Meprin β (Mβ) is a multidomain type-I membrane metallopeptidase that sheds membrane-anchored substrates, releasing their soluble forms. Fetuin-B (FB) is its only known endogenous protein inhibitor. Herein, we analyzed the interaction between the ectodomain of Mβ (MβΔC) and FB, which stabilizes the enzyme and inhibits it with subnanomolar affinity. The MβΔC:FB crystal structure reveals a ∼250-kDa, ∼160-Å polyglycosylated heterotetrameric particle with a remarkable glycan structure. Two FB moieties insert like wedges through a “CPDCP trunk” and two hairpins into the respective peptidase catalytic domains, blocking the catalytic zinc ions through an “aspartate switch” mechanism. Uniquely, the active site clefts are obstructed from subsites S4 to S10′, but S1 and S1′ are spared, which prevents cleavage. Modeling of full-length Mβ reveals an EGF-like domain between MβΔC and the transmembrane segment that likely serves as a hinge to transit between membrane-distal and membrane-proximal conformations for inhibition and catalysis, respectively.
The metalloproteinase ovastacin is released by the mammalian egg upon fertilization and cleaves a distinct peptide bond in zona pellucida protein 2, a component of the enveloping extracellular matrix. This limited proteolysis causes zona pellucida hardening, abolishes sperm binding and thereby regulates fertility. Accordingly, this process is tightly controlled by the plasma protein fetuin-B, an endogenous competitive inhibitor. At present, little is known about how the cleavage characteristics of ovastacin differ from closely related proteases. Physiological implications of ovastacin beyond ZP2 cleavage are still obscure. In this study, we employed N-terminal amine isotopic labeling of substrates (N-TAILS) contained in the secretome of mouse embryonic fibroblasts to elucidate the substrate specificity and the precise cleavage site specificity. Furthermore, we were able to unravel the physicochemical properties governing enzyme-substrate interactions. Eventually, we identified several potential physiological substrates with significance for mammalian fertilization. These data suggest that ovastacin might regulate sperm-oocyte interaction and fertility beyond zona pellucida hardening.
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