Fig 1.
Alignment of rat lysozyme and LYZL proteins.
Green color boxed amino acids represent the active site and black arrows indicate the conserved 8 cysteines. Sequence alignment of some rat LYZLs was reported by us earlier [24] and this figure presents a comprehensive analysis of all rat LYZLs.
Table 1.
General predicted and deduced features of rat Lyzl genes and their encoded proteins$.
Table 2.
Pairwise identity and similarity analysis of rat, mouse and human LYZL proteins.
Fig 2.
Genomic neighbourhood analysis of rat, mouse and human Lyzl genes.
Green arrows with names represent the genes and directions of the arrow represent the direction of sense strand. Black, red and green arrows indicate noncoding, pseudo and hypothetical genes respectively. Numbers in the beginning of each row indicates the chromosome number and are shown in the order of rat, mouse and human.
Fig 3.
Circular dichroism spectra of recombinant LYZL proteins measured in terms of mean residue ellipticity (MRE).
Table 3.
Secondary structure analyses by SOPMA and Circular Dichroism.
Fig 4.
Modelled structure of LYZL proteins showing β- sheets in yellow, helices in red, loop regions in green and disulphide bonds in spheres.
Table 4.
Templates used, G-factor, RMSD value and Ramachandran plot analyses for the 3D structures of rat lysozyme-like proteins.
Fig 5.
Molecular modeling showing the superimposition of rat LYZL and NAG complex with chicken lysozyme-NAG complex.
NAG is represented in the form of stick model and the protein in cartoon model.
Table 5.
Substrate interacting amino acids of chicken lysozyme and rat lysozyme like proteins.
Fig 6.
Expression of Lyzl genes in the male reproductive and non- reproductive tissues.
RNA isolated from different tissues of rats were reverse transcribed and used for gene specific PCR. Gapdh was used as internal control. Cp-caput, Co-corpus, Cd-cauda, T- testes, Sv-seminal vesicle, P-prostate, B-Brain, H- Heart, Lu-Lungs, Li-Liver, K-Kidney, Sp-Spleen, O-Ovary, Ut-Uterus, Ce-Cervix, Gapdh-Glyceraldehyde 3 phosphate. Expression pattern of some rat LYZL genes were reported by us earlier [24]and this figure includes our previously reported data to present a comprehensive analysis of all rat LYZL genes.
Fig 7.
Developmental regulation of Lyzl genes in epididymidis and testes.
RT PCR for Lyzl genes in epididymis and testes collected from rats of different age groups. 10, 20, 30, 40, 50 and 60 are the age of rats in days. cp- caput, cd-cauda. Gapdh was used as the internal control. Expression pattern of some rat LYZL genes were reported by us earlier [24] and this figure includes our previous data to present a comprehensive analysis of all rat LYZL genes.
Fig 8.
LYZL protein expression in male reproductive tract.
Immunoblotting of LYZL proteins in male reproductive tract tissues. The immunoblots were probed with immune serum specific to each protein. Cp-caput, Cd-cauda, T-testes, Sv-seminal vesicles, P-prostate, Spm-sperm, Rec-recombinant protein.
Fig 9.
Serial sections of the rat testes and epididymides were incubated with antigen preadsorbed immune serum (peptide control) or immune serum raised against LYZL1, followed by TRITC (Tetramethylrhodamine-5-(and-6)-Isothiocyanate (5(6)) tagged secondary antibody and counter stained with DAPI (4',6-diamidino-2-phenylindole) nuclear stain. Spermatozoa were stained with FITC (fluorescein Isothiocyanate) tagged secondary antibody.
Fig 10.
Serial sections of the rat testes and epididymides were incubated with antigen preadsorbed immune serum (peptide control) or immune serum raised against LYZL3, followed by TRITC (Tetramethylrhodamine-5-(and-6)-Isothiocyanate (5(6)) tagged secondary antibody and counter stained with DAPI (4',6-diamidino-2-phenylindole) nuclear stain. Spermatozoa were stained with FITC (fluorescein Isothiocyanate) tagged secondary antibody.
Fig 11.
Serial sections of the rat testes and epididymides were incubated with antigen preadsorbed immune serum (peptide control) or immune serum raised against LYZL4, followed by TRITC (Tetramethylrhodamine-5-(and-6)-Isothiocyanate (5(6)) tagged secondary antibody and counter stained with DAPI (4',6-diamidino-2-phenylindole) nuclear stain. Spermatozoa were stained with FITC (fluorescein Isothiocyanate) tagged secondary antibody. Panel D showing LYZL4 localization was reported by us earlier [24] and the same figure is being used for a comprehensive presentation of results.
Fig 12.
Serial sections of the rat testes and epididymides were incubated with antigen preadsorbed immune serum (peptide control) or immune serum raised against LYZL5, followed by TRITC (Tetramethylrhodamine-5-(and-6)-Isothiocyanate (5(6)) tagged secondary antibody and counter stained with DAPI (4',6-diamidino-2-phenylindole) nuclear stain. Spermatozoa were stained with FITC (fluorescein Isothiocyanate) tagged secondary antibody.
Fig 13.
Serial sections of the rat testes and epididymides were incubated with antigen preadsorbed immune serum (peptide control) or immune serum raised against LYZL6, followed by TRITC (Tetramethylrhodamine-5-(and-6)-Isothiocyanate (5(6)) tagged secondary antibody and counter stained with DAPI (4',6-diamidino-2-phenylindole) nuclear stain. Spermatozoa were stained with FITC (fluorescein Isothiocyanate) tagged secondary antibody.
Fig 14.
Serial sections of the rat testes and epididymides were incubated with antigen preadsorbed immune serum (peptide control) or immune serum raised against LYZL7, followed by TRITC (Tetramethylrhodamine-5-(and-6)-Isothiocyanate (5(6)) tagged secondary antibody and counter stained with DAPI (4',6-diamidino-2-phenylindole) nuclear stain. Spermatozoa were stained with FITC (fluorescein Isothiocyanate) tagged secondary antibody.
Fig 15.
Muramidase activity of LYZL proteins.
1 μM (■), 5 μM (▲) and 10 μM (X) of recombinant LYZL proteins were incubated with 2 ml of M. lysodeikticus culture and the O.D monitored at 450nm. Chicken lysozyme (1μM; ♦) was used as a positive control. Values shown are mean ± SD.
Fig 16.
Isopeptidase activity of recombinant LYZL proteins.
1 μM (■), 5 μM (▲) and 10 μM (X) of recombinant LYZL proteins were incubated with 1.75 mM L-γ-Glu-pNA in 0.05 M 3-(N-morpholino) propane sulfonic acid (MOPS) buffer, pH 7, containing 0.01M NaCl and the O.D monitored at 405nm. Chicken lysozyme (1μM; ♦) was used as positive control. Values shown are mean ± SD. The isopeptidase activity of LYZL4 was reported by us earlier [24] and the same data is included in this figure for a comprehensive presentation of results.
Fig 17.
Antibacterial activity of LYZL proteins.
E. coli XL-1 blue grown to mid-log phase were incubated with 0 (♦), 10 (■), 25 (▲), 50 (X) and 100 (*) μg/ml of recombinant protein for 30, 60, 90 and 120 min after the start of incubation. The assay mixtures were diluted, plated on LB agar plates and the colonies hand counted. Values shown are Mean ± S.D. The isopeptidase activity of LYZL4 was reported by us earlier [24] and the same data is included in this figure for a comprehensive presentation of results.
Fig 18.
Membrane permeabilizing ability of LYZL proteins.
(A) Dye based membrane potential measurement in E. coli treated with recombinant LYZL proteins using flow cytometry. A- MFI measurements of the cells in FITC, PE-Texas Red A and PerCP-Cy5-5-A channels. (B) Membrane potential measured in terms of ratio of mean fluorescence intensity of red/green. (C) Effect of recombinant LYZL1 and LYZL6 on the morphology of E. coli. Scanning electron micrographs of E. coli treated with 100 μg/ml recombinant LYZL proteins for 2 h. Chicken Lysozyme (10 μg/ml) was used as positive control.
Fig 19.
Substrate binding ability of LYZL proteins.
(A) ELISA based peptidoglycan binding assay. 40 μg/ml peptidoglycan coated plate was incubated with 0.25, 0.5, 0.75 and 1μM of the recombinant proteins. Affinity of protein bound to peptidoglycan was measured in terms of color produced during development after probing with corresponding primary and secondary antibody. (B) ELISA based hyaluronan binding assay. 40 μg/ml hyaluronan coated into the wells of a microtitre plate was incubated with 0.25, 0.5, 0.75 and 1μM of the recombinant protein. Protein binding to peptidoglycan was measured by ELISA based colour detection. ELISA index was calculated by subtracting the average O.D of negative control and dividing the resultant by the negative control O.D. (C) Hyaluronidase activity of LYZL proteins. Hyaluronan mixed agarose incubated with different concentrations of recombinant LYZL proteins. The amount of cetyl pyridinium chloride precipitate cleared is a measure of hyaluronidase activity. Hyaluronidase was used as positive control. (D) Free radical scavenging assay. DPPH solution was incubated with varying concentrations of recombinant LYZL protein and the decrease in O.D was measured at 517 nm. Lysozyme was used a control. Radical scavenging activity was expressed in terms of percentage. Values shown are mean ± SD. * indicates p<0.05 compared to the corresponding concentration of lysozyme.