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Differences in the activation mechanism between the alpha and beta subunits of human meprin
Christoph Becker1, Markus-N Kruse, Kristina A Slotty
1Institut für Zoophysiologie, Molekulare Physiologie, Westfälische Wilhelms-Universität Münster, Hindenburgplatz 55, D-48143 Münster, Germany.
Abstract:
Meprins are zinc-endopeptidases of the astacin family, which are expressed as membrane-bound or secreted forms in renal and intestinal brush-border membranes of mouse, rat and man. There are two types of meprin subunits, alpha and beta, which form disulfide-bonded homo- and heterodimers; further oligomerization is mediated by non-covalent interactions. Both subunits are translated as proenzymes that have to be activated by removal of an N-terminal propeptide. In the gut, the most probable activator is trypsin. In addition, plasmin has been shown to activate the human alpha subunit in colorectal cancer tissue. In the present study we have overexpressed the human meprin alpha subunit and a His-tagged soluble tail-switch-mutant of meprin beta in Baculovirus-infected insect cells. The recombinant homo-oligomeric proteins were purified by gel filtration and affinity chromatography with yields of up to 10 mg/l cell culture medium and analyzed with regard to their activation mechanism. While both alpha and beta homo-oligomers are activated by trypsin, only meprin alpha homo-oligomers are processed to their mature form by plasmin. These results indicate a different accessibility of the propeptide in meprin homo-oligomers and suggest an explanation for the appearance of meprin hetero-oligomers consisting of active alpha, but latent beta subunits.
Insights
Meprin alpha and beta are enzymes activated by trypsin. However, only meprin alpha is activated by plasmin, suggesting different propeptide accessibility in these important proteases.
Area of Science:
- Biochemistry
- Enzymology
- Protease research
Background:
- Meprins are astacin family zinc-endopeptidases found in renal and intestinal brush borders.
- They exist as alpha and beta subunits forming homo- and heterodimers, functioning as proenzymes requiring activation.
- Trypsin and plasmin are known activators, particularly plasmin for the alpha subunit in cancer tissues.
Purpose of the Study:
- To investigate the activation mechanisms of human meprin alpha and beta subunits.
- To compare the activation of recombinant homo-oligomeric meprins by trypsin and plasmin.
- To elucidate the differential activation contributing to meprin hetero-oligomer composition.
Main Methods:
- Overexpression of human meprin alpha and a soluble tail-switch mutant of meprin beta in insect cells using Baculovirus.
- Purification of recombinant homo-oligomeric proteins via gel filtration and affinity chromatography.
- Analysis of activation by trypsin and plasmin.
Main Results:
- Both meprin alpha and beta homo-oligomers are activated by trypsin.
- Only meprin alpha homo-oligomers are processed to their mature form by plasmin.
- Differential activation suggests varied propeptide accessibility in meprin homo-oligomers.
Conclusions:
- Meprin homo-oligomers exhibit distinct activation profiles for trypsin and plasmin.
- Plasmin selectively activates meprin alpha, while trypsin activates both alpha and beta.
- This differential activation provides a molecular basis for the formation of hetero-oligomers with active alpha and latent beta subunits.