Identification and characterization of neutral endopeptidase (EC 3.4.24.11) from human prostasomes - localization in prostatic tissue and cell lines

BACKGROUND. An antibody directed against a 100 kDa protein was immunoselect ed from a polyvalent antiserum against human prostasomes. The antibody as w ell as biochemical characteristics of the respective antigen were used to s tudy the structural relationship of the latter with prostate membrane speci fic 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-fingerprin ting as well as monosaccharide analysis and lectin binding and identified a s a prostasomal neutral endopeptidase (NEP, EC 3.4.24.11). Immunohistochemi stry, immunoelectron microscopy, in situ hybridization, and RT-PCR were per formed 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 glycosyla ted, integral membrane protein type II. The prevalent glycosyl residues are NeuNAc, GlcNAc, GalNAc, Gal, Man, Fuc. NEP-mRNA is expressed in human pros tatic epithelial and some stromal cells. NEP-immunoreactivity is strong in normal prostatic epithelium and confined to the apical plasma membrane. Dur ing apocrine secretion, the enzyme is released from the secretory cells, co ntributing to the formation of prostasomes. In prostate cancer specimens, i mmunoreactivity of apical plasma membranes is lost, while generalized cytop lasmic immunoreactivity develops. CONCLUSIONS. Prostatic secretory cells contain a membrane-bound, highly gly cosylated neutral endopeptidase which is restricted to the apical plasma me mbrane. The enzyme is released from the cells in an apocrine fashion and co ntributes to the formation of prostasomes. In prostate cancer cells a prefe rential cytoplasmic localization is observed, pointing to alterations in in tracellular targeting. (C) 2001 Wiley-Liss, Inc.