Molecular cloning and sequencing of cDNA encoding for human FA-1 antigen.
The cDNA encoding for the human FA-1 sperm antigen was cloned and sequenced from the in-house constructed subtractive human testis cDNA expression library in lambda Ziplox using the FA-1 monoclonal antibody (mAb). The full--length sequence was obtained by using the 5' rapid amplification of 5'-cDNA end (5'-RACE) procedure. It is 1,576-bp long, and has an open reading frame (ORF) of 283 amino acids (aa) with the first ATG Met start codon at nucleotide (nt) 57 and the stop codon TAG at nt 906. It has two termination codons at the 5' end before the ATG start codon. The translated protein has a calculated molecular weight of 32.1 kDa and estimated isoelectric point (pI) of 11.59. It has one potential N-linked glycosylation site and one tyrosine phosphorylation site, besides several O-linked glycosylation and serine and threonine phosphorylation sites. Hydrophilicity analysis of the deduced aa sequence showed it to be a membrane-anchored protein. Extensive computer search in the database did not identify any known nt/aa sequence having homology with FA-1 cDNA or deduced aa, indicating it to be a novel gene. The Northern blot and reverse transcription-polymerase chain reaction (RT-PCR)-Southern blot analyses indicated the testis-specific expression of FA-1 antigen at the mRNA level. The ORF of the FA-1 was subcloned into pGEX- 1lambda T for expression. The expressed FA-1 recombinant protein had a molecular size of approximately 40 kDa, and was recognized by the FA-1 mAb, and not by the myeloma control Ig. The rabbit antibodies (Ab) raised against the recombinant (r) FA-1 antigen recognized the rFA-1 antigen as well as the native (n) FA-1 antigen. The rFA-1 Ab specifically recognized a protein band of approximately 50 kDa in human testis extract in the Western blot involving 11 types of human tissue extracts, indicating the testis-specific expression of FA-1 at the protein level. The Ab showed binding with live and methanol-fixed human sperm at the post-acrosomal, mid-piece, and tail regions. The Ab caused a significant (P < 0.001) and concentration-dependent inhibition of human sperm capacitation/acrosome reaction by blocking tyrosine phosphorylation of the FA-1 antigen. The sperm-specific human FA-1 recombinant antigen may find applications in immunocontraception, and diagnosis and treatment of immunoinfertility in humans.
Naz
Rajesh K
RK
Division of Research, Department of Obstetrics and Gynecology, Medical College of Ohio, Toledo 43614-5806, USA. Rnaz@mco.edu
Zhu
Xiaolong
X
D020101:Acrosome Reaction / Q000276:immunology; D000595:Amino Acid Sequence; D000906:Antibodies; D000941:Antigens / Q000235:genetics* / Q000276:immunology; D001483:Base Sequence; D018076:DNA, Complementary; D015723:Gene Library; D006801:Humans; D008297:Male; D008969:Molecular Sequence Data; D009928:Organ Specificity; D010766:Phosphorylation; D017422:Sequence Analysis, DNA; D013737:Testis / Q000378:metabolism
D000906:Antibodies; D000941:Antigens; D018076:DNA, Complementary; C054151:fertilization antigen