Cloning and Characterization of a Novel Sperm-Associated Isoantigen (E-3) with Defensin- and Lectin-Like Motifs Expressed in Rat Epididymis1

Abstract

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.

Rat recombinant β-defensin 22 is a heparin-binding protein with antimicrobial activity

“…Therefore, we chose a traditional method to express the protein with His-tag as previously reported. 15 In the SDS-PAGE analysis, the bands of ,14 and ,30 kDa we observed were also reported by other researchers, 15 although their original data were omitted. The high tendency to aggregate might account for the altered migration of proteins in the SDS-PAGE and AU-PAGE analyses.…”

“…13 Immunofluorescence localisation indicated the presence of b-defensin 22 on the entire sperm tail, although a lower intensity of b-defensin 22 is detected in the head of sperm from the corpus and cauda regions. 15 Here, we report that a soluble recombinant protein of b-defensin 22 has antimicrobial and heparin-binding activities, which matches its possession of the defensin-like and lectin-like motifs. 15 Because heparin contributes to sperm chemo-attraction, 16,17 capacitation, 18 acrosome reaction, 19 regulation of sperm-zona pellucida interactions and polyspermy, 20 our findings suggest that b-defensin 22 might function not only in the innate immunity but also in sperm fertilisation abilities via its heparin-binding property.…”

“…15 Primers (forward primer, 59-ccccatgggcaactggtacgttcgaa-39; reverse primer, 59-cccctcgaggggtgcagcagtggtg-39) were synthesized to amplify the cDNA corresponding to the peptide from amino acids 21 to 111 (with no signal peptide). The underlined bases represent restriction sites designed in the primers.…”

“…12 Rat b-defensin 22 was independently identified in different laboratories and is also known as '2D6 glycoprotein' or 'E-3 epididymal fluid protein'. [13][14][15] Using the monoclonal antibody 2D6, b-defensin 22 was localized in principal cells in the distal caput epididymis; immunoblotting also revealed its presence as a soluble high-molecular-weight complex in the secreted fluid from this epididymal region. 13 Immunofluorescence localisation indicated the presence of b-defensin 22 on the entire sperm tail, although a lower intensity of b-defensin 22 is detected in the head of sperm from the corpus and cauda regions.…”

“…13,15 According to a well-accepted theory, charge and hydrophobicity of b-defensins are the most important factors 23 for antimicrobial activity. Whether the glycosylated rat b-defensin 22 retains antimicrobial activity needs further investigation.…”