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Jose M Mato - One of the best experts on this subject based on the ideXlab platform.

  • micrornas regulate methionine adenosyltransferase 1a expression in hepatocellular carcinoma
    Journal of Clinical Investigation, 2013
    Co-Authors: Heping Yang, Michele E Cho, Hui Peng, Jose M Mato
    Abstract:

    MicroRNAs (miRNAs) and methionine adenosyltransferase 1A (MAT1A) are dysregulated in hepatocellular carcinoma (HCC), and reduced MAT1A expression correlates with worse HCC prognosis. Expression of miR-664, miR-485-3p, and miR-495, potential regulatory miRNAs of MAT1A, is increased in HCC. Knockdown of these miRNAs individually in Hep3B and HepG2 cells induced MAT1A expression, reduced growth, and increased apoptosis, while combined knockdown exerted additional effects on all parameters. Subcutaneous and intraparenchymal injection of Hep3B cells stably overexpressing each of this trio of miRNAs promoted tumorigenesis and metastasis in mice. Treatment with miRNA-664 (miR-664), miR-485-3p, and miR-495 siRNAs reduced tumor growth, invasion, and metastasis in an orthotopic liver cancer model. Blocking MAT1A induction significantly reduced the antitumorigenic effect of miR-495 siRNA, whereas maintaining MAT1A expression prevented miRNA-mediated enhancement of growth and metastasis. Knockdown of these miRNAs increased total and nuclear level of MAT1A protein, global CpG methylation, lin-28 homolog B (Caenorhabditis elegans) (LIN28B) promoter methylation, and reduced LIN28B expression. The opposite occurred with forced expression of these miRNAs. In conclusion, upregulation of miR-664, miR-485-3p, and miR-495 contributes to lower MAT1A expression in HCC, and enhanced tumorigenesis may provide potential targets for HCC therapy.

  • inhibition of human methionine adenosyltransferase 1a transcription by coding region methylation
    Journal of Cellular Physiology, 2012
    Co-Authors: Maria Lauda Tomasi, Jose M Mato
    Abstract:

    Two genes (MAT1A and MAT2A) encode for the essential enzyme methionine adenosyltransferase (MAT). MAT1A is silenced in hepatocellular carcinoma (HCC), and absence of MAT1A leads to spontaneous development of HCC in mice. Previous report correlated promoter methylation to silencing of MAT1A but definitive proof was lacking. Here we investigated the role of methylation in regulating MAT1A expression. There are three MspI/HpaII sites from −1,913 to +160 of the human MAT1A gene (numbered relative to the translational start site) at position −977, +10, and +88. Bisulfite treatment and DNA sequencing, and Southern blot analysis showed that methylation at +10 and +88, but not −977, correlated with lack of MAT1A expression. MAT1A promoter construct methylated at −977, +10 or +88 position has 0.7-fold, 3-fold, and 1.6-fold lower promoter activity, respectively. Methylation at −977 and +10 did not inhibit the promoter more than methylation at +10 alone; while methylation at +10 and +88 reduced promoter activity by 60%. Mutation of +10 and +88 sites also resulted in 40% reduction of promoter activity. Reactivation of MAT1A correlated with demethylation of +10 and +88. In vitro transcription assay showed that methylation or mutation of +10 and +88 sites reduced transcription. In conclusion, our data support the novel finding that methylation of the MAT1A coding region can inhibit gene transcription. This represents a key mechanism for decreased MAT1A expression in HCC and a target for therapy. To our knowledge, this is the first example of coding region methylation inhibiting transcription of a mammalian gene. J. Cell. Physiol. 227: 1583–1591, 2012. © 2011 Wiley Periodicals, Inc.

  • methionine adenosyltransferase 1a gene deletion disrupts hepatic very low density lipoprotein assembly in mice
    Hepatology, 2011
    Co-Authors: Ainara Cano, Jose M Mato, Luz M Martinezchantar, Xabier Buque, Maite Martinezuna, Igor Aurrekoetxea, Ariane Menor, Juan L Garciarodriguez, Begona Ochoa, Patricia Aspichueta
    Abstract:

    Very low-density lipoprotein (VLDL) secretion provides a mechanism to export triglycerides (TG) from the liver to peripheral tissues, maintaining lipid homeostasis. In nonalcoholic fatty liver disease (NAFLD), VLDL secretion disturbances are unclear. Methionine adenosyltransferase (MAT) is responsible for S-adenosylmethionine (SAMe) synthesis and MAT I and III are the products of the MAT1A gene. Deficient MAT I and III activities and SAMe content in the liver have been associated with NAFLD, but whether MAT1A is required for normal VLDL assembly remains unknown. We investigated the role of MAT1A on VLDL assembly in two metabolic contexts: in 3-month-old MAT1A-knockout mice (3-KO), with no signs of liver injury, and in 8-month-old MAT1A-knockout mice (8-KO), harboring nonalcoholic steatohepatitis. In 3-KO mouse liver, there is a potent effect of MAT1A deletion on lipid handling, decreasing mobilization of TG stores, TG secretion in VLDL and phosphatidylcholine synthesis via phosphatidylethanolamine N-methyltransferase. MAT1A deletion also increased VLDL– apolipoprotein B secretion, leading to small, lipid-poor VLDL particles. Administration of SAMe to 3-KO mice for 7 days recovered crucial altered processes in VLDL assembly and features of the secreted lipoproteins. The unfolded protein response was activated in 8-KO mouse liver, in which TG accumulated and the phosphatidylcholine-to-phosphatidylethanolamine ratio was reduced in the endoplasmic reticulum, whereas secretion of TG and apolipoprotein B in VLDL was increased and the VLDL physical characteristics resembled that in 3-KO mice. MAT1A deletion also altered plasma lipid homeostasis, with an increase in lipid transport in low-density lipoprotein subclasses and decrease in high-density lipoprotein subclasses. Conclusion:MAT1A is required for normal VLDL assembly and plasma lipid homeostasis in mice. Impaired VLDL synthesis, mainly due to SAMe deficiency, contributes to NAFLD development in MAT1A-KO mice. (HEPATOLOGY 2011

  • forced expression of methionine adenosyltransferase 1a in human hepatoma cells suppresses in vivo tumorigenicity in mice
    American Journal of Pathology, 2010
    Co-Authors: Komal Ramani, Heping Yang, Meng Xia, Zhanfeng Sun, Chishing Zee, Edward Grant, Jose M Mato
    Abstract:

    Methionine adenosyltransferase (MAT) catalyzes the synthesis of S-adenosylmethionine, the principal methyl donor, and is encoded by MAT1A and MAT2A in mammals. Normal liver expresses MAT1A, which is silenced in hepatocellular carcinoma. We have shown that hepatoma cells overexpressing MAT1A grew slower, but whether this is also true in vivo remains unknown. To investigate the effect of overexpressing MAT1A on in vivo tumorigenesis, we generated stable transfectants of Huh7 cells overexpressing either MAT1A or empty vector. Real-time PCR and Western blotting were used to measure expression, and BALB/c nude mice were injected subcutaneously with untransfected or Huh7 cells transfected with empty or MAT1A expression vector to establish tumors. Tumor properties such as proliferation, angiogenesis, and apoptosis were compared, and microarray analysis was performed. Huh7 cells overexpressing MAT1A had higher S-adenosylmethionine levels but lower bromodeoxyuridine incorporation than control cells. Tumor growth rates and weights were lower in MAT1A transfected tumors. In addition, microvessel density and CD31 and Ki-67 staining were lower in MAT1A transfected tumors than control tumors, whereas the apoptosis index was higher in MAT1A-transfected tumors. Forced expression of MAT1A induced genes related to apoptosis and tumor suppression and lowered expression of cell growth and angiogenesis proteins. Our data demonstrate in vivo overexpression of MAT1A in liver cancer cells can suppress tumor growth. They also suggest inducing MAT1A expression might be a strategy to treat hepatocellular carcinoma.

  • role of methionine adenosyltransferase and s adenosylmethionine in alcohol associated liver cancer
    Alcohol, 2005
    Co-Authors: Jose M Mato
    Abstract:

    Two genes (MAT1A and MAT2A) encode for the essential enzyme methionine adenosyltransferase (MAT), which catalyzes the biosynthesis of S-adenosylmethionine (SAMe), the principal methyl donor and, in the liver, a precursor of glutathione. MAT1A is expressed mostly in the liver, whereas MAT2A is widely distributed. MAT2A is induced in the liver during periods of rapid growth and dedifferentiation. In human hepatocellular carcinoma (HCC) MAT1A is replaced by MAT2A. This is important pathogenetically because MAT2A expression is associated with lower SAMe levels and faster growth, whereas exogenous SAMe treatment inhibits growth. Rats fed ethanol intragastrically for 9 weeks also exhibit a relative switch in hepatic MAT expression, decreased SAMe levels, hypomethylation of c-myc, increased c-myc expression, and increased DNA strand break accumulation. Patients with alcoholic liver disease have decreased hepatic MAT activity owing to both decreased MAT1A expression and inactivation of the MAT1A-encoded isoenzymes, culminating in decreased SAMe biosynthesis. Consequences of chronic hepatic SAMe depletion have been examined in the MAT1A knockout mouse model. In this model, the liver is more susceptible to injury. In addition, spontaneous steatohepatitis develops by 8 months, and HCC develops by 18 months. Accumulating evidence shows that, in addition to being a methyl donor, SAMe controls hepatocyte growth response and death response. Whereas transient SAMe depletion is necessary for the liver to regenerate, chronic hepatic SAMe depletion may lead to malignant transformation. It is interesting that SAMe is antiapoptotic in normal hepatocytes, but proapoptotic in liver cancer cells. This should make SAMe an attractive agent for both chemoprevention and treatment of HCC.

Jesús Avila - One of the best experts on this subject based on the ideXlab platform.

  • map1b dependent rac activation is required for ampa receptor endocytosis during long term depression
    The EMBO Journal, 2013
    Co-Authors: Marion Benoist, Jesús Avila, Elena Tortosa, Christian Gonzalezbillault, Rocío Palenzuela, Carlos Rozas, Patricio Rojas, Bernardo Morales, José A. Esteban
    Abstract:

    The microtubule-associated protein 1B (MAP1B) plays critical roles in neurite growth and synapse maturation during brain development. This protein is well expressed in the adult brain. However, its function in mature neurons remains unknown. We have used a genetically modified mouse model and shRNA techniques to assess the role of MAP1B at established synapses, bypassing MAP1B functions during neuronal development. Under these conditions, we found that MAP1B deficiency alters synaptic plasticity by specifically impairing long-term depression (LTD) expression. Interestingly, this is due to a failure to trigger AMPA receptor endocytosis and spine shrinkage during LTD. These defects are accompanied by an impaired targeting of the Rac1 activator Tiam1 at synaptic compartments. Accordingly, LTD and AMPA receptor endocytosis are restored in MAP1B-deficient neurons by providing additional Rac1. Therefore, these results indicate that the MAP1B-Tiam1-Rac1 relay is essential for spine structural plasticity and removal of AMPA receptors from synapses during LTD. This work highlights the importance of MAPs as signalling hubs controlling the actin cytoskeleton and receptor trafficking during plasticity in mature neurons.

  • map1b regulates microtubule dynamics by sequestering eb1 3 in the cytosol of developing neuronal cells
    The EMBO Journal, 2013
    Co-Authors: Elena Tortosa, Jesús Avila, Niels Galjart, Carmen Laura Sayas
    Abstract:

    MAP1B, a structural microtubule (MT)-associated protein highly expressed in developing neurons, plays a key role in neurite and axon extension. However, not all molecular mechanisms by which MAP1B controls MT dynamics during these processes have been revealed. Here, we show that MAP1B interacts directly with EB1 and EB3 (EBs), two core ‘microtubule plus-end tracking proteins' (+TIPs), and sequesters them in the cytosol of developing neuronal cells. MAP1B overexpression reduces EBs binding to plus-ends, whereas MAP1B downregulation increases binding of EBs to MTs. These alterations in EBs behaviour lead to changes in MT dynamics, in particular overstabilization and looping, in growth cones of MAP1B-deficient neurons. This contributes to growth cone remodelling and a delay in axon outgrowth. Together, our findings define a new and crucial role of MAP1B as a direct regulator of EBs function and MT dynamics during neurite and axon extension. Our data provide a new layer of MT regulation: a classical MAP, which binds to the MT lattice and not to the end, controls effective concentration of core +TIPs thereby regulating MTs at their plus-ends.

  • microtubule associated protein 1b function during normal development regeneration and pathological conditions in the nervous system
    Journal of Neurobiology, 2004
    Co-Authors: Francisco Wandosell, Javier Diaznido, Christian Gonzalezbillault, Eva M Jimenezmateos, Alfredo Caceres, Jesús Avila
    Abstract:

    Microtubule-associated protein 1B is the first MAP to be expressed during the development of the nervous system. Several different approaches have revealed that MAP1B function is associated with microtubule and actin microfilament polymerization and dynamics. In recent years, the generation of molecular models to inactivate MAP1B function in invertebrates and mammals has sparked some controversy about the real role of MAP1B. Despite discrepancies between some studies, it is clear that MAP1B plays a principal role in the development of the nervous system. In this article, we summarize the evidence for MAP1B function in a wide variety of cellular processes implicated in the proper construction of the nervous system. We also discuss the role of MAP1B in pathological processes. © 2003 Wiley Periodicals, Inc. J Neurobiol 58: 48–59, 2004

  • The phosphorylated isoform of microtubule associated protein 1B (MAP1B) is expressed in the visual system of the tench (Tinca tinca, L) during optic nerve regeneration.
    Neuroscience letters, 1998
    Co-Authors: Elena Vecino, Luis Ulloa, Jesús Avila
    Abstract:

    By using Western blot analysis and immunohistochemistry we have demonstrated that microtubule associated protein 1B (MAP1B)-phos is present in growing and regenerating axons of retinal ganglion cells of fish (Tinca tinca, L). We have found that the levels of MAP1B-phos substantially increase in regenerating optic nerves. Our observations suggest that MAP1B-phos plays an important role in regeneration processes in the central nervous system (CNS) of the fish. These results are compared in the present paper with that found in the regenerating peripheral nervous system (PNS) of mammals.

  • A protein related to brain microtubule-associated protein MAP1B is a component of the mammalian centrosome.
    Journal of Cell Science, 1994
    Co-Authors: J.e. Dominguez, Eric Karsenti, B. Buendia, Carlos López-otín, Claude Antony, Jesús Avila
    Abstract:

    The centrosome is the main microtubule organizing center of mammalian cells. Structurally, it is composed of a pair of centrioles surrounded by a fibro-granular material (the pericentriolar material) from which microtubules are nucleated. However, the nature of centrosomal molecules involved in microtubules nucleation is still obscure. Since brain microtubule-associated proteins (MAPs) lower the critical tubulin concentration required for microtubule nucleation in tubulin solution in vitro, we have examined their possible association with centrosomes. By immunofluorescence, monoclonal and polyclonal antibodies raised against MAP1B stain the centrosome in cultured cells as well as purified centrosomes, whereas antibodies raised against MAP2 give a completely negative reaction. The MAP1B-related antigen is localized to the pericentriolar material as revealed by immunoelectron microscopy. In preparations of purified centrosomes analyzed on poly-acrylamide gels, a protein that migrates as brain MAP1B is present. After blotting on nitrocellulose, it is decorated by anti-MAP1B antibodies and the amino acid sequence of proteolytic fragments of this protein is similar to brain MAP1B. Moreover, brain MAP1B and its centrosomal counterpart share the same phosphorylation features and have similar peptide maps. These data strongly suggest that a protein homologue to MAP1B is present in centrosomes and it is a good candidate for being involved in the nucleating activity of the pericentriolar material.

Steffen Thiel - One of the best experts on this subject based on the ideXlab platform.

  • map19 the alternative splice product of the masp2 gene
    Journal of Immunological Methods, 2011
    Co-Authors: Soren E Degn, Steffen Thiel, Ole Haagen Nielsen, Annette G Hansen, Rudi Steffensen, Jens C Jensenius
    Abstract:

    The lectin pathway of complement is a central part of innate immunity, but as a powerful inducer of inflammation it needs to be tightly controlled. The MASP2 gene encodes two proteins, MASP-2 and MAp19. MASP-2 is the serine protease responsible for lectin pathway activation. The smaller alternative splice product, MAp19, lacks a catalytic domain but retains two of three domains involved in association with the pattern-recognition molecules (PRMs): mannan-binding lectin (MBL), H-ficolin, L-ficolin and M-ficolin. MAp19 reportedly acts as a competitive inhibitor of MASP-2-mediated complement activation. In light of a ten times lower affinity of MAp19, versus MASP-2, for association with the PRMs, much higher serum concentrations of MAp19 than MASP-2 would be required for MAp19 to exert such an inhibitory activity. Just four amino acid residues distinguish MAp19 from MASP-2, and these are conserved between man, mouse and rat. Nonetheless we generated monoclonal rat anti-MAp19 antibodies and established a quantitative assay. We found the concentration of MAp19 in serum to be 217 ng/ml, i.e., 11nM, comparable to the 7 nM of MASP-2. In serum all MASP-2, but only a minor fraction of MAp19, was associated with PRMs. In contrast to previous reports we found that MAp19 could not compete with MASP-2 for binding to MBL, nor could it inhibit MASP-2-mediated complement activation. Immunohistochemical analyses combined with qRT-PCR revealed that both MAp19 and MASP-2 were mainly expressed in hepatocytes. High levels of MAp19 were found in urine, where MASP-2 was absent.

  • identification of the site of human mannan binding lectin involved in the interaction with its partner serine proteases the essential role of lys55
    Journal of Immunology, 2007
    Co-Authors: Florence Teillet, Steffen Thiel, Monique Lacroix, Dietmar Weilguny, Teit Agger, Gerard J Arlaud, Nicole M Thielens
    Abstract:

    Mannan-binding lectin (MBL) is an oligomeric lectin that binds neutral carbohydrates on pathogens, forms complexes with MBL-associated serine proteases (MASP)-1, -2, and -3 and 19-kDa MBL-associated protein (MAp19), and triggers the complement lectin pathway through activation of MASP-2. To identify the MASP binding site(s) of human MBL, point mutants targeting residues C-terminal to the hinge region were produced and tested for their interaction with the MASPs and MAp19 using surface plasmon resonance and functional assays. Mutation Lys 55 Ala abolished interaction with the MASPs and MAp19 and prevented formation of functional MBL-MASP-2 complexes. Mutations Lys 55 Gln and Lys 55 Glu abolished binding to MASP-1 and -3 and strongly inhibited interaction with MAp19. Conversely, mutation Lys 55 Arg abolished interaction with MASP-2 and MAp19, but only weakened interaction with MASP-1 and -3. Mutation Arg 47 Glu inhibited interaction with MAp19 and decreased the ability of MBL to trigger the lectin pathway. Mutant Arg 47 Lys showed no interaction with the MASPs or MAp19, likely resulting from a defect in oligomerization. In contrast, mutation Arg 47 Ala had no impact on the interaction with the MASPs and MAp19, nor on the ability of MBL to trigger the lectin pathway. Mutation Pro 53 Ala only had a slight effect on the interaction with MASP-1 and -3, whereas mutations at residues Leu 49 and Leu 56 were ineffective. In conclusion, the MASP binding site of MBL involves a sequence stretch centered on residue Lys 55 , which may form an ionic bond representing the major component of the MBL-MASP interaction. The binding sites for MASP-2/MAp19 and MASP-1/3 have common features but are not strictly identical.

  • interaction properties of human mannan binding lectin mbl associated serine proteases 1 and 2 mbl associated protein 19 and mbl
    Journal of Immunology, 2001
    Co-Authors: Nicole M Thielens, Steffen Thiel, Jens C Jensenius, Thomas Vorupjensen, Sandor Cseh, Veronique Rossi, Gerard J Arlaud
    Abstract:

    The mannan-binding lectin (MBL) activation pathway of complement plays an important role in the innate immune defense against pathogenic microorganisms. In human serum, two MBL-associated serine proteases (MASP-1, MASP-2) and MBL-associated protein 19 (MAp19) were found to be associated with MBL. With a view to investigate the interaction properties of these proteins, human MASP-1, MASP-2, MAp19, as well as the N-terminal complement subcomponents C1r/C1s, Uegf, and bone morphogenetic protein-1-epidermal growth factor (CUB-EGF) segments of MASP-1 and MASP-2, were expressed in insect or human kidney cells, and MBL was isolated from human serum. Sedimentation velocity analysis indicated that the MASP-1 and MASP-2 CUB-EGF segments and the homologous protein MAp19 all behaved as homodimers (2.8–3.2 S) in the presence of Ca 2+ . Although the latter two dimers were not dissociated by EDTA, their physical properties were affected. In contrast, the MASP-1 CUB-EGF homodimer was not sensitive to EDTA. The three proteins and full-length MASP-1 and MASP-2 showed no interaction with each other as judged by gel filtration and surface plasmon resonance spectroscopy. Using the latter technique, MASP-1, MASP-2, their CUB-EGF segments, and MAp19 were each shown to bind to immobilized MBL, with K D values of 0.8 nM (MASP-2), 1.4 nM (MASP-1), 13.0 nM (MAp19 and MASP-2 CUB-EGF), and 25.7 nM (MASP-1 CUB-EGF). The binding was Ca 2+ -dependent and fully sensitive to EDTA in all cases. These data indicate that MASP-1, MASP-2, and MAp19 each associate as homodimers, and individually form Ca 2+ -dependent complexes with MBL through the CUB-EGF pair of each protein. This suggests that distinct MBL/MASP complexes may be involved in the activation or regulation of the MBL pathway.

  • interaction of c1q and mannan binding lectin mbl with c1r c1s mbl associated serine proteases 1 and 2 and the mbl associated protein map19
    Journal of Immunology, 2000
    Co-Authors: Steffen Thiel, Steen V Petersen, Thomas Vorupjensen, Misao Matsushita, Teizo Fujita, Cordula M Stover, Wilhelm J Schwaeble, Jens C Jensenius
    Abstract:

    Mannan-binding lectin (MBL) and C1q activate the complement cascade via attached serine proteases. The proteases C1r and C1s were initially discovered in a complex with C1q, whereas the MBL-associated serine proteases 1 and 2 (MASP-1 and -2) were discovered in a complex with MBL. There is controversy as to whether MBL can utilize C1r and C1s or, inversely, whether C1q can utilize MASP-1 and 2. Serum deficient in C1r produced no complement activation in IgG-coated microwells, whereas activation was seen in mannan-coated microwells. In serum, C1r and C1s were found to be associated only with C1q, whereas MASP-1, MASP-2, and a third protein, MAp19 (19-kDa MBL-associated protein), were found to be associated only with MBL. The bulk of MASP-1 and MAp19 was found in association with each other and was not bound to MBL or MASP-2. The interactions of MASP-1, MASP-2, and MAp19 with MBL differ from those of C1r and C1s with C1q in that both high salt concentrations and calcium chelation (EDTA) are required to fully dissociate the MASPs or MAp19 from MBL. In the presence of calcium, most of the MASP-1, MASP-2, and MAp19 emerged on gel-permeation chromatography as large complexes that were not associated with MBL, whereas in the presence of EDTA most of these components formed smaller complexes. Over 95% of the total MASPs and MAp19 found in serum are not complexed with MBL.

Nicole M Thielens - One of the best experts on this subject based on the ideXlab platform.

  • identification of the site of human mannan binding lectin involved in the interaction with its partner serine proteases the essential role of lys55
    Journal of Immunology, 2007
    Co-Authors: Florence Teillet, Steffen Thiel, Monique Lacroix, Dietmar Weilguny, Teit Agger, Gerard J Arlaud, Nicole M Thielens
    Abstract:

    Mannan-binding lectin (MBL) is an oligomeric lectin that binds neutral carbohydrates on pathogens, forms complexes with MBL-associated serine proteases (MASP)-1, -2, and -3 and 19-kDa MBL-associated protein (MAp19), and triggers the complement lectin pathway through activation of MASP-2. To identify the MASP binding site(s) of human MBL, point mutants targeting residues C-terminal to the hinge region were produced and tested for their interaction with the MASPs and MAp19 using surface plasmon resonance and functional assays. Mutation Lys 55 Ala abolished interaction with the MASPs and MAp19 and prevented formation of functional MBL-MASP-2 complexes. Mutations Lys 55 Gln and Lys 55 Glu abolished binding to MASP-1 and -3 and strongly inhibited interaction with MAp19. Conversely, mutation Lys 55 Arg abolished interaction with MASP-2 and MAp19, but only weakened interaction with MASP-1 and -3. Mutation Arg 47 Glu inhibited interaction with MAp19 and decreased the ability of MBL to trigger the lectin pathway. Mutant Arg 47 Lys showed no interaction with the MASPs or MAp19, likely resulting from a defect in oligomerization. In contrast, mutation Arg 47 Ala had no impact on the interaction with the MASPs and MAp19, nor on the ability of MBL to trigger the lectin pathway. Mutation Pro 53 Ala only had a slight effect on the interaction with MASP-1 and -3, whereas mutations at residues Leu 49 and Leu 56 were ineffective. In conclusion, the MASP binding site of MBL involves a sequence stretch centered on residue Lys 55 , which may form an ionic bond representing the major component of the MBL-MASP interaction. The binding sites for MASP-2/MAp19 and MASP-1/3 have common features but are not strictly identical.

  • interaction properties of human mannan binding lectin mbl associated serine proteases 1 and 2 mbl associated protein 19 and mbl
    Journal of Immunology, 2001
    Co-Authors: Nicole M Thielens, Steffen Thiel, Jens C Jensenius, Thomas Vorupjensen, Sandor Cseh, Veronique Rossi, Gerard J Arlaud
    Abstract:

    The mannan-binding lectin (MBL) activation pathway of complement plays an important role in the innate immune defense against pathogenic microorganisms. In human serum, two MBL-associated serine proteases (MASP-1, MASP-2) and MBL-associated protein 19 (MAp19) were found to be associated with MBL. With a view to investigate the interaction properties of these proteins, human MASP-1, MASP-2, MAp19, as well as the N-terminal complement subcomponents C1r/C1s, Uegf, and bone morphogenetic protein-1-epidermal growth factor (CUB-EGF) segments of MASP-1 and MASP-2, were expressed in insect or human kidney cells, and MBL was isolated from human serum. Sedimentation velocity analysis indicated that the MASP-1 and MASP-2 CUB-EGF segments and the homologous protein MAp19 all behaved as homodimers (2.8–3.2 S) in the presence of Ca 2+ . Although the latter two dimers were not dissociated by EDTA, their physical properties were affected. In contrast, the MASP-1 CUB-EGF homodimer was not sensitive to EDTA. The three proteins and full-length MASP-1 and MASP-2 showed no interaction with each other as judged by gel filtration and surface plasmon resonance spectroscopy. Using the latter technique, MASP-1, MASP-2, their CUB-EGF segments, and MAp19 were each shown to bind to immobilized MBL, with K D values of 0.8 nM (MASP-2), 1.4 nM (MASP-1), 13.0 nM (MAp19 and MASP-2 CUB-EGF), and 25.7 nM (MASP-1 CUB-EGF). The binding was Ca 2+ -dependent and fully sensitive to EDTA in all cases. These data indicate that MASP-1, MASP-2, and MAp19 each associate as homodimers, and individually form Ca 2+ -dependent complexes with MBL through the CUB-EGF pair of each protein. This suggests that distinct MBL/MASP complexes may be involved in the activation or regulation of the MBL pathway.

Gerard J Arlaud - One of the best experts on this subject based on the ideXlab platform.

  • identification of the site of human mannan binding lectin involved in the interaction with its partner serine proteases the essential role of lys55
    Journal of Immunology, 2007
    Co-Authors: Florence Teillet, Steffen Thiel, Monique Lacroix, Dietmar Weilguny, Teit Agger, Gerard J Arlaud, Nicole M Thielens
    Abstract:

    Mannan-binding lectin (MBL) is an oligomeric lectin that binds neutral carbohydrates on pathogens, forms complexes with MBL-associated serine proteases (MASP)-1, -2, and -3 and 19-kDa MBL-associated protein (MAp19), and triggers the complement lectin pathway through activation of MASP-2. To identify the MASP binding site(s) of human MBL, point mutants targeting residues C-terminal to the hinge region were produced and tested for their interaction with the MASPs and MAp19 using surface plasmon resonance and functional assays. Mutation Lys 55 Ala abolished interaction with the MASPs and MAp19 and prevented formation of functional MBL-MASP-2 complexes. Mutations Lys 55 Gln and Lys 55 Glu abolished binding to MASP-1 and -3 and strongly inhibited interaction with MAp19. Conversely, mutation Lys 55 Arg abolished interaction with MASP-2 and MAp19, but only weakened interaction with MASP-1 and -3. Mutation Arg 47 Glu inhibited interaction with MAp19 and decreased the ability of MBL to trigger the lectin pathway. Mutant Arg 47 Lys showed no interaction with the MASPs or MAp19, likely resulting from a defect in oligomerization. In contrast, mutation Arg 47 Ala had no impact on the interaction with the MASPs and MAp19, nor on the ability of MBL to trigger the lectin pathway. Mutation Pro 53 Ala only had a slight effect on the interaction with MASP-1 and -3, whereas mutations at residues Leu 49 and Leu 56 were ineffective. In conclusion, the MASP binding site of MBL involves a sequence stretch centered on residue Lys 55 , which may form an ionic bond representing the major component of the MBL-MASP interaction. The binding sites for MASP-2/MAp19 and MASP-1/3 have common features but are not strictly identical.

  • interaction properties of human mannan binding lectin mbl associated serine proteases 1 and 2 mbl associated protein 19 and mbl
    Journal of Immunology, 2001
    Co-Authors: Nicole M Thielens, Steffen Thiel, Jens C Jensenius, Thomas Vorupjensen, Sandor Cseh, Veronique Rossi, Gerard J Arlaud
    Abstract:

    The mannan-binding lectin (MBL) activation pathway of complement plays an important role in the innate immune defense against pathogenic microorganisms. In human serum, two MBL-associated serine proteases (MASP-1, MASP-2) and MBL-associated protein 19 (MAp19) were found to be associated with MBL. With a view to investigate the interaction properties of these proteins, human MASP-1, MASP-2, MAp19, as well as the N-terminal complement subcomponents C1r/C1s, Uegf, and bone morphogenetic protein-1-epidermal growth factor (CUB-EGF) segments of MASP-1 and MASP-2, were expressed in insect or human kidney cells, and MBL was isolated from human serum. Sedimentation velocity analysis indicated that the MASP-1 and MASP-2 CUB-EGF segments and the homologous protein MAp19 all behaved as homodimers (2.8–3.2 S) in the presence of Ca 2+ . Although the latter two dimers were not dissociated by EDTA, their physical properties were affected. In contrast, the MASP-1 CUB-EGF homodimer was not sensitive to EDTA. The three proteins and full-length MASP-1 and MASP-2 showed no interaction with each other as judged by gel filtration and surface plasmon resonance spectroscopy. Using the latter technique, MASP-1, MASP-2, their CUB-EGF segments, and MAp19 were each shown to bind to immobilized MBL, with K D values of 0.8 nM (MASP-2), 1.4 nM (MASP-1), 13.0 nM (MAp19 and MASP-2 CUB-EGF), and 25.7 nM (MASP-1 CUB-EGF). The binding was Ca 2+ -dependent and fully sensitive to EDTA in all cases. These data indicate that MASP-1, MASP-2, and MAp19 each associate as homodimers, and individually form Ca 2+ -dependent complexes with MBL through the CUB-EGF pair of each protein. This suggests that distinct MBL/MASP complexes may be involved in the activation or regulation of the MBL pathway.