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

Abstract

Meprin A and B are highly regulated, secreted, and cell-surface metalloendopeptidases that are abundantly expressed in the kidney and intestine. Meprin oligomers consist of evolutionarily related ␣ and/or ␤ subunits. The work herein was carried out to identify bioactive peptides and proteins that are susceptible to hydrolysis by mouse meprins and kinetically characterize the hydrolysis. Gastrin-releasing peptide fragment 14 -27 and gastrin 17, regulatory molecules of the gastrointestinal tract, were found to be the best peptide substrates for meprin A and B, respectively. Peptide libraries and a variety of naturally occurring peptides revealed that the meprin ␤ subunit has a clear preference for acidic amino acids in the P1 and P1 sites of substrates. The meprin ␣ subunit selected for small (e.g. serine, alanine) or hydrophobic (e.g. phenylalanine) residues in the P1 and P1 sites, and proline was the most preferred amino acid at the P2 position. Thus, although the meprin ␣ and ␤ subunits share 55% amino acid identity within the protease domain and are normally localized at the same tissue cell surfaces, they have very different substrate and peptide bond specificities indicating different functions. Homology models of the mouse meprin ␣ and ␤ protease domains, based on the astacin crystal structure, revealed active site differences that can account for the marked differences in substrate specificity of the two subunits.Meprin A and B are zinc metalloendopeptidases composed of evolutionarily related ␣ and/or ␤ subunits. They are members of the astacin family and are highly expressed in brush border membranes of the intestine and renal proximal tubules (1, 2). Meprins are particularly abundant in mouse juxtamedullary nephrons and constitute ϳ5% of total protein in renal brush border membranes (3). The meprin ␣ and ␤ subunits are expressed early in embryonic development of mouse kidney and intestine, by day 11, and have different patterns of expression in the suckling phase and after weaning (4). Homologous enzymes are found in rat and human kidney and intestine (1,5,6). Meprins are also expressed in leukocytes of intestinal lamina propria and in cancer cells and are consequently implicated in inflammation and cancer growth and metastasis (7,8).Mouse kidney meprin A (EC 3.4.24.18) is a homooligomer of ␣ subunits, or a heterooligomer of ␣ and ␤ subunits (2, 9). Mouse kidney meprin B (EC 3.4.24.63) is a homooligomer of ␤ subunits (10). The multidomain ␣ and ␤ meprin subunits are highly glycosylated and form disulfide-linked dimers and higher order oligomers by noncovalent interactions (11,12). Meprins containing at least one ␤ subunit remain membranebound by virtue of a transmembrane domain located near the carboxyl terminus of ␤ subunits (1). Mature meprin ␣ homooligomers contain no transmembrane domain and are found in mouse urine (13). The expression of the meprin ␣ subunits in mice is strain-dependent (1). Random-bred mice (such as ICR) and many inbred strains of mice (e.g. C57BL/6) express both meprin ␣ and ␤ in...

Genome-wide Analysis of CDX2 Binding in Intestinal Epithelial Cells (Caco-2)

“…This group of proteins are metalloendopeptidases capable of hydrolyzing a variety of peptide and protein substrates (66). MEP1A has via its protease activity on extracellular matrix components been speculated to contribute to tumor progression by facilitating migration (67).…”

Intersubunit and Domain Interactions of the Meprin B Metalloproteinase

“…Although the meprin ␣ and ␤ subunits are 42% identical at the amino acid level, they differ markedly in their ability to self-associate, in proteolytic processing during biosynthesis in the endoplasmic reticulum, and in substrate specificity (1,7,9,10). For example, meprin ␤ has a preference for acidic residues in the P1 and P1Ј sites of the substrate, whereas meprin ␣ selects for small or hydrophobic residues at these sites (10,11).…”

“…The x-ray crystal structure of the 20-kDa crayfish astacin, the prototype of the astacin metalloproteinase family, has been solved (17). Molecular modeling studies of the mouse and rat meprin ␣ and ␤ protease domains indicate that the residues essential for zinc coordination and peptide bond hydrolysis in astacin are conserved in the meprin protease domain (10,11,18). Deletion and truncation studies with the meprin A homooligomer demonstrated that there is an important interdependence of the meprin domains for correct folding of an activable, stable, mature enzyme.…”

“…For example, meprin ␤ has a preference for acidic residues in the P1 and P1Ј sites of the substrate, whereas meprin ␣ selects for small or hydrophobic residues at these sites (10,11). Multimers containing the ␣ subunit (homooligomers or heterooligomers) are designated meprin A (EC 3.4.24.18), whereas homooligomers of the ␤ subunit are designated meprin B (EC 3.4.24.63).…”

Compartmentalised expression of meprin in small intestinal mucosa: enhanced expression in lamina propria in coeliac disease

“…Although both enzymes accommodate a range of different amino acids, there are some preferences, in particular at P9 positions (Bertenshaw et al, 2001). Using a dodecamer peptide library, meprin a was found to prefer serine at P19 and proline at P29 and P39 positions, whereas meprin b preferentially cleaved at sequences containing acidic residues (aspartate and glutamate) at P19 and P29 positions, and proline at P39.…”