Mannan-binding protein blocks the activation of metalloproteases meprin alpha and beta

Mannan-binding protein (MBP) is a C-type serum lectin that is known to be a host defense factor involved in innate immunity, and recognizes mannose, fucose, and N-acetylglucosamine residues. Although some exogenous MBP ligands have been reported, little is known about its endogenous ligands. In the present study, we found that endogenous MBP ligands are highly expressed in the brush border epithelial cells of kidney-proximal tubules by immunohistochemistry, and both meprin alpha and beta (meprins), as novel endogenous MBP ligands, have been identified through affinity chromatography and mass spectrometry. Meprins are membrane-bound and secreted zinc metalloproteases extensively glycosylated and highly expressed in kidney and small intestinal epithelial cells, leukocytes, and certain cancer cells. Meprins are capable of cleaving growth factors, extracellular matrix proteins, and biologically active peptides. Deglycosylation experiments indicated that the MBP ligands on meprins are high mannose- or complex-type N-glycans. The interaction of MBP with meprins resulted in significant decreases in the proteolytic activity and matrix-degrading ability of meprins. Our results suggest that core N-linked oligosaccharides on meprins are associated with the optimal enzymatic activity and that MBP is an important regulator for modulation of the localized meprin proteolytic activity via N-glycan binding. Because meprins are known to be some of the major matrix-degrading metalloproteases in the kidney and intestine, MBP, which functions as a natural and effective inhibitor of meprins, may contribute, as a potential therapeutic target, to tumor progression by facilitating the migration, intravasation, and metastasis of carcinoma cells, and to acute renal failure and inflammatory bowel diseases.     

Probing the active sites and mechanisms of rat metalloproteases meprin A and B

Meprin A and B are highly regulated, secreted and cell-surface homo- and hetero-oligomeric enzymes. Meprins are abundantly expressed in kidney and intestine. The multidomain alpha and beta subunits have high sequence identity, however they have very different substrate specificities, oligomerization potentials and are differentially regulated. Here we describe that meprin subunit activities are modulated differently by physico-chemical factors. Homo-oligomeric meprin B had an acidic pH optimum. The low pH protonation indicated the existence of at least two ionizable groups. An additional ionizable group generated a shoulder in the basic pH range. Homo-oligomeric meprin A had a neutral pH optimum and the activity curve revealed that two ionizable groups might be protonated at acidic pH similar to meprin B. Increasing the concentration of salt generally inhibited meprin B activity. Meprin A was inhibited at low salt concentrations but activated as salt was increased. This work has important implications in the elucidation of the catalytic mechanisms of meprins and other metalloproteases. In addition, the activity of meprin oligomers that arise in tissues will be affected by variations in pH and NaCl. This could have profound implications because meprins are exposed to a range of conditions in the extracellular milieu of renal and intestinal tissues and in inflammation and cancer.     

Marked differences between metalloproteases meprin A and B in substrate and peptide bond specificity

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 alpha and/or beta 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 beta subunit has a clear preference for acidic amino acids in the P1 and P1' sites of substrates. The meprin alpha 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 alpha and beta 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 alpha and beta 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.     

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.     

Regulation of immunological homeostasis in the respiratory tract

The respiratory tract has an approximate surface area of 70 m2 in adult humans, which is in virtually direct contact with the outside environment. It contains a uniquely rich vascular bed containing a large pool of marginated T cells, and harbours a layer of single-cell-thick epithelial tissue through which re-oxygenation of blood must occur uninterrupted for survival. It is therefore not surprising that the respiratory tract is never more than a short step away from disaster. We have only a partial understanding of how immunological homeostasis is maintained in these tissues, but it is becoming clear that the immune system has evolved a range of specific mechanisms to deal with the unique problems encountered in this specialized microenvironment.     

Genetics of meprin in the rat

The locus encoding for meprin activity in kidney was found polymorphic in the rat. The BDIV and COP strains that carried theMep-1 ^a allele had high meprin concentration in the kidney, while the ACI, BN, LE and LEW strains that carried theMep-1 ^b allele had low meprin levels. TheMep-1 ^a allele that controlled the high activity enzyme was expressed in a dominant fashion in the (BN × COP) F1 progeny. The backcross studies showed that the segregation of alleles at theMep-1 and theGlo-1 loci was normal and that the genes were linked (x ² = 4.06,P < 0.05). The map distance between theMep-1 andGlo-1 genes was 41 ± 4 centimorgans.     

Role of the polymeric Ig receptor in mucosal B cell homeostasis

Secretory IgA (SIgA) is the most characteristic component of the mucosal immune system and has long been considered the major protective factor that prevents pathogens from invading hosts through the mucosae. Recent studies, however, have suggested that complete immunity against a range of mucosal bacterial and viral pathogens can be achieved in the absence of IgA. Therefore, to further dissect the role of SIgA, we generated mice deficient in the polymeric Ig receptor (pIgR(-/-) mice). As a result of an inability to transport dimeric IgA to the secretions, pIgR(-/-) mice are deficient in SIgA and accumulate circulating dimeric IgA, with serum levels 100-fold greater than those observed in normal mice. Examination of lamina propria mononuclear cells showed that pIgR(-/-) mice had approximately 3 times as many IgA-secreting cells as C57BL/6 mice. Further analysis showed that these cells displayed the differentiated IgA(+) B220(-) phenotype and accounted for a 2-fold increase in the number of lamina propria blast cells in the pIgR(-/-) mice. Subsequent experiments showed that OVA-specific CD4(+) T cell expansion following OVA feeding was not elevated in pIgR(-/-) mice. Furthermore, no differences in CD8(+) T cell tolerance or induction of influenza virus-specific CD8(+) T cells were detected in pIgR(-/-) mice compared with controls. Therefore, while SIgA is clearly involved in maintaining some parameters of mucosal homeostasis in the intestine, the mechanisms associated with its barrier function and the clinical consequences of its deficiency are yet to be identified.     

Role of calcium homeostasis in gastric mucosal injury and protection.

Using a human gastric mucosal cell line, known as AGS cells, we determined the role that perturbations in intracellular Ca2+ concentration [Ca2+]i might play in cellular injury induced by various damaging agents. For deoxycholate (CD) and ethanol (EtOH) induced damage, a concentration related increase in [Ca2+]i was noted that preceded and closely paralleled the magnitude of injury. Thus, the higher the concentration of DC or EtOH, the more profound were the changes in [Ca2+]i and the resultant degree of cellular injury. Pretreatment with a low concentration of DC (50 microM; called a mild irritant) that was not damaging by itself attenuated injury induced by a damaging concentration (i.e. 250 microM) of DC, and appeared to elicit this protective action through mechanisms that resisted intracellular Ca2+ accumulation. Additional studies indicated that the mechanism of aspirin damage may be similar and that other protective agents such as prostaglandins and growth factors appear to mediate their protective properties through prevention of intracellular Ca2+ alterations. We propose that agents that prevent mucosal injury mediate this activity through a cellular response (involving active Ca2+ efflux) that subsequently provides a protective action by limiting the magnitude of intracellular Ca2+ accumulation.     

Regulation of the mucosal gastrin receptor

The gastrin receptor in rat oxyntic gland mucosa is highly regulated. This regulation has so far only been found to be directed at the total numbers of receptors present. The affinity of the receptor is not altered significantly by agents which affect receptor numbers. Homospecific regulation occurs in that gastrin upregulates its receptor over long periods of time. Upregulation, however, appears to be preceded by a brief period of downregulation. During development corticosterone triggers the synthesis, or at least the appearance of gastrin receptors. Receptor development is maintained by solid food and presumably the gastrin it releases, but the change in diet which occurs at weaning does not in itself induce development. In female rats, estrogens prevent the increase of gastrin receptor levels to those found in males. These results with the gastrin receptor emphasize the importance of studying receptor concentrations as well as hormone levels to the total understanding of an endocrine response. They also suggest that the receptors of the other gastrointestinal hormones are likely to be regulated and that this regulation will probably be important in understanding some of the diseases of the gastrointestinal tract.     

Regulation of phosphatidylcholine homeostasis by Sec14

Phosphatidylcholine is the major phospholipid in eukaryotic cells and serves as both a permeability barrier as well as a modulator of a plethora of cellular and biological functions. This review touches on the importance of proper regulation of phosphatidylcholine metabolism on health, and discusses how yeast genetics has contributed to furthering our understanding of the precise molecular events regulated by alterations in phosphatidylcholine metabolism. Yeast studies have determined that the phosphatidylcholine and (or) phosphatidylinositol binding protein, Sec14, is a major regulator of phosphatidylcholine homeostasis. Sec14 itself regulates vesicular transport from the Golgi, and the interrelationship between phosphatidylcholine metabolism and membrane movement within the cell is described in detail. The recent convergence of the yeast genetic studies with that of mammalian cell biology in how cells maintain phosphatidylcholine homeostasis is highlighted.     

Regulation of ion homeostasis under salt stress

When under salt stress, plants maintain a high concentration of K(+) and a low concentration of Na(+) in the cytosol. They do this by regulating the expression and activity of K(+) and Na(+) transporters and of H(+) pumps that generate the driving force for transport. Although salt-stress sensors remain elusive, some of the intermediary signaling components have been identified. Evidence suggests that a protein kinase complex consisting of the myristoylated calcium-binding protein SOS3 and the serine/threonine protein kinase SOS2 is activated by a salt-stress-elicited calcium signal. The protein kinase complex then phosphorylates and activates various ion transporters, such as the plasma membrane Na(+)/H(+) antiporter SOS1.     

Regulation of neuronal bioenergy homeostasis by glutamate

Bioenergy homeostasis is crucial in maintaining normal cell function and survival and it is thus important to understand cellular mechanisms underlying its regulation. Neurons use a large amount of ATP to maintain membrane potential and synaptic communication, making the brain the most energy consuming organ in the body. Glutamate mediates a large majority of synaptic transmission which is responsible for the expression of neural plasticity and higher brain functions. Most of the energy cost is attributable to the glutamatergic system; under pathological conditions such as stroke and brain ischemia, neural energy depletion is accompanied by a massive release of glutamate. However, the specific cellular processes implicated in glutamate-dependent bioenergy dynamics are not well understood. We find that glutamate induces a rapid and dramatic reduction of ATP levels in neurons, through reduced ATP genesis and elevated consumption. ATP reduction depends on NMDA receptor activity, but is not a result of neuronal firing, gap junction-mediated leaking or intracellular signaling. Similar changes in ATP levels are also induced by synaptic glutamate accumulation following suppression of glutamate transporter activity. Furthermore, the glutamate-induced ATP down-regulation is blocked by the sodium pump inhibitor ouabain, suggesting the sodium pump as the primary energy consumer during glutamate stimulation. These data suggest the important role of glutamate in the control of cellular ATP homeostasis.     

Regulation of lymphoid homeostasis by interleukin-15

Interleukin (IL)-15 is a member of the common gamma chain family of cytokines, and is closely related to IL-2. While these two cytokines share several important biological functions in vitro, recent mouse models have demonstrated unique roles for these two cytokines in supporting lymphoid homeostasis in vivo. IL-15 has been shown to regulate the homeostasis of both innate and adaptive immune cells, and this review will discuss several ways in which this pleiotropic cytokine may support lymphoid homeostasis.