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Gerald J Gleich - One of the best experts on this subject based on the ideXlab platform.
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eosinophil granule Major Basic Protein 1 deposition in eosinophilic esophagitis correlates with symptoms independent of eosinophil counts
Diseases of The Esophagus, 2019Co-Authors: Kathryn A. Peterson, Gerald J Gleich, Frederic Clayton, Hedieh Saffari, John C. Fang, N S Limaye, H Crispin, Jacob Robson, Kristin M LeifermanAbstract:In patients with eosinophilic esophagitis (EoE), symptoms often do not correlate with peak eosinophil counts (PEC) determined on histopathological examination of biopsy specimens. This may be because eosinophils degranulate during active disease and lose their morphological identity as intact cells and, therefore, are not enumerated on microscopic examination. Eosinophil granule Proteins that are released into tissues with degranulation, including Major Basic Protein 1 (eMBP1), likely contribute to disease pathogenesis and, therefore, may correlate with symptoms better than PEC. We sought to determine whether symptoms in patients with EoE more closely relate to eosinophil granule Protein deposition than to eosinophil enumeration, especially in patients with fewer than 15 eosinophils per high power field (HPF). Esophageal biopsy specimens from 34 patients diagnosed with EoE were obtained for histopathological examination and for evaluation of eMBP1 staining by indirect immunofluorescence. PEC by histopathology were compared to extracellular eMBP1 grades by immunostaining. PEC and eMBP1 grades also were analyzed for their relationship to symptoms and clinical course. Biopsy specimens from 19 of the 34 patients had fewer than 15 PEC on histopathological examination, and the other 15 patients had 15 or greater PEC. Positive eMBP1 immunostaining was found in all symptomatic patients. EoE symptoms were related to eMBP1 immunostaining grades (p = 0.0001), but not PEC (P = 0.14). Eosinophil granule Protein deposition, specifically eMBP1, is increased in esophageal biopsy specimens from symptomatic patients with EoE and may be a marker of disease activity, including patients with EoE who have 'resolved' disease.
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Rapid Publication Human Eosinophil Major Basic Protein Induces Airway Constriction and Airway Hyperresponsiveness in Primates
2016Co-Authors: Robert H Gundel, Gordon L. Letts, Gerald J GleichAbstract:We have examined the effects of direct intratracheal instilla-tion of purified eosinophil granule Proteins on pulmonary func-tion and airway responsiveness in primates. The results of this study show for the first time that installation of Major Basic Protein (MBP) directly into the trachea of primates results in a significant and dose-related increase in airway responsiveness to inhaled methacholine. Furthermore, MBP and eosinophil peroxidase (EPO) induce a transient bronchoconstriction imme-diately after instillation that resolves by 1 h postinstillation. In constrast, instillation of other eosinophil granule Proteins had no effect on airway responsiveness or pulmonary function. These data indicate a direct role of the eosinophil in the patho-genesis of airway hyperresponsiveness. We suggest that the MBP of human eosinophils has an effector role in the pathogenesis ofairway hyperresponsiveness which may involve active interaction with resident airway tissue cells. MBP may also mediate altered lung function in various inflammatory lung diseases associated with pulmonary eosino-philia. (J. Clin. Invest. 1991. 87:1470-1473.) Key words: eo-sinophil granule Proteins * airway hyperreactivity- bronchocon-striction * monkeys * asthm
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an immunologic test for chronic rhinosinusitis based on free intranasal eosinophilic Major Basic Protein
International Forum of Allergy & Rhinology, 2015Co-Authors: Jens U Ponikau, Gail M. Kephart, Gerald J Gleich, Laurie A Winter, Diane L Squillace, Matt D Hershcovitch, Seo Moon, David A Sherris, Eugene B Kern, Hirohito KitaAbstract:Background A histologic hallmark of chronic rhinosinusitis (CRS) is an eosinophilic inflammation, present with and without nasal polyposis and independent of atopy. Eosinophils migrate through nasal tissue including the epithelium into the nasal airway mucus, where they form clusters and degranulate, releasing granule Proteins including the toxic Major Basic Protein (MBP). Specific biomarkers for CRS, which could be used as a diagnostic test for CRS with a high sensitivity and specificity, are presently lacking. Recently, an enzyme-linked immunosorbent assay (ELISA)-based test for MBP in nasal airway mucus received regulatory approval. Methods A new assay was specifically developed to detect released MBP in airway mucus. MBP levels in nasal mucus of 85 randomly selected CRS patients diagnosed by endoscopy, computed tomography (CT) scans and symptoms were compared to 13 healthy controls and 5 disease controls (allergic rhinitis). Results Overall, 92% (78/85) of CRS patients’ mucus were positive for MBP (mean 7722 ng/mL) vs none of 13 healthy controls and none of 5 allergic rhinitis patients (<7.8 ng/mL; p < 0.000000000002). In this study, the MBP ELISA had a 92% sensitivity and 100% specificity for CRS. Conclusion Free MBP in nasal mucus can be used as a biomarker to diagnose CRS. The MBP ELISA represents the first immunologically-based test to potentially distinguish CRS from the eosinophilic inflammation in allergic rhinitis.
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LOCALIZATION SIMILAR TO EOSINOPHIL OF A MOLECULE IMMUNOCHEMICALLY IN HUMAN Major Basic Protein PLACENTA
2013Co-Authors: E. Maddox, Gail M. Kephart, Joseph H Butterfield, Carolyn B. Coulam, Kurt Benirschke, Gerald J GleichAbstract:The eosinophil granule Major Basic Protein (MBP) 1 is an arginine rich, highly Basic, 9,300 dalton Protein, which forms the crystalloid core of the granule (1-3) and is present in small amounts in basophils (4). MBP is toxic to parasites (5-7) and is deposited onto the surface of damaged microfiliariae in Onchocerca volvulus infestation (8). MBP is also toxic to cells (5, 9) and causes desquamation of respiratory epithelial cells in vitro (10, 11). Finally, MBP triggers histamine release from human basophils and rat mast cells (12). We have recently reported that MBP circulates in the blood of pregnant women at levels 10 to 20 times those found in normal, nonpregnant women (13). The levels of this immunoreactive MBP increase during the first 3 mo of gestation, plateau during the second and third trimester, and decrease sharply following parturition. Eosinophilia is not present during pregnancy, and other eosinophil-related Proteins are not elevated in the sera of pregnant women. Chromatographic analysis of sera from pregnant women indicates that the immunoreactive MBP has an apparent molecular size greater than the molecul
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Brief Definitive Report ACIDIC PRECURSOR REVEALED IN HUMAN EOSINOPHIL GRANULE Major Basic Protein cDNA
2013Co-Authors: L Barker, Gerald J Gleich, R. PeaseAbstract:Eosinophil granule Major Basic Protein (MBP) comprises the crystalloid core of the eosinophil granule (1) and is a 13.8-kD single polypeptide rich in arginine with a calculated isoelectric point (pI) of 10.9. MBP is a potent toxin for helminths and various cell types in vitro, and it has been localized on damaged helminths and tissues in hypersensitivity diseases including bronchial asthma (2). Amolecule indistinguishable from MBP has been localized to placental X cells and placental-site giant cells (3) and increases in maternal plasma just before labor (4). Human MBP contains 17 arginines, only one carboxylic acid-containing residue, and a high proportion of amino acids with hydrophobic side chains (5). It probably exists in solution as an extended molecule with a Major fraction of its surface occupied by cationic charge. The cationic charge may attract MBP to the negatively charged cell surface of a target, whereupon the apolar residues insert into and perturb the lipid milieu. Previous studies suggest that the cationic nature of MBP is an important determinant of its cytotoxicity (6). Here we report on the nucleotide sequence ofacDNA representing human MBP mRNA from the promyelocytic cell line HL-60, which produces MBP (7). Analysis of the cDNA indicates that MBP is translated as a preproProtein with an acidic pro-portion
Claus Oxvig - One of the best experts on this subject based on the ideXlab platform.
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formation of high molecular weight angiotensinogen during pregnancy is a result of competing redox reactions with the proform of eosinophil Major Basic Protein
Biochemical Journal, 2013Co-Authors: Soren Kloverpris, Michael Christiansen, Kasper Pihl, Simon Glerup, Louise L Skov, Claus OxvigAbstract:The plasma concentration of the placentally derived proMBP (proform of eosinophil Major Basic Protein) increases in pregnancy, and three different complexes containing proMBP have been isolated from pregnancy plasma and serum: a 2:2 complex with the metalloProteinase, PAPP-A (pregnancy-associated plasma Protein-A), a 2:2 complex with AGT (angiotensinogen) and a 2:2:2 complex with AGT and complement C3dg. In the present study we show that during human pregnancy, all of the circulating proMBP exists in covalent complexes, bound to either PAPP-A or AGT. We also show that the proMBP–AGT complex constitutes the Major fraction of circulating HMW (high-molecular weight) AGT in late pregnancy, and that this complex is able to further associate with complement C3 derivatives post-sampling. Clearance experiments in mice suggest that complement C3-based complexes are removed faster from the circulation compared to monomeric AGT and the proMBP–AGT complex. Furthermore, we have used recombinant Proteins to analyse the formation of the proMBP–PAPP-A and the proMBP–AGT complexes, and we demonstrate that they are competing reactions, depending on the same cysteine residue of proMBP, but differentially on the redox potential, potentially important for the relative amounts of the complexes in vivo . These findings may be important physiologically, since the biochemical properties of the Proteins change as a consequence of complex formation.
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the proform of the eosinophil Major Basic Protein binds the cell surface through a site distinct from its c type lectin ligand binding region
Journal of Biological Chemistry, 2006Co-Authors: Simon Glerup, Soren Kloverpris, Claus OxvigAbstract:Abstract The highly Basic eosinophil Major Basic Protein (MBP), present in the crystalloid core of eosinophil leukocyte granules, has both cytotoxic and cytostimulatory properties and is directly implicated in a number of diseases. The crystal structure of MBP resembles that of the C-type lectin (CTL) superfamily, and recent data showed that MBP binds heparan sulfate glycosaminoglycan (GAG), with the CTL ligand-binding region as the binding site. MBP is synthesized as a proform (pro-MBP) containing an acidic propiece believed to neutralize the Basic MBP domain. Using flow cytometry and site-directed mutagenesis, we demonstrate here that the MBP domain of pro-MBP binds to heparan sulfate GAG on the cell surface and that this is independent of GAG covalently bound to pro-MBP. Eight Basic residues located in the CTL ligand-binding region of MBP were hypothesized previously to mediate GAG binding, but we found that surface binding was not compromised by the substitution of these residues with alanine. However, the analysis of a series of mutants with surface-exposed residues substituted with alanine showed that Ser-166, Arg-168, and Arg-171 are involved in surface binding. A binding site formed by these residues is located in the MBP domain between loop 1 and β-strand 5, outside the CTL ligand-binding region. The binding of a cell-surface heparan sulfate proteoglycan may be important in MBP action, and our findings suggest that two regions shown previously to contain the cytotoxic and cytostimulatory properties of MBP are accessible for ligand interaction in cell surface-bound MBP.
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Proteinase inhibition by proform of eosinophil Major Basic Protein pro mbp is a multistep process of intra and intermolecular disulfide rearrangements
Journal of Biological Chemistry, 2005Co-Authors: Simon Glerup, Michael Toft Overgaard, Lars Sottrupjensen, Henning B Boldt, Linda C Giudice, Claus OxvigAbstract:The metzincin metalloProteinase pregnancy-associated plasma Protein A (PAPP-A, pappalysin-1) promotes cell growth by the cleavage of insulin-like growth factor-binding Proteins-4 and -5, causing the release of bound insulin-like growth factors. The proteolytic activity of PAPP-A is inhibited by the proform of eosinophil Major Basic Protein (pro-MBP), which forms a covalent 2:2 Proteinase-inhibitor complex based on disulfide bonds. To understand the process of complex formation, we determined the status of cysteine residues in both of the uncomplexed molecules. A comparison of the disulfide structure of the reactants with the known disulfide structure of the PAPP-A.pro-MBP complex reveals that six cysteine residues of the pro-MBP subunit (Cys-51, Cys-89, Cys-104, Cys-107, Cys-128, and Cys-169) and two cysteine residues of the PAPP-A subunit (Cys-381 and Cys-652) change their status from the uncomplexed to the complexed states. Upon complex formation, three disulfide bonds of pro-MBP, which connect the acidic propiece with the Basic, mature portion, are disrupted. In the PAPP-A.pro-MBP complex, two of these form the basis of both two interchain disulfide bonds between the PAPP-A and the pro-MBP subunits and two disulfide bonds responsible for pro-MBP dimerization, respectively. Based on the status of the reactants, we investigated the role of individual cysteine residues upon complex formation by mutagenesis of specific cysteine residues of both subunits. Our findings allow us to depict a hypothetical model of how the PAPPA.pro-MBP complex is formed. In addition, we have demonstrated that complex formation is greatly enhanced by the addition of micromolar concentrations of reductants. It is therefore possible that the activity in vivo of PAPP-A is controlled by the redox potential, and it is further tempting to speculate that such mechanism operates under pathological conditions of altered redox potential.
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pregnancy associated plasma Protein a and its endogenous inhibitor the proform of eosinophil Major Basic Protein prombp are related to complex stenosis morphology in patients with stable angina pectoris
Circulation, 2004Co-Authors: Juan Cosinsales, Michael Christiansen, Claus Oxvig, Michael Toft Overgaard, Paul Kaminski, Della Cole, David W Holt, Juan Carlos KaskiAbstract:Background— The metalloProteinase pregnancy-associated plasma Protein-A (PAPP-A) has been implicated in coronary plaque disruption. Its endogenous inhibitor, the proform of eosinophil Major Basic Protein (proMBP), may also play a role in this process. Atheromatous plaque disruption often presents as complex angiographic lesions. We sought to assess whether PAPP-A, proMBP, and PAPP-A/ProMBP ratio are markers of angiographic plaque complexity in patients with chronic stable angina. Methods and Results— We studied 396 stable angina patients (age 63±10 years, 230 men) of whom 289 had angiographically documented coronary artery disease (≥75% stenosis). All coronary stenoses ≥30% diameter reduction (n =531 in 322 patients) were assessed and classified as complex (n =228) or smooth (n =303) by previously validated criteria. PAPP-A, proMBP, and C-reactive Protein (hs-CRP) serum levels were measured by ELISA. Patients with complex coronary stenoses had a significantly (P<0.001) higher PAPP-A/proMBP ratio (3.1±1.2 ...
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inhibition of proteolysis by the proform of eosinophil Major Basic Protein prombp requires covalent binding to its target Proteinase
FEBS Letters, 2004Co-Authors: Michael Toft Overgaard, Michael Christiansen, Lars Sottrupjensen, Henning B Boldt, Simon Glerup, Vivien Rodacker, Inger Marie Olsen, Linda C Giudice, Claus OxvigAbstract:By proteolytic cleavage of insulin-like growth factor binding Proteins, the metalloProteinase pregnancy-associated plasma Protein-A (PAPP-A) is able to control the biological activity of insulin-like growth factors. PAPP-A circulates in pregnancy as a proteolytically inactive complex, disulfide bound to the proform of eosinophil Major Basic Protein (proMBP). We here demonstrate that co-transfection of mammalian cells with PAPP-A and proMBP cDNA results in the formation of a covalent PAPP-A/proMBP complex in which PAPP-A is inhibited. Formation of the complex also occurs when PAPP-A and proMBP synthesized separately are incubated. Complex formation was monitored by Western blotting, and by using an immunoassay specific for the complex. Using mutagenesis, we further demonstrate that the complex forms in a specific manner and depends on the presence of two proMBP cysteine residues. Mutated proMBP, in which Cys-51 and -169 are replaced by serine, is unable to form the covalent complex with PAPP-A. Of particular interest, such mutated proMBP further lacks the ability to inhibit PAPP-A. For the first time, this conclusively demonstrates that proMBP is a Proteinase inhibitor. We further conclude that proMBP inhibits PAPP-A in an unusual manner, not paralleled by other Proteinase inhibitors of our knowledge, which requires proMBP to be covalently bound to PAPP-A by disulfide bonds. ProMBP binding to PAPP-A most likely either abrogates substrate access to the active site of PAPP-A or induces a conformational change in the structure of PAPP-A, as we, by further mutagenesis, were able to exclude that the inhibitory mechanism of proMBP is based on a cysteine switch-like mechanism.
Michael Christiansen - One of the best experts on this subject based on the ideXlab platform.
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pregnancy associated plasma Protein a papp a and the proform of the eosinophil Major Basic Protein prombp are associated with increased risk of death in heart failure patients
Scandinavian Journal of Clinical & Laboratory Investigation, 2017Co-Authors: Paula L Hedley, Maja Dembic, Christian Torppedersen, Lars Kober, Michael ChristiansenAbstract:Risk stratification and patient management in heart failure (HF) is difficult due to the unpredictable progression of the disease, necessitating the development of reliable diagnostic biomarkers to facilitate decision-making in clinical practice. Pregnancy-associated plasma Protein-A (PAPP-A) is a marker of arteriosclerotic heart disease. PAPP-A is a serum protease, which is involved in the insulin-like growth factor 1 (IGF-1) axis where it is inhibited by the proform of the eosinophil Major Basic Protein (proMBP). In this study, we evaluated serum PAPP-A and proMBP as long-term prognostic biomarkers of all-cause mortality in HF. Serum PAPP-A and proMBP concentrations were determined in 683 patients with NYHA III-IV HF recruited in the EchoCardiography and Heart Study (ECHOS) in Denmark. The mean age of the patients (73% male) was 70 at admission. During 7 years of follow-up, 516 patients died. In univariate analysis, both PAPP-A and proMBP, divided into quartiles, showed significant association with mortality. Using a Cox proportional hazard model, hazard ratios for continuous values of PAPP-A and proMBP were HR = 1.42 (CI = 1.23-1.64, p < 0.0001) and HR = 1.36 (CI = 1.22-1.51, p <0.0001), respectively. However, neither PAPP-A nor proMBP were significant independent predictors when the model included age, gender, brain-type natriuretic peptide, medical history of HF, ischemic heart disease, chronic obstructive pulmonary disease, and diabetes mellitus. In conclusion, high levels of PAPP-A and proMBP are associated with increased risk of death from all causes in HF and are potential prognostic markers of adverse outcomes in HF patients.
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formation of high molecular weight angiotensinogen during pregnancy is a result of competing redox reactions with the proform of eosinophil Major Basic Protein
Biochemical Journal, 2013Co-Authors: Soren Kloverpris, Michael Christiansen, Kasper Pihl, Simon Glerup, Louise L Skov, Claus OxvigAbstract:The plasma concentration of the placentally derived proMBP (proform of eosinophil Major Basic Protein) increases in pregnancy, and three different complexes containing proMBP have been isolated from pregnancy plasma and serum: a 2:2 complex with the metalloProteinase, PAPP-A (pregnancy-associated plasma Protein-A), a 2:2 complex with AGT (angiotensinogen) and a 2:2:2 complex with AGT and complement C3dg. In the present study we show that during human pregnancy, all of the circulating proMBP exists in covalent complexes, bound to either PAPP-A or AGT. We also show that the proMBP–AGT complex constitutes the Major fraction of circulating HMW (high-molecular weight) AGT in late pregnancy, and that this complex is able to further associate with complement C3 derivatives post-sampling. Clearance experiments in mice suggest that complement C3-based complexes are removed faster from the circulation compared to monomeric AGT and the proMBP–AGT complex. Furthermore, we have used recombinant Proteins to analyse the formation of the proMBP–PAPP-A and the proMBP–AGT complexes, and we demonstrate that they are competing reactions, depending on the same cysteine residue of proMBP, but differentially on the redox potential, potentially important for the relative amounts of the complexes in vivo . These findings may be important physiologically, since the biochemical properties of the Proteins change as a consequence of complex formation.
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the proform of eosinophil Major Basic Protein a new maternal serum marker for adverse pregnancy outcome
Prenatal Diagnosis, 2009Co-Authors: Kasper Pihl, Torben Larsen, Steen Rasmussen, Lone Krebs, Michael ChristiansenAbstract:Objective To establish the first trimester serum levels of the proform of eosinophil Major Basic Protein (proMBP) in pregnancies with adverse outcome. Furthermore, to determine the screening performance using proMBP alone and in combination with other first trimester markers. Methods A case-control study was conducted in a primary hospital setting. The proMBP concentration was measured in cases with small-for-gestational age (SGA) (n = 150), spontaneous preterm delivery (n = 88), preeclampsia (n = 40), gestational hypertension (n = 10) and in controls (n = 500). Concentrations were converted to multiples of the median (MoM) in controls and groups were compared using Mann–Whitney U-test. Logistic regression analysis was used to determine significant factors for predicting adverse pregnancy outcome. Screening performance was assessed using receiver operating characteristic curves. Results The proMBP median was significantly reduced in pregnancies with SGA (0.81 MoM), spontaneous preterm delivery (0.83 MoM), preeclampsia (0.88 MoM) and gestational hypertension (0.60 MoM). The best screening performance was found for preeclampsia including the covariates proMBP and nulliparity yielding an area under the curve equal to 0.737 (p < 0.0005) and a 75% detection rate for a 30% false positive rate. Conclusion The proMBP is a novel first trimester serum marker for adverse pregnancy outcome. Copyright © 2009 John Wiley & Sons, Ltd.
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prognostic value of circulating pregnancy associated plasma Protein a papp a and proform of eosinophil Major Basic Protein pro mbp levels in patients with chronic stable angina pectoris
Clinica Chimica Acta, 2008Co-Authors: Luciano Consuegrasanchez, Michael Christiansen, Juan Cosinsales, David W Holt, Ivana Petrovic, Juan Carlos KaskiAbstract:Abstract Background Pregnancy-associated plasma Protein-A (PAPP-A) concentrations predict outcome in patients with acute coronary syndromes. PAPP-A levels and PAPP-A/pro-MBP ratio are increased in chronic stable angina (CSA) patients with complex coronary artery stenoses. Little is known however, about the long-term prognostic value of PAPP-A and pro-MBP in “real-life” CSA patients. We sought to assess whether PAPP-A, the proform of eosinophil Major Basic Protein (pro-MBP) and PAPP-A/pro-MBP levels predict long-term all-cause mortality in patients with CSA. Methods We recruited 663 consecutive patients (169 women [25.5%]; mean age 62.9 ± 9.7 years) undergoing routine diagnostic coronary angiography. Samples for PAPP-A and pro-MBP were taken at study entry. Patients were followed for a median of 8.8 years (interquartile range 3 – 10.6 years). Results 106 patients (16%) died during follow-up. On a Cox proportional hazards model, increased PAPP-A concentration (> 4.8 mIU/L) was an independent predictor of the occurrence of all-cause mortality (HR 1.953, 95% CI 1.135–3.36, p = .016). Neither pro-MBP nor PAPP-A/pro-MBP ratio were markers of all-cause mortality (p = .45 and .54, respectively). Conclusions High PAPP-A levels (> 4.8 mIU/L) showed an association with all-cause mortality during long-term follow-up in patients with CSA.
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pregnancy associated plasma Protein a and its endogenous inhibitor the proform of eosinophil Major Basic Protein prombp are related to complex stenosis morphology in patients with stable angina pectoris
Circulation, 2004Co-Authors: Juan Cosinsales, Michael Christiansen, Claus Oxvig, Michael Toft Overgaard, Paul Kaminski, Della Cole, David W Holt, Juan Carlos KaskiAbstract:Background— The metalloProteinase pregnancy-associated plasma Protein-A (PAPP-A) has been implicated in coronary plaque disruption. Its endogenous inhibitor, the proform of eosinophil Major Basic Protein (proMBP), may also play a role in this process. Atheromatous plaque disruption often presents as complex angiographic lesions. We sought to assess whether PAPP-A, proMBP, and PAPP-A/ProMBP ratio are markers of angiographic plaque complexity in patients with chronic stable angina. Methods and Results— We studied 396 stable angina patients (age 63±10 years, 230 men) of whom 289 had angiographically documented coronary artery disease (≥75% stenosis). All coronary stenoses ≥30% diameter reduction (n =531 in 322 patients) were assessed and classified as complex (n =228) or smooth (n =303) by previously validated criteria. PAPP-A, proMBP, and C-reactive Protein (hs-CRP) serum levels were measured by ELISA. Patients with complex coronary stenoses had a significantly (P<0.001) higher PAPP-A/proMBP ratio (3.1±1.2 ...
Lars Sottrupjensen - One of the best experts on this subject based on the ideXlab platform.
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Proteinase inhibition by proform of eosinophil Major Basic Protein pro mbp is a multistep process of intra and intermolecular disulfide rearrangements
Journal of Biological Chemistry, 2005Co-Authors: Simon Glerup, Michael Toft Overgaard, Lars Sottrupjensen, Henning B Boldt, Linda C Giudice, Claus OxvigAbstract:The metzincin metalloProteinase pregnancy-associated plasma Protein A (PAPP-A, pappalysin-1) promotes cell growth by the cleavage of insulin-like growth factor-binding Proteins-4 and -5, causing the release of bound insulin-like growth factors. The proteolytic activity of PAPP-A is inhibited by the proform of eosinophil Major Basic Protein (pro-MBP), which forms a covalent 2:2 Proteinase-inhibitor complex based on disulfide bonds. To understand the process of complex formation, we determined the status of cysteine residues in both of the uncomplexed molecules. A comparison of the disulfide structure of the reactants with the known disulfide structure of the PAPP-A.pro-MBP complex reveals that six cysteine residues of the pro-MBP subunit (Cys-51, Cys-89, Cys-104, Cys-107, Cys-128, and Cys-169) and two cysteine residues of the PAPP-A subunit (Cys-381 and Cys-652) change their status from the uncomplexed to the complexed states. Upon complex formation, three disulfide bonds of pro-MBP, which connect the acidic propiece with the Basic, mature portion, are disrupted. In the PAPP-A.pro-MBP complex, two of these form the basis of both two interchain disulfide bonds between the PAPP-A and the pro-MBP subunits and two disulfide bonds responsible for pro-MBP dimerization, respectively. Based on the status of the reactants, we investigated the role of individual cysteine residues upon complex formation by mutagenesis of specific cysteine residues of both subunits. Our findings allow us to depict a hypothetical model of how the PAPPA.pro-MBP complex is formed. In addition, we have demonstrated that complex formation is greatly enhanced by the addition of micromolar concentrations of reductants. It is therefore possible that the activity in vivo of PAPP-A is controlled by the redox potential, and it is further tempting to speculate that such mechanism operates under pathological conditions of altered redox potential.
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inhibition of proteolysis by the proform of eosinophil Major Basic Protein prombp requires covalent binding to its target Proteinase
FEBS Letters, 2004Co-Authors: Michael Toft Overgaard, Michael Christiansen, Lars Sottrupjensen, Henning B Boldt, Simon Glerup, Vivien Rodacker, Inger Marie Olsen, Linda C Giudice, Claus OxvigAbstract:By proteolytic cleavage of insulin-like growth factor binding Proteins, the metalloProteinase pregnancy-associated plasma Protein-A (PAPP-A) is able to control the biological activity of insulin-like growth factors. PAPP-A circulates in pregnancy as a proteolytically inactive complex, disulfide bound to the proform of eosinophil Major Basic Protein (proMBP). We here demonstrate that co-transfection of mammalian cells with PAPP-A and proMBP cDNA results in the formation of a covalent PAPP-A/proMBP complex in which PAPP-A is inhibited. Formation of the complex also occurs when PAPP-A and proMBP synthesized separately are incubated. Complex formation was monitored by Western blotting, and by using an immunoassay specific for the complex. Using mutagenesis, we further demonstrate that the complex forms in a specific manner and depends on the presence of two proMBP cysteine residues. Mutated proMBP, in which Cys-51 and -169 are replaced by serine, is unable to form the covalent complex with PAPP-A. Of particular interest, such mutated proMBP further lacks the ability to inhibit PAPP-A. For the first time, this conclusively demonstrates that proMBP is a Proteinase inhibitor. We further conclude that proMBP inhibits PAPP-A in an unusual manner, not paralleled by other Proteinase inhibitors of our knowledge, which requires proMBP to be covalently bound to PAPP-A by disulfide bonds. ProMBP binding to PAPP-A most likely either abrogates substrate access to the active site of PAPP-A or induces a conformational change in the structure of PAPP-A, as we, by further mutagenesis, were able to exclude that the inhibitory mechanism of proMBP is based on a cysteine switch-like mechanism.
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complex of pregnancy associated plasma Protein a and the proform of eosinophil Major Basic Protein disulfide structure and carbohydrate attachment sites
Journal of Biological Chemistry, 2003Co-Authors: Michael Toft Overgaard, Lars Sottrupjensen, Esben S Sorensen, Damian Stachowiak, Henning B Boldt, Lene Juel Kristensen, Claus OxvigAbstract:Pregnancy-associated plasma Protein-A (PAPP-A) is a metzincin superfamily metalloProteinase responsible for cleavage of insulin-like growth factor-binding Protein-4, thus causing release of bound insulin-like growth factor. PAPP-A is secreted as a dimer of 400 kDa but circulates in pregnancy as a disulfide-bound 500-kDa 2:2 complex with the proform of eosinophil Major Basic Protein (pro-MBP), recently shown to function as a Proteinase inhibitor of PAPP-A. Except for PAPP-A2, PAPP-A does not share global similarity with other Proteins. Three lin-notch (LNR or LIN-12) modules and five complement control Protein modules (also known as SCR modules) have been identified in PAPP-A by sequence similarity with other Proteins, but no data are available that allow unambiguous prediction of disulfide bonds of these modules. To establish the connectivities of cysteine residues of the PAPP-A.pro-MBP complex, biochemical analyses of peptides derived from purified Protein were performed. The PAPP-A subunit contains a total of 82 cysteine residues, of which 81 have been accounted for. The pro-MBP subunit contains 12 cysteine residues, of which 10 have been accounted for. Within the 2:2 complex, PAPP-A is dimerized by a single disulfide bond; pro-MBP is dimerized by two disulfides, and each PAPP-A subunit is connected to a pro-MBP subunit by two disulfide bonds. All other disulfides are intrachain bridges. We also show that of 13 potential sites for N-linked carbohydrate substitution of the PAPP-A subunit, 11 are occupied. The large number of disulfide bonds of the PAPP-A.pro-MBP complex imposes many restraints on polypeptide folding, and knowledge of the disulfide pattern of PAPP-A will facilitate structural studies based on recombinant expression of individual, putative PAPP-A domains. Furthermore, it will allow rational experimental design of functional studies aimed at understanding the formation of the PAPP-A.pro-MBP complex, as well as the inhibitory mechanism of pro-MBP.
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expression of recombinant human pregnancy associated plasma Protein a and identification of the proform of eosinophil Major Basic Protein as its physiological inhibitor
Journal of Biological Chemistry, 2000Co-Authors: Michael Toft Overgaard, Gerald J Gleich, Michael Christiansen, Lars Sottrupjensen, Henning B Boldt, Inger Marie Olsen, Jesper Haaning, Cheryl A Conover, Lisbeth S Laursen, Claus OxvigAbstract:Pregnancy-associated plasma Protein-A (PAPP-A), originally known from human pregnancy serum, has recently been demonstrated to be a metzincin superfamily metalloProteinase involved in normal and pathological insulin-like growth factor (IGF) physiology. PAPP-A specifically cleaves IGF-binding Protein (IGFBP)-4, one of six antagonists of IGF action, which results in release of IGF bound to IGFBP-4. IGFBP-4 is the only known PAPP-A substrate. Its cleavage by PAPP-A uniquely depends on the presence of IGF. We here report mammalian expression and purification of recombinant 1547-residue PAPP-A (rPAPP-A). The recombinant Protein is secreted as a homodimer of about 400 kDa composed of two 200-kDa disulfide-bound subunits. Antigenically and functionally, rPAPP-A behaves like the native Protein. In human pregnancy, PAPP-A is known to circulate as a 500-kDa disulfide-bound 2:2 complex with the proform of eosinophil Major Basic Protein (proMBP), PAPP-A/proMBP. A comparison between rPAPP-A and pregnancy serum PAPP-A/proMBP complex surprisingly reveals a difference greater than 100-fold in proteolytic activity, showing that proMBP functions as a Proteinase inhibitor in vivo. We find that polyclonal antibodies against PAPP-A abrogate all detectable IGFBP-4 proteolytic activity in pregnancy serum, pointing at PAPP-A as the dominating, if not the only, IGFBP-4 Proteinase present in the circulation. We further show that pregnancy serum and plasma contain traces (<1%) of uncomplexed PAPP-A with a much higher specific activity than the PAPP-A/proMBP complex. The measurable activity of the PAPP-A/proMBP complex probably results from the presence of a minor subpopulation of partly inhibited PAPP-A that exists in a 2:1 complex with proMBP. Inhibition of PAPP-A by proMBP represents a novel inhibitory mechanism with the enzyme irreversibly bound to its inhibitor by disulfide bonds.
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the proform of eosinophil Major Basic Protein a new maternal serum marker for down syndrome
Prenatal Diagnosis, 1999Co-Authors: Michael Christiansen, Jill M. Wagner, Gerald J Gleich, Claus Oxvig, Qiu Ping Qin, Tri Huu Nguyen, Michael Toft Overgaard, Severin Olesen Larsen, Lars Sottrupjensen, Bent NorgaardpedersenAbstract:The proform of eosinophil Major Basic Protein (proMBP), the most abundant Protein in the eosinophil specific granule, is synthesized by the placenta and secreted into the maternal circulation, where it is found complex-bound to pregnancy-associated plasma Protein-A (PAPP-A) and other Proteins. We examined the potential of proMBP as a maternal serum marker for fetal Down syndrome (DS) by determining its maternal serum concentration (MSpMBP) in 25 Down syndrome (DS) pregnancies and 152 control pregnancies in the first trimester, and in 105 DS pregnancies and 156 control pregnancies in the second trimester. The median (95 per cent confidence interval) MSpMBP MoM in DS pregnancies (n=15) was 0.66 (0.49–0.79) in gestational weeks 5–9; 1.06 (0.71–1.97) in weeks 10–12 (n=10) and 1.62 (1.18–1.98) in weeks 14–20 (n=105). Using parameterized receiver operator characteristics analysis for proMBP as a single marker for DS, detection rates (DRs) of 22 per cent and 38 per cent, for false-positive rates (FPRs) of 5 per cent, were found in weeks 5–9 (using MSpMBP⩽cut-off) and weeks 14–20 (using MSpMBP⩾cut-off), respectively. When age and MSpMBP were used as markers in combination, a DR of 36.8 per cent for an FPR of 5.5 per cent was obtained in weeks 5–9 using a risk cut-off of 1:250. In weeks 14–20 the DR was 48.4 per cent for an FPR of 5.3 per cent using the same risk cut-off. This makes proMBP a marker comparable in diagnostic efficiency to human chorionic gonadotrophin (hCG), and exceeding that of alpha-fetoProtein (AFP) and unconjugated oestriol (uE3), in the second trimester. Copyright © 1999 John Wiley & Sons, Ltd.
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Proteinase inhibition by proform of eosinophil Major Basic Protein pro mbp is a multistep process of intra and intermolecular disulfide rearrangements
Journal of Biological Chemistry, 2005Co-Authors: Simon Glerup, Michael Toft Overgaard, Lars Sottrupjensen, Henning B Boldt, Linda C Giudice, Claus OxvigAbstract:The metzincin metalloProteinase pregnancy-associated plasma Protein A (PAPP-A, pappalysin-1) promotes cell growth by the cleavage of insulin-like growth factor-binding Proteins-4 and -5, causing the release of bound insulin-like growth factors. The proteolytic activity of PAPP-A is inhibited by the proform of eosinophil Major Basic Protein (pro-MBP), which forms a covalent 2:2 Proteinase-inhibitor complex based on disulfide bonds. To understand the process of complex formation, we determined the status of cysteine residues in both of the uncomplexed molecules. A comparison of the disulfide structure of the reactants with the known disulfide structure of the PAPP-A.pro-MBP complex reveals that six cysteine residues of the pro-MBP subunit (Cys-51, Cys-89, Cys-104, Cys-107, Cys-128, and Cys-169) and two cysteine residues of the PAPP-A subunit (Cys-381 and Cys-652) change their status from the uncomplexed to the complexed states. Upon complex formation, three disulfide bonds of pro-MBP, which connect the acidic propiece with the Basic, mature portion, are disrupted. In the PAPP-A.pro-MBP complex, two of these form the basis of both two interchain disulfide bonds between the PAPP-A and the pro-MBP subunits and two disulfide bonds responsible for pro-MBP dimerization, respectively. Based on the status of the reactants, we investigated the role of individual cysteine residues upon complex formation by mutagenesis of specific cysteine residues of both subunits. Our findings allow us to depict a hypothetical model of how the PAPPA.pro-MBP complex is formed. In addition, we have demonstrated that complex formation is greatly enhanced by the addition of micromolar concentrations of reductants. It is therefore possible that the activity in vivo of PAPP-A is controlled by the redox potential, and it is further tempting to speculate that such mechanism operates under pathological conditions of altered redox potential.
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pregnancy associated plasma Protein a and its endogenous inhibitor the proform of eosinophil Major Basic Protein prombp are related to complex stenosis morphology in patients with stable angina pectoris
Circulation, 2004Co-Authors: Juan Cosinsales, Michael Christiansen, Claus Oxvig, Michael Toft Overgaard, Paul Kaminski, Della Cole, David W Holt, Juan Carlos KaskiAbstract:Background— The metalloProteinase pregnancy-associated plasma Protein-A (PAPP-A) has been implicated in coronary plaque disruption. Its endogenous inhibitor, the proform of eosinophil Major Basic Protein (proMBP), may also play a role in this process. Atheromatous plaque disruption often presents as complex angiographic lesions. We sought to assess whether PAPP-A, proMBP, and PAPP-A/ProMBP ratio are markers of angiographic plaque complexity in patients with chronic stable angina. Methods and Results— We studied 396 stable angina patients (age 63±10 years, 230 men) of whom 289 had angiographically documented coronary artery disease (≥75% stenosis). All coronary stenoses ≥30% diameter reduction (n =531 in 322 patients) were assessed and classified as complex (n =228) or smooth (n =303) by previously validated criteria. PAPP-A, proMBP, and C-reactive Protein (hs-CRP) serum levels were measured by ELISA. Patients with complex coronary stenoses had a significantly (P<0.001) higher PAPP-A/proMBP ratio (3.1±1.2 ...
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inhibition of proteolysis by the proform of eosinophil Major Basic Protein prombp requires covalent binding to its target Proteinase
FEBS Letters, 2004Co-Authors: Michael Toft Overgaard, Michael Christiansen, Lars Sottrupjensen, Henning B Boldt, Simon Glerup, Vivien Rodacker, Inger Marie Olsen, Linda C Giudice, Claus OxvigAbstract:By proteolytic cleavage of insulin-like growth factor binding Proteins, the metalloProteinase pregnancy-associated plasma Protein-A (PAPP-A) is able to control the biological activity of insulin-like growth factors. PAPP-A circulates in pregnancy as a proteolytically inactive complex, disulfide bound to the proform of eosinophil Major Basic Protein (proMBP). We here demonstrate that co-transfection of mammalian cells with PAPP-A and proMBP cDNA results in the formation of a covalent PAPP-A/proMBP complex in which PAPP-A is inhibited. Formation of the complex also occurs when PAPP-A and proMBP synthesized separately are incubated. Complex formation was monitored by Western blotting, and by using an immunoassay specific for the complex. Using mutagenesis, we further demonstrate that the complex forms in a specific manner and depends on the presence of two proMBP cysteine residues. Mutated proMBP, in which Cys-51 and -169 are replaced by serine, is unable to form the covalent complex with PAPP-A. Of particular interest, such mutated proMBP further lacks the ability to inhibit PAPP-A. For the first time, this conclusively demonstrates that proMBP is a Proteinase inhibitor. We further conclude that proMBP inhibits PAPP-A in an unusual manner, not paralleled by other Proteinase inhibitors of our knowledge, which requires proMBP to be covalently bound to PAPP-A by disulfide bonds. ProMBP binding to PAPP-A most likely either abrogates substrate access to the active site of PAPP-A or induces a conformational change in the structure of PAPP-A, as we, by further mutagenesis, were able to exclude that the inhibitory mechanism of proMBP is based on a cysteine switch-like mechanism.
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complex of pregnancy associated plasma Protein a and the proform of eosinophil Major Basic Protein disulfide structure and carbohydrate attachment sites
Journal of Biological Chemistry, 2003Co-Authors: Michael Toft Overgaard, Lars Sottrupjensen, Esben S Sorensen, Damian Stachowiak, Henning B Boldt, Lene Juel Kristensen, Claus OxvigAbstract:Pregnancy-associated plasma Protein-A (PAPP-A) is a metzincin superfamily metalloProteinase responsible for cleavage of insulin-like growth factor-binding Protein-4, thus causing release of bound insulin-like growth factor. PAPP-A is secreted as a dimer of 400 kDa but circulates in pregnancy as a disulfide-bound 500-kDa 2:2 complex with the proform of eosinophil Major Basic Protein (pro-MBP), recently shown to function as a Proteinase inhibitor of PAPP-A. Except for PAPP-A2, PAPP-A does not share global similarity with other Proteins. Three lin-notch (LNR or LIN-12) modules and five complement control Protein modules (also known as SCR modules) have been identified in PAPP-A by sequence similarity with other Proteins, but no data are available that allow unambiguous prediction of disulfide bonds of these modules. To establish the connectivities of cysteine residues of the PAPP-A.pro-MBP complex, biochemical analyses of peptides derived from purified Protein were performed. The PAPP-A subunit contains a total of 82 cysteine residues, of which 81 have been accounted for. The pro-MBP subunit contains 12 cysteine residues, of which 10 have been accounted for. Within the 2:2 complex, PAPP-A is dimerized by a single disulfide bond; pro-MBP is dimerized by two disulfides, and each PAPP-A subunit is connected to a pro-MBP subunit by two disulfide bonds. All other disulfides are intrachain bridges. We also show that of 13 potential sites for N-linked carbohydrate substitution of the PAPP-A subunit, 11 are occupied. The large number of disulfide bonds of the PAPP-A.pro-MBP complex imposes many restraints on polypeptide folding, and knowledge of the disulfide pattern of PAPP-A will facilitate structural studies based on recombinant expression of individual, putative PAPP-A domains. Furthermore, it will allow rational experimental design of functional studies aimed at understanding the formation of the PAPP-A.pro-MBP complex, as well as the inhibitory mechanism of pro-MBP.
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expression of recombinant human pregnancy associated plasma Protein a and identification of the proform of eosinophil Major Basic Protein as its physiological inhibitor
Journal of Biological Chemistry, 2000Co-Authors: Michael Toft Overgaard, Gerald J Gleich, Michael Christiansen, Lars Sottrupjensen, Henning B Boldt, Inger Marie Olsen, Jesper Haaning, Cheryl A Conover, Lisbeth S Laursen, Claus OxvigAbstract:Pregnancy-associated plasma Protein-A (PAPP-A), originally known from human pregnancy serum, has recently been demonstrated to be a metzincin superfamily metalloProteinase involved in normal and pathological insulin-like growth factor (IGF) physiology. PAPP-A specifically cleaves IGF-binding Protein (IGFBP)-4, one of six antagonists of IGF action, which results in release of IGF bound to IGFBP-4. IGFBP-4 is the only known PAPP-A substrate. Its cleavage by PAPP-A uniquely depends on the presence of IGF. We here report mammalian expression and purification of recombinant 1547-residue PAPP-A (rPAPP-A). The recombinant Protein is secreted as a homodimer of about 400 kDa composed of two 200-kDa disulfide-bound subunits. Antigenically and functionally, rPAPP-A behaves like the native Protein. In human pregnancy, PAPP-A is known to circulate as a 500-kDa disulfide-bound 2:2 complex with the proform of eosinophil Major Basic Protein (proMBP), PAPP-A/proMBP. A comparison between rPAPP-A and pregnancy serum PAPP-A/proMBP complex surprisingly reveals a difference greater than 100-fold in proteolytic activity, showing that proMBP functions as a Proteinase inhibitor in vivo. We find that polyclonal antibodies against PAPP-A abrogate all detectable IGFBP-4 proteolytic activity in pregnancy serum, pointing at PAPP-A as the dominating, if not the only, IGFBP-4 Proteinase present in the circulation. We further show that pregnancy serum and plasma contain traces (<1%) of uncomplexed PAPP-A with a much higher specific activity than the PAPP-A/proMBP complex. The measurable activity of the PAPP-A/proMBP complex probably results from the presence of a minor subpopulation of partly inhibited PAPP-A that exists in a 2:1 complex with proMBP. Inhibition of PAPP-A by proMBP represents a novel inhibitory mechanism with the enzyme irreversibly bound to its inhibitor by disulfide bonds.
