Mammalian Sperm MN9 Antigen N-,O-sialoglycoprotein EQUATORIN: Biochemical Characterization and Identification of the Gene. (2008)

MN9 antigen glycoprotein EQUATORIN of mouse epididymal spermatozoa forms broadly ranged heterogeneous band at 40-50kDa. Monoclonal antibody MN9 (MN9) recognizes EQUATORIN in the whole acrosome and became exposed at the equatorial segment after acrosome reaction. The purified MN9 inhibited sperm-egg fusion without affecting sperm motility, zona pellucida binding and penetration into zona pellucida. These previous reports suggested EQUATORIN is a candidate molecule in sperm-egg fusion and mammalian contraception. Here, we present biochemical properties and the corresponding gene. Materials and methods: 1) Purification of EQUATORIN. Spermatozoa removed from cauda epididymes were treated with 0.1% Triton X-100 (Triton) in PBS. After Triton treatment, EQUATORIN was extracted with SDS-EDTA buffer (75mM NaCl, 1 % SDS, 25mM EDTA, pH6.0). After removal of SDS by precipitation with 2D clean-up kit and subsequent dissolution in NP-40 lysis buffer, the lysates were allowed for MN9-immunoprecipitation and subjected to SDS-PAGE and Western blot (WB). 2) MS analyses. After WB analysis, the bands of interests were trypsin-digested and subjected to LC-MS/MS (MS) analysis. 3) Recombinant protein expression assay. Based on the MS analytical data, the corresponding recombinant proteins tagged with EGFP or 6 x His were expressed in E. coli and 293T cells. Produced proteins were analyzed by MN9-WB. 4) EQUATORIN expression. Various tissues including testes and epididymides were extracted with SDS sample buffer. 5) EQUATORIN localization. MN9-indirect immunofluorescence microscopy (IIF) was done for testes and epididymides. 6) Deglycosylation and mobility-shift assay. Spermatozoa denatured by SDS and DTT were treated with PNGase F or sialidase or both, and subjected to WB. Results and Discussion: 1) Broadly ranged EQUATORIN densely focused on about 41kDa after Triton treatment, while 50kDa band reduced the staining intensity. This enabled efficient purification of 41kDa EQUATORIN by immunoprecipitation. 2) MS analysis proposed a candidate gene for isolated 41kDa EQUATORIN. 3) The recombinant protein expression indicated that MN9 did not recognize any recombinant proteins expressed in E. coli, but did recognize the candidate protein at approximately 72kDa and EGFP-tagged recombinant protein at 104kDa expressed in 293T cells; the molecular size difference corresponded to the EGFP (27kDa). Thus, the candidate was confirmed as EQUATORIN gene. 4) MN9-WB for various tissues showed EQUATORIN is expressed specifically in testis and epididymis. 5) MN9-IIF showed EQUATORIN on sperm head in epididymis. 6) PNGase F treatment reduced approximately 2kDa in the two major bands; 50kDa to 48kDa and 41kDa to 39kDa. Sialidase treatment also showed more than 10kDa mobility shift to approximately 27kDa. These results suggest EQUATORIN has not only N-linked carbohydrate but also neuraminic acid on the O-linked carbohydrate. MN9 antigenicity was maintained after these enzyme treatments. Conclusion: EQUATORIN is expressed in testis and become a N-,O-sialoglycoprotein with 41-50kDa in mature spermatozoa. The EQUATORIN gene has been identified and confirmed by recombinant protein analysis. This research was supported by grant from the Japan Society for the Promotion of Science (KT).