Cloning and characterization of a novel sperm-associated isoantigen (E-3) with defensin- and lectin-like motifs expressed in rat epididymis.
In the present study we report the identification of a novel epididymis-specific secretory glycoprotein, E-3, which is a sperm-associated isoantigen containing defensin- and lectin-like motifs. E-3 was detected in rat epididymal fluid and in sperm extracts by two-dimensional (2-D) Western blotting using rat hyperimmune sera raised against rat sperm. The immunoreactive spot of approximately 28 kDa with an isoelectric point (pI) of 3.5 was cored from silver-stained gels. Microsequencing by tandem mass spectrometry and database searches revealed several peptides to be novel sequences. Degenerate deoxyinosine-containing primers corresponding to the novel peptides were used in rapid amplification of cDNA ends and polymerase chain reaction to clone E-3 from a rat epididymal cDNA library. A 449-base pair nucleotide sequence was subsequently obtained consisting of a complete open reading frame (ORF) of 111 amino acids, which showed similarity to the defensin and lectin families. The first 21 amino acids constituted a putative signal peptide, suggesting that E-3 is a secretory protein. Mature E-3 protein corresponding to amino acids 22-111 was expressed in E. coli, and chickens were immunized with recombinant E-3 (rE-3). The resulting anti-rE-3 antisera recognized the recombinant immunogen as well as a "native" protein of 28 kDa, pI 2.5-3.5 in both epididymal fluid and in sperm extracts on 2-D Western blots. Northern hybridization indicated that E-3 mRNA was present in the epididymis but not in testis or other tissues, and that E-3 mRNA was predominantly expressed in the corpus and cauda of the epididymis, but not in the initial segment or caput. Similarly, Western blots detected the E-3 protein only in the epididymal fluid and sperm from the corpus and caudal regions. Finally, indirect immunofluorescence localized E-3 on the entire tail, and with less intensity on the head of the sperm. These observations indicate that E-3 is a secreted epididymal protein that becomes associated with the sperm as it transits through the corpus and cauda. The presence of a defensin-like motif suggests that E-3 may play a role in protecting the sperm from microbial infections in the epididymis and in the female reproductive tract.
Rao
Jayasimha
J
Department of Cell Biology and the Center for Research in Contraceptive and Reproductive Health, University of Virginia, Charlottesville, Virginia 22908, USA.
Herr
John C
JC
Reddi
P Prabhakara
PP
Wolkowicz
Michael J
MJ
Bush
Leigh Ann
LA
Sherman
Nicholas E
NE
Black
Michael
M
Flickinger
Charles J
CJ
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D023082:Defensins / Q000737:chemistry / Q000235:genetics; D030501:Epididymal Secretory Proteins / Q000737:chemistry / Q000235:genetics*; D004822:Epididymis / Q000378:metabolism*; D005260:Female; D015870:Gene Expression; D007519:Isoantigens / Q000737:chemistry / Q000235:genetics; D037102:Lectins / Q000737:chemistry / Q000235:genetics; D008297:Male; D008969:Molecular Sequence Data; D051381:Rats; D011917:Rats, Inbred Lew; D011994:Recombinant Proteins / Q000235:genetics; D017386:Sequence Homology, Amino Acid; D013094:Spermatozoa / Q000378:metabolism
D018076:DNA, Complementary; D023082:Defensins; D030501:Epididymal Secretory Proteins; D007519:Isoantigens; D037102:Lectins; D011994:Recombinant Proteins