Expression of the alpha subunit of PABA peptide hydrolase (EC 3.4.24.18) in MDCK cells

Abstract

In this paper, we report the expression of PPH alpha in the polarized cell line MDCK (Madin Darby canine kidney). In these cells, the enzyme was synthesized in an inactive proform, which upon treatment with trypsin was activated. The enzyme isolated from cell extracts was core-glycosylated and appeared to be retained in the ER as a homodimer. No PPH alpha was detectable on the surface of intact cells by immunofluorescence. However, a complex glycosylated soluble but inactive form was present in the culture medium, suggesting that proteolytic removal of the C-terminal membrane anchoring peptide leads to the secretion of PPH alpha.

Polarised Expression of Human Intestinal N‐benzoyl‐l‐tyroxyl‐p‐aminobenzoic Acid Hydrolase (Human Meprin) α and β Subunits in Madin‐Darby Canine Kidney Cells

“…However, the cell-associated a subunit was an immature and transport-incompetent protein which did not reach the cell surface. Activation of both the soluble and cell-bound forms of the enzyme could be achieved by limited treatment with trypsin through the proteolytic removal of the propeptide [20].…”

Compartmentalised expression of meprin in small intestinal mucosa: enhanced expression in lamina propria in coeliac disease

“…In this respect, the foetal gut resembles the colon in the adult, where meprin b is not expressed and meprin a is secreted (Lottaz et al, 1999a). Secretion of meprin a in the absence of meprin b has been described previously in cell culture systems (Grü nberg et al, 1993;Eldering et al, 1997;Lottaz et al, 1999a) and meprin b knockout animals (Norman et al, 2003). The predominant presence of meprin a protein in the human foetal gut points to independent regulation of meprin a and b during development and maturation of the intestine.…”

“…The protease consists of homo-and heterooligomeric complexes of two homologous isoforms, meprin a and meprin b, which accumulate at the brush border membrane of villus enterocytes and are partially secreted into the gut lumen. Biosynthesis of meprin has been studied in intestinal biopsies in organ culture (Sterchi et al, 1988a) and in transfected mammalian cells (Grü nberg et al, 1993;Eldering et al, 1997;Pischitzis et al, 1999). Different forms were observed, generated by posttranslational modifications and proteolytic processing along the secretory pathway.…”

“…We analysed the expression of meprin in inflamed mucosa of untreated coeliac disease patients using Western blotting and probing with polyclonal antibodies generated in our laboratory and previously characterised (Grü nberg et al, 1993;Lottaz et al, 1999a). The analysis of human clinical specimens was approved by the Hackney and District Health Authority (London, UK) and the Ethics Committee of the Medical Faculty, University of Bern (Bern, Switzerland).…”

“…Multiple forms were detected in cell lysates, corresponding to precursors and proteolytically processed forms, as previously described (Grü nberg et al, 1993;Eldering et al, 1997;Pischitzis et al, 1999). Meprin a is constitutively expressed in foetal gut organ cultures and accumulates in culture supernatants.…”