Genomic Organization, Tissue Distribution and Functional Characterization of the Rat Pate Gene Cluster

The cysteine rich prostate and testis expressed (Pate) proteins identified till date are thought to resemble the three fingered protein/urokinase-type plasminogen activator receptor proteins. In this study, for the first time, we report the identification, cloning and characterization of rat Pate gene cluster and also determine the expression pattern. The rat Pate genes are clustered on chromosome 8 and their predicted proteins retained the ten cysteine signature characteristic to TFP/Ly-6 protein family. PATE and PATE-F three dimensional protein structure was found to be similar to that of the toxin bucandin. Though Pate gene expression is thought to be prostate and testis specific, we observed that rat Pate genes are also expressed in seminal vesicle and epididymis and in tissues beyond the male reproductive tract. In the developing rats (20–60 day old), expression of Pate genes seem to be androgen dependent in the epididymis and testis. In the adult rat, androgen ablation resulted in down regulation of the majority of Pate genes in the epididymides. PATE and PATE-F proteins were found to be expressed abundantly in the male reproductive tract of rats and on the sperm. Recombinant PATE protein exhibited potent antibacterial activity, whereas PATE-F did not exhibit any antibacterial activity. Pate expression was induced in the epididymides when challenged with LPS. Based on our results, we conclude that rat PATE proteins may contribute to the reproductive and defense functions

Role of Gingipains in Growth of Porphyromonas gingivalis in the Presence of Human Serum Albumin

Porphyromonas gingivalis, a bacterium associated with active chronic periodontitis lesions, produces several proteolytic enzymes that are thought to be involved in host colonization, perturbation of the immune system, and tissue destruction. The aim of the present study was to investigate the contribution of Arg- and Lys-gingipains produced by P. gingivalis to its growth. Although all of the proteins studied were degraded by P. gingivalis, only human serum albumin and transferrin supported growth during serial transfers in a chemically defined medium (CDM). Growth studies with site-directed gingipain-deficient mutants of P. gingivalis revealed that inactivation of both gingipains prevents growth, whereas inactivation of either Arg- or Lys-gingipain activity extended the doubling times to 33 or 13 h, respectively, compared to 9 h for the parent strain. Growth of the mutants and the parent strain was similar when the CDM was supplemented with a protein hydrolysate instead of human serum albumin. Incubation of resting P. gingivalis ATCC 33277 cells with fluorophore-labeled albumin indicated that the proteolytic fragments generated by the gingipains were internalized by the bacterial cells. Internalization of fluorophore-labeled albumin fragments was reduced or completely inhibited in the proteinase-deficient mutants. Interestingly, gingival crevicular fluid samples from diseased periodontal sites contained low-molecular-mass albumin fragments, whereas samples from healthy sites did not. The critical role of proteinases in the growth of P. gingivalis was further investigated using specific Arg- and Lys-gingipain inhibitors. Adding the inhibitors to CDM containing albumin revealed that leupeptin (Arg-gingipain A and B inhibitor) was more efficient at inhibiting growth than cathepsin B inhibitor II (Lys-gingipain inhibitor). Our study suggests that Arg-gingipains and, to a lesser extent, Lys-gingipain play an important role in the growth of P. gingivalis in a defined medium containing a human protein as the sole carbon and nitrogen source