/or subsequent transport to the epididymis. On two-dimensional (2D) SDS-PAGE, testicular SPESP1 isoforms resolved as a train of pI values from 4.9 to 5.2. Immunoprecipitated 77-kDa SPESP1 from testis reacted with the glycoprofile stain after one-dimensional and 2D gel electrophoresis, indicating that the 77-kDa testicular isoform was highly glycosylated. One charge variant of the 67-kDa isoform was also glycoprofile positive after 2D gel resolution. The 47- and 43-kDa isoforms of SPESP1 from epididymal sperm did not stain with glycoprofile, suggesting an absence of, or few, glycoprofile-sensitive glycoconjugates in epididymal SPESP1. Treatment of testicular extracts with a variety of glycosidases resulted in mass shifts in immunoreactive SPESP1, indicating that testicular SPESP1 was glycosylated and that terminal sialic acid, N- and O-glycans were present. A mixture of deglycosidase enzymes (including PNGase-F, neuraminidase, beta1-4 galactosidase, endo-alpha-N-acetylgalactosaminidase, and beta N-acetyl-glucosaminidase) completely eliminated the 77- and 67-kDa SPESP1 bands and resulted in the appearance of 75-, 60-, 55-, 50-, 47-, and 43-kDa forms, confirming that both the 77- and 67-kDa testicular forms of SPESP1 contain complex carbohydrate residues. Treatment of caudal epididymal sperm with PNGase-F enzymes showed a faint deglycosylated band at 30 kDa, but neuraminidase did not result in any molecular shift, indicating that epididymal sperm SPESP1 did not contain sialic acid/N-acetylglucosamine residues. These findings are consistent with the hypothesis that SPSPESP1 undergoes significant glycosylation in the testis and that the majority of these glycoconjugates are removed by the time sperm reach the caput epididymis. Studies of the fate of SPESP1 after the acrosome reaction localized SPESP1 to the equatorial segment region in both noncapacitated and capacitated, acrosome-reacted sperm. During capacitation, SPESP1 underwent proteolysis, resulting in a 27-kDa fragment. Zona-free oocytes incubated with recSPESP1 protein showed complementary binding sites on the microvillar oolemmal domain. Both recSPESP1 and anti-recSPESP1 antibody inhibited in vitro fertilization.

Oocyte specific oolemmal SAS1B involved in sperm binding through intra-acrosomal SLLP1 during fertilization.

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.

MEIG1 is essential for spermiogenesis in mice.

Spermatogenesis can be divided into three stages: spermatogonial mitosis, meiosis of spermatocytes, and spermiogenesis. During spermiogenesis, spermatids undergo dramatic morphological changes including formation of a flagellum and chromosomal packaging and condensation of the nucleus into the sperm head. The genes regulating the latter processes are largely unknown. We previously discovered that a bi-functional gene, Spag16, is essential for spermatogenesis. SPAG16S, the 35 kDa, testis-specific isoform derived from the Spag16 gene, was found to bind to meiosis expressed gene 1 product (MEIG1), a protein originally thought to play a role in meiosis. We inactivated the Meig1 gene and, unexpectedly, found that Meig1 mutant male mice had no obvious defect in meiosis, but were sterile as a result of impaired spermatogenesis at the stage of elongation and condensation. Transmission electron microscopy revealed that the manchette, a microtubular organelle essential for sperm head and flagellar formation was disrupted in spermatids of MEIG1-deficient mice. We also found that MEIG1 associates with the Parkin co-regulated gene (PACRG) protein, and that testicular PACRG protein is reduced in MEIG1-deficient mice. PACRG is thought to play a key role in assembly of the axonemes/flagella and the reproductive phenotype of Pacrg-deficient mice mirrors that of the Meig1 mutant mice. Our findings reveal a critical role for the MEIG1/PARCG partnership in manchette structure and function and the control of spermiogenesis.

CABYR isoforms expressed in late steps of spermiogenesis bind with AKAPs and ropporin in mouse sperm fibrous sheath.

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Identification of calcium-binding proteins associated with the human sperm plasma membrane.

BACKGROUND: The precise composition of the human sperm plasma membrane, the molecular interactions that define domain specific functions, and the regulation of membrane associated proteins during the capacitation process, still remain to be fully understood. Here, we investigated the repertoire of calcium-regulated proteins associated with the human sperm plasma membrane. METHODS: Surface specific radioiodination was combined with two-dimensional gel electrophoresis, a 45Ca-overlay assay, computer assisted image analysis and mass spectrometry to identify calcium-binding proteins exposed on the human sperm surface. RESULTS: Nine acidic 45Ca-binding sperm proteins were excised from stained preparative 2D gels and identified by mass spectrometry. Five of the calcium binding proteins; HSPA2 (HSP70-1), HSPA5 (Bip), HYOU1 (ORP150), serum amyloid P-component (SAP) and protein kinase C substrate 80K-H (80K-H) were found to be accessible to Iodo-Bead catalyzed 125I-labelling on the surface of intact human sperm. Agglutination and immunofluorescence analysis confirmed that SAP is situated on the plasma membrane of intact, motile sperm as well as permeabilized cells. Western blot analysis showed increased phosphorylation of human sperm 80K-H protein following in vitro capacitation. This is the first demonstration of the 80K-H protein in a mammalian sperm. CONCLUSION: The presence of SAP on the surface of mature sperm implies that SAP has a physiological role in reproduction, which is thought to be in the removal of spermatozoa from the female genital tract via phagocytosis. Since 80K-H is a Ca2+-sensor recently implicated in the regulation of both inositol 1,4,5-trisphosphate receptor and transient receptor potential (TRP) cation channel activities, its detection in sperm represents the first direct signaling link between PKC and store-operated calcium channels identified in human sperm.

Distribution of RNA binding protein MOEP19 in the oocyte cortex and early embryo indicates pre-patterning related to blastomere polarity and trophectoderm specification.

We report the cloning and characterization of MOEP19, a novel 19 kDa RNA binding protein that marks a defined cortical cytoplasmic domain in oocytes and provides evidence of mammalian oocyte polarity and a form of pre-patterning that persists in zygotes and early embryos through the morula stage. MOEP19 contains a eukaryotic type KH-domain, typical of the KH-domain type I superfamily of RNA binding proteins, and both recombinant and native MOEP19 bind polynucleotides. By immunofluorescence, MOEP19 protein was first detected in primary follicles throughout the ooplasm. As oocytes expanded in size during oogenesis, MOEP19 increased in concentration. MOEP19 localized in the ovulated egg and early zygote as a symmetrical spherical cortical domain underlying the oolemma, deep to the zone of cortical granules. MOEP19 remained restricted to a cortical cytoplasmic crescent in blastomeres of 2-, 4- and 8-cell embryos. The MOEP19 domain was absent in regions underlying cell contacts. In morulae, the MOEP19 domain was found at the apex of outer, polarized blastomeres but was undetectable in blastomeres of the inner cell mass. In early blastocysts, MOEP19 localized in both mural and polar trophectoderm and a subset of embryos showed inner cell mass localization. MOEP19 concentration dramatically declined in late blastocysts. When blastomeres of 4- to 8-cell stages were dissociated, the polarized MOEP19 domain assumed a symmetrically spherical localization, while overnight culture of dissociated blastomeres resulted in formation of re-aggregated embryos in which polarity of the MOEP19 domain was re-established at the blastomere apices. MOEP19 showed no evidence of translation in ovulated eggs, indicating that MOEP19 is a maternal effect gene. The persistence during early development of the MOEP19 cortical oocyte domain as a cortical crescent in blastomers suggests an intrinsic pre-patterning in the egg that is related to the apical-basolateral polarity of the embryo. Although the RNAs bound to MOEP19 are presently unknown, we predict that the MOEP19 domain directs RNAs essential for normal embryonic development to specific locations in the oocyte and early embryo.

Targeted deletion of Tssk1 and 2 causes male infertility due to haploinsufficiency.

Targeted deletion of Tssk1 and 2 resulted in male chimeras which produced sperm/spermatogenic cells bearing the mutant allele, however this allele was never transmitted to offspring, indicating infertility due to haploinsufficiency. Morphological defects in chimeras included failure to form elongated spermatids, apoptosis of spermatocytes and spermatids, and the appearance of numerous round cells in the epididymal lumen. Characterization of TSSK2 and its interactions with the substrate, TSKS, were further investigated in human and mouse. The presence of both kinase and substrate in the testis was confirmed, while persistence of both proteins in spermatozoa was revealed for the first time. In vivo binding interactions between TSSK2 and TSKS were established through co-immunoprecipitation of TSSK2/TSKS complexes from both human sperm and mouse testis extracts. A role for the human TSKS N-terminus in enzyme binding was defined by deletion mapping. TSKS immunoprecipitated from both mouse testis and human sperm extracts was actively phosphorylated. Ser281 was identified as a phosphorylation site in mouse TSKS. These results confirm both TSSK 2 and TSKS persist in sperm, define the critical role of TSKS' N-terminus in enzyme interaction, identify Ser 281 as a TSKS phosphorylation site and indicate an indispensable role for TSSK 1 and 2 in spermiogenesis.

TSKS concentrates in spermatid centrioles during flagellogenesis.

Centrosomal coiled-coil proteins paired with kinases play critical roles in centrosomal functions within somatic cells, however knowledge regarding gamete centriolar proteins is limited. In this study, the substrate of TSSK1 and 2, TSKS, was localized during spermiogenesis to the centrioles of post-meiotic spermatids, where it reached its greatest concentration during the period of flagellogenesis. This centriolar localization persisted in ejaculated human spermatozoa, while centriolar TSKS diminished in mouse sperm, where centrioles are known to undergo complete degeneration. In addition to the centriolar localization during flagellogenesis, mouse TSKS and the TSSK2 kinase localized in the tail and acrosomal regions of mouse epididymal sperm, while TSSK2 was found in the equatorial segment, neck and the midpiece of human spermatozoa. TSSK2/TSKS is the first kinase/substrate pair localized to the centrioles of spermatids and spermatozoa. Coupled with the infertility due to haploinsufficiency noted in chimeric mice with deletion of Tssk1 and 2 (companion paper) this centriolar kinase/substrate pair is predicted to play an indispensable role during spermiogenesis.

Biochemical and structural characterization of apolipoprotein A-I binding protein, a novel phosphoprotein with a potential role in sperm capacitation.

The physiological changes that sperm undergo in the female reproductive tract rendering them fertilization-competent constitute the phenomenon of capacitation. Cholesterol efflux from the sperm surface and protein kinase A (PKA)-dependent phosphorylation play major regulatory roles in capacitation, but the link between these two phenomena is unknown. We report that apolipoprotein A-I binding protein (AI-BP) is phosphorylated downstream to PKA activation, localizes to both sperm head and tail domains, and is released from the sperm into the media during in vitro capacitation. AI-BP interacts with apolipoprotein A-I, the component of high-density lipoprotein involved in cholesterol transport. The crystal structure demonstrates that the subunit of the AI-BP homodimer has a Rossmann-like fold. The protein surface has a large two compartment cavity lined with conserved residues. This cavity is likely to constitute an active site, suggesting that AI-BP functions as an enzyme. The presence of AI-BP in sperm, its phosphorylation by PKA, and its release during capacitation suggest that AI-BP plays an important role in capacitation possibly providing a link between protein phosphorylation and cholesterol efflux.

Clinical and consumer trial performance of a sensitive immunodiagnostic home test that qualitatively detects low concentrations of sperm following vasectomy.

PURPOSE: Compliance with post-vasectomy semen analysis could be improved with the availability of a simple, rapid and accurate home test. SpermCheck Vasectomy, a highly sensitive lateral flow immunochromatographic diagnostic device, was designed to detect extreme oligospermia or azoospermia in men after vasectomy. We report the results of clinical and consumer testing of SpermCheck. MATERIALS AND METHODS: A prospective, noncomparative observational study assessed the ability of SpermCheck Vasectomy to predict post-vasectomy sperm counts obtained using a hemacytometer procedure based on standard World Health Organization methodology. Consumer studies evaluated ease of use. RESULTS: A cohort of 144 post-vasectomy semen samples was tested in the clinical trial. SpermCheck was 96% accurate in predicting whether sperm counts were greater or less than a threshold of 250,000 sperm per ml, a level associated with little or no risk of pregnancy. Sensitivity was 93% (95% CI 79% to 98%) and specificity was 97% (91% to 99%). The positive predictive value of the test was 93% (79% to 98%), and most importantly the negative predictive value was 97% (91% to 99%). The test gave a positive result 100% of the time at sperm concentrations of 385,000/ml or greater. Consumer studies with 109 lay volunteers showed that SpermCheck was easy to use. Volunteers obtained the correct or expected test result in every case and the correct response rate on a 20 question survey about the test was 97%. CONCLUSIONS: SpermCheck Vasectomy, a simple and reliable immunodiagnostic test that can provide evidence of vasectomy success or failure, offers a useful alternative to improve compliance with post-vasectomy sperm monitoring. It is currently the only Food and Drug Administration approved test for this purpose.

Immunogenicity of a multi-component recombinant human acrosomal protein vaccine in female Macaca fascicularis.

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.

Radial spoke protein 44 (human meichroacidin) is an axonemal alloantigen of sperm and cilia.

To identify novel sperm alloantigens relevant to immune infertility, sera from infertile men containing antisperm antibodies (ASA) were employed on 2-D immunoblots of human sperm proteins. An immunoreactive protein spot (MW: 44 kDa, pI: 4.5) was microsequenced and the related cDNA was cloned yielding a 309 amino acid sequence corresponding to a gene currently annotated in Genbank as TSGA2 homolog (mouse) to signify 'testis specific gene A2'. In Genbank the protein deduced from this gene is currently named human meichroacidin, an orthologue of meichroacidin previously identified in mouse spermatocytes. Human TSGA2 mapped to chromosome 21q22.3. Human meichroacidin (hMCA) contained a single potential tyrosine phosphorylation site and five casein kinase phosporylation motifs. The N-terminus contained a Membrane Occupation Recognition Nexus (MORN) motif found in the lipid kinase-phosphatidylinositol 4-phosphate 5-kinase (PIP5K) family and junctophilins. However hMCA lacked the characteristic kinase homology domain of PIP5K. Northern blot analysis revealed 1.5 kb hMCA transcripts in testis and trachea with lower levels in thyroid and spinal cord. A semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) analysis demonstrated occurrence of the mRNA messages in all the ciliated tissues tested with highest levels of messages in testis and trachea. Western blot analysis showed the presence of hMCA protein in brain, thyroid and trachea at the identical mass, 44 kDa, as in human testis. However, this immunoreactive pattern differed from that of sperm in which a 38 kDa form was also evident suggesting that hMCA undergoes proteolytic processing. In human testis, hMCA localized to the tails of developing spermatids and did not localize to the nucleus of either spermatocytes or spermatids. EM immunocytochemistry localized hMCA within the radial spokes of the axonemal complex of the sperm flagellum, and immunofluorescence studies revealed h-meichroacidin in the cilia of epithelial cells in the trachea and ependyma. Bioinformatic identification of orthologues of meichroacidin in several lower organisms including ciliates and flagellates suggest the protein plays a role in flagellar motility across phyla. We propose the term radial spoke protein 44 as an accurate designation, preferable to human meichroacidin because it denotes the restricted localization of the protein to the radial spokes of the axonemes of both sperm and cilia. Further, since the human gene is expressed in brain, thyroid, trachea and lung in addition to testis, we suggest that the gene name be changed from TSGA2 [testis specific gene A2] to radial spoke protein 44 [RSP44].

Genomic organization, incidence, and localization of the SPAN-x family of cancer-testis antigens in melanoma tumors and cell lines.

PURPOSE: Members of the SPAN-X (sperm protein associated with the nucleus mapped to the X chromosome) family of cancer-testis antigens are promising targets for tumor immunotherapy because they are normally expressed exclusively during spermiogenesis on the adluminal side of the blood-testis barrier, an immune privileged compartment. EXPERIMENTAL DESIGN AND RESULTS: This study analyzed the human SPANX genomic organization, as well as SPAN-X mRNA and protein expression in somatic and cancer cells. The SPANX family consists of five genes, one of which is duplicated, all located in a gene cluster at Xq27.1. From the centromere, the arrangement of the five SPANX genes mapped on one contiguous sequence is SPANXB, -C, -A1, -A2, and -D. Reverse transcription-PCR analyses demonstrated expression of SPAN-X mRNA in melanoma and ovarian cell lines, and virtual Northern analysis established SPANX gene expression in numerous cancer cell lines. Immunoblot analysis using polyclonal antisera raised against recombinant SPAN-X confirmed the translation of SPAN-X proteins in melanoma and ovarian tumor cell lines. The immunoreactive proteins migrated between M(r) 15,000 and M(r) 20,000 similar to those observed in spermatozoa. Immunoperoxidase labeling of melanoma cells and tissue sections demonstrated SPAN-X protein localization in the nucleus, cytoplasm, or both. Ultrastructurally, in melanoma cells with nuclear SPAN-X, the protein was associated with the nuclear envelope, a localization similar to that observed in human spermatids and spermatozoa. Significantly, the incidence of SPAN-X-positive immunostaining was greatest in the more aggressive skin tumors, particularly in distant, nonlymphatic metastatic melanomas. CONCLUSIONS: The data herein suggest that the SPAN-X protein may be a useful target in cancer immunotherapy.

Membrane associated cancer-oocyte neoantigen SAS1B/ovastacin is a candidate immunotherapeutic target for uterine tumors.

The metalloproteinase SAS1B [ovastacin, ASTL, astacin-like] was immunolocalized on the oolemma of ovulated human oocytes and in normal ovaries within the pool of growing oocytes where SAS1B protein was restricted to follicular stages spanning the primary-secondary follicle transition through ovulation. Gene-specific PCR and immunohistochemical studies revealed ASTL messages and SAS1B protein in both endometrioid [74%] and malignant mixed Mullerian tumors (MMMT) [87%] of the uterus. A MMMT-derived cell line, SNU539, expressed cell surface SAS1B that, after binding polyclonal antibodies, internalized into EEA1/LAMP1-positive early and late endosomes. Treatment of SNU539 cells with anti-SAS1B polyclonal antibodies caused growth arrest in the presence of active complement. A saporin-immunotoxin directed to SAS1B induced growth arrest and cell death. The oocyte restricted expression pattern of SAS1B among adult organs, cell-surface accessibility, internalization into the endocytic pathway, and tumor cell growth arrest induced by antibody-toxin conjugates suggest therapeutic approaches that would selectively target tumors while limiting adverse drug effects in healthy cells. The SAS1B metalloproteinase is proposed as a prototype cancer-oocyte tumor surface neoantigen for development of targeted immunotherapeutics with limited on-target/off tumor effects predicted to be restricted to the population of growing oocytes.

Splicing in murine CABYR and its genomic structure.

Human calcium-binding tyrosine-phosphorylation regulated protein (CABYR) is a polymorphic, testis-specific, calcium binding protein that undergoes tyrosine phosphorylation during in vitro capacitation. A protein kinase A (PKA) regulatory subunit type II alpha (RII-alpha) homologous domain in the N-terminus, phosphorylation dependent Ca(++) binding isoforms, and localization to the principal piece of the human sperm tail suggest that CABYR may be involved in sperm motility. In this paper, four mouse orthologous cDNAs and the genomic DNA of CABYR were cloned, nucleotide and protein sequences of mouse and humans were compared, and the genomic organization of the mCABYR gene was analyzed. Human and mouse CABYR conserve potential functional motifs including a domain homologous to the dimerization interface of cyclic adenosine monophosphate dependent PKA RII-alpha, 14 PXXP motifs, and regions of homology with extensins and src homology-3-binding protein 1. mCABYR is arranged into six exons spanning about 14 kb of DNA. Mouse CABYR showed several similarities with human CABYR: (1) the protein was localized to the principal piece of mouse epididymal spermatozoa; (2) mouse CABYR has two coding regions (CR-A and CR-B), with 66 and 82% identity, respectively to human; and (3) mCABYR showed the presence of two testis-specific transcripts of approximately 1.4 and approximately 2.4 kb. Three murine splice variants were identified, two of which spliced into CR-B. Exon 4, present in all human and mouse variants and comprising 85% of CR-A appears suitable for targeted deletion. The overall 81% nucleotide identity between mouse and human CABYR, the common genomic organization, presence of similar testis-specific transcripts, localization in the principal piece of tail and occurrence of homologous splice variants indicate an authentic murine orthologue of CABYR has been identified.

CABYR binds to AKAP3 and Ropporin in the human sperm fibrous sheath.

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.

FSCB, a novel protein kinase A-phosphorylated calcium-binding protein, is a CABYR-binding partner involved in late steps of fibrous sheath biogenesis.

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.

Translation and assembly of CABYR coding region B in fibrous sheath and restriction of calcium binding to coding region A.

CABYR is a highly polymorphic, sperm flagellar calcium-binding protein that is tyrosine as well as serine/threonine phosphorylated during capacitation. Six alternative splice variants of human CABYR (I-VI) have previously been identified, involving two coding regions, CR-A and CR-B, separated by an intervening stop codon. It is presently unknown if proteins encoded by the predicted coding region B of CABYR are translated during spermiogenesis, where they localize, or which CABYR isoforms bind calcium. Immunofluorescent and electron microscopic studies using polyclonal antibodies generated to the recombinant c-terminal 198 aa CABYR-B localized the isoforms containing CABYR-B to the ribs and longitudinal columns of the fibrous sheath in the principal piece of the flagellum. Antisera to recombinant CABYR-A and CABYR-B proteins recognized distinct populations of CABYR isoforms encoded by either CR-A alone and/or CR-B as well as a common population of CABYR isoforms. Only the recombinant CABYR-A and not the CABYR-B bound calcium in vitro, which is consistent with the hypothesis that CABYR-A is the only form that binds calcium in sperm. These observations confirmed that, despite the presence of the stop codon in CR-A, splice variants containing CR-B are expressed during spermiogenesis and assemble into the fibrous sheath of the principal piece; however, calcium binding occurs only to those CABYR isoforms containing CABYR-A.