The ontogeny of a 57-Kd cationic antimicrobial protein of human polymorphonuclear leukocytes: localization to a novel granule population

Pereira, HA; Jk, Spitznagel; Winton, EF; Shafer, W M; Martin, Larry E.; Guzman, GS; Pohl, Jan; Scott, RW; Marra, MN; Kinkade, JM Jr

doi:10.1182/blood.v76.4.825.825

Cited by 30 publications

(6 citation statements)

References 35 publications

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“…In the present study, three bactericidal proteins were purified from the granules of PMNL, and all are candidates for a role in the microbicidal mechanism. When compared in terms of their bactericidal activity toward P. aeruginosa, BP 55 was 70fold more active than BP 30 and 120-fold more active than cathepsin G. We also found that the N-terminal 21 amino acid residues of BP 55 were identical to those of B/PI protein (11,30) and the 57-kDa cationic antimicrobial protein (CAP57) (32,45), which have been studied with S. typhimurium and E. coli as targets. The extended sequence of CAP57 (45) and the deduced sequence from a cDNA for B/PI (11) have a T instead of an R at residue 22, which we identified.…”

Section: Discussionmentioning

confidence: 72%

Comparison of granule proteins from human polymorphonuclear leukocytes which are bactericidal toward Pseudomonas aeruginosa

1991

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Killing of Pseudomonas aeruginosa by a 55-kDa bactericidal protein (BP 55), a 30-kDa protein (BP 30), cathepsin G, elastase, and proteinase 3 has been compared. P. aeruginosa was resistant to killing by elastase and proteinase 3. BP 55 at a 50% lethal dose (LD50) of 0.23 ,ug of protein per 5 x 106 bacteria per ml killed P. aeruginosa and was far more active than BP 30 and cathepsin G. The LD5s of BP 30 and cathepsin G were 16.9 and 28.3 ,ug of protein per 5 x 106 bacteria per ml, respectively. Preincubation of BP 55 or BP 30 with lipopolysaccharide (LPS) from P. aeruginosa inhibited bactericidal activity. The N-terminal amino acid sequence of BP 55 and BP 30 revealed no relationship between the two proteins. However, a monoclonal antibody (AHN-15) reacted with both proteins by Western immunoblot. The bactericidal activity of cathepsin G toward P. aeruginosa appeared to be dependent on the availability of the active site of the enzyme; bactericidal activity was inhibited by phenylmethylsulfonyl fluoride (PMSF) and by the specific cathepsin G inhibitor, Z-Gly-Leu-Phe-CH2Cl. The enzyme and bactericidal activities of cathepsin G were also inhibited by LPS from P. aeruginosa. LPS from P. aeruginosa was shown to be a competitive inhibitor of the enzyme activity of cathepsin G. Elastase enzyme activity was also inhibited noncompetitively by LPS, but the enzyme was not bactericidal. We have concluded that all three bactericidal proteins (BP 55, BP 30, and cathepsin G) may bind to the LPS of the outer membrane of P. aeruginosa. It appears that the enzyme active site must be available for cathepsin G to kill P. aeruginosa and that the active site may be involved in the binding of cathepsin G to P. aeruginosa.Polymorphonuclear leukocytes (PMNL) occupy a central role in host defense and destroy intracellular microorganisms by both oxygen-dependent and oxygen-independent mechanisms. As reviewed by Spitznagel (45) and Thomas et al. (47), several proteins are implicated in oxygen-independent killing of microorganisms. Prominent among the cationic bactericidal proteins thus far purified from human PMNL is a 58to 60-kDa bactericidal/permeability-increasing protein (B/PI) described by Weiss et al. (51). B/PT is bactericidal toward Salmonella typhimurium and Escherichia coli. Shafer et al. have described two cationic antibacterial proteins (CAP) of 57 kDa (CAP57) and 37 kDa (CAP37) that are bactericidal toward S. typhimurium (37). We have described a 55-kDa glycoprotein (BP 55) that was isolated from normal human PMNL and was bactericidal toward Pseudomonas aeruginosa (19,20). The smaller cationic defensins (3.5 to 4.0 kDa) kill a spectrum of microorganisms, including P. aeruginosa (10, 16).More recently, reports by Gabay et al. (9) and Campanelli et al. (6) of a 30-kDa protein, called azurocidin, which is bactericidal for E. coli and Streptococcus faecalis and fungicidal for Candida albicans, have generated a great deal of interest. These same investigators have recalled attention to the bactericidal activity of serine proteinase...

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Section: Discussionmentioning

confidence: 72%

Comparison of granule proteins from human polymorphonuclear leukocytes which are bactericidal toward Pseudomonas aeruginosa

1991

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“…It has been suggested that P. mirabilis is resistant to BPI (also called CAP 57 [26,27] and BP 55 [9, 31]) because an ester-linked phosphate group within the lipid A region is substituted with a positively charged moiety, 4-amino-4deoxy-L-arabinopyranosyl (29). However, we have shown here that BPI (both holo-BPI and rBPI-23) has potent antibacterial activity against a deep rough mutant of P. mirabilis that also possesses this cationic substitution within the lipid A region.…”

Section: R45 Lpsmentioning

confidence: 99%

“…It has been proposed that this substitution may account for the resistance of this microorganism to cationic antibiotics, such as polymyxin B (12), and antibacterial proteins, such as BPI (also called CAP 57 [26,27] and BP 55 [9,31]).…”

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confidence: 99%

Effect of lipopolysaccharide (LPS) chain length on interactions of bactericidal/permeability-increasing protein and its bioactive 23-kilodalton NH2-terminal fragment with isolated LPS and intact Proteus mirabilis and Escherichia coli

et al. 1994

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The target-specific cytotoxicity for gram-negative bacteria and the endotoxin-neutralizing activity of the 55-kDa bactericidal/Permeability-increasing protein (BPI) and its bioactive 23-kDa NH2-terminal fragment depend on the strong attraction of BPI for the lipid A region of lipopolysaccharides (LPS). We have shown before that smooth gram-negative bacteria with long-chain LPS are more resistant to BPI (especially holo-BPI) than are rough strains. It has been suggested that the high BPI resistance of some gram-negative bacteria, such as Proteus mirabilis, might also reflect the structural diversity of lipid A. To explore this possibility, we compared the antibacterial activity and binding of natural and recombinant holo-BPI and a recombinant NH2-terminal fragment (rBPI-23) to an isogenic rough (Re-LPS chemotype) and a smooth (S-LPS chemotype) strain ofP. mirabilis and to LPS isolated from the two strains. Holo-BPI and rBPI-23 were both potently active against the Re strain of P. mirabilis (90%o lethal dose, 20 nM). In contrast, the smooth strain was .100 times more resistant to holo-BPI but only 10 times more resistant to rBPI-23. rBPI-23 was also more potent against several Escherichia coli strains from clinical bacteremia isolates. Differences in the antibacterial potency of BPI toward the Re and S strains of P. mirabilis correlated with differences in the binding of holo-BPI and rBPI-23 to these bacteria. In contrast, the binding of biosynthetically (in vitro transcribed and translated) 35S-labeled holo-BPI and NH2-terminal fragment to isolated Reand S-LPS from P. mirabiis in solution was similar. Moreover, in the Limulus amebocyte lysate assay, holo-BPI and rBPI-23 potently neutralized both forms of LPS with equal effectiveness. Together, these results strongly suggest that BPI recognizes Proteus lipid A and that the relative resistance of (smooth) P. mirabilis to holo-BPI is due to the inhibitory effect of long polysaccharide chains of tightly packed LPS in the envelope.

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“…Clinical details of the patients, with respect to whether or not they had vasculitis, and if so, the distribution, as well as any further information about the role of CAP57 as an ANCA antigen, have not been published. Recently it has been shown that CAP57 and the 55-kD BPI share N-terminal amino acid sequence homology, this homology having been further supported in studies demonstrating that a monoclonal raised against CAP57 could also bind BPI [24].…”

Section: Discussionmentioning

confidence: 85%

Bactericidal/permeability-increasing protein (BPI) is an important antigen for anti-neutrophil cytoplasmic autoantibodies (ANCA) in vasculitis

Zhao

Jones

Lockwood

1995

Clinical and Experimental Immunology

142

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SUMMARYIndirect immunofluorescence (IIF) techniques have shown that ANCA are useful serological markers for some small vessel vasculitides, and ELISA assays, using purified molecules as solidphase ligand, have helped to identify proteinase 3 (PR3) and myeloperoxidase (MPO) as two of the major ANCA antigens. There remain a substantial number of serum samples, which are positive by IIF, yet recognize neither PR3 nor MPO (double-negative samples). We found, by Western blot analysis of soluble neutrophil granule proteins, that certain ofthese double-negative samples recognized a 55-kD doublet of which the first eight residues shared N-terminal amino acid sequence homology with BPI, a potent antibiotic towards Gram-negative bacteria. We developed a simple, quick and robust two-step immunobiochemical method to purify BPI. This was then employed to detect anti-BPI autoantibodies by ELISA and Western blot analysis. We tested 100 double-negative samples and 400 consecutive new samples sent for routine ANCA testing in the anti-BPI ELISA. We found that 45 of the 100 double-negative and 44 of the 400 new routine samples recognized BPI. By Western blot analysis 20/20 positive anti-BPI samples blotted the 55-kD protein. Inhibition assays confirmed the specificity of binding. Review of the 89 anti-BPI-positive patients showed a male dominance (M: F ratio 55:34), a mean age of 60 4 years and clinical diagnoses ranging from organ limited vasculitis to widespread systemic vasculitis.

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The ontogeny of a 57-Kd cationic antimicrobial protein of human polymorphonuclear leukocytes: localization to a novel granule population

Cited by 30 publications

References 35 publications

Comparison of granule proteins from human polymorphonuclear leukocytes which are bactericidal toward Pseudomonas aeruginosa

Comparison of granule proteins from human polymorphonuclear leukocytes which are bactericidal toward Pseudomonas aeruginosa

Effect of lipopolysaccharide (LPS) chain length on interactions of bactericidal/permeability-increasing protein and its bioactive 23-kilodalton NH2-terminal fragment with isolated LPS and intact Proteus mirabilis and Escherichia coli

Bactericidal/permeability-increasing protein (BPI) is an important antigen for anti-neutrophil cytoplasmic autoantibodies (ANCA) in vasculitis

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