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

ClustalW2 score for rat PATE proteins.

<p>ClustalW2 score for rat PATE proteins.</p

Antibacterial activities of rat PATE and PATE-F and their response to endotoxin challenge.

<p><b>A,</b> Mid-log phase <i>E. coli</i> were incubated with 0 (○), 10 (▪), 25 (▴), 50 (•) and 100 (♦) µg/ml rat recombinant PATE or 100 µg/ml of PATE-F (◊) protein for 0–180 min. After incubation, the assay mixture was serially diluted with 10 mM phosphate buffered saline (pH 7.4), plated on LB plates and incubated overnight. Colonies were hand counted. Values shown are Mean ± S.D. * indicates p<0.05. <b>B,</b> Rat epididymides were maintained in nutritive medium (136.89 mM NaCl, 5.63 mM KCl, 1.80 mM CaCl₂, 0.36 mM NaH₂PO₄, 14.88 mM NaHCO₃ and 5.55 mM glucose pH 7.6–7.8) and challenged with LPS (1 µg/ml) for 0, 3, 6 and 9 h. RNA was isolated after LPS treatment and the expression of rat <i>Pate</i> in the epididymides was analyzed by RT-PCR. <b>C,</b> Rats were challenged with a single intraperitoneal dose (1 mg/kg body weight) of LPS and epididymides were collected at 0, 3, 6, 9, 15 and 24 h after injection. RNA isolated from the epididymides was used for <i>Pate</i> gene expression.</p

Multiple sequence alignment of PATE proteins.

<p>Alignment of rat PATE proteins. Signal peptide is shown in blue. The characteristic ten cysteines are indicated in red and underlined. The conserved amino acid residues are shaded.</p

<i>Pate</i> gene expression in the epididymides of developing rats.

<p>RNA was isolated from the epididymides of 20–60 day old rats, reverse transcribed and PCR for <i>Pate</i> genes performed. Caput, corpus and cauda obtained from 50 and 60 day old animals are indicated as <b>Cp</b>, <b>Co</b> and <b>Cu</b> respectively.</p

Gene specific primers used in this study.

<p>Gene specific primers used in this study.</p

Immunofluorescence detection of PATE and PATE-F on rat sperm.

<p>Cauda epididymides from adult rats were dissected out and the spermatozoa collected were air dried and fixed on glass slides by methanol. PATE and PATE-F localization was carried out by incubating with PATE and PATE-F polyclonal antibodies raised in rabbit followed by FITC conjugated secondary antibodies against rabbit IgG raised in goat. Counter staining was carried out using DAPI. <b>A–C</b>, preimmune serum. <b>D–F</b>, immune serum. Magnification – 60×.</p

Expression of <i>Pate</i> genes in the male reproductive tract of rat.

<p>Total RNA isolated from the caput, corpus, cauda, testis, seminal vesicle and prostate of adult rats was reverse transcribed and <i>Pate</i> mRNAs amplified using gene specific primers. Arrows indicate the representative alternate transcripts of <i>Pate</i> and <i>Pate-2</i>.</p

Tissue distribution of <i>Pate</i> genes in the rat.

<p>RT-PCR analysis was performed using total RNA isolated from <b>B</b>rain, <b>H</b>eart, <b>L</b>ung, <b>Li</b>ver, <b>K</b>idney, <b>Sp</b>leen, <b>O</b>vary, <b>Ut</b>erus and <b>Ce</b>rvix of adult rats.</p

Immunolocalization of rat PATE and PATE-F in the epididymis.

<p>Serial sections of the rat tissues were subjected to antigen retrieval in citrate buffer pH 6.0. They were then probed with polyclonal antibodies (1∶250 dilution) raised in rabbit against PATE and PATE-F followed by TRIC (for PATE) or FITC (for PATE-F) conjugated secondary antibody (1∶500 dilution) against rabbit IgG raised in goat. Sections were counter-stained with DAPI. Panels A–C – preimmune serum; D–F – immune serum. Magnification – 10×.</p