N‐Benzoyl‐l‐tyrosyl‐p‐aminobenzoic acid hydrolase (PPH, human meprin), is a peptidase found in the microvillus membrane of human small intestinal epithelial cells. PPH belongs to the astacin family ...of zinc‐metalloendopeptaidases and is a protein complex composed of two glycosylated subunits, α and β. The present report describes the cloning of the complete β subunit and the remaining N2‐terminal end of the a subunit for analysis of their primary structures in addition to the examination of their biogenesis in transfected cell cultures. The complete open reading frame of the PPHβ cDNA translates into 700 amino acid residues compared with 746 residues for the PPHα cDNA. The primary structure of β and α subunits are 44% identical and 61% similar. As predicted from their primary structure, the two subunits of PPH have identical modular structures; starting at the N2‐terminus both contain a signal peptide, a propeptide, a protease domain containing the astacin signature, a meprin A5 protein tyrosine phospatase μ (MAM) and a meprin and TRAF homology domain (MATH) domain, an epidermal growth factor(EGF)‐like domain, a putative transmembrane anchor domain and a short cytosolic tail. Pulsekhase labelling and immuno‐Gold lectronmicroscopy of recombinant PPH β and α subunits expressed in transfected Madin‐Darby canine kidney (MDCK) cells show that post‐translational processing and transport of the two subunits are very different. When expressed alone, the β subunit acquired complex glycan residues, readily formed homodimers and was transported to the plasma membrane. Small amounts of PPHP were found in the culture medium. In contrast, the cell‐bound a subunit, when expressed alone, remained primarily in the high‐mannose form, was aggregated and not expressed at the cell surface. However, the bulk of mostly endo‐β‐N‐acetylglucosaminidase H‐resistant α subunit was found in the filtered culture medium. The proteolytic event that leads to the formation of this soluble transport‐competent form occurs in the endoplasmic reticulum (ER). Coexpression of the α subunit with the β subunit allowed the localisation of the a subunit to the plasma membrane. These studies indicate that assembly of the two subunits of PPH is required for the localisation of the α subunit to the plasma membrane. In contrast to rodent meprin, both PPH subunits are apically secreted from MDCK cells.
An ovarian carcinoma cell line (OTN 11) was produced from the ascitic fluid of a patient with a moderately to well differentiated papilliferous cystadenocarcinoma of the ovary. The cell line was ...characterized using electron microscopy karyotyping, immunohistochemical techniques with monoclonal antibodies against keratins as epithelial markers, and the monoclonal antibodies OV-TL 3 and OC 125 as ovarian carcinoma markers. These techniques revealed the epithelial and adenocarcinomatous nature of the cell line and the presence of ovarian carcinoma-related surface markers. The adenocarcinomatous nature of the cell line also became apparent after heterotransplantation of cell suspensions into nude mice and nude rats, in which adenomatous tumor structures were formed. These xenografts had the same ultrastructural and immunohistochemical properties as the cell line. Despite the adenocarcinomatous character of the tumor the cultured cells release estradiol into the culture medium. We may conclude that OTN 11 is an ovarian carcinoma cell line which has retained highly differentiated functions, such as the production of an ovarian hormone.
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