The Experts below are selected from a list of 837 Experts worldwide ranked by ideXlab platform
Hans Flodgaard - One of the best experts on this subject based on the ideXlab platform.
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heparin binding protein hbp cap37 a missing link in neutrophil evoked alteration of vascular permeability
Nature Medicine, 2001Co-Authors: Narinder Gautam, Hans Flodgaard, Lars Fogh Iversen, Evy Lundgrenakerlund, Maria A Olofsson, Per Hedqvist, Heiko Herwald, Karl-e. Arfors, Lennart LindbomAbstract:Polymorphonuclear leukocyte infiltration into tissues in host defense and inflammatory diseasecauses increased vascular permeability and edema formation through unknown mechanisms.Here, we report the involvement of a paracrine mechanism in neutrophil-evoked alteration inendothelial barrier function. We show that upon neutrophil adhesion to the endothelial lining,leukocytic 2 integrin signaling triggers the release of neutrophil-borne heparin-binding protein(HBP), also known as CAP37/Azurocidin, a member of the serprocidin family of neutrophilcationic proteins. HBP induced Ca++-dependent cytoskeletal rearrangement and intercellular gapformation in endothelial-cell monolayers in vitro, and increased macromolecular efflux in microvesselsin vivo. Moreover, selective inactivation of HBP prevented the neutrophils from inducingendothelial hyperpermeability. Our data suggest a fundamental role of neutrophil-derivedHBP in the vascular response to neutrophil trafficking in inflammation. Targeting this moleculein inflammatory disease conditions offers a new strategy for prevention of endothelial barrierdysfunction caused by misdirected leukocyte activation. (Less)
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Heparin-binding protein (HBP/CAP37): a missing link in neutrophil-evoked alteration of vascular permeability.
Nature Medicine, 2001Co-Authors: Narinder Gautam, Hans Flodgaard, Lars Fogh Iversen, A. Maria Olofsson, Evy Lundgren-Åkerlund, Per Hedqvist, Heiko Herwald, Karl-e. Arfors, Lennart LindbomAbstract:Polymorphonuclear leukocyte infiltration into tissues in host defense and inflammatory diseasecauses increased vascular permeability and edema formation through unknown mechanisms.Here, we report the involvement of a paracrine mechanism in neutrophil-evoked alteration inendothelial barrier function. We show that upon neutrophil adhesion to the endothelial lining,leukocytic 2 integrin signaling triggers the release of neutrophil-borne heparin-binding protein(HBP), also known as CAP37/Azurocidin, a member of the serprocidin family of neutrophilcationic proteins. HBP induced Ca++-dependent cytoskeletal rearrangement and intercellular gapformation in endothelial-cell monolayers in vitro, and increased macromolecular efflux in microvesselsin vivo. Moreover, selective inactivation of HBP prevented the neutrophils from inducingendothelial hyperpermeability. Our data suggest a fundamental role of neutrophil-derivedHBP in the vascular response to neutrophil trafficking in inflammation. Targeting this moleculein inflammatory disease conditions offers a new strategy for prevention of endothelial barrierdysfunction caused by misdirected leukocyte activation. (Less)
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heparin binding protein hbp cap37 a missing link in neutrophil evoked alteration of vascular permeability
Nature Medicine, 2001Co-Authors: Narinder Gautam, Hans Flodgaard, Evy Lundgrenakerlund, Per Hedqvist, Heiko Herwald, Karl-e. Arfors, Maria Olofsson, Lars Iversen, Lennart LindbomAbstract:Polymorphonuclear leukocyte infiltration into tissues in host defense and inflammatory disease causes increased vascular permeability and edema formation through unknown mechanisms. Here, we report the involvement of a paracrine mechanism in neutrophil-evoked alteration in endothelial barrier function. We show that upon neutrophil adhesion to the endothelial lining, leukocytic β2 integrin signaling triggers the release of neutrophil-borne heparin-binding protein (HBP), also known as CAP37/Azurocidin, a member of the serprocidin family of neutrophil cationic proteins. HBP induced Ca++-dependent cytoskeletal rearrangement and intercellular gap formation in endothelial-cell monolayers in vitro, and increased macromolecular efflux in microvessels in vivo. Moreover, selective inactivation of HBP prevented the neutrophils from inducing endothelial hyperpermeability. Our data suggest a fundamental role of neutrophil-derived HBP in the vascular response to neutrophil trafficking in inflammation. Targeting this molecule in inflammatory disease conditions offers a new strategy for prevention of endothelial barrier dysfunction caused by misdirected leukocyte activation.
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Heparin-binding protein (HBP/CAP37): a missing link in neutrophil-evoked alteration of vascular permeability.
Nature medicine, 2001Co-Authors: Narinder Gautam, Hans Flodgaard, Lars Fogh Iversen, A. Maria Olofsson, Evy Lundgren-Åkerlund, Per Hedqvist, Heiko Herwald, Karl-e. Arfors, Lennart LindbomAbstract:Polymorphonuclear leukocyte infiltration into tissues in host defense and inflammatory disease causes increased vascular permeability and edema formation through unknown mechanisms. Here, we report the involvement of a paracrine mechanism in neutrophil-evoked alteration in endothelial barrier function. We show that upon neutrophil adhesion to the endothelial lining, leukocytic β2 integrin signaling triggers the release of neutrophil-borne heparin-binding protein (HBP), also known as CAP37/Azurocidin, a member of the serprocidin family of neutrophil cationic proteins. HBP induced Ca++-dependent cytoskeletal rearrangement and intercellular gap formation in endothelial-cell monolayers in vitro, and increased macromolecular efflux in microvessels in vivo. Moreover, selective inactivation of HBP prevented the neutrophils from inducing endothelial hyperpermeability. Our data suggest a fundamental role of neutrophil-derived HBP in the vascular response to neutrophil trafficking in inflammation. Targeting this molecule in inflammatory disease conditions offers a new strategy for prevention of endothelial barrier dysfunction caused by misdirected leukocyte activation.
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Characterization of the biosynthesis, processing, and sorting of human HBP/CAP37/Azurocidin
Journal of leukocyte biology, 1999Co-Authors: Anders Lindmark, Hans Flodgaard, Daniel Garwicz, Poul Baad Rasmussen, Urban GullbergAbstract:Azurocidin is a multifunctional endotoxin-binding serine protease homolog synthesized during the promyelocytic stage of neutrophil development. To characterize the biosynthesis and processing of Azurocidin, cDNA encoding human preproAzurocidin was stably transfected to the rat basophilic leukemia cell line RBL-1 and the murine myeloblast-like cell line 32D cl3; cell lines previously utilized to study the related proteins cathepsin G and proteinase 3. After 30 min of pulse radiolabeling, two forms of newly synthesized proAzurocidin (34.5 and 37 kDa), differing in carbohydrate content but with protein cores of identical sizes, were recognized. With time, the 34.5-kDa form disappeared, while the 37-kDa form was further processed proteolytically, as judged by digestion with N-glycosidase F. Conversion of high-mannose oligosaccharides into complex forms was shown by acquisition of complete resistance to endoglycosidase H. Radiosequence analysis demonstrated that the amino-terminal seven amino acid propeptide of proAzurocidin was removed in a stepwise manner during processing; initial removal of five amino acids was followed by cleavage of a dipeptide. Presence of the protease inhibitors Gly-Phe-diazomethyl ketone, bestatin, or leupeptin inhibited only the cleavage of the dipeptide, thus indicating the involvement of at least two amino-terminal processing enzymes. Translocation of Azurocidin to granules was shown by subcellular fractionation. Similar results, with efficient biosynthesis, processing, and targeting to granules in both cell lines, were obtained with a mutant form of human preproAzurocidin lacking the amino-terminal heptapropeptide. In conclusion, this investigation is an important addition to our previous studies on related azurophil granule proteins, and provides novel information concerning the biosynthesis and distinctive amino-terminal processing of human Azurocidin.
Joelle E. Gabay - One of the best experts on this subject based on the ideXlab platform.
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Ciba Foundation Symposium 186 - Antimicrobial Peptides - Antimicrobial proteins with homology to serine proteases.
Ciba Foundation symposium, 2007Co-Authors: Joelle E. GabayAbstract:The azurophil granule, a specialized lysosome of human neutrophils, contains a family of antimicrobial proteins with structural homology to serine proteases, the serprocidins. Three members of this family are serine proteases (cathepsin G, elastase and proteinase-3) and one is a proteolytically inactive homologue (Azurocidin). They are synthesized as preproproteins with a characteristic leader peptide and a propiece, both of which are removed by processing enzymes to yield the mature protein. The functional genes for three serprocidins (elastase, proteinase-3 and Azurocidin) are grouped in a single genetic locus on chromosome 19 and are coordinately expressed and regulated during haemopoietic differentiation. Multiple and sometimes overlapping biological functions are a feature of this family, yet they all seem to pertain to host immunity. The structural requirements for the function of one member of this group (Azurocidin), particularly its antibiotic function, are under investigation.
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basic residues in Azurocidin hbp contribute to both heparin binding and antimicrobial activity
Journal of Biological Chemistry, 2002Co-Authors: Denise Mccabe, Tali Cukierman, Joelle E. GabayAbstract:Azurocidin/CAP37/HBP is an antimicrobial and chemotactic protein that is part of the innate defenses of human neutrophils. In addition, Azurocidin is an inactive serine protease homolog with binding sites for diverse ligands including heparin and the bovine pancreatic trypsin inhibitor (BPTI). The structure of the protein reveals a highly cationic domain concentrated on one side of the molecule and responsible for its strong polarity. To investigate the role of this highly basic region, we produced three recombinant Azurocidin mutant proteins that were altered in either one or both of two clusters of 4 basic residues located symmetrically on each side of a central cleft in the cationic domain. Two of the mutant proteins (Loop 3: R5Q, K6Q, R8Q, and R10Q; Loop 4: R61Q, R62Q, R63Q, and R65Q) exhibited little or no change in heparin and BPTI binding or in antimicrobial function. In contrast, the Loop 3/Loop 4 mutant (R5Q, K6Q, R8Q, R10Q, R61Q, R62Q, R63Q, and R65Q) in which all 8 basic residues were replaced showed greatly decreased ability to bind heparin and to kill Escherichia coli and Candida albicans. Thus, we report that the 8 basic residues that were altered in the Loop 3/Loop 4 mutant contribute to the ability of the wild-type Azurocidin molecule to bind heparin and to kill E. coli and C. albicans. Because BPTI binding was comparable in wild-type and Loop 3/Loop 4 mutant protein, we conclude that the same 8 basic residues are not involved in the binding of BPTI to Azurocidin, supporting the notion that the binding site for BPTI is distinct from the site involved in heparin binding and antimicrobial activity. Finally, we show that removal of all 4 positively charged amino acids in the 20-44 Azurocidin sequence (DMC1: R23Q,H24S,H32S,R34Q), a region previously thought to contain an antimicrobial domain, does not affect the activity of the protein against E. coli, Streptococcus faecalis, and C. albicans.
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Basic residues in Azurocidin/HBP contribute to both heparin binding and antimicrobial activity.
The Journal of biological chemistry, 2002Co-Authors: Denise Mccabe, Tali Cukierman, Joelle E. GabayAbstract:Azurocidin/CAP37/HBP is an antimicrobial and chemotactic protein that is part of the innate defenses of human neutrophils. In addition, Azurocidin is an inactive serine protease homolog with binding sites for diverse ligands including heparin and the bovine pancreatic trypsin inhibitor (BPTI). The structure of the protein reveals a highly cationic domain concentrated on one side of the molecule and responsible for its strong polarity. To investigate the role of this highly basic region, we produced three recombinant Azurocidin mutant proteins that were altered in either one or both of two clusters of 4 basic residues located symmetrically on each side of a central cleft in the cationic domain. Two of the mutant proteins (Loop 3: R5Q, K6Q, R8Q, and R10Q; Loop 4: R61Q, R62Q, R63Q, and R65Q) exhibited little or no change in heparin and BPTI binding or in antimicrobial function. In contrast, the Loop 3/Loop 4 mutant (R5Q, K6Q, R8Q, R10Q, R61Q, R62Q, R63Q, and R65Q) in which all 8 basic residues were replaced showed greatly decreased ability to bind heparin and to kill Escherichia coli and Candida albicans. Thus, we report that the 8 basic residues that were altered in the Loop 3/Loop 4 mutant contribute to the ability of the wild-type Azurocidin molecule to bind heparin and to kill E. coli and C. albicans. Because BPTI binding was comparable in wild-type and Loop 3/Loop 4 mutant protein, we conclude that the same 8 basic residues are not involved in the binding of BPTI to Azurocidin, supporting the notion that the binding site for BPTI is distinct from the site involved in heparin binding and antimicrobial activity. Finally, we show that removal of all 4 positively charged amino acids in the 20-44 Azurocidin sequence (DMC1: R23Q,H24S,H32S,R34Q), a region previously thought to contain an antimicrobial domain, does not affect the activity of the protein against E. coli, Streptococcus faecalis, and C. albicans.
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Azurocidin, a natural antibiotic from human neutrophils: expression, antimicrobial activity, and secretion.
Protein expression and purification, 1996Co-Authors: Roque P. Almeida, Anne Vanet, Véronique Witko-sarsat, Maxine Melchior, Denise Mccabe, Joelle E. GabayAbstract:The azurophil granules of human PMN contain four antibiotic proteins, the serprocidins, which have extensive homology to one another and to serine proteases. Azurocidin, a member of this family, is a 29-kDa glycoprotein with broad spectrum antimicrobial activity and chemotactic activity toward monocytes. Insect cells transfected with a baculovirus vector carrying Azurocidin cDNA produced a recombinant Azurocidin protein. We purified the recombinant Azurocidin protein from the culture medium of the infected cells and showed that it retained the antimicrobial activity of the native neutrophil-derived molecule. In addition, we present evidence that a 49-amino-acid region of the recombinant Azurocidin protein is required for its secretion from insect cells.
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Antibiotic peptides and serine protease homologs in human polymorphonuclear leukocytes: defensins and Azurocidin
Current opinion in immunology, 1993Co-Authors: Joelle E. Gabay, Roque P. AlmeidaAbstract:The azurophil granule, a specialized lysosome of neutrophils, contains two families of antimicrobial proteins, each with four members. They are the defensins, comprising human neutrophil protein 1, -2, -3 and -4, on the one hand and the serprocidins, comprising cathepsin G, elastase, proteinase 3 and Azurocidin, on the other. Defensins appear to contribute to mammalian as well as invertebrate immunity. Recent studies show that defensins and structurally related peptides are found not only in phagocytes but also in intestinal and respiratory cells. Aside from their antibiotic function, members of the defensin family may also act as hormonal agents. Within the serprocidin family the genes encoding the novel antibiotics and serine protease homologs Azurocidin and proteinase 3 have been identified recently.
Anders Lindmark - One of the best experts on this subject based on the ideXlab platform.
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Characterization of the biosynthesis, processing, and sorting of human HBP/CAP37/Azurocidin
Journal of leukocyte biology, 1999Co-Authors: Anders Lindmark, Hans Flodgaard, Daniel Garwicz, Poul Baad Rasmussen, Urban GullbergAbstract:Azurocidin is a multifunctional endotoxin-binding serine protease homolog synthesized during the promyelocytic stage of neutrophil development. To characterize the biosynthesis and processing of Azurocidin, cDNA encoding human preproAzurocidin was stably transfected to the rat basophilic leukemia cell line RBL-1 and the murine myeloblast-like cell line 32D cl3; cell lines previously utilized to study the related proteins cathepsin G and proteinase 3. After 30 min of pulse radiolabeling, two forms of newly synthesized proAzurocidin (34.5 and 37 kDa), differing in carbohydrate content but with protein cores of identical sizes, were recognized. With time, the 34.5-kDa form disappeared, while the 37-kDa form was further processed proteolytically, as judged by digestion with N-glycosidase F. Conversion of high-mannose oligosaccharides into complex forms was shown by acquisition of complete resistance to endoglycosidase H. Radiosequence analysis demonstrated that the amino-terminal seven amino acid propeptide of proAzurocidin was removed in a stepwise manner during processing; initial removal of five amino acids was followed by cleavage of a dipeptide. Presence of the protease inhibitors Gly-Phe-diazomethyl ketone, bestatin, or leupeptin inhibited only the cleavage of the dipeptide, thus indicating the involvement of at least two amino-terminal processing enzymes. Translocation of Azurocidin to granules was shown by subcellular fractionation. Similar results, with efficient biosynthesis, processing, and targeting to granules in both cell lines, were obtained with a mutant form of human preproAzurocidin lacking the amino-terminal heptapropeptide. In conclusion, this investigation is an important addition to our previous studies on related azurophil granule proteins, and provides novel information concerning the biosynthesis and distinctive amino-terminal processing of human Azurocidin.
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characterization of the biosynthesis processing and sorting of human hbp cap37 Azurocidin
Journal of Leukocyte Biology, 1999Co-Authors: Anders Lindmark, Hans Flodgaard, Daniel Garwicz, Poul Baad Rasmussen, Urban GullbergAbstract:Azurocidin is a multifunctional endotoxin-binding serine protease homolog synthesized during the promyelocytic stage of neutrophil development. To characterize the biosynthesis and processing of Azurocidin, cDNA encoding human preproAzurocidin was stably transfected to the rat basophilic leukemia cell line RBL-1 and the murine myeloblast-like cell line 32D cl3; cell lines previously utilized to study the related proteins cathepsin G and proteinase 3. After 30 min of pulse radiolabeling, two forms of newly synthesized proAzurocidin (34.5 and 37 kDa), differing in carbohydrate content but with protein cores of identical sizes, were recognized. With time, the 34.5-kDa form disappeared, while the 37-kDa form was further processed proteolytically, as judged by digestion with N-glycosidase F. Conversion of high-mannose oligosaccharides into complex forms was shown by acquisition of complete resistance to endoglycosidase H. Radiosequence analysis demonstrated that the amino-terminal seven amino acid propeptide of proAzurocidin was removed in a stepwise manner during processing; initial removal of five amino acids was followed by cleavage of a dipeptide. Presence of the protease inhibitors Gly-Phe-diazomethyl ketone, bestatin, or leupeptin inhibited only the cleavage of the dipeptide, thus indicating the involvement of at least two amino-terminal processing enzymes. Translocation of Azurocidin to granules was shown by subcellular fractionation. Similar results, with efficient biosynthesis, processing, and targeting to granules in both cell lines, were obtained with a mutant form of human preproAzurocidin lacking the amino-terminal heptapropeptide. In conclusion, this investigation is an important addition to our previous studies on related azurophil granule proteins, and provides novel information concerning the biosynthesis and distinctive amino-terminal processing of human Azurocidin.
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On the Biosynthesis and Processing of Cathepsin G, Leukocyte Elastase, and Azurocidin neutrophil granule members of a hematopoietic serine protease superfamily
1997Co-Authors: Anders LindmarkAbstract:The serine proteases cathepsin G, leukocyte elastase, proteinase 3, and the inactive protease homologue Azurocidin, stored in the azurophil granules of neutrophils, belong to a hematopoietic serine protease superfamily with members in mature effector cells of bone marrow origin. Promonocytic U-937 cells were used for studying the biosynthesis of cathepsin G and leukocyte elastase by biosynthetic radiolabelling. The proteases were found to be synthesized as precursor forms that were subjected to processing, including acquisition of N-linked complex oligosaccharides. Targeting to a granule compartment was also demonstrated. Using brefeldin A, monensin, and ammonium chloride, the subcellular localization of distinct processing events was characterized. To make possible an evaluation of the importance of distinct protein motifs, a transfectable cellular model for studying the processing was established. cDNA encoding human cathepsin G was transfected to the rat RBL cell line, after which the model was proven valid by characterizing the activation, processing, and sorting of cathepsin G. Mutant cDNAs encoding cathepsin G and leukocyte elastase lacking carboxyl-terminal peptide extensions, normally removed during processing, were transfected to RBL cells. The mutant proteins were found to be activated and sorted to granules, approximately as efficiently as the wild type proteins, hence ruling out a critical role of the propeptides for folding, stability or molecular targeting. The granule thiol protease dipeptidyl peptidase I (DPP I) has been suggested as responsible for the activation of all hematopoietic serine proteases. It was, however, not possible to activate procathepsin G, expressed in COS-7 cells, neither by co-transfection of rat DPP I cDNA nor by in vitro incubation with purified bovine DPP I. After transfection to RBL cells, a novel amino-terminal processing pattern distinct from that of other granule serine proteases was demonstrated for the synthesis and processing of Azurocidin.
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on the biosynthesis and processing of cathepsin g leukocyte elastase and Azurocidin neutrophil granule members of a hematopoietic serine protease superfamily
1997Co-Authors: Anders LindmarkAbstract:The serine proteases cathepsin G, leukocyte elastase, proteinase 3, and the inactive protease homologue Azurocidin, stored in the azurophil granules of neutrophils, belong to a hematopoietic serine protease superfamily with members in mature effector cells of bone marrow origin. Promonocytic U-937 cells were used for studying the biosynthesis of cathepsin G and leukocyte elastase by biosynthetic radiolabelling. The proteases were found to be synthesized as precursor forms that were subjected to processing, including acquisition of N-linked complex oligosaccharides. Targeting to a granule compartment was also demonstrated. Using brefeldin A, monensin, and ammonium chloride, the subcellular localization of distinct processing events was characterized. To make possible an evaluation of the importance of distinct protein motifs, a transfectable cellular model for studying the processing was established. cDNA encoding human cathepsin G was transfected to the rat RBL cell line, after which the model was proven valid by characterizing the activation, processing, and sorting of cathepsin G. Mutant cDNAs encoding cathepsin G and leukocyte elastase lacking carboxyl-terminal peptide extensions, normally removed during processing, were transfected to RBL cells. The mutant proteins were found to be activated and sorted to granules, approximately as efficiently as the wild type proteins, hence ruling out a critical role of the propeptides for folding, stability or molecular targeting. The granule thiol protease dipeptidyl peptidase I (DPP I) has been suggested as responsible for the activation of all hematopoietic serine proteases. It was, however, not possible to activate procathepsin G, expressed in COS-7 cells, neither by co-transfection of rat DPP I cDNA nor by in vitro incubation with purified bovine DPP I. After transfection to RBL cells, a novel amino-terminal processing pattern distinct from that of other granule serine proteases was demonstrated for the synthesis and processing of Azurocidin.
Lars C Petersen - One of the best experts on this subject based on the ideXlab platform.
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Binding of bovine pancreatic trypsin inhibitor to heparin binding protein/CAP37/Azurocidin
FEBS Journal, 1993Co-Authors: Lars C Petersen, Jens J. Birktoft, Hans FlodgaardAbstract:Heparin-binding protein (HBP; also known as CAP37 or Azurocidin) is a member of the serine proteinase family. Evolution, however, has reverted this protein into a non-proteolytic form by mutation of two of the three residues of the active-site triad. Although proteolytically inactive, the human heparin-binding protein (hHBP) is still capable of binding bovine pancreatic trypsin inhibitor (BPTI). This was demonstrated by affinity chromatography to BPTI immobilized on a solid matrix and by studies on plasmin inhibition kinetics. hHBP competes with plasmin for BPTI and this effect on plasmin inhibition has been analyzed in terms of a kinetic model. A dissociation constant, Kd= 0.1 μM, was found for the interaction between BPTI and hHBP. The hHBP provides an example of a serine proteinase which has lost its catalytic function by reverting residues of the active center while still preserving its capability of specific interactions with Kunitz inhibitors. pHBP, the porcine counterpart to hHBP, on the other hand, was incapable of BPTI binding. The structural basis for the BPTI binding to the human protein and the species difference is discussed in terms of putative three-dimensional structures of the proteins derived by comparative molecular modelling methods.
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Binding of bovine pancreatic trypsin inhibitor to heparin binding protein/CAP37/Azurocidin. Interaction between a Kunitz-type inhibitor and a proteolytically inactive serine proteinase homologue.
European journal of biochemistry, 1993Co-Authors: Lars C Petersen, Jens J. Birktoft, Hans FlodgaardAbstract:Heparin-binding protein (HBP; also known as CAP37 or Azurocidin) is a member of the serine proteinase family. Evolution, however, has reverted this protein into a non-proteolytic form by mutation of two of the three residues of the active-site triad. Although proteolytically inactive, the human heparin-binding protein (hHBP) is still capable of binding bovine pancreatic trypsin inhibitor (BPTI). This was demonstrated by affinity chromatography to BPTI immobilized on a solid matrix and by studies on plasmin inhibition kinetics. hHBP competes with plasmin for BPTI and this effect on plasmin inhibition has been analyzed in terms of a kinetic model. A dissociation constant, Kd = 0.1 microM, was found for the interaction between BPTI and hHBP. The hHBP provides an example of a serine proteinase which has lost its catalytic function by reverting residues of the active center while still preserving its capability of specific interactions with Kunitz inhibitors. pHBP, the porcine counterpart to hHBP, on the other hand, was incapable of BPTI binding. The structural basis for the BPTI binding to the human protein and the species difference is discussed in terms of putative three-dimensional structures of the proteins derived by comparative molecular modelling methods.
Urban Gullberg - One of the best experts on this subject based on the ideXlab platform.
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Functional dissociation between proforms and mature forms of proteinase 3, Azurocidin, and granzyme B in regulation of granulopoiesis
Experimental hematology, 2002Co-Authors: Stefan Sköld, Lennart Zeberg, Urban Gullberg, Tor OlofssonAbstract:Abstract Objective We previously demonstrated that secreted proform(s) of the neutrophil serine protease PR3 (proteinase 3) can down-modulate the fraction of normal human colony-forming unit granulocyte-macrophage (CFU-GM) in S-phase, whereas PR3 extracted from mature neutrophils lacks this ability. The objective of this study was to characterize the structural and functional dissociation between secreted proforms and granule-stored mature forms and to extend the investigation to other related hematopoietic serine proteases. Materials and Methods Conditioned media containing secreted proteases from transfectant cell lines with stable expression of human PR3, neutrophil elastase, cathepsin G, Azurocidin, and granzymes A, B, H, K, and M were tested for their ability to reduce the fraction of normal human CFU-GM in S phase. Furthermore, recombinant PR3, Azurocidin, and granzyme B with defined N-terminal propeptides, and the respective mature forms without propeptide, were functionally characterized. Results In addition to PR3, secreted proforms of Azurocidin and granzymes A, B, H, K, and M, but not cathepsin G or neutrophil elastase, have S-phase reducing activity. This activity is restricted to the dipeptide proforms, whereas mature forms without propeptide have no S-phase reducing activity. On the other hand, only the mature forms of PR3 and granzyme B could bind the serine protease inhibitor diisopropylfluorophosphate (DFP), or aprotinin in the case of Azurocidin. We also demonstrate that granulocyte colony-stimulating factor–stimulated CD34 + cells and interleukin-2–stimulated lymphocytes secrete active proforms of PR3 and granzyme B, respectively. Conclusion These results demonstrate distinctive functional and conformational differences between proforms and mature forms of these hematopoietic serine proteases and suggest novel growth regulatory mechanisms in granulopoiesis.
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Characterization of the biosynthesis, processing, and sorting of human HBP/CAP37/Azurocidin
Journal of leukocyte biology, 1999Co-Authors: Anders Lindmark, Hans Flodgaard, Daniel Garwicz, Poul Baad Rasmussen, Urban GullbergAbstract:Azurocidin is a multifunctional endotoxin-binding serine protease homolog synthesized during the promyelocytic stage of neutrophil development. To characterize the biosynthesis and processing of Azurocidin, cDNA encoding human preproAzurocidin was stably transfected to the rat basophilic leukemia cell line RBL-1 and the murine myeloblast-like cell line 32D cl3; cell lines previously utilized to study the related proteins cathepsin G and proteinase 3. After 30 min of pulse radiolabeling, two forms of newly synthesized proAzurocidin (34.5 and 37 kDa), differing in carbohydrate content but with protein cores of identical sizes, were recognized. With time, the 34.5-kDa form disappeared, while the 37-kDa form was further processed proteolytically, as judged by digestion with N-glycosidase F. Conversion of high-mannose oligosaccharides into complex forms was shown by acquisition of complete resistance to endoglycosidase H. Radiosequence analysis demonstrated that the amino-terminal seven amino acid propeptide of proAzurocidin was removed in a stepwise manner during processing; initial removal of five amino acids was followed by cleavage of a dipeptide. Presence of the protease inhibitors Gly-Phe-diazomethyl ketone, bestatin, or leupeptin inhibited only the cleavage of the dipeptide, thus indicating the involvement of at least two amino-terminal processing enzymes. Translocation of Azurocidin to granules was shown by subcellular fractionation. Similar results, with efficient biosynthesis, processing, and targeting to granules in both cell lines, were obtained with a mutant form of human preproAzurocidin lacking the amino-terminal heptapropeptide. In conclusion, this investigation is an important addition to our previous studies on related azurophil granule proteins, and provides novel information concerning the biosynthesis and distinctive amino-terminal processing of human Azurocidin.
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characterization of the biosynthesis processing and sorting of human hbp cap37 Azurocidin
Journal of Leukocyte Biology, 1999Co-Authors: Anders Lindmark, Hans Flodgaard, Daniel Garwicz, Poul Baad Rasmussen, Urban GullbergAbstract:Azurocidin is a multifunctional endotoxin-binding serine protease homolog synthesized during the promyelocytic stage of neutrophil development. To characterize the biosynthesis and processing of Azurocidin, cDNA encoding human preproAzurocidin was stably transfected to the rat basophilic leukemia cell line RBL-1 and the murine myeloblast-like cell line 32D cl3; cell lines previously utilized to study the related proteins cathepsin G and proteinase 3. After 30 min of pulse radiolabeling, two forms of newly synthesized proAzurocidin (34.5 and 37 kDa), differing in carbohydrate content but with protein cores of identical sizes, were recognized. With time, the 34.5-kDa form disappeared, while the 37-kDa form was further processed proteolytically, as judged by digestion with N-glycosidase F. Conversion of high-mannose oligosaccharides into complex forms was shown by acquisition of complete resistance to endoglycosidase H. Radiosequence analysis demonstrated that the amino-terminal seven amino acid propeptide of proAzurocidin was removed in a stepwise manner during processing; initial removal of five amino acids was followed by cleavage of a dipeptide. Presence of the protease inhibitors Gly-Phe-diazomethyl ketone, bestatin, or leupeptin inhibited only the cleavage of the dipeptide, thus indicating the involvement of at least two amino-terminal processing enzymes. Translocation of Azurocidin to granules was shown by subcellular fractionation. Similar results, with efficient biosynthesis, processing, and targeting to granules in both cell lines, were obtained with a mutant form of human preproAzurocidin lacking the amino-terminal heptapropeptide. In conclusion, this investigation is an important addition to our previous studies on related azurophil granule proteins, and provides novel information concerning the biosynthesis and distinctive amino-terminal processing of human Azurocidin.
