Molecular cloning and sequencing of cDNA encoding for a novel testis-specific antigen

cDNA encoding for a sperm antigen, designated NZ-1, was cloned and sequenced from murine testis cDNA-lambda gt11 expression library using antibodies to human sperm surface antigens belonging to 14-18 kD molecular region. These sperm antigens are involved in zona pellucida binding and have tyrosine phyosphorylation activity. Computer generated translation analysis of 1395-bp cDNA yielded an open reading frame (ORF) of 152 aa with first ATG, Met start codon at nt 32 and the stop codon TGA at nt 487. The translated protein has a calculated molecular weight of 17.9 kD and a potential tyrosine phosphorylation site at aa 46-54, besides at least two O-linked glycosylation sites. The hydropathy plot generated from the deduced aa sequence indicated it to be a membrane-anchored peptide with a hydrophobic NH2-terminus that is characteristic of a signal peptide. Extensive computer search in the GenBank, NBRF, and Swiss sequence banks, indicating it to be a novel protein. Northern blot analysis indicated testis-specific expression of NZ-1 antigen. The NZ-1 cDNA was subcloned into pGEX-1 lambda T vector and expressed in glutathione-S-transferase gene fusion system to obtain the recombinant protein. The recombinant protein specifically reacted with the original antibodies raised against the native 14-18 kD sperm proteins. These findings suggest that the sperm-specific recombinant NZ-1 may find applications in the development of a contraceptive vaccine, and in studying the normal and abnormal sperm function and the signal transduction mechanism.