Characterization of the 84-kDa protein with ABH activity in human seminal plasma

In order to identify the blood group substance detected on the sperm plasma membrane (SPM), a plasma membrane preparation was obtained from the sperm soluble protein and then injected into a rabbit to produce an anti-SPM antibody. An anti-SPM antibody-binding affinity column specifically bound 6 polypeptides with molecular masses of 135 kDa, 84 kDa, 78 kDa, 67 kDa, 54 kDa and 20 kDa from seminal plasma. Among these polypeptides, the 84-kDa protein (p 84) showed ABH antigenic activity upon immunoblotting. When viable, motile sperm were incubated at 37 degrees C in the culture medium, they became capacitated and p 84 was released into the medium from the sperm surface after 3 h of incubation, indicating that p 84 is a sperm-coating antigen. Immunoblotting of sexual glands revealed that this protein is originated from the seminal vesicle. Its immunological properties were similar to those of lactoferrin. When seminal plasma was immunoprecipitated with anti-human lactoferrin antiserum, the immunoprecipitates contained both p 84 and ABH antigenic activity. The amino acid sequence of the N-terminus of p 84 was determined to be: G-R-?-R-?-S-V-Q-W-?-A-V-S-Q-P-E-A-D-K-?-F-Q-W-Q-R-N-M-R-K-V-R-G-P-?-V or P-S?-?-I. Although this sequence is highly homologous to lactoferrin, the 18th residue is different (p 84, D; lactoferrin, T). These data suggest that p 84 is the protein which has not been identified and bears the ABH antigen. A sandwich ELISA using anti-SPM antibody was able to bind p 84 and allowed determination of the ABO blood type of semen and saliva, but detected no ABH antigenic activity in breast milk, vaginal fluid, erythrocytes, serum or urine. These results suggest that p 84 is the best candidate for ABO blood typing of semen when contaminating vaginal fluid is present.