Expression of meprin subunit precursors. Membrane anchoring through the beta subunit and mechanism of zymogen activation

• Published in: The Journal of biological chemistry Vol. 269; no. 10; pp. 7682 - 7688
• Main Authors: JOHNSON, G. D, HERSH, L. B
• Format: Journal Article
• Language: English
• Published: Bethesda, MD American Society for Biochemistry and Molecular Biology 11-03-1994
• Subjects: Amino Acid Sequence; Analytical, structural and metabolic biochemistry; Animals; Base Sequence; Biological and medical sciences; Cell Line; Cell Membrane - metabolism; DNA, Complementary; Enzyme Activation; Enzyme Precursors - metabolism; Enzymes and enzyme inhibitors; Epitopes; Fundamental and applied biological sciences. Psychology; Humans; Hydrolases; Metalloendopeptidases - metabolism; Molecular Sequence Data; Proto-Oncogene Proteins c-myc - immunology; Rats; Recombinant Proteins - metabolism; Molecular processing; Rat; Enzyme; Meprin A; Rodentia; Metalloendopeptidases; Subunit; Vertebrata; Mammalia; Plasma membrane; Molecular association; Hydrolases; Biosynthesis precursor; Recombinant protein; Proteinases
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(17)37341-6

The biosynthesis of the membrane-bound metalloendopeptidase meprin was studied after the introduction of cDNAs encoding the
rat meprin alpha and beta subunits into human 293 cells. When expressed individually the meprin alpha subunit was found to
be primarily secreted into the culture medium, while the beta subunit was determined to be an integral membrane protein. Coexpression
of the alpha and beta subunits results in the localization of both subunits to the plasma membrane. In this case the alpha
subunit is specifically released from the cell surface by dithiothreitol, indicating the alpha subunit is associated with
the membrane via disulfide bond(s) to the beta subunit. Meprin expressed in 293 cells is similar to the rat kidney enzyme
in that it forms disulfide-linked dimers and has a similar pattern of glycosylation. However, the expressed protein displayed
no detectable peptidase activity against four meprin substrates. Processing of the alpha subunit was followed after the introduction
of sequences coding for the human c-myc peptide epitope EQKLISEEDL into its cDNA in the region of its prosequence and at the
COOH terminus. Detection of the resulting proteins using a monoclonal antibody specific for the c-myc epitope indicates the
alpha subunit is processed at its COOH terminus but retains the prosequence which is absent from the enzyme purified from
rat kidney. Limited trypsin digestion of meprin precursors expressed in 293 cells results in both the activation of the enzyme
and the removal of the prosequence. This result supports the hypothesis of Bode et al. (Bode W., Gomis-Ruth, F. X., Huber,
R., Zwilling, R., and Stocker, W. (1992) Nature 358, 164-167) that meprin and other astacin family proteases require removal
of NH2-terminal prosequences at the junction of the astacin protease domain for zymogen activation. Trypsin-activated meprin
was assayed with the protein substrate azocasein and three synthetic peptide substrates. The membrane-bound beta subunit was
found to be more active than the secreted alpha subunit against azocasein but much less active toward the synthetic peptide
substrates.