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Homo- and heterotetrameric forms of the membrane-bound metalloendopeptidases meprin A and B
C M Gorbea1, A V Flannery, J S Bond
1Department of Biochemistry, Virginia Polytechnic Institute and State University, Blacksburg 24061-0308.
Abstract:
Meprin A and B are disulfide-linked, tetrameric metalloendopeptidases in renal brush border membranes. Meprin A contains 90-kDa subunits (alpha subunits) and is expressed in random-bred and some inbred strains of mice. Meprin B contains subunits of 110 kDa (beta subunits) in situ, and the enzyme from C3H mice, a strain that does not express alpha subunits, has been characterized. Evidence from this and previous studies indicate that beta subunits are expressed in all mouse strains. The tetrameric organization of these meprins was examined in brush border membrane fractions from a random-bred strain (ICR) and two inbred strains of mice (C57BL/6 and C3H/He). Lectin blotting using biotinylated concanavalin A revealed that membranes from the random-bred strain contained three oligomeric complexes of approximately 390, 440, and 490 kDa as determined after sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in the absence of reducing agents. The subunits in all three oligomers were linked by disulfide bridges. Western blotting using an anti-alpha monoclonal antibody indicated that alpha subunits (90 kDa) were present in the 390- and 440-kDa complexes. Western blotting with a polyclonal antibody specific for beta subunits (110 kDa) indicated the presence of these subunits in the 440- and 490-kDa complexes. Electroelution of the individual oligomers followed by SDS-PAGE under reducing conditions confirmed that the 390- and 490-kDa molecules are homotetramers of alpha and beta subunits, respectively, and that the 440-kDa molecule is a heterotetramer consisting of disulfide-bridged alpha and beta subunits. C57BL/6 mice expressed both alpha and beta subunits and contained tetramers composed of alpha 4 and alpha 2 beta 2. C3H/He mice expressed only the 110-kDa beta subunits and the beta 4 oligomer. This type of multimeric organization of disulfide-linked subunits is unique for the known endopeptidases.
Insights
Meprin A and B metalloendopeptidases exhibit unique tetrameric structures in mouse renal membranes. These enzymes form homo- and heterotetramers through disulfide linkages, revealing novel multimeric organization in endopeptidases.
Area of Science:
- Biochemistry
- Molecular Biology
- Enzymology
Background:
- Meprin A and B are metalloendopeptidases found in renal brush border membranes.
- Meprin A consists of 90-kDa alpha subunits, while Meprin B has 110-kDa beta subunits.
- Beta subunits are expressed in all mouse strains, but alpha subunits are strain-specific.
Purpose of the Study:
- To investigate the tetrameric organization of meprin A and B in different mouse strains.
- To characterize the subunit composition of meprin oligomers.
Main Methods:
- Lectin blotting with biotinylated concanavalin A to detect oligomeric complexes.
- Western blotting using specific antibodies for alpha and beta subunits.
- Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing and non-reducing conditions.
- Electroelution of oligomers for subunit analysis.
Main Results:
- Three oligomeric complexes (390, 440, and 490 kDa) were identified in random-bred mice.
- The 390-kDa complex is a homotetramer of alpha subunits (α4).
- The 490-kDa complex is a homotetramer of beta subunits (β4).
- The 440-kDa complex is a heterotetramer of alpha and beta subunits (α2β2).
- C57BL/6 mice expressed α4 and α2β2 tetramers, while C3H/He mice only expressed β4 tetramers.
Conclusions:
- Meprin A and B form unique disulfide-linked tetrameric structures.
- The multimeric organization varies based on the expression of alpha and beta subunits in different mouse strains.
- This tetrameric organization is a novel characteristic among known endopeptidases.