Intersubunit and Domain Interactions of the Meprin B Metalloproteinase

Abstract

Meprins, multimeric metalloproteases expressed in kidney and intestinal epithelial cells as well as in certain leukocytes and cancer cells, have the ability to hydrolyze a variety of growth factors, vasoactive peptides, cytokines, and extracellular matrix proteins. The meprin B isoform exists primarily as a cell-surface homooligomer composed of disulfide-linked, multidomain ␤-subunits. To gain insight into how the tertiary and quaternary structure of meprin B affects function, the disulfide-bonding pattern and sites of domain-domain interactions were investigated using sedimentation equilibrium ultracentrifugation, cross-linking, and mass spectrometry techniques. Three symmetrical intersubunit disulfide bonds were identified in the noncatalytic interaction domains; two in the MAM (meprin, A-5 protein, protein-tyrosine phosphatase ) domain and one in the TRAF (tumor necrosis factor receptorassociated factor) domain. These disulfide bridges are unique for the known homophilic interactions of these domains. Mutation of any of the intersubunit cysteine residues resulted in the inability of meprin B to form disulfide-linked dimers. The four cysteines of the protease domain formed intradomain disulfide bonds. The MAM domain also had one intradomain disulfide bond and one free cysteine. Cross-linking studies of the meprin B dimer with the amine-reactive cross-linker disuccinimidyl suberate revealed inter-and intradomain contacts within the protein, including prosequence-prosequence, protease-TRAF, protease-epidermal growth factor, and TRAF-TRAF interactions. From these observations, a model of the meprin B dimer structure is proposed that provides insight into the relationship between structure and function of this isoform.

Diet1 Functions in the FGF15/19 Enterohepatic Signaling Axis to Modulate Bile Acid and Lipid Levels

“…Accounting for a large portion of the protein are 9 copies of the MAM (meprin-A5-protein tyrosine phosphatase mu) domain, a 170 amino acid domain that contains 4 conserved cysteine residues that likely form two disulfide bridges (Beckmann and Bork, 1993). The MAM domain is a component of numerous proteins with diverse functions, and has been implicated in protein-protein interactions, signal transduction, and cell-cell adhesion (Cismasiu et al, 2004; Ishmael et al, 2005). Also present in Diet1 are 9 low density lipoprotein receptor (class A) domains, an epidermal growth factor-like (subtype 2) domain, and a putative transmembrane domain near the C-terminus (Figure 1E).…”

Protease Domain Glycans Affect Oligomerization, Disulfide Bond Formation, and Stability of the Meprin A Metalloprotease Homo-oligomer

“…In this process, nonnative disulfide pairings form first and are subsequently rearranged by an oxidoreductase (37). If cysteine residues are missing, as in the case of meprin cysteine mutants, the shuffling process is stalled in nonnative pairings and intersubunit disulfide bonds do not form (10,13,31). Misfolding and/or the inability to form the two intradomain disulfide bonds in the protease domain could trap the protein in a conformation that does not allow crucial cysteines in the protease and/or MAM domains to interact with the oxidoreductase or to find their native bonding partners.…”

“…Depending on the orientation of the glycans with respect to the surface of the protein, the glycans may also shield vulnerable regions of the oligomer from other proteases in the extracellular milieu, as was demonstrated for the prion glycoprotein (42). The glycans of the MAM and TRAF domains were originally hypothesized to be the most likely candidates to influence the oligomeric state of meprin A, given that these domains mediate oligomerization in other proteins and contain other elements that direct meprin A oligomerization (10,12,13,31). However, the glycans in these domains were not necessary by themselves to form the large molecular mass oligomers.…”

“…There were no ions corresponding to either the unmodified or glycosylated peptides for Asn-330 and Asn-426; therefore, the occupancy of these sites could not be determined from this experiment. Based on mutagenesis and structural studies of meprins and protein-tyrosine phosphatase , MAM domains are highly disulfide-bonded (31)(32)(33). Incomplete cleavage of the protein in this region because of the disulfide bonds may be one reason why the MAM domain peptides were not observed.…”

Prointerleukin-18 Is Activated by Meprin β in Vitro and in Vivo in Intestinal Inflammation

“…Both subunits contain signal sequences that direct the proteins to the secretory pathway, prosequences that keep the enzymes inactive, proteolytic domains of the "astacin family of metalloproteinases," and other C-terminal non-catalytic domains (13). The subunits form homo-or heteromeric dimers that are linked by disulfide bonds (14). During biosynthesis, the meprin ␣ subunit is proteolytically cleaved near the C terminus and thus loses its epidermal growth factor-like, transmembrane, and cytoplasmic domains.…”