Abstract
Secreted forms of the alpha subunit of recombinant mouse meprin A include an NH2-terminal prosequence, a catalytic domain, and three COOH-terminal domains designated as MAM (meprin, A-5 protein, receptor protein-tyrosine phosphatase mu), MATH (meprin and TRAF homology), and AM (after MATH). In this study, the importance of these COOH-terminal domains for biosynthesis of secreted, activable forms of the protease was investigated. Transcripts of the meprin subunit truncated after the protease (alpha(1-275)), MAM (alpha(1-452)), and MATH (alpha(1-528)) domains or with individual domains deleted (DeltaMAM, DeltaMATH, and DeltaAM), were transfected into human embryonic kidney 293 cells. The wild-type subunit, DeltaMATH, DeltaAM, alpha(1-452), and alpha(1-528) were secreted into the media, although the DeltaAM mutant was secreted at very low levels. The DeltaMATH and alpha(1-452) mutants were not activable by limited proteolysis. The alpha(1-528) mutant was as active as wild-type meprin alpha against a bradykinin substrate, but had no activity against azocasein, and it, as all other mutants, was more vulnerable to extensive degradation by proteases than the wild-type protein. Pulse-chase experiments revealed that the DeltaMAM and alpha(1-275) mutants were rapidly degraded within cells. Treatment with lactacystin, a specific inhibitor of the proteasome, significantly decreased the degradation, indicating that the mutants lacking the MAM domain are degraded by the proteasome as misfolded proteins. These results indicate that the MAM domain is necessary for correct folding and transport through the secretory pathway, the MATH domain is required for folding of an activable zymogen, and the AM domain is important for activity against proteins and efficient secretion of the protein. The work demonstrates the interdependence of the domains for correct folding of an activable, stable, mature enzyme.
Highlights
Meprins belong to the “astacin family” of metalloendopeptidases and the “metzincin superfamily” (1, 2)
Wild-type Meprin ␣ and Mutants ␣(1–528), ␣(1– 452), and ⌬MATH Were Secreted into the Media—When the wild-type mouse meprin ␣ subunit cDNA was transfected into human embryonic kidney 293 cells, the protein was secreted into the medium with a molecular mass of approximately 95 kDa (Fig. 2A, lane 2)
The work clearly demonstrates that the MAM, MATH, and AM domains of meprin ␣ are essential for efficient transport of the protein to the cell surface and/or correct folding to generate an enzymatically active protease
Summary
Reagents and Materials—[35S]Methionine/cysteine was purchased from ICN Pharmaceuticals, Inc. Plasmid Construction and Mutagenesis—The pcDNA I/Amp plasmid expressing full-length wild-type mouse meprin subunit cDNA was described previously (9, 10). The ⌬MAM mutant was constructed with Met1–His[275] and Gly446– Glu[760] using the PCR amplification primers 5Ј-TACAACTGCACCGCAACACATGGGGTTTGGACCACTCGGAAATA-3Ј and 5Ј-TATTCCGGATGGTCCAAACCCCATGTGTTGCGGTGCAGTTG-3Ј These NH2-terminal and the COOH-terminal fragments were separately constructed, and both fragments were extended with Pfu DNA polymerase (Stratagene). The recombinant meprin ␣ wild-type or mutants were expressed in human embryonic kidney 293 cells after transfection by the calcium phosphate precipitation method using 10 g of expression plasmid and 1 g of helper plasmid pVA1/ 100-mm tissue culture plate (9). The sedimented cells were suspended in PBS containing 0.1% Triton X-100, sonicated for 1 min at 4 °C, and subjected to centrifugation at 100,000 ϫ g for 1 h; the supernatant fraction is referred to as the cell lysate.
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
