Identification and characterization of neutral endopeptidase (EC 3. 4. 24. 11) from human prostasomes--localization in prostatic tissue and cell lines.

摘要

BACKGROUND: An antibody directed against a 100 kDa protein was immunoselected from a polyvalent antiserum against human prostasomes. The antibody as well as biochemical characteristics of the respective antigen were used to study the structural relationship of the latter with prostate membrane specific antigen (PMSA), another 100 kDa membrane protein of the prostate. METHODS: The isolated purified 100 kDa protein was characterized by tryptic degradation, aminoacid-sequencing and mass spectroscopy peptide-fingerprinting as well as mono-saccharide analysis and lectin binding and identified as a prostasomal neutral endopeptidase (NEP, EC 3.4.24.11). Immunohistochemistry, immunoelectron microscopy, in situ hybridization, and RT-PCR were performed to analyze the expression and distribution of the protein in normal and malignant human prostatic tissues and cell lines. RESULTS: Prostatic NEP, which has no relationship with PMSA, is a glycosylated, integral membrane protein type II. The prevalent glycosyl residues are NeuNAc, GlcNAc, GalNAc, Gal, Man, Fuc. NEP-mRNA is expressed in human prostatic epithelial and some stromal cells. NEP-immunoreactivity is strong in normal prostatic epithelium and confined to the apical plasma membrane. During apocrine secretion, the enzyme is released from the secretory cells, contributing to the formation of prostasomes. In prostate cancer specimens, immunoreactivity of apical plasma membranes is lost, while generalized cytoplasmic immunoreactivity develops. CONCLUSIONS: Prostatic secretory cells contain a membrane-bound, highly glycosylated neutral endopeptidase which is restricted to the apical plasma membrane. The enzyme is released from the cells in an apocrine fashion and contributes to the formation of prostasomes. In prostate cancer cells a preferential cytoplasmic localization is observed, pointing to alterations in intracellular targeting. Copyright 2001 Wiley-Liss, Inc.