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2007 | 54 | 3 | 567-573
Article title

Analysis of individual azurocidin N-glycosylation sites in regard to its secretion by insect cells, susceptibility to proteolysis and antibacterial activity

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EN
Abstracts
EN
Azurocidin is an inactive serine protease homolog with primary sequence similarity to neutrophil elastase, cathepsin G, and proteinase 3. The aim of this study was to investigate possible consequences of differential glycosylation of azurocidin in regard to its secretion, protein stability as measured by susceptibility to proteolysis, and antibacterial activity. Site-directed mutagenesis was employed to generate mutant azurocidin variants lacking individual N-glycosylation sites. Our results show that N-linked glycans may play a role in proper azurocidin folding and subsequent secretion by insect cells. We also demonstrate that N-linked glycosylation contributes to azurocidin stability by protecting it from proteolysis. The lack of N-glycosylation at individual sites does not significantly influence the azurocidin antibacterial activity.
Year
Volume
54
Issue
3
Pages
567-573
Physical description
Dates
published
2007
received
2007-02-24
revised
2007-06-21
accepted
2007-07-06
(unknown)
2007-07-26
References
  • Almeida RP, Vanet A, Witko-Sarsat V, Melchior M, McCabe D, Gabay JE (1996) Azurocidin, a natural antibiotic from human neutrophils: expression, antimicrobial activity, and secretion. Protein Expr Purif 7: 355-366.
  • van Berkel PHC, Geerts MEJ, van Veen HA, Kooiman PM, Pieper FR, de Boer HA, Nuijens JH (1995) Glycosylated and unglycosylated human lactoferrins both bind iron and show identical affinities towards human lysozyme and bacterial lipopolysaccharide, but differ in their susceptibilities towards tryptic proteolysis. Biochem J 312: 107-114.
  • Brackett DJ, Lerner MR, Lacquement MA, He R, Pereira HA (1997) A synthetic lipopolisaccharide-binding peptide based on the neutrophil-derived protein CAP37 prevents endotoxin-induced responses in consious rats. Infect Immun 65: 2803-2811.
  • Campanelli D, Detmers PA, Nathan CF, Gabay JE (1990) Azurocidin and a homologous serine protease from neutrophils. Differential antimicrobial and proteolytic properties. J Clin Invest 85: 904-915.
  • Gabay JE, Scott RW, Campanelli D, Griffith J, Wilde C, Marra MN, Seeger M, Nathan CF (1989) Antibiotic proteins of human polymorphonuclear leukocytes. Proc Natl Acad Sci USA 86: 5610-5614.
  • Gronborg M, Bunkenborg J, Kristiansen TZ, Jensen ON, Yeo CJ, Hruban RH, Maitra A, Goggins MG, Pandey A (2004) Comprehensive proteomic analysis of human pancreatic juice. J Proteome Res 3: 1042-1055.
  • Iversen LF, Kastrup JS, Bjorn SE, Rasmussen PB, Wiberg FC, Flodgaard HJ, Larsen IK (1997) Structure of HBP, a multifunctional protein with a serine proteinase fold. Nat Struct Biol 4: 265-268.
  • Iversen LF, Kastrup JS, Bjorn SE, Wiberg FC, Larsen IK, Flodgaard HJ, Rasmussen PB (1999) Structure and function of the N-linked glycans of HBP/CAP37/azurocidin: crystal structure determination and biological characterization of nonglycosylated HBP. Protein Sci 8: 2019-2026.
  • Heinzelmann M, Mercer-Jones MA, Flodgaard H, Miller FN (1998) Heparin-binding protein (CAP37) is internalized in monocytes and increases LPS-induced monocyte activation. J Immunol 160: 5530-5536.
  • Heinzelmann M, Platz A, Flodgaard H, Polk HC Jr, Miller FN (1999) Endocytosis of heparin-binding protein (CAP37) is essential for the enhancement of lipopolysaccharide-induced TNF-α production in human monocytes. J Immunol 162: 4240-4245.
  • Karlsen S, Iversen LF, Larsen IK, Flodgaard HJ, Kastrup JS (1998) Atomic resolution structure of human HBP/CAP37/azurocidin. Acta Crystallogr D Biol Crystallogr 54: 598-609.
  • Kastrup JS, Linde V, Pedersen AK, Stoffer B, Iversen LF, Larsen IK, Rasmussen PB, Flodgaard HJ, Bjorn SE (2001) Two mutants of human heparin binding protein (CAP37): toward the understanding of the nature of lipid A/LPS and BPTI binding. Proteins 42: 442-451.
  • Lee TD, Gonzalez ML, Kumar P, Chary-Reddy S, Grammas P, Pereira HA (2002) CAP37, a novel inflammatory mediator. Its expression in endothelial cells and localization to atherosclerotic lesions. Am J Pathol 160: 841-848.
  • Lehrer RI, Rosenman M, Harwig SS, Jackson R, Eisenhauer P (1991) Ultrasensitive assays for endogenous antimicrobial polypeptides. J Immunol Methods 137: 167-173.
  • Mak P, Siwek M, Pohl J, Dubin A (2007) Menstrual hemocidin HbB115-146 is an acidophilic antibacterial peptide potentiating the activity of human defensins, cathelicidin and lysozyme. Am J Reprod Immunol 57: 81-91.
  • McCabe D, Cukierman T, Gabay JE (2002) Basic residues in azurocidin/HBP contribute to both heparin binding and antimicrobial activity. J Biol Chem 277: 27477-27488.
  • Olczak M, Olczak T (2006) Comparison of different signal peptides for protein secretion in nonlytic insect cell system. Anal Biochem 359: 45-53.
  • Olczak M, Watorek W (2002) Structural analysis of N-glycans from human neutrophil azurocidin. Biochem Biophys Res Commun 293: 213-219.
  • Ostergaard E, Flodgaard H (1992) A neutrophil-derived proteolytic inactive elastase homologue (hHBP) mediates reversible contraction of fibroblasts and endothelial cell monolayers and stimulates monocyte survival and thrombospondin secretion. J Leukoc Biol 51: 316-323.
  • Pereira HA (1995) CAP37, a neutrophil-derived multifunctional inflammatory mediator. J Leukoc Biol 57: 805-812.
  • Pereira HA, Spitznagel JK, Pohl J, Wilson DE, Morgan J, Palings I, Larrick JW (1990) CAP37, a 37 kD human neutrophil granule cationic protein shares homology with inflammatory proteinases. Life Sci 46: 189-196.
  • Pereira HA, Erdem I, Pohl J, Spitznagel JK (1993) Synthetic bactericidal peptide based on CAP37: a 37-kDa human neutrophil granule-associated cationic antimicrobial protein chemotactic for monocytes. Proc Natl Acad Sci USA 90: 4733-4737.
  • Pereira HA, Kumar P, Grammas P (1996a) Expression of CAP37, a novel inflammatory mediator, in Alzheimer's disease. Neurobiol Aging 17: 753-759.
  • Pereira HA, Moore P, Grammas P (1996b) CAP37, a neutrophil granule-derived protein stimulates protein kinase C activity in endothelial cells. J Leukoc Biol 60: 415-422.
  • Pereira HA, Ruan X, Kumar P (2003) Activation of microglia: a neuroinflammatory role for CAP37. Glia 41: 64-72.
  • Polikandritou LM, Sheu E, Lin JS, Pereira HA (1997) Interaction of a synthetic peptide based on the neutrophil-derived antimicrobial protein CAP37 with dipalmitoyl phosphatidylcholine membranes. Biochim Biophys Acta 1329: 285-290.
  • Rasmussen PB, Bjorn S, Hastrup S, Nielsen PF, Norris K, Thim L, Wiberg FC, Flodgaard H (1996) Characterization of recombinant human HBP/CAP37/azurocidin, a pleiotropic mediator of inflammation-enhancing LPS-induced cytokine release from monocytes. FEBS Lett 390: 109-112.
  • Ruan X, Chodosh J, Callegan MC, Booth MC, Lee TD, Kumar P, Gilmore MS, Pereira HA (2002) Corneal expression of the inflammatory mediator CAP37. Invest Ophthalmol Vis Sci 43: 1414-1421.
  • Shafer WM, Martin LE, Spitznagel JK (1984) Cationic antimicrobial proteins isolated from human neutrophil granulocytes in the presence of diisopropyl fluorophosphate. Infect Immun 45: 29-35.
  • Shafer WM, Martin LE, Spitznagel JK (1986) Late intraphagosomal hydrogen ion concentration favors the in vitro antimicrobial capacity of a 37-kilodalton cationic granule protein of human neutrophil granulocytes. Infect Immun 53: 651-655.
  • Valmu L, Kalkkinen N, Husa A, Rye PD (2005) Differential susceptibility of transferrin glycoforms to chymotrypsin: a proteomics approach to the detection of carbohydrate-deficient transferrin. Biochemistry 44: 16007-16013.
  • van Veen HA, Geerts MEJ, van Berkel PHC, Nuijens JH (2004) The role of N-linked glycosylation in the protection of human and bovine lactoferrin against tryptic proteolysis. Eur J Biochem 271: 678-684.
  • Watorek W (2003) Azurocidin - inactive serine proteinase homolog acting as a multifunctional inflammatory mediator. Acta Biochim Polon 50: 743-752.
  • Watorek W, Polanowski A, Wilusz T (1996) The use of sequential affinity chromatography for separation of human neutrophil elastase, cathepsin G and azurocidin. Acta Biochim Polon 43: 503-506.
  • Zor T, Selinger Z (1996) Linearization of the Bradford protein assay increases its sensitivity: theoretical and experimental studies. Anal Biochem 236: 302-308.
Document Type
Publication order reference
YADDA identifier
bwmeta1.element.bwnjournal-article-abpv54p567kz
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