Meprin is a zinc endopeptidase of the astacin family, which is expressed as a membrane-bound or secreted protein in mammalian epithelial cells, in intestinal leucocytes and in certain cancer cells. There are two types of meprin subunits, alpha and beta, which form disulphide-bonded homo- and hetero-oligomers. Here we report on the cleavage of matrix proteins by hmeprin (human meprin) alpha and beta homo-oligomers, and on the interactions of these enzymes with inhibitors. Despite their completely different cleavage specificities, both hmeprin alpha and beta are able to hydrolyse basement membrane components such as collagen IV, nidogen-1 and fibronectin. However, they are inactive against intact collagen I. Hence the matrix-cleaving activity of hmeprin resembles that of gelatinases rather than collagenases. Hmeprin is inhibited by hydroxamic acid derivatives such as batimastat, galardin and Pro-Leu-Gly-hydroxamate, by TAPI-0 (tumour necrosis factor alpha protease inhibitor-0) and TAPI-2, and by thiol-based compounds such as captopril. Therapeutic targets for these inhibitors are MMPs (matrix metalloproteases), TACE (tumour necrosis factor alpha-converting enzyme) and angiotensin-converting enzyme respectively. The most effective inhibitor of hmeprin alpha in the present study was the naturally occurring hydroxamate actinonin ( K(i)=20 nM). The marked variance in the cleavage specificities of hmeprin alpha and beta is reflected by their interaction with the TACE inhibitor Ro 32-7315, whose affinity for the beta subunit (IC50=1.6 mM) is weaker by three orders of magnitude than that for the alpha subunit ( K(i)=1.6 microM). MMP inhibitors such as the pyrimidine-2,4,6-trione derivative Ro 28-2653 that are more specific for gelatinases do not bind to hmeprin, presumably due to the subtle differences in the mode of zinc binding and active-site structure between the astacins and the MMPs.

Human meprin alpha and beta homo-oligomers: cleavage of basement membrane proteins and sensitivity to metalloprotease inhibitors.

Removal and replacement of metal ions in metallopeptidases.

Astacin, a zinc-endopeptidase from the crayfish Astacus astacus L., represents a structurally distinct group of metalloproteinases termed the 'astacin family'. This protein family includes oligomeric membrane-bound proteins with zinc proteinase domains found in rodent kidneys (meprins A and B) and human small intestine (N-benzoyl-L-tyrosyl-4-aminobenzoate hydrolase). Another branch of this family comprises morphogenetically active proteins, which induce bone formation (human bone morphogenetic protein 1), or which play specific roles during the embryonic development of amphibians, fishes, echinoderms, and insects. The X-ray crystal structure of astacin has recently been solved to a resolution of 0.18 nm [Bode et al. (1992) Nature 358, 164-167]. This structure is different from hitherto known metalloendopeptidase structures and has been used in the present study to analyze the structures of the other members of the astacin protein family. Computer-assisted modelling of the proteolytic domain of the alpha-subunit of meprin A based on the astacin structure is possible if five single and one double residue deletions and three single residue insertions are implied. The proteinase domains of the other astacins can be included in the model-based sequence alignment by introducing additionally three insertions and one deletion. All of these insertions and deletions are observed in loop segments connecting regular secondary structure elements and should leave the overall structure unaltered. The topology of residues forming the zinc-binding active site of astacin corresponds to almost identical arrangements in all other astacins, suggesting that these are likewise metalloproteinases. Based on this similarity, it is proposed that the active-site metal ion of the astacins is penta-coordinated by three histidine residues, a tyrosine residue and a water molecule in a trigonal bipyramidal geometry. Other remarkable common features are a hydrophobic cluster in the N-terminal domain and a conserved, solvent-filled cavity buried in the C-terminal domain. Most interestingly, the amino-termini of all astacins can be modelled to start in a corresponding internal water cavity as seen in the astacin template, where the terminal alanine residue forms a water-linked salt bridge to Glu103, directly adjacent to His102, the third zinc ligand. Therefore, an activation mechanism for the astacins reminiscent of that of the trypsin-like proteinases had been suggested, which now seems to be probable also for the other astacins. Besides these common traits, there are some minor differences which may have important consequences on the function of the astacins.(ABSTRACT TRUNCATED AT 400 WORDS)

https://febs.onlinelibrary.wiley.com/doi/pdf/10.1111/j.1432-1033.1993.tb17915.x

Implications of the three‐dimensional structure of astacin for the structure and function of the astacin family of zinc‐endopeptidases

Characterization of the proteolytic enzymes in the midgut of the European Cockchafer, Melolontha melolontha (Coleoptera: Scarabaeidae).

Crystal structures, spectroscopic features, and catalytic properties of cobalt(II), copper(II), nickel(II), and mercury(II) derivatives of the zinc endopeptidase astacin. A correlation of structure and proteolytic activity.

Russell L. Wolz

Papers

Thiol containing compounds and amino acid hydroxamates as reversible synthetic inhibitors of Astacus protease.

Kinetic evidence for cooperative binding of two orthophenanthroline molecules to astacus protease during metal removal