Abstract
Defensins are important elements in innate immunity that can also trigger adaptive immune responses. The defensins form a family of small cationic antimicrobial peptides with six characteristic cysteine residues, whose pairing pattern in forming three intramolecular disulfide bonds defines the alpha- and beta-defensin subfamilies. In a search for new beta-defensin genes, we performed computational analysis using the Celera mouse genome data base and found exons encoding 23 different beta-defensins, including the eight previously characterized members of this family. Among the new beta-defensins, nine of them form two groups of phylogenetically related sequences that were characterized in greater detail. Northern blot, reverse transcription PCR, and in situ hybridization analysis showed that expression of these genes is restricted to the epididymis, with a specific regional expression pattern. One of the new beta-defensins (Defb38) was chemically synthesized; in in vitro assays on Gram-positive and -negative bacterial strains, Defb38 showed the characteristic salt-dependent antimicrobial activity of beta-defensins. The results demonstrate the existence of a relatively large number of beta-defensins with specific expression in distinct regions of the murine epididymis and suggest complex roles for these proteins in host defense and other physiological processes of the male reproductive tract.
Highlights
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AJ489588, AJ575424, AJ575425, AJ575426, AJ575427, and AJ575428
Reverse transcription PCR, and in situ hybridization analysis showed that expression of these genes is restricted to the epididymis, with a specific regional expression pattern
The results demonstrate the existence of a relatively large number of -defensins with specific expression in distinct regions of the murine epididymis and suggest complex roles for these proteins in host defense and other physiological processes of the male reproductive tract
Summary
Bioinformatics—The TBLASTN program [7] was used to query the Celera mouse genome assembly (www.celera.com) with the protein sequences of known -defensins. -Defensin mRNA Characterization and Expression—Total RNA from mouse organs was extracted with Tri-reagent (Sigma). DNA fragments corresponding to partial new -defensin cDNAs were obtained by PCR amplification of murine cDNA (15 s at 94 °C and 60 s at 68 °C for 35 cycles). Prehybridization of the membranes and hybridization with 32P-labeled DNA fragments containing partial coding sequences of the putative new -defensin genes were carried out in Rapid-Hyb buffer (Amersham Biosciences) as recommended by the supplier. Quantitative analyses using primer pairs that amplify genomic DNA were performed with DNase I-treated RNA (Ambion, Austin, TX) prior to cDNA synthesis. In situ hybridization was performed on 10-m-thick frozen sagittal epididymis sections using digoxigenin-labeled sense or antisense RNA probes for Defb and Defb. Mid-logarithmic phase Escherichia coli (ATCC 11303 strain), Pseudomonas aeruginosa (PAO1 strain) [14], and Enterococcus faecium (CECT 4102 strain) were suspended in 10 mM sodium phosphate buffer (15 mM sodium concentration), adjusted to a density of 5 ϫ 108 colony-forming units/ml,
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