Meprin Alpha Research Articles

A preliminary study of possible fibrotic role of meprin metalloproteases in scleroderma patients.

Objectives This study aims to investigate the possible fibrotic role of meprin metalloproteases and possible fibrotic effects of activator protein-1 (AP-1) in scleroderma patients.Patients and methods Between April 2018 and April 2019, a total of 85 scleroderma patients (9 males, 76 females; mean age: 54.9 years; range, 22 to 80 years) who met the 2013 American College of Rheumatology/European League Against Rheumatism criteria and 80 healthy control individuals (10 males, 70 females; mean age 42.9 years; range, 19 to 65 years) were included. Patients’ data and blood samples were collected. Messenger ribonucleic acid expressions of interleukin (IL)-6, AP-1 subunits, and tumor necrosis factor-alpha (TNF-α) were analyzed by quantitative real-time polymerase chain reaction. Serum meprin alpha and beta protein levels were analyzed using the enzyme-linked immunosorbent assay. Results Meprin alpha and meprin beta protein levels increased in scleroderma patients. The AP-1 subunits (c-Fos, c-Jun), IL-6, and TNF-α increased in scleroderma patients, compared to controls.Conclusion Our results provide evidence showing that increased meprins levels may be related to AP-1 levels and increased meprins levels may responsible for increased inflammatory TNF-α and IL-6 levels. All these data suggest meprins as promising therapeutic targets to restore the balance between inflammation and extracellular matrix deposition in scleroderma.

Metalloproteinase meprin α regulates migration and invasion of human hepatocarcinoma cells and is a mediator of the oncoprotein Reptin.

Hepatocellular carcinoma is associated with a high rate of intra-hepatic invasion that carries a poor prognosis. Meprin alpha (Mep1A) is a secreted metalloproteinase with many substrates relevant to cancer invasion. We found that Mep1A was a target of Reptin, a protein that is oncogenic in HCC. We studied Mep1A regulation by Reptin, its role in HCC, and whether it mediates Reptin oncogenic effects.MepA and Reptin expression was measured in human HCC by qRT-PCR and in cultured cells by PCR, western blot and enzymatic activity measurements. Cell growth was assessed by counting and MTS assay. Cell migration was measured in Boyden chambers and wound healing assays, and cell invasion in Boyden chambers.Silencing Reptin decreased Mep1A expression and activity, without affecting meprin β. Mep1A, but not meprin β, was overexpressed in a series of 242 human HCC (2.04 fold, p < 0.0001), and a high expression correlated with a poor prognosis. Mep1A and Reptin expressions were positively correlated (r = 0.39, p < 0.0001). Silencing Mep1A had little effect on cell proliferation, but decreased cell migration and invasion of HuH7 and Hep3B cells. Conversely, overexpression of Mep1A or addition of recombinant Mep1A increased migration and invasion. Finally, overexpression of Mep1A restored a normal cell migration in cells where Reptin was depleted.Mep1A is overexpressed in most HCC and induces HCC cell migration and invasion. Mep1A expression is regulated by Reptin, and Mep1A mediates Reptin-induced migration. Overall, we suggest that Mep1A may be a useful target in HCC.

Understanding the Exacerbating Role of the Metalloproteinase Meprin during AKI, an &lt;i&gt;In Silico&lt;/i&gt; Approach

Acute kidney injury (AKI) is a syndrome characterised by the rapid loss of the kidney’s excretory function and is typically diagnosed by the accumulation of end products of nitrogen metabolism (urea and creatinine) or decreased urine output, or both. It is the clinical manifestation of several disorders that affect the kidney acutely. No specific therapies have yet emerged that can attenuate AKI or expedite recovery; thus, the only treatment is supportive therapies and intensive care. The present study was aimed to provide an insight into the importance of a metalloproteinase involved in the pathological conditions of AKI and potentially is a unique target for therapeutic intervention during the disease; Meprin. The data obtained using literature search from PubMed and interaction networks analysis software STRING strongly support the concept that meprin acts as a major matrix degrading enzyme in the kidney, and thus creating an environment that leads to impairment in cellular function rather than cellular stability in response to AKI. The present study discerns the structure of meprin alpha subunit using in silico tools SWISS-MODE, Phyre2 web server and identify the active site and critical amino acid residues in the active site using AADS (IIT Delhi), 3DLigandSite and DoGSiteScorer. Further it is documented that actinonin, a naturally occurring antibacterial agent as a pharmacologically active intervention for the metalloproteinase’s α subunit by blocking its active sites from the environment which was validated using molecular docking algorithms of SWISS-DOCK and FlexX.

Understanding the Exacerbating Role of the Metalloproteinase Meprin during AKI, an &lt;i&gt;In Silico&lt;/i&gt; Approach

Acute kidney injury (AKI) is a syndrome characterised by the rapid loss of the kidney’s excretory function and is typically diagnosed by the accumulation of end products of nitrogen metabolism (urea and creatinine) or decreased urine output, or both. It is the clinical manifestation of several disorders that affect the kidney acutely. No specific therapies have yet emerged that can attenuate AKI or expedite recovery; thus, the only treatment is supportive therapies and intensive care. The present study was aimed to provide an insight into the importance of a metalloproteinase involved in the pathological conditions of AKI and potentially is a unique target for therapeutic intervention during the disease; Meprin. The data obtained using literature search from PubMed and interaction networks analysis software STRING strongly support the concept that meprin acts as a major matrix degrading enzyme in the kidney, and thus creating an environment that leads to impairment in cellular function rather than cellular stability in response to AKI. The present study discerns the structure of meprin alpha subunit usingin silicotools SWISS-MODE, Phyre2 web server and identify the active site and critical amino acid residues in the active site using AADS (IIT Delhi), 3DLigandSite and DoGSiteScorer. Further it is documented that actinonin, a naturally occurring antibacterial agent as a pharmacologically active intervention for the metalloproteinase’s α subunit by blocking its active sites from the environment which was validated using molecular docking algorithms of SWISS-DOCK and FlexX.

Meprin Metalloproteases Inactivate Interleukin 6

Meprins have been implicated in the pathogenesis of several inflammatory diseases, including inflammatory bowel disease, in which the cytokine IL-6 is a prominent effector molecule. Because IL-6 levels are elevated markedly in meprin α and α/β knockout mice in an experimental model of inflammatory bowel disease, the interaction between meprins and IL-6 was studied. The results demonstrate that rodent and human meprin A and B cleave IL-6 to a smaller product and, subsequently, are capable of extensive degradation of the cytokine. Analysis of the limited degradation product formed by meprin A indicated that three to five amino acids are removed from the C terminus of the cytokine. Meprin A and meprin B cleaved IL-6 with micromolar affinities (Km of 4.7 and 12.0 μM, respectively) and with high efficiencies (kcat/Km of 0.2 and 2.5 (M(-1)/s(-1)) × 10(6), respectively). These efficiency constants are among the highest for known meprin substrates. Madin-Darby canine kidney cells transiently transfected with meprin α or meprin β constructs also cleave exogenous IL-6. Both human and murine IL-6 cleaved by meprin A or B are inactivated, as demonstrated by their decreased capability to stimulate proliferation of B9 cells. These results are consistent with the proposition that one function of meprin metalloproteases is to modulate inflammation by inactivating IL-6.

IL‐6 is a Newly‐Discovered Substrate for Meprin Metalloproteinases

Meprins are unique metalloproteinases that are highly expressed in intestinal, kidney, and skin epithelial cells, and also in certain leukocytes (eg., monocytes and natural killer cells). Meprins are secreted and membrane‐bound proteases comprised of alpha and/or beta subunits. These proteases have been implicated in the pathogenesis of several inflammatory diseases such as: Inflammatory Bowel Disease (IBD), Urinary Tract Infections (UTI), Acute Renal Failure (ARF), and cancer. Previous work has demonstrated, using experimental mouse models, that meprin knock‐out mice have altered cytokine profiles compared to their wild‐type counterparts in the IBD, UTI, and ARF models. IL‐6 was one of the cytokines significantly increased in meprin alpha and alpha/beta knock‐out mice with IBD, leading us to hypothesize that IL‐6 is a meprin substrate and that meprin plays a role in modulating inflammation by inactivating IL‐6. So far, we have determined through SDS‐PAGE gel‐shift analysis that meprins do cleave IL‐6, which is the first direct in vitro evidence that IL‐6 is a meprin substrate. Additionally, the homomeric meprin A isoform fragmented IL‐6 differently than the homomeric meprin B isoforms. Subsequent studies are being done to measure the affinity of IL‐6 as a meprin substrate and determine whether meprin‐generated IL‐6 products are bioactive. (Supported by NIH DK 19691).

Let It Flow: Morpholino Knockdown in Zebrafish Embryos Reveals a Pro-Angiogenic Effect of the Metalloprotease Meprin α2

BackgroundMeprin metalloproteases are thought to be involved in basic physiological functions such as cell proliferation and tissue differentiation. However, the specific functions of these enzymes are still ambiguous, although a variety of growth factors and structural proteins have been identified as meprin substrates. The discovery of meprins α1, α2 and β in teleost fish provided the basis for uncovering their physiological functions by gene silencing in vivo.Methodology/Principal FindingsA Morpholino knockdown in zebrafish embryos targeting meprin α1 and β mRNA caused defects in general tissue differentiation. But meprin α2 morphants were affected more specifically and showed severe failures in the formation of the vascular system provoking the hypothesis of a pro-angiogenic effect. The blood circulation was largely diminished resulting in erythrocyte accumulation. These phenotypes mimic a previously described VEGF-A morphant, revealing a possible role of meprin α in VEGF-A activation. Indeed, human recombinant meprin α processed the vascular endothelial growth factor-A (VEGF-A) specifically, revealing the same cleavage products detectable for VEGF from zebrafish whole lysate.Conclusions/SignificanceOur results demonstrate that meprin metalloproteases are important for cell differentiation and proliferation already during embryogenesis, predominantly by the activation of growth factors. Thus, we conclude that meprins play a significant role in VEGF-A processing, subsequently regulating angiogenesis. Therefore, meprin α might be a new therapeutic target in cardiovascular diseases or in tumor growth inhibition.

Disruption of the meprin α and β genes in mice alters homeostasis of monocytes and natural killer cells

Meprin metalloproteases are implicated in inflammatory bowel disease, which involves dysfunction of immune cells. However, the roles of meprins in the immune and hematological system remain uncharacterized. In this report, we demonstrate that meprins were expressed in the hematological system, and meprin alpha/beta null (alpha(-/-)/beta(-/-)) mice had decreased prevalence of resident monocytes and natural killer (NK) cells in blood, with a concomitant accumulation of inflammatory monocytes and NK cells in bone marrow. In contrast, T and B lymphocytes were not affected by meprin deficiency. In response to acute inflammation induced by intraperitoneal injection of thioglycollate, meprin-deficient mice exhibited higher body temperature than wild-type mice, which was correlated with retention of inflammatory monocytes, but persistent low prevalence of NK cells in blood. These results indicate that meprin metalloproteases play important roles in the homeostasis of monocytes and NK cells, and possibly are involved in egress of these two type cells from bone marrow and homing to the periphery. Our findings are the first report to demonstrate that metalloproteases affect homeostasis of leukocytes, which have important implications for understanding physiology of and pathogenesis in the hematological system.

Activation of the Epidermal Growth Factor Receptor (EGFR) by a Novel Metalloprotease Pathway

Neutrophil Elastase (NE) is a pro-inflammatory protease present at higher than normal levels in the lung during inflammatory disease. NE regulates IL-8 production from airway epithelial cells and can activate both EGFR and TLR4. TACE/ADAM17 has been reported to trans-activate EGFR in response to NE. Here, using 16HBE14o-human bronchial epithelial cells we demonstrate a new mechanism by which NE regulates both of these events. A high molecular weight soluble metalloprotease activity detectable only in supernatants from NE-treated cells by gelatin and casein zymography was confirmed to be meprin alpha by Western immunoblotting. In vitro studies demonstrated the ability of NE to activate meprin alpha, which in turn could release soluble TGFalpha and induce IL-8 production from 16HBE14o- cells. These effects were abrogated by actinonin, a specific meprin inhibitor. NE-induced IL-8 expression was also inhibited by meprin alpha siRNA. Immunoprecipitation studies detected EGFR/TLR4 complexes in NE-stimulated cells overexpressing these receptors. Confocal studies confirmed colocalization of EGFR and TLR4 in 16HBE14o- cells stimulated with meprin alpha. NFkappaB was also activated via MyD88 in these cells by meprin alpha. In bronchoalveolar lavage fluid from NE knock-out mice infected intra-tracheally with Pseudomonas aeruginosa meprin alpha was significantly decreased compared with control mice, and was significantly increased and correlated with NE activity, in bronchoalveolar lavage fluid from individuals with cystic fibrosis but not healthy controls. The data describe a previously unidentified lung metalloprotease meprin alpha, and its role in NE-induced EGFR and TLR4 activation and IL-8 production.

The α and β Subunits of the Metalloprotease Meprin Are Expressed in Separate Layers of Human Epidermis, Revealing Different Functions in Keratinocyte Proliferation and Differentiation

The zinc endopeptidase meprin (EC 3.4.24.18) is expressed in brush border membranes of intestine and kidney tubules, intestinal leukocytes, and certain cancer cells, suggesting a role in epithelial differentiation and cell migration. Here we show by RT-PCR and immunoblotting that meprin is also expressed in human skin. As visualized by immunohistochemistry, the two meprin subunits are localized in separate cell layers of the human epidermis. Meprin alpha is expressed in the stratum basale, whereas meprin beta is found in cells of the stratum granulosum just beneath the stratum corneum. In hyperproliferative epidermis such as in psoriasis vulgaris, meprin alpha showed a marked shift of expression from the basal to the uppermost layers of the epidermis. The expression patterns suggest distinct functions for the two subunits in skin. This assumption is supported by diverse effects of recombinant meprin alpha and beta on human adult low-calcium high-temperature keratinocytes. Here, beta induced a dramatic change in cell morphology and reduced the cell number, indicating a function in terminal differentiation, whereas meprin alpha did not affect cell viability, and may play a role in basal keratinocyte proliferation.

Sex Difference in Ischemic Acute Renal Failure in Rats: Approach by Proteomic Analysis

It is known that female rats are resistant to ischemic acute renal failure (ARF), compared with male rats. To elucidate sex differences in ischemic ARF, we searched global protein expression in post-ischemic kidneys using proteomic techniques. Ischemic ARF was induced by 45-min ischemia followed by reperfusion. By proteomic analysis, many male- or female-dominant proteins were detected in sham-operated rat kidneys, and significantly increased or decreased proteins were found in post-ischemic kidneys 2 h after reperfusion, at which there were no significant deterioration in renal function of both sexes. We detected 86 proteins showing more than 1.5-fold significant alterations (p<0.01) in both sexes by ischemia/reperfusion (I/R) treatment. Among the altered proteins, we identified a significantly up-regulated protein in male rat kidneys, meprin alpha, a subunit of meprin which had been reported to play a role in the pathophysiology of I/R-induced ARF. In addition, it is known that a potent meprin alpha inhibitor, actinonin, can protect against I/R-induced renal injury when administered to male rats. We therefore compared the effect of actinonin on I/R-induced renal dysfunction between male and female rats. Renal function of both males and females showed significant deterioration when measured at 24 h after the reperfusion, although the degree of renal dysfunction was much less in females than in males. Pre-ischemic treatment with actinonin (30 mg/kg, i.v.) prevented the I/R-induced renal dysfunction in males but not in females. Our results provide information on differences in protein expression at an early phase after the reperfusion between male and female rats. Moreover, the present study suggests that up-regulation of meprin alpha in the post-ischemic kidney is at least partly involved in aggravation of I/R-induced renal injury in male rats. The possibility that meprin alpha is a key component of the sex difference in ischemic ARF, warrants further attention.

Targeted disruption of the meprin beta gene results in decreased renal ischemia/reperfusion injury in mice

Meprins are metalloproteinases consisting of alpha and/or beta subunits and are highly expressed in kidneys of mice. Inbred mouse strains that express very low levels of meprin alpha have been shown to be less susceptible to renal ischemia reperfusion (I/R) injury than mice expressing normal levels of meprin alpha. Bilateral renal I/R in normal meprin expressing mice resulted in a rapid [3 h] redistribution of meprin alpha and beta in cells in S3 segments of proximal tubules, followed by shedding of apical cell membrane and detachment of cells. Meprin redistribution and juxtamedullary tubular damage co-localized with activated leukocytes. To explore the role of meprins in disease, mice with targeted disruption of the meprin beta gene were generated. These mice expressed the same levels of meprin alpha mRNA as wild-type mice, however, the meprin alpha protein was not localized on the cell membrane and was secreted in an inactive form. By six hours of reperfusion, meprin beta deficient mice had better preservation of renal function than wild-type mice as determined by lower plasma blood urea nitrogen (BUN) and lower Levels of IL-6 and osteopontin mRNA. BUN levels were significantly lower in KO mice at all subsequent time points. At 96 hr post ischemia, mRNA expression levels for iNOS, TNF alpha, and HSP-27 (indicators of inflammation) were significantly lower in the meprin beta deficient mice. These results indicate that meprins are important in the pathogenesis of renal injury following ischemia/reperfusion. We propose that the redistribution of active meprin alpha is a major contributor during I/R to renal injury and subsequent inflammation. (Supported by NIH DK 19691)

Meprin metalloproteases confer resistance to urinary tract infections

Meprins are membrane-bound and secreted zinc metalloproteases that are expressed by kidney proximal tubules, intestinal epithelial cells, and leukocytes. They are found in the urinary tract, the intestinal lumen, and at sites of inflammation. The secreted form of meprin, composed of alpha subunits, is a large multimer capable of degrading bacterial proteins and shedding proteins from the cell surface. It was hypothesized that high levels of urinary meprin confer resistance to urinary tract infections (UTIs). Urine samples from healthy and UTI infected women were collected for analysis. Quantitative Western blotting showed that 89% of healthy women had low or undetectable levels of urinary meprin. In contrast, the majority (86%) of women suffering from an acute UTI had high levels of meprin protein in the urine. To examine susceptibility of different meprin genotypes to UTI, both wild-type and meprin alpha knockout mice were infected with Escherichia coli (E. coli) via transurethral catheterization. Bacterial counts in bladder show that meprin alpha knockout mice have 50 times greater number of E. coli versus wild-type. In addition, kidney bacterial counts in meprin alpha knockout mice were 13 fold greater in comparison to wild-type. These data indicate that meprin expression confers resistance to infections of the urinary tract. (NIH Grant DK 19691)

Inhibitors of Polyamine Biosynthesis Decrease the Expression of the Metalloproteases Meprin α and MMP-7 in Hormone-independent Human Breast Cancer Cells

Inhibition of ornithine decarboxylase (ODC), a key enzyme in polyamine biosynthesis, by the irreversible inhibitor alpha-difluoromethylornithine (DFMO) has been shown to decrease the invasiveness of metastatic human breast cancer cell lines. However, the mechanism by which DFMO acts to reduce invasiveness is unclear. Using the human breast cancer cell line MDA-MB-435, the effect of DFMO on metalloprotease gene expression was investigated. DFMO treatment decreases the expression of the metalloprotease meprin alpha, while concurrent treatment with DFMO and the polyamine putrescine partially restored meprin alpha expression levels. Expression of MMP-7 mRNA was reduced by DFMO, while MMPs-1, -2, -3, -14, and meprin beta were unaffected. Treatment of cells with a second inhibitor of polyamine biosynthesis, the S-adenosylmethionine decarboxylase (SAMDC) inhibitor SAM486A, also resulted in a dosage dependent decrease in meprin alpha and MMP-7 mRNA. In addition, DFMO treatment decreased meprin alpha at the protein level by 2 days of treatment, and MMP-7 protein levels at 4 and 6 days. Previous studies have shown that DFMO treatment increases ERK phosphorylation and signaling through the MAP kinase pathway. The decrease in meprin alpha expression was reversed with the MEK inhibitor PD98059, demonstrating that MAP kinase signaling mediates the effect of DFMO and SAM486A. MDA-MB-435 cells treated with the meprin alpha inhibitor actinonin (5 nM) were less invasive in vitro, indicating that meprin alpha is mechanistically involved in invasion. The decrease in meprin alpha expression in DFMO and SAM486A-treated cells indicates a means by which these compounds can decrease the invasiveness of metastatic breast cancer cells.

Transport of Meprin Subunits through the Secretory Pathway: ROLE OF THE TRANSMEMBRANE AND CYTOPLASMIC DOMAINS AND OLIGOMERIZATION

The meprin alpha subunit, a multidomain metalloproteinase, is synthesized as a type I membrane protein and proteolytically cleaved during biosynthesis in the endoplasmic reticulum (ER), consequently losing its membrane attachment and COOH-terminal domains. The meprin alpha subunit is secreted as a disulfide-linked dimer that forms higher oligomers. By contrast, the evolutionarily related meprin beta subunit retains the COOH-terminal domains during biosynthesis and travels to the plasma membrane as a disulfide-linked integral membrane dimer. Deletion of a unique 56-amino acid inserted domain (the I domain) of meprin alpha prevents COOH-terminal proteolytic processing and results in the retention of this subunit within the ER. To determine elements responsible for this retention versus transport to the cell surface, mutagenesis experiments were performed. Replacement of the meprin alpha transmembrane (alphaT) and cytoplasmic (alphaC) domains with their beta counterparts allowed rapid movement of the alpha subunit to the cell surface. The meprin alphaT and alphaC domains substituted into meprin beta delayed movement of this chimera through the secretory pathway. Replacement of glycines in the meprin alphaT domain GXXXG motif with leucine residues, alanine insertions in the meprin alphaT domain, and mutagenesis of basic residues within the meprin alphaC domain did not enhance the movement of the alpha subunit through the secretory pathway. By contrast, a mutant of meprin alpha (C320AalphaDeltaI) that did not form disulfide-linked dimers or higher order oligomers was transported through the secretory pathway, although more slowly than meprin beta. Taken together, the data indicate that the meprin alphaT and alphaC domains together contain a weak signal for retention within the ER/cis-Golgi compartments that is strengthened by oligomerization.

Critical Amino Acids in the Active Site of Meprin Metalloproteinases for Substrate and Peptide Bond Specificity

The protease domains of the evolutionarily related alpha and beta subunits of meprin metalloproteases are approximately 55% identical at the amino acid level; however, their substrate and peptide bond specificities differ markedly. The meprin beta subunit favors acidic residues proximal to the scissile bond, while the alpha subunit prefers small or aromatic amino acids flanking the scissile bond. Thus gastrin, a peptide that contains a string of five Glu residues, is an excellent substrate for meprin beta, while it is not hydrolyzed by meprin alpha. Work herein aimed to identify critical amino acids in the meprin active sites that determine the substrate specificity differences. Sequence alignments and homology models, based on the crystal structure of the crayfish astacin, showed electrostatic differences within the meprin active sites. Site-directed mutagenesis of active site residues demonstrated that replacement of a hydrophobic residue by a basic amino acid enabled the meprin alpha protease to cleave gastrin. The meprin alphaY199K mutant was most effective; the corresponding mutation of meprin betaK185Y resulted in decreased activity toward gastrin. Peptide cleavage site determinations and kinetic analyses using a variety of peptides extended evidence that meprin alphaTyr-199/betaLys-185 are substrate specificity determinants in meprin active sites. These studies shed light on the molecular basis for the substrate specificity differences of astacin metalloproteinases.

Meprin proteolytic complexes at the cell surface and in extracellular spaces.

Meprins are metalloproteinases of the astacin family and metzincin superfamily that are composed of evolutionarily related alpha and beta subunits, which exist as homo- and hetero-oligomeric complexes. These complexes are abundant at the brush border membranes of kidney proximal tubule cells and epithelial cells of the intestine, and are also expressed in certain leucocytes and cancer cells. Meprins cleave bioactive peptides such as gastrin, cholecystokinin and parathyroid hormone, cytokines such as osteopontin and monocyte chemotactic peptide-1, as well as proteins such as gelatin, collagen IV, fibronectin and casein. Database predictions and initial data indicate that meprins are also capable of shedding proteins, including itself, from the cell surface. Membrane-bound meprin subunits are composed of dimeric meprin beta subunits or tetrameric hetero-oligomeric alpha beta complexes of approx. 200-400 kDa, and can be activated at the cell surface; secreted forms of homo-oligomeric meprin alpha are zymogens that form high-molecular-mass complexes of 1-6 MDa. These are among the largest extracellular proteases identified thus far. The latent (self-associating) homo-oligomeric complexes can move through extracellular spaces in a non-destructive manner, and deliver a concentrated form of the metalloproteinase to sites that have activating proteases, such as sites of inflammation, infection or cancerous growth. Meprins provide examples of novel ways of concentrating proteolytic activity at the cell surface and in the extracellular milieu, which may be critical to proteolytic function.

Differences in the activation mechanism between the alpha and beta subunits of human meprin.

Meprins are zinc-endopeptidases of the astacin family, which are expressed as membrane-bound or secreted forms in renal and intestinal brush-border membranes of mouse, rat and man. There are two types of meprin subunits, alpha and beta, which form disulfide-bonded homo- and heterodimers; further oligomerization is mediated by non-covalent interactions. Both subunits are translated as proenzymes that have to be activated by removal of an N-terminal propeptide. In the gut, the most probable activator is trypsin. In addition, plasmin has been shown to activate the human alpha subunit in colorectal cancer tissue. In the present study we have overexpressed the human meprin alpha subunit and a His-tagged soluble tail-switch-mutant of meprin beta in Baculovirus-infected insect cells. The recombinant homo-oligomeric proteins were purified by gel filtration and affinity chromatography with yields of up to 10 mg/l cell culture medium and analyzed with regard to their activation mechanism. While both alpha and beta homo-oligomers are activated by trypsin, only meprin alpha homo-oligomers are processed to their mature form by plasmin. These results indicate a different accessibility of the propeptide in meprin homo-oligomers and suggest an explanation for the appearance of meprin hetero-oligomers consisting of active alpha, but latent beta subunits.

Structure of Homo- and Hetero-oligomeric Meprin Metalloproteases: DIMERS, TETRAMERS, AND HIGH MOLECULAR MASS MULTIMERS

Meprin A and B, metalloproteases consisting of evolutionarily related alpha and/or beta subunits, are membrane-bound and secreted enzymes expressed by kidney and intestinal epithelial cells, leukocytes, and cancer cells. Previous work established that the multidomain meprin subunits (each approximately 80 kDa) form disulfide-bridged homo- and 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 beta subunits only, was dimeric under a wide range of conditions. By contrast, meprin alpha homodimers formed heterogeneous multimers (ring-, circle-, spiral-, and tube-like structures) containing up to 100 subunits, with molecular masses at protein peaks ranging from approximately 1.0 to 6.0 MDa. The size of the meprin alpha homo-oligomers was dependent on protein concentration, ionic strength, and activation state. Meprin alphabeta heterodimers tended to form tetramers but not higher oligomers. Thus, the presence of meprin beta, 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 alpha 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.

Zinc ligands in an astacin family metalloprotease meprin A.

A conserved tyrosine residue in the 'astacin family' of metalloproteases is one of five ligands proposed to coordinate zinc at the active site. Site-directed mutagenesis of the conserved Tyr (Y226) of recombinant mouse meprin alpha was used to test the hypothesis that this residue is essential for zinc binding and enzymatic activity. In addition, another proposed zinc binding ligand, H167, in the conserved (HEXXH) zinc binding motif of the meprin alpha protease domain was replaced by an alanine residue. Both mutants were expressed and secreted with the same subunit mass as wild type (90 kDa). The Y226F mutant retained the capacity to oligomerize to higher covalently and noncovalently-linked oligomers as the wild type, whereas H167A was predominantly a monomer. The kcat/Km for Y226F against a fluorgenic bradykinin substrate analog was approximately 15% of the wild type, while the H167A mutant had no detectable activity. Both Y226F and H167A were more susceptible to extensive degradation by trypsin compared with the wild-type protein. The zinc content in the wild-type and Y226F mutant proteins were similar, one molecule of zinc per subunit. The results indicate that Y226 is not essential for zinc binding, but Y226 and H167 are essential for full enzymatic activity and stability of the metalloproteinase.