Purification Of Mannose Receptor Of Human Sperm And Identification By Peptide Mass Fingerprinting

The mechanism of fertilization, especially of sperm-oocyte fusion, has not been explained thoroughly until now. The discovery of the sperm mannose receptor(MR) gave us a chance for further understanding human fertilization.Previous studies were involved in the distribution, biochemical characteristics and physiological function of MR. In order to investigate molecular characterization of MR, the protein was isolated from motile human sperm by mannose-agarose gel affinity chromatography. The specific mannose-binding activity of purified protein was identified by DMA-Biotin dot blot and western blot. Then, peptide mass fingerprinting was used for the identification from tryptic fragment sizes of purified protein by using the MASCOT search engine based on the Swiss-Prot protein database. The protein obtained from protein database was identified by immunoblotting .The results were as follows: (1)The home-made DMA-Biotin probe was proved to be effective by dot blot(;2)Dot blot showed high specific mannose-binding activity of purified protein; (3)Under nonreduce condition, 27kDa, 34kDa, 72kDa and 121kDa protein bands were identified by SDS-PAGE, Under reduce condition 27kDa, 34kDa, and 72kDa protein bands were identified by SDS-PAGE, But the stain of 27 kDa band became deeper and of 72kDa were very shallow. Only 27kDa and 72kDa proteins was proved to have specific mannose-binding activity by western blot; (4)Peptide mass fingerprinting identification of 27kDa, 72kDa and 121kDa proteins were shown to be serum amyloid P component(SAP)precursor,suggesting that the 72 kDa protein and 121kDa protein may be 27kDa protein polymer;(5)by immunoblotting, 27kDa band was proved to be SAP;(6)Peptide mass fingerprinting identification of 34kDa protein was shown to be annexin (ANXA5, ANXA1, ANXA2), but 34kDa protein was proved to be not ANXA5 by immunoblotting.In conclusion, to our knowledge, from human sperm by mannose-agarose gel affinity chromatography, it's the first time to isolate SAP which was proved to have the specific mannose-binding activity and most likely to be MR of human sperm.