Identification, cloning and functional characterization of novel beta-defensins in the rat (Rattus norvegicus).
BACKGROUND
Beta-defensins are small cationic peptides that exhibit broad spectrum antimicrobial properties. The majority of beta-defensins identified in humans are predominantly expressed in the male reproductive tract and have roles in non-immunological processes such as sperm maturation and capacitation. Characterization of novel defensins in the male reproductive tract can lead to increased understanding of their dual roles in immunity and sperm maturation.


METHODS
In silico rat genomic analyses were used to identify novel beta-defensins related to human defensins 118-123. RNAs isolated from male reproductive tract tissues of rat were reverse transcribed and PCR amplified using gene specific primers for defensins. PCR products were sequenced to confirm their identity. RT-PCR analysis was performed to analyze the tissue distribution, developmental expression and androgen regulation of these defensins. Recombinant defensins were tested against E. coli in a colony forming unit assay to analyze their antimicrobial activities.


RESULTS
Novel beta-defensins, Defb21, Defb24, Defb27, Defb30 and Defb36 were identified in the rat male reproductive tract. Defb30 and Defb36 were the most restricted in expression, whereas the others were expressed in a variety of tissues including the female reproductive tract. Early onset of defensin expression was observed in the epididymides of 10-60 day old rats. Defb21-Defb36 expression in castrated rats was down regulated and maintained at normal levels in testosterone supplemented animals. DEFB24 and DEFB30 proteins showed potent dose and time dependent antibacterial activity.


CONCLUSIONS
Rat Defb21, Defb24, Defb27, Defb30 and Defb36 are abundantly expressed in the male reproductive tract where they most likely protect against microbial invasion. They are developmentally regulated and androgen is required for full expression in the adult epididymis.
Yenugu
Suresh
S
Department of Pediatrics, University of North Carolina, Chapel Hill, North Carolina 27599, USA. ysnaidu@yahoo.com
Chintalgattu
Vishnu
V
Wingard
Christopher J
CJ
Radhakrishnan
Yashwanth
Y
French
Frank S
FS
Hall
Susan H
SH
D000595:Amino Acid Sequence; D000818:Animals; D000900:Anti-Bacterial Agents / Q000378:metabolism / Q000494:pharmacology; D002874:Chromosome Mapping; D003001:Cloning, Molecular; D004822:Epididymis / Q000378:metabolism; D005260:Female; D015870:Gene Expression; D018507:Gene Expression Regulation, Developmental; D005837:Genitalia, Male / Q000276:immunology / Q000378:metabolism*; D008297:Male; D008969:Molecular Sequence Data; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology; D051381:Rats; D017208:Rats, Wistar; D016415:Sequence Alignment; D013737:Testis / Q000378:metabolism; D014018:Tissue Distribution; D023083:beta-Defensins / Q000235:genetics* / Q000378:metabolism / Q000494:pharmacology
D000900:Anti-Bacterial Agents; D020033:Protein Isoforms; D023083:beta-Defensins