Lysozyme-like proteins (LYZLs) belong to the class of c-type lysozymes and are not well characterized in many species including the rat. In this study, using in silico and molecular biology techniques, we report the identification, cloning and characterization of rat Lyzl4 gene and also determine the expression pattern of Lyzl1, Lyzl3 and Lyzl6. The rat Lyzl genes were found to be distributed on three chromosomes and all of them retained the characteristic eight cysteine signature of c-type lysozyme. Homology modeling of rat LYZL4 indicated that its structure is similar to that of the mouse SLLP1. In the male reproductive tract of rat, Lyzl gene expression was confined to the testis. Lyzl1 and Lyzl4 were found to be expressed in tissues beyond the male reproductive tract, whereas Lyzl3 and Lyzl6 were not. Lyzl expression in the developing (10–60 day old) rats was androgen dependent in the testis. Immunodetection using antibodies against rat LYZL4 revealed the presence of LYZL4 protein in the germinal layer of the testes and on the sperm tail. Recombinant LYZL4 did not exhibit antibacterial, muramidase and isopeptidase activities characteristic to c-type lysozyme. To the best of our knowledge, for the first time we report the characterization of Lyzl genes in the rat. Results of our study indicate that rat LYZL proteins may have an important role in male reproductive tract function.

Reproductive tract infections pose a serious threat to health and fertility. Due to the emergence of antibiotic resistant pathogens, antimicrobial proteins and peptides of the reproductive tract are extensively characterized in recent years toward developing newer strategies to treat genital tract infections. Pathogen growth inhibition using a combination of naturally occurring male reproductive tract antimicrobial peptides and commonly used antibiotics has not been reported. Checker board analyses were carried out to determine the nature of interaction (synergistic, additive and antagonistic) between HE2alpha and HE2beta2 peptides and the commonly used antibiotics. Using Escherichia coli as the target organism, the minimal inhibitory concentration and fractional inhibitory concentration indices were determined. We demonstrate for the first time that the human male reproductive tract antimicrobial peptides HE2alpha and HE2beta2 act synergistically with the commonly used antibiotics to inhibit E. coli growth. A combination of HE2alpha and HE2beta2 peptides resulted in an additive effect. Interestingly, the synergistic effects of HE2 peptides were highest with doxycycline and ciprofloxacin, antibiotics generally used to treat epididymitis. Results of this study demonstrate the potential of endogenous HE2 peptides to be pharmacologically important in designing novel strategies to treat reproductive tract infections.

Tlr5, Tlr10, and Tlr11 are ubiquitously expressed in the rat. Tlrs seem to be expressed during male reproductive tract development and under conditions of androgen ablation, suggesting the preparedness of the male reproductive tract to detect an infection under all conditions of androgen status.

BackgroundSpermatogenesis and sperm maturation in the male reproductive tract is dictated by a variety of proteins secreted in the testis and epididymis. Though the proteome of these tissues is known, the functional role of many of these proteins remains uncharacterized. In this study, we characterize the rat Lysozyme-like (Lyzl) genes and proteins.MethodsIn silico tools were used to predict the primary, secondary and tertiary structures. Reverse transcription PCR, immunofluorescence and immunoblotting were used to determine the expression pattern. Lysozyme like enzyme activity was assessed by standard assays.ResultsSix rat Lyzl genes namely Lyzl1, Lyzl3, Lyzl4, Lyzl5, Lyzl6 and Lyzl7 were found to be highly conserved among the vertebrates with higher homology to mouse counterparts than with human counterparts. All the LYZL proteins contained the characteristic 4 disulfide bridges similar to c-type lysozyme. Only LYZL 1 and 6, conserved the active site amino acids of the lysozyme. Molecular modeling studies indicated that LYZL proteins exhibit strikingly similar three-dimensional structures among themselves. The secondary structure analysis of the recombinant LYZL proteins indicated the presence of α-helix, β-sheet and random coil with α-helix being the majority. Docking studies indicated the peptidoglycan binding nature of LYZL proteins. All the rat Lyzl mRNA transcripts (Lyzl1, Lyzl3, Lyzl4, Lyzl5, Lyzl6 and Lyzl7) are predominantly expressed in testes though some of them are expressed in tissues other than reproductive tract. Their expression was androgen independent. The rat LYZL proteins are localized in the germinal epithelium and on the spermatozoa. Recombinant LYZL1 and 6 possessed muramidase, isopeptidase and antibacterial activities. The mechanism of antibacterial action of LYZL1 and LYZL6 involved bacterial membrane damage and leakage of cellular contents. Only LYZL1 and 6 possess peptidoglycan binding ability, whereas LYZL3, LYZL4 and LYZL5 possess hyaluronan binding ability suggesting a possible functional divergence of these proteins. LYZL3, LYZL4 and LYZL7 possessed free radical scavenging property, suggesting that they may act as antioxidants.ConclusionThe divergent properties of LYZL proteins indicate that they may have a role in sperm function, innate immunity and other physiological process as well.

Antimicrobial proteins and peptides are ubiquitous in nature with diverse structural and biological properties. Among them, the human beta-defensins are known to contribute to the innate immune response. Besides the defensins, a number of defensin-like proteins and peptides are expressed in many organ systems including the male reproductive system. Some of the protein isoforms encoded by the sperm associated antigen 11B (SPAG11) gene in humans are beta-defensin-like and exhibit structure dependent and salt tolerant antimicrobial activity, besides contributing to sperm maturation. Though some of the functional roles of these proteins are reported, the structural and molecular features that contribute to their antimicrobial activity is not yet reported. In this study, using in silico tools, we report the three dimensional structure of the human SPAG11B proteins and their C-terminal peptides. web-based hydropathy, amphipathicity, and topology (WHAT) analyses and grand average of hydropathy (GRAVY) indices show that these proteins and peptides are amphipathic and highly hydrophilic. Self-optimized prediction method with alignment (SOPMA) analyses and circular dichroism data suggest that the secondary structure of these proteins and peptides primarily contain beta-sheet and random coil structure and alpha-helix to a lesser extent. Ramachandran plots show that majority of the amino acids in these proteins and peptides fall in the permissible regions, thus indicating stable structures. The secondary structure of SPAG11B isoforms and their peptides were not perturbed with increasing NaCl concentration (0-300 mM) and at different pH (3, 7, and 10), thus reinforcing our previously reported observation that their antimicrobial activity is salt tolerant. To the best of our knowledge, for the first time, results of our study provide vital information on the structural features of SPAG11B protein isoforms and their contribution to antimicrobial activity.