Abstract
Meprin A secreted from kidney and intestinal epithelial cells is capable of cleaving growth factors, extracellular matrix proteins, and biologically active peptides. The secreted form of meprin A is a homo-oligomer composed of alpha subunits, a multidomain protease of 582 amino acids coded for near the major histocompatibility complex of the mouse and human genome. Analyses of the recombinant homo-oligomeric form of mouse meprin A by gel filtration, nondenaturing gel electrophoresis, and cross-linking (with disuccinimidyl suberate or N-(4-azido-2,3,5,6-tetraflourobenzyl)-3-maleimidylpropionamide) indicate that the secreted enzyme forms high molecular weight multimers, with a predominance of decamers. The multimers are composed of disulfide-linked dimers attached noncovalently by interactions involving the meprin, A5 protein, receptor protein-tyrosine phosphatase mu (MAM) domain. The active protomer is the noncovalently linked dimer. Linkage of active protomers by disulfide-bonds results in an oligomer of approximately 900 kDa, which is unique among proteases and distinguishes meprin A as the largest known secreted protease. Electron microscopy revealed that the protein was present in two states, a crescent-shaped structure and a closed ring. It is concluded from this and other data that the covalent attachment of the protomers enables noncovalent associations of the native enzyme to form higher oligomers that are critical for hydrolysis of protein substrates.
Highlights
Meprins are zinc endopeptidases that are secreted from or found in brush border membranes of kidney and intestinal epithelial cells of humans and rodents
Meprin ␣ cDNA (Structure I) encodes a 760-amino acid protein with an NH2-terminal signal sequence followed by a prosequence, the protease domain, a MAM1 domain, a MATH domain, followed by an AM domain, an EGF-like domain, a putative COOH-terminal transmembrane domain (TM), and a cytoplasmic (C) tail
The Meprin A Homo-oligomer Is Capable of Forming High Molecular Weight Structures—When purified recombinant mouse meprin A homo-oligomer was subjected to nondenaturing, nonreducing gel electrophoresis, the native protein migrated as a species larger than the 669,000 molecular weight marker (Fig. 1A)
Summary
Meprins are zinc endopeptidases that are secreted from or found in brush border membranes of kidney and intestinal epithelial cells of humans and rodents. This work indicated that the oligomeric state is critical for activity and stability of the enzyme but could not determine whether the properties exhibited by the monomeric mutant were due to lack of formation of the disulfide-linked dimer or the inability of the subunits to associate noncovalently.
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