Cloning and identification of a novel sperm binding protein, HEL-75, with antibacterial activity and expressed in the human epididymis.
BACKGROUND
The HEL-75 protein is a beta-defensin that was identified by analyzing a human epididymis cDNA library. Studying its function may not only elucidate the mechanisms of host defense, but may also provide new alternatives for novel therapeutic drugs for reproductive tract infections.


METHODS
The HEL-75 gene was amplified by PCR, and its structure and function were predicted and analyzed with bioinformatics tools. Polyclonal serum was raised against recombinant HEL (rHEL)-75 protein. The gene expression pattern was analyzed with RT-PCR and immunofluorescent staining. Finally, the antimicrobial activity and function during fertilization of HEL-75 were analyzed using a colony-forming unit assay and IVF, respectively.


RESULTS
The human HEL-75 gene is located on chromosome 20p13 and encodes a 95 amino acid protein with a predicted N-terminal signal peptide of 22 amino acids. The protein has six conserved cysteine residues, characteristic of members of the beta-defensin superfamily, as well as several potential post-translational modification sites. At the transcriptional level, HEL-75 was expressed in the epididymis and lung, but only in the epididymis at the translational level. Immunofluorescent staining showed that HEL-75 protein bound spermatozoa in the epididymis. RHEL-75 protein could kill Escherichia coli in vitro in a dose- and time-dependent fashion. However, no effect was observed on sperm motility nor fertilization when spermatozoa were blocked with anti-rHEL-75 polyclonal serum.


CONCLUSIONS
HEL-75 is a new beta-defensin expressed in the epididymis and on sperm; it may play an important role in host defense.
Lin
Y Q
YQ
Shandong Research Center of Stem Cell Engineering, Yantai Yu-huang-ding Hospital, No. 20, Yu-huang-ding Dong Road, 264000 Yantai, People's Republic of China.
Li
J Y
JY
Wang
H Y
HY
Liu
J
J
Zhang
C L
CL
Wang
W T
WT
Liu
J
J
Li
N
N
Jin
S H
SH
D000595:Amino Acid Sequence; D000900:Anti-Bacterial Agents / Q000378:metabolism / Q000494:pharmacology; D001483:Base Sequence; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000737:chemistry; D047108:Embryonic Development; D004822:Epididymis / Q000378:metabolism*; D004926:Escherichia coli / Q000187:drug effects; D006801:Humans; D008168:Lung / Q000378:metabolism; D008297:Male; D008826:Microbial Sensitivity Tests; D008969:Molecular Sequence Data; D012333:RNA, Messenger / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000032:analysis; D029589:Seminal Plasma Proteins / Q000737:chemistry / Q000494:pharmacology / Q000502:physiology*; D013081:Sperm Motility; D013094:Spermatozoa / Q000378:metabolism*; D023083:beta-Defensins / Q000737:chemistry / Q000494:pharmacology / Q000502:physiology*
D000900:Anti-Bacterial Agents; D018076:DNA, Complementary; C532923:HEL-75 protein, human; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D029589:Seminal Plasma Proteins; D023083:beta-Defensins