Structural basis for the sheddase function of human meprin ß metalloproteinase at the plasma membrane.

Ectodomain shedding at the cell surface is a major mechanism to regulate the extracellular and circulatory concentration or the activities of signaling proteins at the plasma membrane. Human meprin ß is a 145-kDa disulfide-linked homodimeric multidomain type-I membrane metallopeptidase that sheds membrane-bound cytokines and growth factors, thereby contributing to inflammatory diseases, angiogenesis, and tumor progression. In addition, it cleaves amyloid precursor protein (APP) at the ß-secretase site, giving rise to amyloidogenic peptides. We have solved the X-ray crystal structure of a major fragment of the meprin ß ectoprotein, the first of a multidomain oligomeric transmembrane sheddase, and of its zymogen. The meprin ß dimer displays a compact shape, whose catalytic domain undergoes major rearrangement upon activation, and reveals an exosite and a sugar-rich channel, both of which possibly engage in substrate binding. A plausible structure-derived working mechanism suggests that substrates such as APP are shed close to the plasma membrane surface following an "N-like" chain trace.

The substrate degradome of meprin metalloproteases reveals an unexpected proteolytic link between meprin ß and ADAM10.

The in vivo roles of meprin metalloproteases in pathophysiological conditions remain elusive. Substrates define protease roles. Therefore, to identify natural substrates for human meprin α and ß we employed TAILS (terminal amine isotopic labeling of substrates), a proteomics approach that enriches for N-terminal peptides of proteins and cleavage fragments. Of the 151 new extracellular substrates we identified, it was notable that ADAM10 (a disintegrin and metalloprotease domain-containing protein 10)-the constitutive α-secretase-is activated by meprin ß through cleavage of the propeptide. To validate this cleavage event, we expressed recombinant proADAM10 and after preincubation with meprin ß, this resulted in significantly elevated ADAM10 activity. Cellular expression in murine primary fibroblasts confirmed activation. Other novel substrates including extracellular matrix proteins, growth factors and inhibitors were validated by western analyses and enzyme activity assays with Edman sequencing confirming the exact cleavage sites identified by TAILS. Cleavages in vivo were confirmed by comparing wild-type and meprin(-/-) mice. Our finding of cystatin C, elafin and fetuin-A as substrates and natural inhibitors for meprins reveal new mechanisms in the regulation of protease activity important for understanding pathophysiological processes.

The metalloprotease meprin ß generates amino terminal-truncated amyloid ß peptide species.

The amyloid ß (Aß) peptide, which is abundantly found in the brains of patients suffering from Alzheimer disease, is central in the pathogenesis of this disease. Therefore, to understand the processing of the amyloid precursor protein (APP) is of critical importance. Recently, we demonstrated that the metalloprotease meprin ß cleaves APP and liberates soluble N-terminal APP (N-APP) fragments. In this work, we present evidence that meprin ß can also process APP in a manner reminiscent of ß-secretase. We identified cleavage sites of meprin ß in the amyloid ß sequence of the wild type and Swedish mutant of APP at positions p1 and p2, thereby generating Aß variants starting at the first or second amino acid residue. We observed even higher kinetic values for meprin ß than BACE1 for both the wild type and the Swedish mutant APP form. This enzymatic activity of meprin ß on APP and Aß generation was also observed in the absence of BACE1/2 activity using a ß-secretase inhibitor and BACE knock-out cells, indicating that meprin ß acts independently of ß-secretase.

Metalloprotease meprin beta generates nontoxic N-terminal amyloid precursor protein fragments in vivo.

Identification of physiologically relevant substrates is still the most challenging part in protease research for understanding the biological activity of these enzymes. The zinc-dependent metalloprotease meprin ß is known to be expressed in many tissues with functions in health and disease. Here, we demonstrate unique interactions between meprin ß and the amyloid precursor protein (APP). Although APP is intensively studied as a ubiquitously expressed cell surface protein, which is involved in Alzheimer disease, its precise physiological role and relevance remain elusive. Based on a novel proteomics technique termed terminal amine isotopic labeling of substrates (TAILS), APP was identified as a substrate for meprin ß. Processing of APP by meprin ß was subsequently validated using in vitro and in vivo approaches. N-terminal APP fragments of about 11 and 20 kDa were found in human and mouse brain lysates but not in meprin ß(-/-) mouse brain lysates. Although these APP fragments were in the range of those responsible for caspase-induced neurodegeneration, we did not detect cytotoxicity to primary neurons treated by these fragments. Our data demonstrate that meprin ß is a physiologically relevant enzyme in APP processing.