Activation of the epithelial sodium channel by the metalloprotease meprin ß subunit.

The Epithelial Na(+) Channel (ENaC) is an apical heteromeric channel that mediates Na(+) entry into epithelial cells from the luminal cell surface. ENaC is activated by proteases that interact with the channel during biosynthesis or at the extracellular surface. Meprins are cell surface and secreted metalloproteinases of the kidney and intestine. We discovered by affinity chromatography that meprins bind γ-ENaC, a subunit of the ENaC hetero-oligomer. The physical interaction involves NH(2)-terminal cytoplasmic residues 37-54 of γ-ENaC, containing a critical gating domain immediately before the first transmembrane domain, and the cytoplasmic COOH-terminal tail of meprin ß (residues 679-704). This potential association was confirmed by co-expression and co-immunoprecipitation studies. Functional assays revealed that meprins stimulate ENaC expressed exogenously in Xenopus oocytes and endogenously in epithelial cells. Co-expression of ENaC subunits and meprin ß or α/ß in Xenopus oocytes increased amiloride-sensitive Na(+) currents approximately two-fold. This increase was blocked by preincubation with an inhibitor of meprin activity, actinonin. The meprin-mediated increase in ENaC currents in oocytes and epithelial cell monolayers required meprin ß, but not the α subunit. Meprin ß promoted cleavage of α and γ-ENaC subunits at sites close to the second transmembrane domain in the extracellular domain of each channel subunit. Thus, meprin ß regulates the activity of ENaC in a metalloprotease-dependent fashion.

Syk expression in peripheral blood leukocytes, CD34+ progenitors, and CD34-derived basophils.

In human basophils from different subjects, maximum IgE-mediated histamine release and the level of syk protein expression correlate well. It is not clear when in the basophil's lifetime the set-point for syk expression is reached or how expression levels are determined for a given individual. An examination of syk expression in peripheral blood eosinophils, neutrophils, monocytes, B and T cells, DCs, and NK cells showed that with the exception of T cells, basophils were unique in expressing low levels of syk. No correlations were observed between syk expression in basophils and other types of leukocytes, suggesting a unique mechanism of regulation for basophils. The expression level of syk in CD34+ progenitors was approximately 11-fold higher than in peripheral blood basophils, and it remained at this level during maturation of the cells in IL-3 to a cell with characteristics of peripheral blood basophils. Down-regulation of syk expression in the culture-derived basophils was induced by culturing under conditions of chronic aggregation of FcepsilonRI. Syk was down-regulated to peripheral blood basophil levels in 50% of the cells. Despite the chronic aggregation of FcepsilonRI, the cells retained the same expression of FcepsilonRI, histamine content, and morphological staining of granules as cells not experiencing chronic aggregation. These results suggest that chronic stimulation through FcepsilonRI during basophil maturation might be a mechanism for down-regulating syk expression, while retaining other characteristics associated with mature peripheral blood basophils.

Protease domain glycans affect oligomerization, disulfide bond formation, and stability of the meprin A metalloprotease homo-oligomer.

The meprin A homo-oligomer is a highly glycosylated, secreted zinc metalloprotease of the astacin family and metzincin superfamily. This isoform of meprin is composed of disulfide-bonded dimers of alpha subunits that further associate to form large, secreted megadalton complexes of 10 or more subunits. The aim of this study was to determine the sites of glycan attachment and to assess their ability to affect the formation and stability of the homo-oligomer. Nine of the ten potential N-linked glycosylation sites (Asn-41, Asn-152, Asn-234, Asn-270, Asn-330, Asn-426, Asn-452, Asn-546, and Asn-553) were found to be glycosylated in recombinant mouse meprin A using chemical and enzymatic deglycosylation methods and electrospray ionization mass spectrometry. Chemical cross-linking demonstrated that carbohydrates are at or near the noncovalent subunit interface. The removal of two glycans in the protease domain at Asn-234 and Asn-270, as well as one in the tumor necrosis factor receptor-associated factor domain at Asn-452, by a deglycosidase under nondenaturing conditions decreased the chemical and thermal stability of the homo-oligomer without affecting quaternary structure. Site-directed mutagenesis demonstrated that no single glycan was essential for oligomer formation; however, the combined absence of the glycans at Asn-152 and Asn-270 in the protease domain hindered intersubunit disulfide bond formation, prevented noncovalent associations, and abolished enzymatic activity. These studies provide insights into the role of glycans in the biosynthesis, activity, and stability of this extracellular protease.

Intersubunit and domain interactions of the meprin B metalloproteinase. Disulfide bonds and protein-protein interactions in the MAM and TRAF domains.

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 beta-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 mu) domain and one in the TRAF (tumor necrosis factor receptor-associated 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.