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There is nothing quite like the excitement of discovery in science—of finding something no one else knew and seeing a story unfold. One has to be part of an emerging picture to feel the elation. These moments in a lifetime are few and far between, but they fuel enthusiasm and keep one going. They are embedded in struggles and joys of everyday life, years of establishing what Louis Pasteur called “the prepared mind,” working with mentors, trainees, and colleagues, failures and successes. This article recalls 1) how I got to be a biochemist; 2) my contributions as an educator and researcher, especially regarding meprin metalloproteases; and 3) my participation in communities of science. Perhaps my reflections will help an aspiring scientist see how fulfilling a career in science can be.

The Journal of Biological Chemistry (JBC) has been a major vehicle for disseminating and recording the discovery and characterization of proteolytic enzymes. The pace of discovery in the protease field accelerated during the 1971–2010 period that Dr. Herb Tabor served as the JBC's editor-in-chief. When he began his tenure, the fine structure and kinetics of only a few proteases were known; now thousands of proteases have been characterized, and over 600 genes for proteases have been identified in the human genome. In this review, besides reflecting on Dr. Tabor's invaluable contributions to the JBC and the American Society for Biochemistry and Molecular Biology (ASBMB), I endeavor to provide an overview of the extensive history of protease research, highlighting a few discoveries and roles of proteases in vivo. In addition, metalloproteinases, particularly meprins of the astacin family, will be discussed with regard to structural characteristics, regulation, mechanisms of action, and roles in health and disease. Proteases and protein degradation play crucial roles in living systems, and I briefly address future directions in this highly diverse and thriving research area.

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.

Meprin metalloproteases are highly expressed at the luminal interface of the intestine and kidney and in certain leukocytes. Meprins cleave a variety of substrates in vitro, including extracellular matrix proteins, adherens junction proteins, and cytokines, and have been implicated in a number of inflammatory diseases. The linkage between results in vitro and pathogenesis, however, has not been elucidated. The present study aimed to determine whether meprins are determinative factors in disrupting the barrier function of the epithelium. Active meprin A or meprin B applied to Madin-Darby canine kidney (MDCK) cell monolayers increased permeability to fluorescein isothiocyanate-dextran and disrupted immunostaining of the tight junction protein occludin but not claudin-4. Meprin A, but not meprin B, cleaved occludin in MDCK monolayers. Experiments with recombinant occludin demonstrated that meprin A cleaves the protein between Gly100and Ser101on the first extracellular loop. In vivo experiments demonstrated that meprin A infused into the mouse bladder increased the epithelium permeability to sodium fluorescein. Furthermore, monocytes from meprin knockout mice on a C57BL/6 background were less able to migrate through an MDCK monolayer than monocytes from their wild-type counterparts. These results demonstrate the capability of meprin A to disrupt epithelial barriers and implicate occludin as one of the important targets of meprin A that may modulate inflammation.

Meprins are multimeric proteases that are implicated in inflammatory bowel disease by both genetic association studies and functional studies in knock-out mice. Patients with inflammatory bowel disease show decreased colonic expression of meprin α, although regulation of expression, particularly under inflammatory stimuli, has not been studied. The studies herein demonstrate that the human meprin α transcript is bound and stabilized by Hu antigen R at baseline, and that treatment with the inflammatory stimulus phorbol 12-myristate 13-acetate downregulates meprin α expression by inducing tristetraprolin. The enhanced binding of tristetraprolin to the MEP1A 3'-UTR results in destabilization of the transcript and occurs at a discrete site from Hu antigen R. This is the first report to describe a mechanism for post-transcriptional regulation of meprin α and will help clarify the role of meprins in the inflammatory response and disease.

MEP1A, which encodes the α subunit of meprin metalloproteinases, is a susceptibility gene for inflammatory bowel disease (IBD), and decreased intestinal meprin-α expression is associated with enhanced IBD in humans. Mice lacking meprin α (α knockout, αKO) have more severe colitis induced by dextran sulfate sodium (DSS) than wild-type (WT) mice, indicating an anti-inflammatory role for meprin A. Previous studies and those herein indicate the meprin B has proinflammatory activities. Therefore, mice lacking both meprin A and B (dKO mice) were generated to determine how their combined absence alters the inflammatory response to DSS. Unchallenged dKO mice grow and reproduce normally and have no obvious abnormal phenotype, except for a slightly elevated plasma albumin in both males and females and a lower urine creatinine level in dKO males. Upon oral administration of 3.5% DSS, the dKO mice have more severe colitis than the WT and βKO mice but significantly less than the αKO mice. The dKO mice lose more weight and have elevated MPO and IL-6 activities in the colon compared with WT mice. Systemic inflammation, monitored by plasma nitric oxide levels, is absent in DSS-treated dKO mice, unlike WT mice. The severity of experimental IBD in dKO mice is intermediate between αKO and WT mice. The data indicate that the absence of meprin A aggravates chronic inflammation and the lack of meprin B affords some protection from injury. Manipulation of the expression of meprin gene products may have therapeutic potential.

The Epithelial Na(+) Channel (ENaC) is an apical heteromeric channel that mediates Na(+) entry into epithelial cells from the luminal cell surface. ENaC is activated by proteases that interact with the channel during biosynthesis or at the extracellular surface. Meprins are cell surface and secreted metalloproteinases of the kidney and intestine. We discovered by affinity chromatography that meprins bind γ-ENaC, a subunit of the ENaC hetero-oligomer. The physical interaction involves NH(2)-terminal cytoplasmic residues 37-54 of γ-ENaC, containing a critical gating domain immediately before the first transmembrane domain, and the cytoplasmic COOH-terminal tail of meprin β (residues 679-704). This potential association was confirmed by co-expression and co-immunoprecipitation studies. Functional assays revealed that meprins stimulate ENaC expressed exogenously in Xenopus oocytes and endogenously in epithelial cells. Co-expression of ENaC subunits and meprin β or α/β in Xenopus oocytes increased amiloride-sensitive Na(+) currents approximately two-fold. This increase was blocked by preincubation with an inhibitor of meprin activity, actinonin. The meprin-mediated increase in ENaC currents in oocytes and epithelial cell monolayers required meprin β, but not the α subunit. Meprin β promoted cleavage of α and γ-ENaC subunits at sites close to the second transmembrane domain in the extracellular domain of each channel subunit. Thus, meprin β regulates the activity of ENaC in a metalloprotease-dependent fashion.

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.

Meprin metalloproteases, composed of alpha and/or beta subunits, consist of membrane-bound and secreted forms that are abundantly expressed in proximal tubules of the kidney as well as secreted into the urinary tract. Previous studies indicated that meprin metalloproteases play a role in pathological conditions such as ischemic acute renal failure and urinary tract infection. The aim of this work was to examine the role of meprins in endotoxemic acute renal failure using meprin alpha knockout (alphaKO), meprin beta knockout (betaKO), and wild-type (WT) mice. Differences among the responses of the genotypes were observed as early as 1 h after challenge with 2.5 mg/kg ip Escherichia coli LPS, establishing roles for meprins in the endotoxemic response. Meprin alphaKO mice displayed lower blood urea nitrogen levels and decreased nitric oxide levels, indicative of a decreased systemic response to LPS compared with WT and meprin betaKO mice. Serum cytokine profiles showed lower levels of IL-1beta and TNF-alpha in the meprin alphaKO mice within 3 h after LPS challenge and confirmed a role for meprins in the early phases of the host response. Meprin alphaKO mice were also hyporesponsive to LPS administered to the bladder, exhibiting significantly less bladder edema, leukocyte infiltration, and bladder permeability than WT mice. These data indicate that meprin A contributes to the renal and urogenital pathogenesis of endotoxicity.

Interleukin-18 (IL-18), a pro-inflammatory cytokine, is a key factor in inflammatory bowel disease (IBD). Caspase-1 activates this cytokine, but other proteases are likely involved in maturation. Because meprin metalloproteinases have been implicated in IBD, the interaction of these proteases with proIL-18 was studied. The results demonstrate that the meprin beta subunit of meprins A and B cleaves proIL-18 into a smaller 17-kDa product. The cleavage is at the Asn51-Asp52 bond, a site C-terminal to caspase-1 cleavage. The cleavage occurred in vitro with a Km of 1.3 microm and in Madin-Darby canine kidney cells transfected with meprin beta when proIL-18 was added to the culture medium. The product of meprin B cleavage of proIL-18 activated NF-kappaB in EL-4 cells, indicating that it was biologically active. To determine the physiological significance of the interactions of meprins with proIL-18, an experimental model of IBD was produced by administering dextran sulfate sodium (DSS) to wild-type and meprin beta knock-out (betaKO) mice, and the serum levels of active IL-18 were determined. DSS-treated meprin betaKO mice had lower levels of the active cytokine in the serum compared with wild-type mice. Furthermore, in meprin alphaKO mice, which express meprin beta but not alpha, active IL-18 was elevated in the serum of DSS-treated mice compared with wild-type mice, indicating that the meprin isoforms have opposing effects on the IL-18 levels in vivo. This study identifies proIL-18 as a biologically important substrate for meprin beta and implicates meprins in the modulation of inflammation.

Meprins are membrane-bound and secreted metalloproteinases consisting of alpha- and/or beta-subunits that are highly expressed in mouse kidney proximal tubules. Previous studies have implied that the meprin alpha/beta-isoform is deleterious when renal tissue is subjected to ischemia-reperfusion (I/R). To delineate the roles of the meprin isoforms in renal disease, we subjected mice deficient in meprin-beta (KO) and their wild-type (WT) counterparts to I/R. WT mice were markedly more susceptible to renal injury after I/R than the meprin-beta KO mice as determined by blood urea nitrogen levels. Urinary levels of inflammatory cytokines IL-6 and KC (CXCL1) were significantly higher in WT compared with meprin-beta KO mice by 6 h post-I/R. At 96 h postischemia, kidney mRNA expression levels for tumor necrosis factor-alpha, transforming growth factor-beta, inducible nitric oxide synthase, and heat shock protein-27 were significantly higher in the WT than meprin-beta KO mice. For WT mice subjected to I/R, there was a rapid (3 h) redistribution of meprin beta-subunits in cells in S3 segments of proximal tubules, followed by shedding of apical cell membrane and detachment of cells. These studies indicate that meprin-beta is important in the pathogenesis of renal injury following I/R and that the redistribution of active meprin-alpha/beta is a major contributor to renal injury and subsequent inflammation.

Meprin metalloproteinases have been implicated in the susceptibility to and progression of diabetic nephropathy and inflammatory bowel diseases. Our studies with experimental models of these diseases in mice are congruent with the conclusion that meprins modulate the inflammatory responses and tissue damage. To determine whether the mouse and human enzymes differ, recombinant forms of meprin A from the two species were compared with respect to structure, substrates and inhibitors. Human homo-oligomeric meprin A formed oligomers ranging from 950 000 to 1 500 000 Da vs. 900 000 Da for mouse meprin A. Human and mouse meprin A exhibited similar activity against azocasein, fibronectin, collagen IV, and peptides such as parathyroid hormone, ghrelin, and gastrin-releasing peptide. The human enzyme had lower activity against gelatin, bradykinin, α-melanocyte-stimulating hormone and neurotensin, and higher activity against secretin and orcokinin. Human meprin A showed a preference for acidic residues in the P1′ position of the substrate, unlike mouse meprin A. Several metalloproteinase inhibitors had IC50 values in the nanomolar range, but potency ranged from similar values to a difference of several orders of magnitude for meprins from the two species. This work provides valuable data to improve predictability for human systems based on meprin functions in mouse models.

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The meprin A homo-oligomer is a highly glycosylated, secreted zinc metalloprotease of the astacin family and metzincin superfamily. This isoform of meprin is composed of disulfide-bonded dimers of alpha subunits that further associate to form large, secreted megadalton complexes of 10 or more subunits. The aim of this study was to determine the sites of glycan attachment and to assess their ability to affect the formation and stability of the homo-oligomer. Nine of the ten potential N-linked glycosylation sites (Asn-41, Asn-152, Asn-234, Asn-270, Asn-330, Asn-426, Asn-452, Asn-546, and Asn-553) were found to be glycosylated in recombinant mouse meprin A using chemical and enzymatic deglycosylation methods and electrospray ionization mass spectrometry. Chemical cross-linking demonstrated that carbohydrates are at or near the noncovalent subunit interface. The removal of two glycans in the protease domain at Asn-234 and Asn-270, as well as one in the tumor necrosis factor receptor-associated factor domain at Asn-452, by a deglycosidase under nondenaturing conditions decreased the chemical and thermal stability of the homo-oligomer without affecting quaternary structure. Site-directed mutagenesis demonstrated that no single glycan was essential for oligomer formation; however, the combined absence of the glycans at Asn-152 and Asn-270 in the protease domain hindered intersubunit disulfide bond formation, prevented noncovalent associations, and abolished enzymatic activity. These studies provide insights into the role of glycans in the biosynthesis, activity, and stability of this extracellular protease.

Meprin (MEP) A is a metalloendopeptidase that is present in the renal proximal tubule brush-border membrane (BBM) and that colocalizes with angiotensin-converting enzyme (ACE). The MEP beta-chain gene locus on chromosome 18 has been linked to a heightened risk of diabetic nephropathy (DN) in patients with type 2 diabetes. This study evaluated 1) whether MEP-alpha and MEP-beta gene and protein expression are altered in db/db mice before the onset of DN and 2) the role of MEP-alpha in the pathogenesis of DN and the impact of the renin-angiotensin system on this interaction in two experimental models of diabetes. MEP-alpha and MEP-beta gene and protein expression were evaluated in db/db mice, 13-14 wk of age, compared with lean C57BLKS/J littermate animals. A treatment study was then performed in which db/db mice and controls were assigned to one of three groups: control (C) water, no therapy; ACE inhibitor therapy, enalapril (EN)-treated water, 50 mg/l; ANG II receptor type 1 blocker (ARB) therapy, losartan (LOS)-treated water, 500 mg/l. Treatment was started at 8 wk of age and continued for 52 wk. Male Sprague-Dawley rats with diabetes for 52 wk following a single dose of streptozocin (STZ; 60 mg/kg) were also studied. At 13.5 wk of age, MEP-alpha and MEP-beta kidney mRNA abundance and protein expression were significantly lower in db/db mice compared with lean controls, with greater changes in MEP-beta (P < 0.05). In the treatment study, EN ameliorated and LOS exacerbated DN in db/db mice. BBM MEP A enzymatic activity and MEP-alpha protein content were lower in db/db mice vs. control nonobese mice at 52 wk (P < 0.02). EN-treated db/db mice showed increased MEP A activity, MEP-alpha content in BBM, decreased urinary MEP-alpha excretion, and enhanced BBM staining for MEP-alpha protein vs. C and LOS-treated db/db mice. In nonobese mice, EN and LOS treatment had no effect on MEP-alpha expression. In rats with STZ-induced diabetes for 52 wk, urinary MEP-alpha excretion was increased and MEP A activity and MEP-alpha protein content per milligram of BBM protein were decreased compared with age-matched control animals (P < 0.05). These results indicate that db/db mice manifest decreased MEP-alpha and MEP-beta gene and protein expression, before the development of overt kidney disease. Moreover, in db/db mice with DN and rats with STZ-diabetes, there was an inverse relationship between renal MEP-alpha content and the severity of the renal injury. Treatment with an ACE inhibitor was more effective than ARB in ameliorating DN in db/db mice, a change that correlated with alterations in urinary excretion and BBM content of MEP-alpha. MEP-alpha may play a role in the pathogenesis of DN and the benefits of ACE inhibitor therapy on the progression of diabetic kidney disease may be related, in part, to its impact on renal MEP-alpha expression.

There is evidence that susceptibility to diabetic nephropathy has a significant genetic component. This investigation tested the hypothesis that variations in the structural or regulatory regions of the MEP1B gene are related to susceptibility to diabetic nephropathy in the Pima Indian population. The structure of the human MEP1B gene on chromosome 18 was determined by polymerase chain reaction (PCR) amplification. Samples from 154 diabetic individuals were analyzed for polymorphisms. These individuals belonged to 65 sibships with at least one sibling pair discordant for diabetic nephropathy. Approximately half of the individuals had diabetic nephropathy. Of the 154 samples, there were 91 discordant sibling pairs. Sequencing revealed 19 single nucleotide polymorphisms (SNPs) in the MEP1B gene. SNPs 1-5 were in the 5' region upstream of the start site for transcription; SNPs 6, 7, 9, 11-15, 17, and 19 were within introns; SNPs 8, 10, 16, and 18 were in exons 4, 9, 12, and 14. SNP 18 was the only one that results in an amino acid change (proline to leucine in the cytoplasmic tail). No overall associations were found for individual SNPs. Within-family association tests found significant results for SNPs 1, 3, 4, 5, 6, 9, 11, 18, and 19 such that the more common allele was more frequently observed in those with nephropathy than in their unaffected siblings. The present study demonstrates significant within-family association for SNPs in MEP1B gene with diabetic nephropathy. These results could be explained by functional effects of one or more of these SNPs or by linkage disequilibrium with a nearby functional locus.

Introduction Polyamines affect proliferation, differentiation, migration and apoptosis of cells, indicating their potential as a target for cancer chemotherapy. Ornithine decarboxylase converts ornithine to putrescine and is the rate-limiting step in polyamine synthesis. α-Difluoromethylornithine (DFMO) irreversibly inhibits ornithine decarboxylase and MDA-MB-435 human breast cancer metastasis to the lung without blocking orthotopic tumor growth. This study tested the effects of DFMO on orthotopic tumor growth and lung colonization of another breast cancer cell line (MDA-MB-231) and the effects on bone metastasis of MDA-MB-435 cells. Methods MDA-MB-231 cells were injected into the mammary fat pad of athymic mice. DFMO treatment (2% per orally) began at the day of tumor cell injection or 21 days post injection. Tumor growth was measured weekly. MDA-MB-231 cells were injected into the tail vein of athymic mice. DFMO treatment began 7 days prior to injection, or 7 or 14 days post injection. The number and incidence of lung metastases were determined. Green fluorescent protein-tagged MDA-MB-435 cells were injected into the left cardiac ventricle in order to assess the incidence and extent of metastasis to the femur. DFMO treatment began 7 days prior to injection. Results DFMO treatment delayed MDA-MB-231 orthotopic tumor growth to a greater extent than growth of MDA-MB-435 tumors. The most substantial effect on lung colonization by MDA-MB-231 cells occurred when DFMO treatment began 7 days before intravenous injection of tumor cells (incidence decreased 28% and number of metastases per lung decreased 35–40%). When DFMO treatment began 7 days post injection, the incidence and number of metastases decreased less than 10%. Surprisingly, treatment initiated 14 days after tumor cell inoculation resulted in a nearly 50% reduction in the number of lung metastases without diminishing the incidence. After intracardiac injection, DFMO treatment decreased the incidence of bone metastases (55% vs 87%) and the area occupied by the tumor (1.66 mm2 vs 4.51 mm2, P < 0.05). Conclusion Taken together, these data demonstrate that DFMO exerts an anti-metastatic effect in more than one hormone-independent breast cancer, for which no standard form of biologically-based treatment exists. Importantly, the data show that DFMO is effective against metastasis to multiple sites and that treatment is generally more effective when administered early.

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.

Meprins, multimeric metalloproteases expressed in kidney and intestinal epithelial cells as well as in certain leukocytes and cancer cells, have the ability to hydrolyze a variety of growth factors, vasoactive peptides, cytokines, and extracellular matrix proteins. The meprin B isoform exists primarily as a cell-surface homooligomer composed of disulfide-linked, multidomain beta-subunits. To gain insight into how the tertiary and quaternary structure of meprin B affects function, the disulfide-bonding pattern and sites of domain-domain interactions were investigated using sedimentation equilibrium ultracentrifugation, cross-linking, and mass spectrometry techniques. Three symmetrical intersubunit disulfide bonds were identified in the noncatalytic interaction domains; two in the MAM (meprin, A-5 protein, protein-tyrosine phosphatase mu) domain and one in the TRAF (tumor necrosis factor receptor-associated factor) domain. These disulfide bridges are unique for the known homophilic interactions of these domains. Mutation of any of the intersubunit cysteine residues resulted in the inability of meprin B to form disulfide-linked dimers. The four cysteines of the protease domain formed intradomain disulfide bonds. The MAM domain also had one intradomain disulfide bond and one free cysteine. Cross-linking studies of the meprin B dimer with the amine-reactive cross-linker disuccinimidyl suberate revealed inter- and intradomain contacts within the protein, including prosequence-prosequence, protease-TRAF, protease-epidermal growth factor, and TRAF-TRAF interactions. From these observations, a model of the meprin B dimer structure is proposed that provides insight into the relationship between structure and function of this isoform.

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.

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.

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.

Meprins are multidomain zinc metalloproteases that are highly expressed in mammalian kidney and intestinal brush border membranes and in leukocytes and certain cancer cells. Mature meprins are oligomers of evolutionarily related, separately encoded alpha and/or beta subunits. Homooligomers of meprin alpha are secreted; oligomers containing meprin beta are plasma membrane associated. Meprin substrates include bioactive peptides and extracellular matrix proteins. Meprins have been implicated in cancer and intestinal inflammation. Additionally, meprin beta is a candidate gene for diabetic nephropathy. To elucidate in vivo functions of these metalloproteases, meprin beta null mice were generated by targeted disruption of the meprin beta gene on mouse chromosome 18q12. Analyses of meprin beta knockout mice indicated that (i) 50% fewer null mice are born than the Mendelian distribution predicts, (ii) null mice that survive develop normally and are viable and fertile, (iii) meprin beta knockout mice lack membrane-associated meprin alpha in kidney and intestine, and (iv) null mice have changes in renal gene expression profiles compared to wild-type mice as assessed by microarray analyses. Thus, disruption of the meprin beta allele in mice affects embryonic viability, birth weight, renal gene expression profiles, and the distribution of meprin alpha in kidney and intestine.

Meprin A and B, metalloproteases consisting of evolutionarily related α and/or β 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 ∼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 β subunits only, was dimeric under a wide range of conditions. By contrast, meprin α homodimers formed heterogeneous multimers (ring-, circle-, spiral-, and tube-like structures) containing up to 100 subunits, with molecular masses at protein peaks ranging from ∼1.0 to 6.0 MDa. The size of the meprin α homo-oligomers was dependent on protein concentration, ionic strength, and activation state. Meprin αβ heterodimers tended to form tetramers but not higher oligomers. Thus, the presence of meprin β, 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 α 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.

The secreted form of mouse meprin A is a homooligomer of meprin alpha subunits that contain a prosequence, a catalytic domain, and three domains designated as MAM (meprin, A5 protein, receptor protein-tyrosine phosphatase mu), MATH (meprin and TRAF homology), and AM (AfterMath). Previous studies indicated that wild-type mouse meprin alpha is predominantly a secreted protein, while the MAM deletion mutant (DeltaMAM) is degraded intracellularly. The work herein indicates that the DeltaMAM mutant is ubiquitinated and degraded via the proteasomal pathway. Both wild-type meprin alpha and the DeltaMAM mutant interact with the molecular chaperones calnexin and calreticulin in the endoplasmic reticulum. The interactions of the chaperones with the DeltaMAM mutant were significantly prolonged in the presence of lactacystin, a specific inhibitor of the proteasome, whereas those with the wild type were not affected by this inhibitor. Trimming of the Asn-linked core oligosaccharides of meprin subunits was required for interactions with the chaperones. The data indicated that folding of the wild-type protein was accelerated by chaperones, whereas the rate of dimerization was unaffected. Thus, calnexin and calreticulin are intimately involved in the correct folding and transport of meprin to the plasma membrane, as well as in retrograde transport of the DeltaMAM mutant to the ubiquitin-dependent proteasomal degradative pathway in the cytosol.

The mucus that covers and protects the epithelium of the intestine is built around its major structural component, the gel-forming MUC2 mucin. The gel-forming mucins have traditionally been assumed to be secreted as nonattached. The colon has a two-layered mucus system where the inner mucus is attached to the epithelium, whereas the small intestine normally has a nonattached mucus. However, the mucus of the small intestine of meprin β-deficient mice was now found to be attached. Meprin β is an endogenous zinc-dependent metalloprotease now shown to cleave the N-terminal region of the MUC2 mucin at two specific sites. When recombinant meprin β was added to the attached mucus of meprin β-deficient mice, the mucus was detached from the epithelium. Similar to meprin β-deficient mice, germ-free mice have attached mucus as they did not shed the membrane-anchored meprin β into the luminal mucus. The ileal mucus of cystic fibrosis (CF) mice with a nonfunctional cystic fibrosis transmembrane conductance regulator (CFTR) channel was recently shown to be attached to the epithelium. Addition of recombinant meprin β to CF mucus did not release the mucus, but further addition of bicarbonate rendered the CF mucus normal, suggesting that MUC2 unfolding exposed the meprin β cleavage sites. Mucus is thus secreted attached to the goblet cells and requires an enzyme, meprin β in the small intestine, to be detached and released into the intestinal lumen. This process regulates mucus properties, can be triggered by bacterial contact, and is nonfunctional in CF due to poor mucin unfolding.

Meprins are secreted and membrane-bound metalloendopeptidases highly expressed in kidney and intestinal epithelial cells. They are oligomeric glycoproteins composed of evolutionarily related alpha and/or beta subunits. The present work revealed that the messages for both meprin subunits were expressed in intestine and kidney in ICR and C3H/He mouse embryos (as early as day 11), indicating developmental functions for both subunits. During the first 2 weeks after birth, the mRNA levels for both subunits increased in ICR mice, but between 10 days and 3 weeks (time of weaning) the alpha subunit level in the intestine fell markedly. In adult ICR mice, meprin beta mRNA was consistently expressed in both kidney and intestine, whereas meprin alpha mRNA was highly expressed in kidney but only present at low levels in intestine. In C3H/He mice, the pattern of meprin alpha and beta subunit mRNA expression was similar to that of ICR mice, except that meprin alpha was barely detectable in kidney after birth. The results of postnatal studies indicate that the meprin alpha subunit has a role in the intestine during suckling but is not essential after weaning, and that the beta homooligomer is the major meprin form after weaning in both kidney and intestine.

A novel mRNA isoform (meprin beta') of the cell-surface protease subunit meprin beta was previously identified in human colon cancer cells. The study reported here revealed that this mRNA isoform was identical within the protein coding region and at the 3' end to the beta isoform of normal intestine but that it contained an extended 5' untranslated region. Meprin beta' mRNA was expressed in the human breast cancer cell lines MCF-7 and SK-BR-3, in the human osteosarcoma cell line U2 Os, and in the human pancreatic cancer cell line BxPC-3. Meprin beta mRNA, but not beta' mRNA, was expressed in human fetal kidney cells. We cloned and sequenced genomic DNA encoding portions of the promoter region of the meprin beta gene. The unique sequences present in the beta' mRNA were present in the human genomic DNA immediately upstream of the transcription start site for the beta mRNA. The human meprin promoter sequence was searched for potential transcription-factor binding sites, and putative activator protein-1, polyoma enhancer activator 3 (PEA3), CCAAT enhancer-binding protein beta, and estrogen-receptor binding sites were identified along with binding sites for the intestine-specific cdx-2 transcription factor. The activity of meprin promoter/luciferase reporter gene constructs transfected into U2 Os cells was highest with constructs containing 83 and 639 bp of promoter DNA. These regions of the promoter each contain a putative PEA3 element. Treatment of the human colon adenocarcinoma cell line HT29-18C1 with 50 or 100 ng/mL phorbol myristal acetate for 8 h increased meprin beta' mRNA levels. Likewise, U2 Os cells transfected with the -639/luciferase or -1800/luciferase constructs showed a phorbol myristal acetate-inducible increase in reporter gene activity, indicating that the PEA3 element within the -639 construct or other elements further upstream respond to phorbol ester.

Secreted forms of the alpha subunit of recombinant mouse meprin A include an NH2-terminal prosequence, a catalytic domain, and three COOH-terminal domains designated as MAM (meprin, A-5 protein, receptor protein-tyrosine phosphatase mu), MATH (meprin and TRAF homology), and AM (after MATH). In this study, the importance of these COOH-terminal domains for biosynthesis of secreted, activable forms of the protease was investigated. Transcripts of the meprin subunit truncated after the protease (alpha(1-275)), MAM (alpha(1-452)), and MATH (alpha(1-528)) domains or with individual domains deleted (DeltaMAM, DeltaMATH, and DeltaAM), were transfected into human embryonic kidney 293 cells. The wild-type subunit, DeltaMATH, DeltaAM, alpha(1-452), and alpha(1-528) were secreted into the media, although the DeltaAM mutant was secreted at very low levels. The DeltaMATH and alpha(1-452) mutants were not activable by limited proteolysis. The alpha(1-528) mutant was as active as wild-type meprin alpha against a bradykinin substrate, but had no activity against azocasein, and it, as all other mutants, was more vulnerable to extensive degradation by proteases than the wild-type protein. Pulse-chase experiments revealed that the DeltaMAM and alpha(1-275) mutants were rapidly degraded within cells. Treatment with lactacystin, a specific inhibitor of the proteasome, significantly decreased the degradation, indicating that the mutants lacking the MAM domain are degraded by the proteasome as misfolded proteins. These results indicate that the MAM domain is necessary for correct folding and transport through the secretory pathway, the MATH domain is required for folding of an activable zymogen, and the AM domain is important for activity against proteins and efficient secretion of the protein. The work demonstrates the interdependence of the domains for correct folding of an activable, stable, mature enzyme.

The alpha subunit of meprin A is synthesized as a type I integral membrane protein; however, the mature form contains no membrane-spanning region due to COOH-terminal proteolytic cleavage during biosynthesis. Previous studies with transfected mouse meprin alpha subunit cDNA, showed that the inserted (I) domain in the COOH-terminus was essential for proteolytic processing and transport of the subunit out of the endoplasmic reticulum. A furin site in the I domain was implicated as the site of cleavage in the rat meprin alpha subunit, however, this site was shown not to be required in the homologous mouse subunit. The present studies were designed to determine whether there is a species difference in the use of the furin site for processing of the subunit, and whether the different mutations used in the previous studies could account for the different conclusions regarding the importance of the furin site. When the furin sites in mouse and rat cDNAs were mutated, using similar amino acid substitutions, and expressed in human 293 cells, all mutants were secreted, and had comparable activities compared to the wild-types against a protein (azocasein) and peptide (bradykinin analog) substrate. These data revealed no difference between processing of the rat and mouse subunits, and indicated that the furin site is not essential for COOH-terminal processing in either species. Additional transfection investigations with brefeldin A or low temperature confirmed that COOH-terminal processing occurs in the endoplasmic reticulum, further supporting the contention that furin-type enzymes localized to the Golgi apparatus are not responsible for processing this subunit. COOH-terminal amino acid sequence analysis of the mature detergent-purified membrane form of meprin alpha from ICR mouse kidney indicated that the subunit ends at Arg615, which is NH2-terminal to the I domain in the After-MATH domain. Mutation of Arg615 to an Ala did not affect secretion of the protein from 293 cells. The results indicate that the I domain enables or directs the final COOH-terminal processing of meprin alpha to a region NH2-terminal to the I domain, and that there are no essential dibasic, furin, or single base processing sites.

Meprins are mammalian zinc metalloendopeptidases with protease domains structurally related to astacin, the prototype of the "astacin family" of metalloproteases. Mature, active astacins are produced by proteolytic removal of an activation peptide to generate a new NH2-terminal residue. Structural studies indicate that the NH2-terminal ammonium group inserts into a water-filled cavity adjacent to the active site to form a salt bridge with a Glu residue that is conserved in all astacins. A similar interaction is known to play a crucial role in the activation of trypsin, resulting in the hypothesis that this salt bridge is required for the activation of astacin-like proteases. In this study, we have used the mouse meprin alpha subunit as a model to test this hypothesis of zymogen activation of the astacins. Mutants were generated to vary the NH2-terminal residue of the mature meprin alpha subunit (Asn78) and its putative salt bridge partner (Glu178). In addition, mutants creating NH2-terminal extensions and truncations were expressed in human embryonic kidney 293 cells. The recombinant proteins were activated by limited protease digestion and assayed for enzymatic activity and thermal stability. Point mutations of Asn78 resulted in enzymes with activity comparable to the wild-type enzyme, indicating that the structure of this side chain is not essential for activity. NH2-terminal extension mutants of meprin alpha retained partial activity, with greater decreases against peptide relative to protein substrates. A mutant with a deletion of Asn78 to disrupt salt bridge formation with Glu178 had full activity, indicating that the putative salt bridge with Glu178 is not essential for enzyme activity. However, all changes in meprin alpha subunit NH2-terminal structure were found to decrease the thermal stability of the enzyme. These observations and additional data indicate that the zymogen activation mechanism of meprin and other astacins differs from that of the trypsin family of enzymes, and has some features in common with matrixins. It is proposed that prosequence removal of astacins allows the formation of hydrogen bonds involving the two NH2-terminal residues that are critical for enzyme structure.

Author(s): Kennelly, Peter J; Bond, Judith S; Masters, Bettie Sue; Dennis, Edward A; Brenner, Charles; Raben, Daniel M

Type I fibrillar collagen is the most abundant protein in the human body, crucial for the formation and strength of bones, skin, and tendon. Proteolytic enzymes are essential for initiation of the assembly of collagen fibrils by cleaving off the propeptides. We report that Mep1a −/− and Mep1b −/− mice revealed lower amounts of mature collagen I compared with WT mice and exhibited significantly reduced collagen deposition in skin, along with markedly decreased tissue tensile strength. While exploring the mechanism of this phenotype, we found that cleavage of full-length human procollagen I heterotrimers by either meprin α or meprin β led to the generation of mature collagen molecules that spontaneously assembled into collagen fibrils. Thus, meprin α and meprin β are unique in their ability to process and release both C- and N-propeptides from type I procollagen in vitro and in vivo and contribute to the integrity of connective tissue in skin, with consequent implications for inherited connective tissue disorders.

The digestive system works in concert with the other organ systems of the body, and with the external environment, in particular ingested material and microorganisms. The pathway from the point of ingestion to the point of excretion is long and tortuous, and is made up of dozens of niches with specialized physiologic roles and constellations of proteases. In each of these niches, there is a characteristic array of molecules from the lumen to the surface of the cells lining the alimentary tract (Hopfer 2011). The cellular level is a heterogeneous mosaic of resident and transient cells with special functions and arrays of proteases. This chapter will focus on the mammalian proteases found in the lumen and on the cell surface of the alimentary tract, both membrane-bound and secreted proteases. The proteases of the intestinal bacteria, mainly in the lower intestine, are dealt with in separate chapters, but structures and products of bacteria will be addressed as substrates and as factors that affect protease expression and activity. Similarly, the proteases of immune cells, which are transient members of the digestive system, are dealt with separately in other chapters, but their roles in the alimentary tract are inextricably enmeshed in normal physiologic processes of digestion and pathologic responses to injury. Accordingly, this review of proteases in the digestive system will focus on the human system beginning anatomically at the oral cavity, and proceed to the esophagus, stomach, and the niches of the intestinal tract, ending with the colon. Representative protease functions in intestinal pathobiology are discussed in the anatomical sections and in separate sections at the end of the chapter. There is detailed information on the many proteases mentioned in this chapter in the Handbook of Proteolytic Enzymes, second Edition (Barrett et al. 2004) and the third Edition (Rawlings and Salvesen 2013), and in the MEROPs database (http://www.merops.sanger.ac.uk).