Molecular cloning and characterization of prostase, an androgen-regulated serine protease with prostate-restricted expression

Abstract

The identification of genes with selective expression in specific organs or cell types provides an entry point for understanding biological processes that occur uniquely within a particular tissue. Using a subtraction approach designed to identify genes preferentially expressed in specific tissues, we have identified prostase, a human serine protease with prostate-restricted expression. The prostase cDNA encodes a putative 254-aa polypeptide with a conserved serine protease catalytic triad and an amino-terminal pre-propeptide sequence, indicating a potential secretory function. The genomic sequence comprises five exons and four introns and contains multiple copies of a chromosome 19q-specific minisatellite repeat. Northern analysis indicates that prostase mRNA is expressed in hormonally responsive normal and neoplastic prostate epithelial tissues, but not in prostate stromal constituents. Prostase shares 35% amino acid identity with prostate-specific antigen (PSA) and 78% identity with the porcine enamel matrix serine proteinase 1, an enzyme involved in enamel matrix degradation and with a putative role in the disruption of intercellular junctions. Radiation-hybrid-panel mapping localized prostase to chromosome 19q13, a region containing several other serine proteases, including protease M, pancreatic/renal kallikrein hK1, and the prostate-specific kallikreins hK2 and hK3 (PSA). The sequence homology between prostase and other well-characterized serine proteases suggests several potential functional roles for the prostase protein that include the degradation of extracellular matrix and the activation of PSA and other proteases.

Figure 1

The sequences of prostase intron/exon boundaries.

Figure 2

The DNA and derived amino acid sequence of human prostase. The number of the last nucleotide in each row is indicated on the right. The putative signal peptide sequence is underlined with the predicted cleavage site indicated by an upward arrow. The propeptide sequence is boxed with the predicted cleavage site indicated by a downward arrow. Cysteine residues involved in putative disulfide bonds are marked with an ∗. The catalytic triad active-site residues (H, D, S) are boxed. One potential N-linked glycosylation site (N¹¹⁰) is circled. The polyadenylation signal is underlined twice.

Figure 3

Alignment of the deduced amino acid sequence of prostase with the serine proteases EMSP1, SCCE, protease M, hK1, hK2, hK3, rat KLK8, mouse KLK1, and trypsin. GenBank accession numbers are listed. Amino acid sequence identity (black) or similarity (gray) between prostase and at least one other protein is shaded. Dashes represent gaps to bring the sequences to better alignment. The serine protease catalytic triad of histidine, aspartic acid, and serine are boxed vertically and denoted with an ∗. The 29 invariant serine protease residues are indicated by ●. The Asn residue conferring trypsin cleavage specificity is indicated by a cross. The hatched horizontal bar indicates the kallikrein loop sequence.

Figure 4

Multiple tissue Northern analysis of prostase. (A) Prostase is expressed specifically in normal prostate tissue with a transcript size of approximately 1.3 kb. (B) A human glyceraldehyde-3-phosphate dehydrogenase probe was used as a control for equivalent RNA loading. Tissue types are indicated at the top of each lane.

Figure 5

Androgen regulation of prostase expression. (A) Northern analysis by using a prostase probe with RNA extracted from normal prostate (NP), LNCaP at steady state (SS), LNCaP after 24 hr of androgen deprivation (0), LNCaP at specified hours after androgen exposure (1, 2, 4, 8, 24, and 48), the PC3 (PC3) and DU145 (DU145) prostate cancer cell lines, the androgen dependent (PXe-AD) and androgen independent (PXe-AI) prostate cancer xenografts, and prostate stroma (PS). A probe for β-actin is used to estimate RNA loading. (B) Relative prostase expression levels. Expression ratios are determined by normalizing all samples for β-actin intensity relative to the LNCaP steady-state (SS) sample. Prostase expression for each sample is shown in PhosphorImager intensity units relative to the SS LNCaP sample. (C) Northern analysis of prostase expression in LNCaP cells grown either with (+) or without (−) androgen (1 nM R1881) for 72 or 144 hr. A probe for β-actin is used to estimate RNA loading.