Structure of Homo- and Hetero-oligomeric Meprin Metalloproteases

Abstract

Meprin A and B, metalloproteases consisting of evolutionarily related ␣ and/or ␤ subunits, are membranebound and secreted enzymes expressed by kidney and intestinal epithelial cells, leukocytes, and cancer cells. Previous work established that the multidomain meprin subunits (each ϳ80 kDa) form disulfide-bridged homoand heterodimers, and differ in substrate and peptide bond specificities. The work herein clearly demonstrates that meprin dimers differ markedly in their ability to oligomerize. Electrophoresis, light scattering, size exclusion chromatography, and electron microscopy were used to characterize quaternary structures of recombinant rat meprins. Meprin B, consisting of meprin ␤ subunits only, was dimeric under a wide range of conditions. By contrast, meprin ␣ homodimers formed heterogeneous multimers (ring-, circle-, spiral-, and tube-like structures) containing up to 100 subunits, with molecular masses at protein peaks ranging from ϳ1.0 to 6.0 MDa. The size of the meprin ␣ homo-oligomers was dependent on protein concentration, ionic strength, and activation state. Meprin ␣␤ heterodimers tended to form tetramers but not higher oligomers. Thus, the presence of meprin ␤, which has a transmembrane domain in vivo, restricts the oligomerization potential of meprin molecules and localizes meprins to the plasma membrane. By contrast, the propensity of secreted meprin ␣ homodimers to self-associate concentrates proteolytic potential into high molecular mass multimers and thus allows for autocompartmentalization. The work indicates that different mechanisms exist to localize and concentrate the proteolytic activity of membrane-bound and secreted meprin metalloproteinases.Proteolytic enzymes are essential components of many cellular and extracellular processes from maturation of proteins to cell death (1). Their activities and localities, however, must be highly regulated because of their destructive potential. Regulation of proteases is accomplished through several mechanisms including zymogen formation, inhibition, localization to specific compartments, and transcriptional regulation. The structures of proteases themselves have revealed mechanisms to regulate proteolytic activity, and this has been amply demonstrated in the high molecular mass oligomeric structures of the proteasome, tripeptidyl peptidase II, and the tricorn protease (2-4). These multimeric serine and threonine proteolytic complexes are homo-or hetero-oligomeric, have molecular masses of 0.7-9 MDa, and are found intracellularly. The structures serve to restrict, localize, and concentrate proteolytic activity. Proteases at the cell surface are known to form transient oligomeric complexes, such as those between membranetype 1 matrix metalloproteinase, tissue inhibitor of metalloproteinase 2, and matrix metalloproteinase 2 that lead to the activation of matrix metalloproteinase 2 (5). However, stable secreted, multimeric proteolytic complexes were not described until recently, when homo-oligomers of meprin A were found to form multimers of ϳ0.9 MD...

Intersubunit and Domain Interactions of the Meprin B Metalloproteinase

“…Mutation of meprin ␤Cys-492 to an alanine was accomplished using the primers 5Ј-TCAATTAC-AGTGGCCGGCTCCTTGGCAAGC-3Ј and 5Ј-GCTTGCTGCCAAGGAG-CCGGCCACTGTAATTGA-3Ј. Polymerase chain reaction established the mutation in the rat meprin ␤ cDNA, and transformation of the construct was conducted as previously described (7,29). This DNA was then stably transfected into human embryonic kidney 293 cells as described previously (30).…”

“…Analytical Ultracentrifugation-Recombinant rat meprin B was purified as described previously and dialyzed into ultracentrifugation buffer containing 50 mM potassium phosphate, pH 7.5, 150 mM potassium chloride (7). Sedimentation equilibrium measurements of meprin B were conducted using a Beckman XL-I analytical ultracentrifuge in absorbance mode.…”

“…Only dimers of homooligomeric meprin ␤ have been observed thus far. However, the local concentrations of meprin B in kidney brush border membranes have been estimated to be in the high micromolar range, higher than conditions used for in vitro experiments (7,8). Thus, it is possible that higher oligomers are formed under these conditions.…”

“…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).…”

“…Meprins are unique among mammalian metalloproteinases in that they are composed of multidomain, evolutionarily related ␣ and ␤ subunits that form disulfide-linked homo-or heterooligomeric dimers (6,7). The heterodimers tend to form tetramers (␣ 2 ,␤ 2 ; ␣ 3 ,␤ 1 ), whereas the meprin ␣ homodimers form decameric 900-kDa complexes to very higher molecular mass multimers of up to 6 MDa, among the largest extracellular proteolytic complexes known.…”