Characterization of meprin, a membrane-bound metalloendopeptidase from mouse kidney

Abstract

Meprin is an intrinsic protein of the brush border, a specialized plasma membrane, of the mouse kidney. It is a metalloendopeptidase that contains 1 mol of zinc and 3 mol of calcium per mol of the 85,000-Mr subunit. The enzyme is isolated, and active, as a tetramer. The behaviour of the enzyme on SDS/polyacrylamide gels in the presence and absence of beta-mercaptoethanol indicates that the subunits are of the same Mr (approx. 85,000) and held together by intersubunit S--S bridges. Eight S-carboxymethyl-L-cysteine residues were detected after reduction of the enzyme with beta-mercaptoethanol and carboxymethylation with iodoacetate. The enzyme is a glycoprotein and contains approx. 18% carbohydrate. Most of the carbohydrate is removed by endoglycosidase F, indicating that the sugar residues are N-linked. The isoelectric point of the enzyme is between pH 4 and 5, and the purified protein yields a pattern of evenly spaced bands in this range on isoelectric focusing. The peptide-bond specificity of the enzyme has been determined by using the oxidized B-chain of insulin as substrate. In all, 15 peptide degradation products were separated by h.p.l.c. and analysed for their amino acid content and N-terminal amino acid residue. The prevalent peptide-bond cleavages were between Gly20 and Glu21, Phe24 and Phe25 and between Phe25 and Tyr26. Other sites of cleavage were Leu6-Cysteic acid7, Ala14-Leu15, His10-Leu11, Leu17-Val18, Gly8-Ser9, Leu15-Tyr16, His5-Leu6. These results indicate that meprin has a preference for peptide bonds that are flanked by hydrophobic or neutral amino acid residues, but hydrolysis is not limited to these bonds. The ability of meprin to hydrolyse peptide bonds between small neutral and negatively charged amino acid residues distinguishes it from several other metalloendopeptidases.

Marked Differences between Metalloproteases Meprin A and B in Substrate and Peptide Bond Specificity

“…Both proteases have extended binding sites and prefer substrates of at least 6 amino acids (Ref. 28 and Table V). The different specificities of the two subunits implicate diverse functions.…”

“…The Hydrolytic Efficiency of Meprin B Is Dependent on Peptide Length-Previous work indicated that meprin A has a preference for substrates with a minimum of eight amino acids (28). To test the peptide length requirement for efficient hydrolysis by meprin B, derivatives of sCCK8 NH2 were used as substrates (Table V).…”

The astacin family of metalloendopeptidases

“…Zinc has been established as the active site metal in meprin A (1.1 mol z i n c h o l subunit), astacin (0.97 k 0.03 mol zinc/mol protein), and HCEILCE (approximately 1.3 pg/mg protein) (Butler et al, 1987;Stocker et al, 1988;Yasumasu et al, 1989aYasumasu et al, , 1989b. Reactivation studies have indicated that Zn2+, Cu2+, and Co2+ can reactivate astacin apoenzyme; Zn2+, and Cu2+ can reactivate the fish enzymes and meprin A apoenzyme (Wolz & Bond, 1995).…”

Astacin family metallopeptidases and serine peptidase inhibitors in spider digestive fluid

“…2B). Like astacin, meprin A has a considerably lower pI (4-5) (Butler et al 1987) than the bulk of the peptidases in SDF. It remains to be established which peptidases, by the designations used in this paper, correspond to the four Argiope peptidase fractions (A-D) of Kavanagh (1979) and Kavanagh and Tillinghast (1983), and in particular to the apparently homogeneous Argiope protease B and Argiope protease D. Similarities with respect to molecular mass, pI, and relative abundance make it likely that p16 is one of these two peptidases.…”