SMART MODE:

NORMAL GENOMIC

• Hubacek JA et al.
• Gene variants of the bactericidal/permeability increasing protein and lipopolysaccharide binding protein in sepsis patients: gender-specific genetic predisposition to sepsis.
• Crit Care Med. 2001; 29: 557-61
• Display abstract

• OBJECTIVES: To determine whether the genotype frequencies of the five bi-allelic polymorphisms in the bactericidal/permeability increasing protein (BPI) (Lys216 --> Glu; PstI polymorphism in intron 5; silent mutation G545 --> C) and the lipopolysaccharide binding protein (LBP) (Cys98 --> Gly; Pro436 --> Leu) are associated with the incidence and lethality of sepsis. DESIGN: Case control study of patients with sepsis. SETTING: Intensive care units within university hospitals. PATIENTS: A total of 204 patients diagnosed with sepsis and 250 healthy blood donors. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Short DNA fragments containing the polymorphic sites of the LBP and BPI locus were amplified by the polymerase chain reaction or mismatched polymerase chain reaction. The individual polymorphisms were determined with the appropriate restriction enzyme digestions and subsequent agarose gel electrophoresis. The presence of LBP genotypes with the less frequent Gly98 allele was found to be associated with sepsis (p < .02) in male patients, but not in females. Patients which were homozygote for either of the rare Gly98 (n = 6) and/or Leu436 (n = 5) LBP alleles, furthermore, exclusively were nonsurvivors of sepsis. The genotype frequencies in the BPI gene did not differ between patients and control individuals. CONCLUSIONS: Our findings suggest that common polymorphisms in the gene for LBP in combination with male gender are associated with an increased risk for the development of sepsis and, furthermore, may be linked to an unfavorable outcome. These data support the important immunomodulatory role of LBP in Gram-negative sepsis and suggest that genetic testing may be helpful for the identification of patients with an unfavorable response to Gram-negative infection.

• Desrumaux C et al.
• A hydrophobic cluster at the surface of the human plasma phospholipid transfer protein is critical for activity on high density lipoproteins.
• J Biol Chem. 2001; 276: 5908-15
• Display abstract

• The plasma phospholipid transfer protein (PLTP) belongs to the lipid transfer/lipopolysaccharide binding protein (LT/LBP) family, together with the cholesteryl ester transfer protein, the lipopolysaccharide binding protein (LBP) and the bactericidal permeability increasing protein (BPI). In the present study, we used the crystallographic data available for BPI to build a three-dimensional model for PLTP. Multiple sequence alignment suggested that, in PLTP, a cluster of hydrophobic residues substitutes for a cluster of positively charged residues found on the surface of LBP and BPI, which is critical for interaction with lipopolysaccharides. According to the PLTP model, these hydrophobic residues are situated on an exposed hydrophobic patch at the N-terminal tip of the molecule. To assess the role of this hydrophobic cluster for the functional activity of PLTP, single point alanine mutants were engineered. Phospholipid transfer from liposomes to high density lipoprotein (HDL) by the W91A, F92A, and F93A PLTP mutants was drastically reduced, whereas their transfer activity toward very low density lipoprotein and low density lipoprotein did not change. The HDL size conversion activity of the mutants was reduced to the same extent as the PLTP transfer activity toward HDL. Based on these results, we propose that a functional solvent-exposed hydrophobic cluster in the PLTP molecule specifically contributes to the PLTP transfer activity on HDL substrates.

• Yamashita S et al.
• Roles of plasma lipid transfer proteins in reverse cholesterol transport.
• Front Biosci. 2001; 6: 36687-36687
• Display abstract

• Plasma lipid transfer proteins include plasma cholesteryl ester transfer protein (CETP) and phospholipid transfer protein (PLTP). Plasma CETP facilitates the transfer of cholesteryl ester (CE) from high-density lipoprotein (HDL) to apolipoprotein (apo) B-containing lipoproteins, and is a key protein in reverse cholesterol transport which protects vessel walls from atherosclerosis. The importance of plasma CETP in lipoprotein metabolism was highlighted by the discovery of CETP-deficient subjects with a marked hyperalphalipoproteinemia (HALP). The deficiency of CETP causes various abnormalities in the concentration, composition, and functions of both HDL and low-density lipoprotein (LDL). Although the significance of CETP in terms of atherosclerosis has been controversial, the in vitro evidence showed that large CE-rich HDL particles in CETP deficiency are defective in cholesterol efflux. Recent epidemiological studies in Japanese-Americans and in Omagari area where HALP subjects with the intron 14 splicing defect of CETP gene are markedly frequent, have demonstrated an increased incidence of coronary atherosclerosis in CETP-deficient patients. Similarly, scavenger receptor BI (SR-BI) knockout mice show a marked increase in HDL-cholesterol but accelerated atherosclerosis in atherosclerosis-susceptible mice. Thus, CETP deficiency is a state of impaired reverse cholesterol transport which may possibly lead to the development of atherosclerosis. PLTP transfers phospholipids from triglyceride (TG)-rich lipoproteins to HDL during lipolysis. Human plasma PLTP has a 20% sequence homology to human CETP and human PLTP gene has a marked similarity in the exon-intron organization. Both CETP and PLTP belong to the lipid transfer/lipopolysaccharide binding protein (LBP) gene family, which also includes LBP and bactericidal/permeability-increasing protein (BPI). Although these 4 proteins possess different physiological functions, they share marked biochemical similarities. The current review will also focus on the molecular genetics and function of plasma lipid transfer proteins, including CETP and PLTP.

• Vesy CJ, Kitchens RL, Wolfbauer G, Albers JJ, Munford RS
• Lipopolysaccharide-binding protein and phospholipid transfer protein release lipopolysaccharides from gram-negative bacterial membranes.
• Infect Immun. 2000; 68: 2410-7
• Display abstract

• Although animals mobilize their innate defenses against gram-negative bacteria when they sense the lipid A moiety of bacterial lipopolysaccharide (LPS), excessive responses to this conserved bacterial molecule can be harmful. Of the known ways for decreasing the stimulatory potency of LPS in blood, the binding and neutralization of LPS by plasma lipoproteins is most prominent. The mechanisms by which host lipoproteins take up the native LPS that is found in bacterial membranes are poorly understood, however, since almost all studies of host-LPS interactions have used purified LPS aggregates. Using native Salmonella enterica serovar Typhimurium outer membrane fragments (blebs) that contained (3)H-labeled lipopolysaccharide (LPS) and (35)S-labeled protein, we found that two human plasma proteins, LPS-binding protein (LBP) and phospholipid transfer protein (PLTP), can extract [(3)H]LPS from bacterial membranes and transfer it to human high-density lipoproteins (HDL). Soluble CD14 (sCD14) did not release LPS from blebs yet could facilitate LBP-mediated LPS transfer to HDL. LBP, but not PLTP, also promoted the activation of human monocytes by bleb-derived LPS. Whereas depleting or neutralizing LBP significantly reduced LPS transfer from blebs to lipoproteins in normal human serum, neutralizing serum PLTP had no demonstrable effect. Of the known lipid transfer proteins, LBP is thus most able to transfer LPS from bacterial membranes to the lipoproteins in normal human serum.

• Yamashita S, Hirano K, Sakai N, Matsuzawa Y
• Molecular biology and pathophysiological aspects of plasma cholesteryl ester transfer protein.
• Biochim Biophys Acta. 2000; 1529: 257-75
• Display abstract

• Plasma cholesteryl ester transfer protein (CETP) facilitates the transfer of cholesteryl ester (CE) from high density lipoprotein (HDL) to apolipoprotein B-containing lipoproteins. Since CETP regulates the plasma levels of HDL cholesterol and the size of HDL particles, CETP is considered to be a key protein in reverse cholesterol transport, a protective system against atherosclerosis. CETP, as well as plasma phospholipid transfer protein, belongs to members of the lipid transfer/lipopolysaccharide-binding protein (LBP) gene family, which also includes the lipopolysaccharide-binding protein (LBP) and bactericidal/permeability-increasing protein. Although these four proteins possess different physiological functions, they share marked biochemical and structural similarities. The importance of plasma CETP in lipoprotein metabolism was demonstrated by the discovery of CETP-deficient subjects with a marked hyperalphalipoproteinemia (HALP). Two common mutations in the CETP gene, intron 14 splicing defect and exon 15 missense mutation (D442G), have been identified in Japanese HALP patients with CETP deficiency. The deficiency of CETP causes various abnormalities in the concentration, composition, and functions of both HDL and low density lipoprotein. Although the pathophysiological significance of CETP in terms of atherosclerosis has been controversial, the in vitro experiments showed that large CE-rich HDL particles in CETP deficiency are defective in cholesterol efflux. Epidemiological studies in Japanese-Americans and in the Omagari area where HALP subjects with the intron 14 splicing defect of CETP gene are markedly frequent, have shown an increased incidence of coronary atherosclerosis in CETP-deficient patients. The current review will focus on the recent findings on the molecular biology and pathophysiological aspects of plasma CETP, a key protein in reverse cholesterol transport.

• Kawano K, Qin SC, Lin M, Tall AR, Jiang XC
• Cholesteryl ester transfer protein and phospholipid transfer protein have nonoverlapping functions in vivo.
• J Biol Chem. 2000; 275: 29477-81
• Display abstract

• Plasma phospholipid transfer protein (PLTP) and cholesteryl ester transfer protein (CETP) are homologous molecules that mediate neutral lipid and phospholipid exchange between plasma lipoproteins. Biochemical experiments suggest that only CETP can transfer neutral lipids but that there could be overlap in the ability of PLTP and CETP to transfer or exchange phospholipids. Recently developed PLTP gene knock-out (PLTP0) mice have complete deficiency of plasma phospholipid transfer activity and markedly reduced high density lipoprotein (HDL) levels. To see whether CETP can compensate for PLTP deficiency in vivo, we bred the CETP transgene (CETPTg) into the PLTP0 background. Using an in vivo assay to measure the transfer of [(3)H]PC from VLDL into HDL or an in vitro assay that determined [(3)H]PC transfer from vesicles into HDL, we could detect no phospholipid transfer activity in either PLTP0 or CETPTg/PLTP0 mice. On a chow diet, HDL-PL, HDL-CE, and HDL-apolipoprotein AI in CETPTg/PLTP0 mice were significantly lower than in PLTP0 mice (45 +/- 7 versus 79 +/- 9 mg/dl; 9 +/- 2 versus 16 +/- 5 mg/dl; and 51 +/- 6 versus 100 +/- 9, arbitrary units, respectively). Similar results were obtained on a high fat, high cholesterol diet. These results indicate 1) that there is no redundancy in function of PLTP and CETP in vivo and 2) that the combination of the CETP transgene with PLTP deficiency results in an additive lowering of HDL levels, suggesting that the phenotype of a human PLTP deficiency state would include reduced HDL levels.

• Haudek SB, Natmessnig BE, Redl H, Schlag G, Hatlen LE, Tobias PS
• Isolation, partial characterization, and concentration in experimental sepsis of baboon lipopolysaccharide-binding protein.
• J Lab Clin Med. 2000; 136: 363-70
• Display abstract

• Lipopolysaccharide-binding protein (LBP) is important for mediating host responses to lipopolysaccharide (LPS). The structure and properties of human, rabbit, and murine LBP have been previously described. In this study we partially characterized baboon LBP and investigated its appearance in experimental sepsis. Recurrent bacteremia was induced in baboons by infusion of live Escherichia coli organisms over a 2-hour period at 0, 24, and 48 hours. To assay baboon plasma LBP levels, an enzyme-linked immunosorbent assay with cross-reactive antibodies to human LBP was developed. Control baboon plasma LBP concentrations were 2 to 5 microg/mL. During experimental sepsis, baboon plasma LBP levels increased to between 200 and 350 microg/mL and in parallel with the increase in C-reactive protein levels. Baboon LBP was isolated from acute phase serum by ion-exchange chromatography followed by immuno-affinity chromatography. Its NH2-terminal sequence (XNPGLVARTTNKGLEYSAQE) and its molecular weight (approximately 60 kd) were determined and were proved to be highly homologous to human LBP.

• Bulow E, Gullberg U, Olsson I
• Structural requirements for intracellular processing and sorting of bactericidal/permeability-increasing protein (BPI): comparison with lipopolysaccharide-binding protein.
• J Leukoc Biol. 2000; 68: 669-78
• Display abstract

• The bactericidal/permeability-increasing protein (BPI), which is stored in the azurophil granules of neutrophils, and the circulating lipopolysaccharide-binding protein (LBP) share the same structure. Both bind lipopolysaccharide of gram-negative bacteria through their amino-terminal domains. The carboxy-terminal domain of BPI promotes bacterial attachment to phagocytes, whereas the corresponding domain of LBP delivers lipopolysaccharide to monocytes/macrophages. Our aim was to investigate the role of the amino-and carboxy-terminal domains of BPI and LBP for sorting and storage in myeloid cells after transfection of cDNA to two rodent hematopoietic cell lines. Full-length BPI and LBP were both targeted for storage in these cells. Deletion of the carboxy-terminal half of BPI resulted in storage followed by degradation while the reciprocal deletion of the amino-terminal half led to retention in the endoplasmic reticulum for proteasomal degradation. Chimeras between halves of BPI and LBP were also targeted for storage, but those containing carboxy-terminal BPI had the highest stability, again indicating a role for the carboxy-terminal domain of BPI in protection against degradation. Therefore, we propose a critical stability function for the hydrophobic carboxy-terminal domain of BPI during intracellular sorting for storage while the amino-terminal domain may confer targeting for storage.

• Huuskonen J, Wohlfahrt G, Jauhiainen M, Ehnholm C, Teleman O, Olkkonen VM
• Structure and phospholipid transfer activity of human PLTP: analysis by molecular modeling and site-directed mutagenesis.
• J Lipid Res. 1999; 40: 1123-30
• Display abstract

• The plasma phospholipid transfer protein (PLTP) is an important regulator of high density lipoprotein (HDL) metabolism. We have here, based on sequence alignments of the plasma LPS-binding/lipid transfer protein family and the X-ray structure of the bactericidal/permeability increasing protein (BPI), modeled the structure of PLTP. The model predicts a two-domain architecture with conserved lipid-binding pockets consisting of apolar residues in each domain. By site-directed mutagenesis of selected amino acid residues and transient expression of the protein variants in HeLa cells, the pockets are shown to be essential for PLTP-mediated phospholipid transfer. A solid phase ligand binding assay was used to determine the HDL-binding ability of the mutants. The results suggest that the observed decreases in phospholipid transfer activity of the N-terminal pocket mutants cannot be attributed to altered HDL-binding, but the C-terminal lipid-binding pocket may be involved in the association of PLTP with HDL. Further, the essential structural role of a disulfide bridge between cysteine residues 146 and 185 is demonstrated. The structural model and the mutants characterized here provide powerful tools for the detailed analysis of the mechanisms of PLTP function.

• Beamer LJ, Carroll SF, Eisenberg D
• The three-dimensional structure of human bactericidal/permeability-increasing protein: implications for understanding protein-lipopolysaccharide interactions.
• Biochem Pharmacol. 1999; 57: 225-9
• Display abstract

• Gram-negative bacterial infections are often complicated by the inflammatory properties of lipopolysaccharides (LPS) on or released from the bacterial outer membrane. When present in the mammalian bloodstream, LPS can trigger a series of pathological changes, sometimes resulting in septic shock. Two related mammalian proteins, bactericidal/permeability-increasing protein (BPI) and lipopolysaccharide-binding protein (LBP), are known to affect the LPS-induced inflammatory response and are, therefore, of clinical interest. The recently determined three-dimensional structure of human BPI provides information on the overall protein fold, domain organization, and conserved regions of these two proteins. In addition, the discovery of two apolar lipid binding pockets in BPI indicates a possible site of interaction with LPS. The BPI structure is a powerful tool for the design of site-directed mutants, peptide mimetics/inhibitors, and BPI/LBP chimeras. These studies should help further define the functions of BPI and LBP, and their mechanism of interaction with LPS.

• Ingalls RR, Monks BG, Golenbock DT
• Membrane expression of soluble endotoxin-binding proteins permits lipopolysaccharide signaling in Chinese hamster ovary fibroblasts independently of CD14.
• J Biol Chem. 1999; 274: 13993-8
• Display abstract

• The activation of phagocytes by lipopolysaccharide (LPS) has been implicated in the pathogenesis of Gram-negative sepsis. Although the interaction between CD14 and LPS is a key event in the signaling cascade, the molecular mechanism by which cellular activation occurs remains obscure. We hypothesized that the main function of CD14 was to bind LPS and transfer it to a second receptor, which then initiates the subsequent signal for cellular activation. Thus, surface binding of LPS to the cell membrane would be the critical step that CD14 carries out. To test this hypothesis, we examined the activity of two other proteins known to bind LPS, lipopolysaccharide-binding protein and bactericidal/permeability-increasing protein. We found that when these normally soluble proteins were expressed in Chinese hamster ovary-K1 fibroblasts as glycosylphosphatidylinositol-anchored proteins, both could substitute for CD14 in initiating LPS signaling. Pharmacological studies with synthetic lipid A analogues demonstrated that these surface expressed LPS-binding proteins had characteristics that were qualitatively identical to membrane CD14. These data support the hypothesis that a receptor distinct from CD14 functions as the actual signal transducer and suggest that surface binding of LPS to the cell membrane is the crucial first step for initiating downstream signaling events.

• Guyard-Dangremont V et al.
• Immunochemical evidence that cholesteryl ester transfer protein and bactericidal/permeability-increasing protein share a similar tertiary structure.
• Protein Sci. 1999; 8: 2392-8
• Display abstract

• Cholesteryl ester transfer protein (CETP) plays an important role in plasma lipoprotein metabolism through its ability to transfer cholesteryl ester, triglyceride, and phospholipid between lipoproteins. CETP is a member of a gene family that also includes bactericidal/permeability-increasing protein (BPI). The crystal structure of BPI shows it to be composed of two domains that share a similar structural fold that includes an apolar ligand-binding pocket. As structurally important residues are conserved between BPI and CETP, it is thought that CETP and BPI may have a similar overall conformation. We have previously proposed a model of CETP structure based on the binding characteristics of anti-CETP monoclonal antibodies (mAbs). We now present a refined epitope map of CETP that has been adapted to a structural model of CETP that uses the atomic coordinates of BPI. Four epitopes composed of CETP residues 215-219, 219-223, 223-227, and 444-450, respectively, are predicted to be situated on the external surface of the central beta-sheet and a fifth epitope (residues 225-258) on an extended linker that connects the two domains of the molecule. Three other epitopes, residues 317-331, 360-366, and 393-410, would form part of the putative carboxy-terminal beta-barrel. The ability of the corresponding mAbs to compete for binding to CETP is consistent with the proximity of the respective epitopes in the model. These results thus provide experimental evidence that is consistent with CETP and BPI having similar surface topologies.

• Lengacher S, Reed D, Heumann D, Jongeneel CV
• Genomic organization and chromosomal localization of the mouse lipopolysaccharide binding protein gene.
• Immunogenetics. 1999; 49: 553-6

• Schumann RR, Zweigner J
• A novel acute-phase marker: lipopolysaccharide binding protein (LBP).
• Clin Chem Lab Med. 1999; 37: 271-4
• Display abstract

• Acute phase proteins are extremely helpful markers for indicating a disturbance of the homeostasis within the organism and for monitoring the course of a disease. Despite the availability of several serum acute phase markers, a better and more specific prediction of sepsis and related disorders, such as systemic inflammatory response syndrome (SIRS) is still needed, as these diseases still have a high mortality rate and have to be detected early and with high specificity. Here a novel acute-phase protein is introduced, that has certain biological functions in host defense and that may be a useful addition for the diagnosis and monitoring of sepsis. Lipopolysaccharide (LPS or endotoxin), binding protein (LBP) is a class 1 acute-phase protein with the ability to bind and transfer bacterial LPS. Changes in serum levels of LBP have profound effects on the host's ability to react to endotoxin stimulation and to defend itself against sepsis. Results obtained from in vitro studies and from an animal model are reviewed here and a perspective on ongoing clinical studies is given. There is evidence that LBP, along with other LPS-recognizing molecules, is an important parameter for monitoring the acute phase and the ability of the host to react to LPS-challenge.

• Tu AY, Albers JJ
• DNA sequences responsible for reduced promoter activity of human phospholipid transfer protein by fibrate.
• Biochem Biophys Res Commun. 1999; 264: 802-7
• Display abstract

• Phospholipid transfer protein (PLTP) plays an important role in plasma lipid and lipoprotein metabolism. We have previously cloned and characterized the promoter region of the human PLTP gene. The present study was conducted to determine if the promoter activity of the human PLTP gene is affected by fibrate, a hypolipidemic drug, and to identify DNA sequences that are responsible for the effect. The results indicated that the promoter activity of the PLTP gene was significantly reduced by fenofibrate, and the area that was mainly responsive to the reducing effect by fibrate was located between -377 and -230 of the 5'-flanking region. The DNA sequence analysis suggested that each area of the DNA sequences from -342 to -323 and from -322 to -299 has two repeated sequences, which are inverted and homologous to the recognition motif of peroxisome proliferator-activated receptor (PPAR), namely the PPAR-responsive element (PPRE). Mutagenesis of these PPRE-like sequences, especially that at -322 to -299, abolished most of the reducing effects of fibrate on the PLTP promoter activity. These findings strongly suggest that the PPRE-like elements are responsible for the reduced promoter activity of the human PLTP gene by fibrate.

• Qu SJ, Fan HZ, Kilinc C, Pownall HJ
• Role of cysteine residues in human plasma phospholipid transfer protein.
• J Protein Chem. 1999; 18: 193-8
• Display abstract

• Phospholipid transfer protein (PLTP) belongs to a family of human plasma lipid transfer proteins that bind to small amphophilic molecules. PLTP contains cysteines at residues 5, 129, 168, and 318. Bactericidal/permeability-increasing protein, which is a member of the same gene family, contains an essential disulfide bond between Cys135 and Cys175; these residues, which correspond to Cys129 and Cys168 in PLTP, are conserved among all known members of the gene family. To identify the importance of these and the remaining cysteine residues to PLTP secretion and activity, each was replaced by a glycine by site-directed mutagenesis. The mutant as well as wild-type PLTP cDNAs were cloned into the mammalian expression vector pSV.SPORT1, and the PLTP cDNAs were transfected to COS-6 cells for expression. PLTP Cys129 --> Gly and PLTP Cys168 --> Gly were secretion incompetent. Neither PLTP mass nor activity was detectable in cell lysates and culture medium. Relative to wild-type PLTP, PLTP Cys5 --> Gly and PLTP Cys318 --> Gly exhibited similar specific activities but partially impaired PLTP synthesis and secretion. Intracellular PLTP appeared as two bands of 75 and 51 kDa corresponding to reported molecular masses for the glycosylated and nonglycosylated forms. The specific activities of PLTP Cys5 --> Gly and PLTP Cys318 --> Gly were similar in the cell lysates and medium, suggesting that glycosylation does not affect transfer activity.

• Elsbach P
• The bactericidal/permeability-increasing protein (BPI) in antibacterial host defense.
• J Leukoc Biol. 1998; 64: 14-8
• Display abstract

• The bactericidal/permeability-increasing protein (BPI) is a 456-residue cationic protein produced only by precursors of polymorphonuclear leukocytes (PMN) and is stored in the primary granules of these cells. The potent (nM) cytotoxicity of BPI is limited to gram-negative bacteria (GNB), reflecting the high affinity (<10 nM) of BPI for bacterial lipopolysaccharides (LPS). The biological effects of isolated BPI are linked to complex formation with LPS. Binding of BPI to live bacteria via LPS causes immediate growth arrest. Actual killing coincides with later damage to the inner membrane. Complex formation of BPI with cell-associated or cell-free LPS inhibits all LPS-induced host cell responses. BPI-blocking antibodies abolish the potent activity of whole PMN lysates and inflammatory fluids against BPI-sensitive GNB. The antibacterial and the anti-endotoxin activities of BPI are fully expressed by the amino terminal half of the molecule. These properties of BPI have prompted preclinical and subsequent clinical testing of recombinant amino-terminal fragments of BPI. In animals, human BPI protein products protect against lethal injections of isolated LPS and inocula of GNB. Phase I trials in healthy human volunteers and multiple Phase I/II clinical trials have been completed or are in progress (severe pediatric meningococcemia, hemorrhagic trauma, partial hepatectomy, severe peritoneal infections, and cystic fibrosis) and two phase III trials (meningococcemia and hemorrhagic trauma) have been initiated. In none of >900 normal and severely ill individuals have issues of safety or immunogenicity been encountered. Preliminary evidence points to overall benefit in BPI-treated patients. These results suggest that BPI may have a place in the treatment of life-threatening infections and conditions associated with bacteremia and endotoxemia.

• Beamer LJ, Fischer D, Eisenberg D
• Detecting distant relatives of mammalian LPS-binding and lipid transport proteins.
• Protein Sci. 1998; 7: 1643-6
• Display abstract

• In mammals, a family of four lipid binding proteins has been previously defined that includes two lipopolysaccharide binding proteins and two lipid transfer proteins. The first member of this family to have its three-dimensional structure determined is bactericidal/permeability-increasing protein (BPI). Using both the sequence and structure of BPI, along with recently developed sequence-sequence and sequence-structure similarity search methods, we have identified 13 distant members of the family in a diverse set of eukaryotes, including rat, chicken, Caenorhabditis elegans, and Biomphalaria galbrata. Although the sequence similarity between these 13 new members and any of the 4 original members of the BPI family is well below the "twilight zone," their high sequence-structure compatibility with BPI indicates they are likely to share its fold. These findings broaden the BPI family to include a member found in retina and brain, and suggest that a primitive member may have contained only one of the two similar domains of BPI.

• Calafat J et al.
• The bactericidal/permeability-increasing protein (BPI) is present in specific granules of human eosinophils.
• Blood. 1998; 91: 4770-5
• Display abstract

• Eosinophils participate in the inflammatory response seen in allergy and parasitic infestation, but a role in host defense against bacterial infection is not settled. The bactericidal/permeability-increasing protein (BPI) has been demonstrated in neutrophils and it exerts bacteriostatic and bactericidal effects against a wide variety of Gram-negative bacterial species. Using the Western blot technique, a 55-kD band, corresponding to BPI, was detected in lysates from both neutrophils and eosinophils. The localization of BPI in immature and mature eosinophils was investigated using immunoelectron microscopy. BPI was found in immature and mature specific granules of eosinophils and was detected in phagosomes as well, indicating release of the protein from the granules into the phagosomes. Using a specific enzyme-linked immunosorbent assay, eosinophils were shown to contain 179 ng of BPI/5 x 10(6) eosinophils compared with 710 ng BPI/5 x 10(6) neutrophils. The presence of BPI in eosinophils suggests a role for these cells in host defense against Gram-negative bacterial invasion or may suggest a role for BPI against parasitic infestation.

• Elass-Rochard E et al.
• Lactoferrin inhibits the endotoxin interaction with CD14 by competition with the lipopolysaccharide-binding protein.
• Infect Immun. 1998; 66: 486-91
• Display abstract

• Human lactoferrin (hLf), a glycoprotein released from neutrophil granules during inflammation, and the lipopolysaccharide (LPS)-binding protein (LBP), an acute-phase serum protein, are known to bind to the lipid A of LPS. The LPS-binding sites are located in the N-terminal regions of both proteins, at amino acid residues 28 to 34 of hLf and 91 to 108 of LBP. Both of these proteins modulate endotoxin activities, but they possess biologically antagonistic properties. In this study, we have investigated the competition between hLf and recombinant human LBP (rhLBP) for the binding of Escherichia coli 055:B5 LPS to the differentiated monocytic THP-1 cell line. Our studies revealed that hLf prevented the rhLBP-mediated binding of LPS to the CD14 receptor on cells. Maximal inhibition of LPS-cell interactions by hLf was raised when both hLf and rhLBP were simultaneously added to LPS or when hLf and LPS were mixed with cells 30 min prior to the incubation with rhLBP. However, when hLf was added 30 min after the interaction of rhLBP with LPS, the binding of the rhLPS-LBP complex to CD14 could not be reversed. These observations indicate that hLf competes with rhLBP for the LPS binding and therefore interferes with the interaction of LPS with CD14. Furthermore, experiments involving competitive binding of the rhLBP-LPS complex to cells with two recombinant mutated hLfs show that in addition to residues 28 to 34, another basic cluster which contains residues 1 to 5 of hLf competes for the binding to LPS. Basic sequences homologous to residues 28 to 34 of hLf were evidenced on LPS-binding proteins such as LBP, bactericidal/permeability-increasing protein, and Limulus anti-LPS factor.

• Bruce C, Beamer LJ, Tall AR
• The implications of the structure of the bactericidal/permeability-increasing protein on the lipid-transfer function of the cholesteryl ester transfer protein.
• Curr Opin Struct Biol. 1998; 8: 426-34
• Display abstract

• The cholesteryl ester transfer protein (CETP) is evolutionarily related to the bactericidal/permeability-increasing protein (BPI). The recently solved structure of BPI shows an elongated, boomerang-shaped molecule, with two hydrophobic pockets opening to its concave side. These pockets each contain a phospholipid molecule. A model of CETP, based on the recently solved crystal structure of BPI, provides the basis for interpreting functional studies on CETP. In this model, C-terminal residues 461-476, which were shown to be required for neutral lipid transfer between plasma lipoproteins, from an amphipathic helix covering the opening of the N-terminal pocket. A possible lipid-transfer mechanism for CETP, with the initial step involving the disordering of lipids in the lipoprotein surface, followed by the flipping and entry of a lipid molecule into the hydrophobic lipid-binding pocket, is hypothesized in light of structural evidence and recent studies.

• Beamer LJ, Carroll SF, Eisenberg D
• The BPI/LBP family of proteins: a structural analysis of conserved regions.
• Protein Sci. 1998; 7: 906-14
• Display abstract

• Two related mammalian proteins, bactericidal/permeability-increasing protein (BPI) and lipopolysaccharide-binding protein (LBP), share high-affinity binding to lipopolysaccharide (LPS), a glycolipid found in the outer membrane of gram-negative bacteria. The recently determined crystal structure of human BPI permits a structure/function analysis, presented here, of the conserved regions of these two proteins sequences. In the seven known sequences of BPI and LBP, 102 residues are completely conserved and may be classified in terms of location, side-chain chemistry, and interactions with other residues. We find that the most highly conserved regions lie at the interfaces between the tertiary structural elements that help create two apolar lipid-binding pockets. Most of the conserved polar and charged residues appear to be involved in inter-residue interactions such as H-bonding. However, in both BPI and LBP a subset of conserved residues with positive charge (lysines 42, 48, 92, 95, and 99 of BPI) have no apparent structural role. These residues cluster at the tip of the NH2-terminal domain, and several coincide with residues known to affect LPS binding; thus, it seems likely that these residues make electrostatic interactions with negatively charged groups of LPS. Overall differences in charge and electrostatic potential between BPI and LBP suggest that BPI's bactericidal activity is related to the high positive charge of its NH2-terminal domain. A model of human LBP derived from the BPI structure provides a rational basis for future experiments, such as site-directed mutagenesis and inhibitor design.

• Guo N, Mogues T, Weremowicz S, Morton CC, Sastry KN
• The human ortholog of rhesus mannose-binding protein-A gene is an expressed pseudogene that localizes to chromosome 10.
• Mamm Genome. 1998; 9: 246-9

• Yu B, Hailman E, Wright SD
• Lipopolysaccharide binding protein and soluble CD14 catalyze exchange of phospholipids.
• J Clin Invest. 1997; 99: 315-24
• Display abstract

• Lipopolysaccharide binding protein (LBP) is a plasma protein known to facilitate the diffusion of bacterial LPS (endotoxin). LBP catalyzes movement of LPS monomers from LPS aggregates to HDL particles, to phospholipid bilayers, and to a binding site on a second plasma protein, soluble CD14 (sCD14). sCD14 can hasten transfer by receiving an LPS monomer from an LPS aggregate, and then surrendering it to an HDL particle, thus acting as a soluble "shuttle" for an insoluble lipid. Here we show that LBP and sCD14 shuttle not only LPS, but also phospholipids. Phosphatidylinositol (PI), phosphatidylcholine, and a fluorescently labeled derivative of phosphatidylethanolamine (R-PE) are each transferred by LBP from membranes to HDL particles. The transfer could be observed using recombinant LBP and sCD14 or whole human plasma, and the plasma-mediated transfer of PI could be blocked by anti-LBP and partially inhibited by anti-CD14. sCD14 appears to act as a soluble shuttle for phospholipids since direct binding of PI and R-PE to sCD14 was observed and because addition of sCD14 accelerated transfer of these lipids. These studies define a new function for LBP and sCD14 and describe a novel mechanism for the transfer of phospholipids in blood. In further studies, we show evidence suggesting that LBP transfers LPS and phospholipids by reciprocal exchange: LBP-catalyzed binding of R-PE to LPS x sCD14 complexes was accompanied by the exit of LPS from sCD14, and LBP-catalyzed binding of R-PE to sCD14 was accelerated by prior binding of LPS to sCD14. Binding of one lipid is thus functionally coupled with the release of a second. These results suggest that LBP acts as a lipid exchange protein.

• Tobias PS, Soldau K, Iovine NM, Elsbach P, Weiss J
• Lipopolysaccharide (LPS)-binding proteins BPI and LBP form different types of complexes with LPS.
• J Biol Chem. 1997; 272: 18682-5
• Display abstract

• Lipopolysaccharide (LPS)-binding protein (LBP) and bactericidal/permeability-increasing protein (BPI) are closely related LPS-binding proteins whose binding to LPS has markedly different functional consequences. To gain better insight into the possible basis of these functional differences, the physical properties of LBP-LPS and BPI-LPS complexes have been compared in this study by sedimentation, light scattering, and fluorescence analyses. These studies reveal dramatic differences in the physical properties of LPS complexed to LBP versus BPI. They suggest that of the two proteins, only LBP can disperse LPS aggegates. However, BPI can enhance both the sedimentation velocity and apparent size of LPS aggregates while inhibiting LPS-LBP binding even at very low (1:40 to 1:20) BPI:LPS molar ratios.

• Iovine NM, Elsbach P, Weiss J
• An opsonic function of the neutrophil bactericidal/permeability-increasing protein depends on both its N- and C-terminal domains.
• Proc Natl Acad Sci U S A. 1997; 94: 10973-8
• Display abstract

• The host response to Gram-negative bacterial infection is influenced by two homologous lipopolysaccharide (LPS)-interactive proteins, LPS-binding protein (LBP) and the bacteridical/permeability-increasing protein (BPI). Both proteins bind LPS via their N-terminal domains but produce profoundly different effects: BPI and a bioactive N-terminal fragment BPI-21 exert a selective and potent antibacterial effect upon Gram-negative bacteria and suppress LPS bioactivity whereas LBP is not toxic toward Gram-negative bacteria and potentiates LPS bioactivity. The latter effect of LBP requires the C-terminal domain for delivery of LPS to CD14, so we postulated that the C-terminal region of BPI may serve a similar delivery function but to distinct targets. LBP, holoBPI, BPI-21, and LBP/BPI chimeras were compared for their ability to promote uptake by human phagocytes of an encapsulated, phagocytosis-resistant strain of Escherichia coli. We show that only bacteria preincubated with holoBPI are ingested by neutrophils and monocytes. These findings suggest that, when extracellular holoBPI is bound via its N-terminal domain to Gram-negative bacteria, the C-terminal domain promotes bacterial attachment to neutrophils and monocytes, leading to phagocytosis. Therefore, analogous to the role of the C-terminal domain of LBP in delivery of LPS to CD14, the C-terminal domain of BPI may fulfill a similar function in BPI-specific disposal pathways for Gram-negative bacteria.

• Schumann RR, Lamping N, Hoess A
• Interchangeable endotoxin-binding domains in proteins with opposite lipopolysaccharide-dependent activities.
• J Immunol. 1997; 159: 5599-605
• Display abstract

• Host defense against microorganisms involves proteins that bind specifically to bacterial endotoxins (LPS), causing different cellular effects. Although LPS-binding protein (LBP) can enhance LPS activities, while bactericidal/permeability-increasing protein (BPI) and Limulus anti-LPS factor (LALF) neutralize LPS, it has been proposed that their LPS-binding domains possess a similar structure. Here, we provide evidence that the LBP/LPS-binding domain is, as in the LALF structure, solvent exposed and therefore available for LPS binding. Our investigations into the activity of LPS-binding domains of different LPS-binding proteins, in the context of LBP, provide the first functional analysis of these domains in a whole protein. We constructed domain exchange hybrid proteins by substituting 12 amino acids of the LBP/LPS-binding domain with those of BPI and LALF and expressed them in Chinese hamster ovary cells. Although discrete point mutations within the LPS-binding domain of LBP disrupted its specific functions, the hybrid proteins were still able to bind LPS and, in addition, retained the wild-type LBP activity of enhancing LPS priming for FMLP-induced oxygen radical production by neutrophils and transferring LPS aggregates to CD14. Although BPI and LALF display opposite activities to LBP, and LALF does not share any sequence homology with LBP, our data provide strong evidence that LBP, BPI, and LALF possess a solvent-exposed, interchangeable LPS binding motif that is functionally independent of LPS transport or neutralization.

• Abrahamson SL et al.
• Biochemical characterization of recombinant fusions of lipopolysaccharide binding protein and bactericidal/permeability-increasing protein. Implications in biological activity.
• J Biol Chem. 1997; 272: 2149-55
• Display abstract

• The physiological response to endotoxin (lipopolysaccharide (LPS)) can be regulated by two closely related LPS-binding proteins, LPS-binding protein (LBP), which potentiates LPS' inflammatory activity via interaction with the monocytic antigen CD14, and bactericidal/permeability-increasing protein (BPI), which neutralizes LPS. Both proteins bind LPS with high affinity sites in their N-terminal domains, whereas interaction between LBP and CD14 is dependent upon the LBP C-terminal domain. We have created fusions of the N- and C-terminal domains from each protein and compared the functional activities and pharmacokinetics of these fusions, the individual N-terminal domains, and the parent proteins. The N-terminal domains of BPI and LBP bound lipid A with their characteristic apparent affinity constants, regardless of the C-terminal fusion partner. In addition, the C-terminal domain of LBP allowed transfer of LPS to CD14 in conjunction with either N-terminal LPS binding domain. Proteins containing a BPI N-terminal domain had greater heparin binding capacities in vitro and were cleared more rapidly from the plasma of whole animals. Taken together, these data better define how closely related proteins such as BPI and LBP can have opposing effects on the body's response to LPS.

• Pussinen PJ, Olkkonen VM, Jauhiainen M, Ehnholm C
• Molecular cloning and functional expression of cDNA encoding the pig plasma phospholipid transfer protein.
• J Lipid Res. 1997; 38: 1473-81
• Display abstract

• Humans and the pig show marked similarities in lipoprotein metabolism; therefore, the pig has been used as a model in numerous nutritional studies. Pig plasma displays no activity of cholesteryl ester transfer protein (CETP), which is known to be responsible for half of the phospholipid mass transfer in human plasma, the other half being accounted for by the plasma phospholipid transfer protein (PLTP). This makes the pig an ideal subject for the study of PLTP structure and function. Here we report the molecular cloning of pig PLTP and the eukaryotic cell expression of its complementary DNA. Pig PLTP was found to share 93% amino acid sequence identity with human PLTP and 81% with mouse PLTP. Tissue expression of PLTP mRNA was examined by a method based on reverse transcription-polymerase chain reaction (RT-PCR) and solid-phase minisequencing in nine pig tissues. The highest PLTP mRNA levels were found in the pancreas, brain, lung, and liver. Medium from COS-1 cells expressing PLTP possessed phospholipid transfer activity, and the secreted recombinant PLTP was detectable by Western blotting in the culture supernatant. A mutant protein with a substitution of Cys at position 22 by Arg was found to display impaired secretion into growth medium indicating a role for cysteines in the correct folding of PLTP. This study forms the basis for future work on the structure-function relationships in pig PLTP.

• Kirschning CJ et al.
• Similar organization of the lipopolysaccharide-binding protein (LBP) and phospholipid transfer protein (PLTP) genes suggests a common gene family of lipid-binding proteins.
• Genomics. 1997; 46: 416-25
• Display abstract

• The transfer of lipids in aqueous environments such as serum has been attributed to a recently characterized class of proteins. Abnormal regulation of serum lipids by these proteins is thought to be a key event in the pathophysiology of cardiovascular diseases. Lipopolysaccharide (endotoxin) binding protein (LBP) was identified by virtue of its ability to bind bacterial lipid A. We have analyzed the exon-intron organization of the LBP gene and the nucleotide sequence of its approximately 20 kb spanning 5'- and 3'-untranslated regions. When comparing the genomic organization of LBP with that of two other genes coding for lipid transfer proteins, significant homologies were found. The LBP gene includes 15 exons, and the 2-kb promoter contains recognition elements of acute phase-typical reactants and a repetitive 12-mer motif with an as yet unknown protein-binding property. Detailed sequence comparison revealed a closer relatedness of LBP with PLTP than with CETP as demonstrated by an almost identical intron positioning. This high degree of similarity supports functional studies by others suggesting that like LBP, PLTP may also be able to bind and transport bacterial lipopolysaccharide.

• Uknis ME et al.
• Design of a potent novel endotoxin antagonist.
• Surgery. 1997; 122: 380-5
• Display abstract

• BACKGROUND: Bactericidal permeability increasing protein (BPI) binds to and neutralizes lipopolysaccharide (LPS, endotoxin). Small synthetic peptides based on the amino acid sequence of the LPS binding domain of BPI neutralize LPS, albeit inefficiently. Although the LPS binding domain of native BPI possesses a beta-turn secondary structure, this structure is not present in small derivative peptides. The purpose of this study was to determine whether the addition of a beta-turn to a BPI-derived peptide is associated with more potent endotoxin antagonism. METHODS: We generated a hybrid peptide (BU3) on the basis of (1) a portion of the LPS binding domain from BPI and (2) amino acids known to initiate a beta-turn. BU3 folds with a beta-turn, and we tested its effects on LPS neutralization and LPS-induced tumor necrosis factor-alpha secretion, comparing it with BPI-derived peptide BG22 that lacks a beta-turn and to an irrelevant peptide (BG16). RESULTS: Compared with BG22, BU3 demonstrated enhanced LPS neutralization and inhibition of LPS-induced tumor necrosis factor-alpha secretion in vitro and a similar diminution of endotoxemia and tumor necrosis factor-alpha secretion in a murine model of endotoxemia. CONCLUSIONS: These data demonstrate the potential for enhancing the biologic activity of a BPI-derived peptide endotoxin antagonist via manipulation of its conformational structure.

• Capodici C, Weiss J
• Both N- and C-terminal regions of the bioactive N-terminal fragment of the neutrophil granule bactericidal/permeability-increasing protein are required for stability and function.
• J Immunol. 1996; 156: 4789-96
• Display abstract

• An N-terminal fragment (residues 1-199) of the 456-residue human bactericidal/permeability-increasing protein (BPI), isolated after limited proteolysis, exhibits antibacterial and LPS-neutralizing activities equal to or greater than those of holo-BPI. To assess minimal structural requirements for bioactivity, mutant species of BPI were expressed in vivo by transient transfection and in vitro by cellfree transcription/translation. BPI1-456 and BPI1-193 demonstrated the expected antibacterial and LPS-binding activities. Deletion of the N-terminal 12 residues did not diminish BPI function. However, further truncation either from the C-terminus to residue 169 (BPI1-169) or from the N-terminus (BPIdelta15-56) yielded in vitro products with little or no LPS-binding activity and in vivo products that could not be recovered from the culture medium or cellular acid extracts. The possible role of cysteine-175 (the three cysteines in human BPI are at residues 132, 135, and 175) in BPI stability/function was examined by substitution of Cys(175) with serine. Recovery of C175S BPI from extracellular medium was reduced 10-fold, and C175S BPI produced in vitro had little LPS-binding activity. Compared with wild-type holo-BPI and BPI1-193, BPI1-169, BPIdelta15-56, and C175S BPI showed increased susceptibility to cleavage by elastase in the region 1-193 (but not in the region 200-456), indicating conformational changes that may account for the loss of function. These findings suggest that the proteolytic N-terminal fragment of BPI corresponds closely to the minimum functional (antibacterial/anti-LPS) domain of BPI and that residues near both ends of this fragment are essential for structural stability and functional integrity.

• Albers JJ, Tu AY, Wolfbauer G, Cheung MC, Marcovina SM
• Molecular biology of phospholipid transfer protein.
• Curr Opin Lipidol. 1996; 7: 88-93
• Display abstract

• Lipid transfer proteins play an essential role in the intravascular dynamics of lipids among lipoproteins and between lipoproteins and cell membranes. Phospholipid transfer protein has been known for over a decade but, unlike cholesteryl ester transfer protein, has been investigated relatively little with regard to its physiological importance. The recent determination of the phospholipid transfer protein complementary DNA sequence as well as the further characterization of its gene structure will direct future studies toward the understanding of its structure-function correlations, physiological regulation, and clinical assessment at the molecular level. As a member of the lipid-transfer lipopolysaccharide-binding protein gene family, phospholipid transfer protein will attract investigators to studying its possible involvement in lipopolysaccharide or endotoxin interactions in addition to its phospholipid transfer activity.

• Tu AY, Deeb SS, Iwasaki L, Day JR, Albers JJ
• Organization of human phospholipid transfer protein gene.
• Biochem Biophys Res Commun. 1995; 207: 552-8
• Display abstract

• We have determined the exon/intron organization of the human phospholipid transfer protein gene. The gene, which spans approximately 13.3 kilobases, is comprised of 16 exons. The organization of the phospholipid transfer protein gene strikingly resembles that encoding another plasma lipid transfer protein, the human cholesterol ester transfer protein. The exon-intron junctions in these two genes are highly conserved, with eight out of fifteen junctions interrupting the same codons, while the remaining junctions lie within 5 residues of each other. The similarity in gene structure and homology in coding sequences suggests that these two genes most likely evolved from a common ancestral gene.

• Jiang XC, Bruce C, Cocke T, Wang S, Boguski M, Tall AR
• Point mutagenesis of positively charged amino acids of cholesteryl ester transfer protein: conserved residues within the lipid transfer/lipopolysaccharide binding protein gene family essential for function.
• Biochemistry. 1995; 34: 7258-63
• Display abstract

• The cholesteryl ester transfer protein (CETP) binds to plasma lipoproteins and transfers neutral lipids between them. Previous studies showed that lipoprotein binding involves ionic interactions between CETP and lipoproteins, with increased binding of CETP to lipoproteins carrying increased negative charge. In order to understand the molecular determinants of lipoprotein binding in CETP, site-directed mutagenesis was carried out on positively charged amino acids within and outside regions of conserved sequence in the putative family of lipid transfer/lipopolysaccharide (LPS) binding proteins (LT/LBP). Within the conserved regions, two mutant proteins, K233A and R259D, were well secreted by the transfected cells but showed markedly reduced cholesteryl ester transfer activity. Separating the bound from free CETP by gel filtration after incubation with HDL, HDL binding by K233A was found to be impaired, suggesting that the binding deficiency of the mutant may be responsible for decreased transfer activity. Kinetic analysis showed a marked increase in the apparent Km but no change in Vmax, consistent with a lipoprotein binding defect. Thus, within CETP, K233 and R259 play an essential role in cholesteryl ester transfer activity probably by mediating binding of CETP to lipoproteins. Sequence alignment of CETP, phospholipid transfer protein, LPS binding protein, and bactericidal permeability-inducing protein showed that K223 and R259 were strictly conserved as positively charged amino acids, suggesting a common function within the LT/LBP gene family.

• McCabe KM, Wheeler DA, Adams V, McCabe ER
• Comparison of human VDAC1 with streptococcal streptokinase and bovine bactericidal permeability increasing protein: role of structural information in identifying functionally significant domains.
• Biochem Mol Med. 1995; 56: 176-9
• Display abstract

• Comparison of the primary amino acid sequence of the human X-linked voltage-dependent anion channel, with other sequences in data base searches, identified regions of similarity in streptococcal streptokinase and bovine bactericidal permeability increasing protein. These regions of similarity were in different areas of the protein and were relatively short. However, examination of an empirically derived structural model of the channel showed that each region of similarity in streptokinase and bovine bactericidal permeability increasing protein corresponded to contiguous transmembrane domains within the channel protein. We speculate that these transmembrane domains may be functionally significant for streptokinase and bovine bactericidal permeability increasing protein. These investigations demonstrate the need for incorporation of information regarding secondary, tertiary, and quaternary structures, as well as function, in algorithms used for database searches.

• Battafaraono RJ et al.
• Peptide derivatives of three distinct lipopolysaccharide binding proteins inhibit lipopolysaccharide-induced tumor necrosis factor-alpha secretion in vitro.
• Surgery. 1995; 118: 318-24
• Display abstract

• BACKGROUND. Bactericidal permeability increasing protein (BPI), Limulus anti-lipopolysaccharide factor (LALF), and lipopolysaccharide binding protein (LBP) are three distinct proteins that bind to lipopolysaccharide (LPS). Intriguingly, binding of BPI and LALF to LPS results in neutralization of LPS activity, whereas the binding of LBP to LPS creates a complex that results in augmentation of LPS activity. Despite their different effector functions, we hypothesized that peptides based on the sequences of the proposed LPS-binding motif from each protein would neutralize LPS in vitro. METHODS. Three peptide sequences, each 27 amino acids in length, of the proposed LPS-binding motif of BPI (BG38), LALF (BG42), and LBP (BG43) were synthesized. These peptides were then tested for their: (1) ability to inhibit macrophage secretion of TNF-alpha after stimulation by LPS derived from Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Serratia marcescens; and (2) bactericidal activity against these same four gram-negative bacteria in vitro. RESULTS. Synthetic peptides BG38 (BPI-derived), BG42 (LALF-derived), and BG43 (LBP-derived) but not control peptide significantly inhibited LPS-induced tumor necrosis factor-alpha secretion by macrophages and mediated the lysis of gram-negative bacteria in vitro. In addition, preincubation of LPS with peptide BG38 mediated complete protection subsequent to lethal endotoxin challenge. CONCLUSIONS. These data demonstrate that small peptides derived from BPI, LALF, and LBP retained significant endotoxin-neutralizing and bactericidal activity against many different gram-negative bacteria in vitro. Identification of this conserved LPS-binding region within each protein may aid in the development of new immunomodulatory reagents for use as adjuvant therapy in the treatment of gram-negative bacterial sepsis.

• Horwitz AH, Williams RE, Nowakowski G
• Human lipopolysaccharide-binding protein potentiates bactericidal activity of human bactericidal/permeability-increasing protein.
• Infect Immun. 1995; 63: 522-7
• Display abstract

• Human bactericidal/permeability-increasing protein (BPI) from neutrophils and a recombinant amino-terminal fragment, rBPI23, bind to and are cytotoxic for gram-negative bacteria both in vitro and ex vivo in plasma or whole blood. To function in vivo as an extracellular bactericidal agent, rBPI23 must act in the presence of the lipopolysaccharide-binding protein (LBP), which also binds to but has no reported cytotoxicity for gram-negative bacteria. LBP, which is present at 5 to 10 micrograms/ml in healthy humans and at much higher levels in septic patients, mediates proinflammatory host responses to gram-negative infection. On the basis of these previous observations, we have examined the effect of recombinant LBP (rLBP) on the bactericidal activity of rBPI23 against Escherichia coli J5 in vitro. Physiological concentrations of rLBP (5 to 20 micrograms/ml) had little or no bactericidal activity but reduced by up to approximately 10,000-fold the concentration of BPI required for bactericidal or related activities in assays which measure (i) cell viability as CFUs on solid media or growth in broth culture and (ii) protein synthesis following treatment with BPI. LBP also potentiated BPI-mediated permeabilization of the E. coli outer membrane to actinomycin D by about 100-fold but had no permeabilizing activity of its own. Under optimal conditions for potentiation, fewer than 100 BPI molecules were required to kill a single E. coli J5 bacterium.

• Opal SM, Palardy JE, Marra MN, Fisher CJ Jr, McKelligon BM, Scott RW
• Relative concentrations of endotoxin-binding proteins in body fluids during infection.
• Lancet. 1994; 344: 429-31
• Display abstract

• Endotoxin initiates the systemic inflammatory response, haemodynamic changes, and multi-organ failure that may occur as a consequence of systemic gram-negative bacterial infection. The serum protein lipopolysaccharide-binding protein (LBP) binds to the lipid A component of bacterial endotoxin and facilitates its delivery to the CD14 antigen on the macrophage, where inflammatory cytokines are released and a cascade of host mediators is initiated. The neutrophil granular protein bactericidal/permeability-increasing protein (BPI) competes with LBP for endotoxin binding and functions as a molecular antagonist of LBP-endotoxin interactions. We have measured concentrations of both proteins in body fluids from 49 consecutive patients. In 16 of 17 samples of fluid from closed-space infections, BPI was present in greater concentration than LBP (median BPI/LBP ratio 7.6 [95% CI 2.32-22.1]). The ratio of BPI and LBP was not significantly different from 1.0 in abdominal fluid from 10 patients with peritonitis (ratio 0.235 [0.18-0.47]), whereas the BPI/LBP ratio was low in 22 non-infected body fluids (0.01 [0.001-0.04]) and concentrations of both proteins approached those in normal human plasma. BPI concentrations were directly correlated with the quantity of neutrophils within clinical samples (rs = 0.81, p < 0.0001). Thus, within abscess cavities BPI is available in sufficient quantities for effective competition with LBP for endotoxin. BPI may attenuate the local inflammatory response and the systemic toxicity of endotoxin release during gram-negative infections.

• Calvano SE et al.
• Changes in polymorphonuclear leukocyte surface and plasma bactericidal/permeability-increasing protein and plasma lipopolysaccharide binding protein during endotoxemia or sepsis.
• Arch Surg. 1994; 129: 220-6
• Display abstract

• OBJECTIVE: To evaluate changes in levels of polymorphonuclear leukocyte surface bactericidal/permeability-increasing protein (BPI), plasma BPI, and plasma lipopolysaccharide (LPS) binding protein (LBP) in normal human volunteers administered Escherichia coli LPS and in patients with sepsis and gram-negative infections. DESIGN: Survey; case series. SETTING: Clinical research center and surgical intensive care unit of a medical school and an associated tertiary care hospital. PATIENTS OR OTHER PARTICIPANTS: Volunteers (n = 10) screened prior to study by history and physical examination to exclude those with underlying diseases or hematologic abnormalities. Consecutive sample of surgical intensive care unit patients (n = 10) meeting criteria for sepsis syndrome with gram-negative infection. An additional patient with systemic inflammatory response syndrome but no gram-negative infection. All patients were studied on meeting the criteria. Three of the patients with sepsis syndrome and the patient with systemic inflammatory response syndrome were evaluated on recovery (approximately 25 days after initial study). Because these studies in volunteers and patients overlapped temporally, the control values were those of volunteers evaluated prior to LPS administration. No matching was employed. MEASUREMENTS AND RESULTS: Compared with controls, LPS-challenged volunteers and patients with sepsis both exhibited significant granulocytosis (P < .01) and increased concentrations of polymorphonuclear leukocyte surface BPI (P < .01) and of plasma LBP (P < .01). Plasma BPI concentrations were increased (P < .01) in volunteers following LPS administration. There was a trend toward increased concentrations of plasma BPI in patients, but this was not significant relative to controls. Maximum concentrations of plasma LBP were approximately 250- and 3000-fold higher than plasma BPI concentrations in endotoxemic volunteers and in patients, respectively. CONCLUSIONS: Circulating polymorphonuclear leukocytes increase expression of BPI in response to LPS or gram-negative sepsis. Subsequently, concentrations of plasma BPI and LBP increase. Because both LBP and BPI bind to LPS, it is suggested that endogenously derived plasma levels of BPI are likely to be inadequate to compete for LPS binding to the much more abundant LBP in the circulation.

• Wilde CG et al.
• Bactericidal/permeability-increasing protein and lipopolysaccharide (LPS)-binding protein. LPS binding properties and effects on LPS-mediated cell activation.
• J Biol Chem. 1994; 269: 17411-6
• Display abstract

• We have previously shown that human bactericidal/permeability-increasing protein (BPI) is able to inhibit serum-dependent lipopolysaccharide (LPS)-mediated activation of human monocytes and neutrophils in vitro, and to counteract the lethal effects of LPS challenge in vivo. Lipopolysaccharide-binding protein (LBP) is a serum protein which participates in LPS-mediated activation of cells (Tobias, P. S., Mathison, J., Mintz, D., Lee, J. D., Kravchenko, V., Kato, K., Pugin, J., and Ulevitch, R. J. (1992) Am. J. Respir. Cell. Mol. Biol. 7, 239-245). We have proposed that BPI functions in a negative feedback loop which opposes this activation (Marra, M. N., Wilde, C. G., Collins, M. S., Snable, J. L., Thornton, M. B., and Scott, R. W. (1992) J. Immunol. 148, 532-537). We have now cloned and expressed recombinant forms of human BPI and LBP. Here we demonstrate that purified recombinant human LBP can replace the serum requirement for both LPS binding to human monocytes and LPS-mediated secretion of tumor necrosis factor alpha from these cells. These activities of LBP are inhibited by a neutralizing anti-CD14 monoclonal antibody. We further demonstrate that purified recombinant human BPI can inhibit LBP-mediated LPS binding to cells and their subsequent activation. Comparison of the LPS binding properties of BPI and LBP in enzyme-linked immunosorbent type assays and in the Limulus amebocyte lysate assay suggest that BPI has a stronger affinity for LPS than does LBP. Direct competition between BPI and LBP for LPS may explain the inhibition by BPI of the proinflammatory effects of LBP in the presence of LPS.

• Marra MN et al.
• Regulation of the response to bacterial lipopolysaccharide by endogenous and exogenous lipopolysaccharide binding proteins.
• Blood Purif. 1993; 11: 134-40
• Display abstract

• Bactericidal/permeability-increasing protein (BPI) is a natural constituent of human neutrophils. Recombinant BPI has been shown to bind to bacterial lipopolysaccharide (LPS), and to neutralize the ability of LPS to stimulate inflammatory cells in vitro and in vivo. BPI shares sequence homology and immunocrossreactivity with another endogenous LPS binding protein, lipopolysaccharide binding protein (LBP). Despite the homology, these proteins have opposite effects on LPS. LBP mediates cell activation by low, otherwise nonstimulatory concentrations, while BPI neutralizes LPS bioactivity. Exogenous LPS binding proteins in the form of monoclonal antibodies have been developed with the goal of generating antiendotoxin therapeutics to treat gram-negative sepsis and related syndromes. Here we show that LPS-binding and neutralizing properties of BPI compare favorably with two monoclonal antibodies tested, HA-1A and XMMEN-OE5. BPI also competes effectively with LBP for LPS. Thus, BPI may represent an endogenous LPS-regulatory molecule suitable for use as a potent antiendotoxin therapeutic.

• Dentener MA, Von Asmuth EJ, Francot GJ, Marra MN, Buurman WA
• Antagonistic effects of lipopolysaccharide binding protein and bactericidal/permeability-increasing protein on lipopolysaccharide-induced cytokine release by mononuclear phagocytes. Competition for binding to lipopolysaccharide.
• J Immunol. 1993; 151: 4258-65
• Display abstract

• Serum proteins play an important role in LPS-induced cell activation. The LPS binding protein (LBP) enhances cellular responses to LPS, whereas the polymorphonuclear leukocyte product bactericidal/permeability-increasing protein (BPI) inhibits LPS-induced cell activation. In this study the influences of LBP and BPI, two proteins with opposite effects, but with considerable sequence homology, on LPS-induced mononuclear phagocytic cell cytokine release was studied. LBP was shown to enhance LPS-induced TNF-alpha, IL-6, and IL-8 release by mononuclear phagocytic cells, whereas BPI inhibited the release of these cytokines. Furthermore, the effects of LBP and BPI on LPS-induced cytokine release by mononuclear phagocytic cells were shown to be counteractive. BPI interfered with the enhancing effect of LBP on the LPS-induced cytokine release. At high LBP to BPI ratios, BPI could no longer inhibit LBP-induced enhancement. In accordance, increasing concentrations of BPI abrogated the LBP effect. Next, it was shown that LBP and BPI compete for binding to LPS by using an assay system that detects binding of free BPI to an anti-BPI mAb. LPS prevented binding of BPI to anti-BPI mAb, whereas preincubation of LPS with LBP prevented the LPS-induced inhibition. Also, it was observed that both BPI and LBP inhibited LPS activity in the chromogenic LAL assay. We conclude from this study that LBP and BPI have counteractive effects on LPS-induced mononuclear phagocytic cell cytokine release by competing for binding to LPS.

• Gray PW, Corcorran AE, Eddy RL Jr, Byers MG, Shows TB
• The genes for the lipopolysaccharide binding protein (LBP) and the bactericidal permeability increasing protein (BPI) are encoded in the same region of human chromosome 20.
• Genomics. 1993; 15: 188-90
• Display abstract

• The lipopolysaccharide binding protein is an acute-phase reactant produced during gram-negative bacterial infections. The bactericidal/permeability increasing protein is associated with human neutrophil granules and has bactericidal activity on gram-negative organisms. In addition to their functional relationship, both proteins share extensive structural similarity. This article demonstrates that the genes for both proteins are in the same region of human chromosome 20, between q11.23 and q12.

• Heumann D, Gallay P, Betz-Corradin S, Barras C, Baumgartner JD, Glauser MP
• Competition between bactericidal/permeability-increasing protein and lipopolysaccharide-binding protein for lipopolysaccharide binding to monocytes.
• J Infect Dis. 1993; 167: 1351-7
• Display abstract

• The bactericidal/permeability-increasing protein (BPI) inhibits the lipopolysaccharide (LPS)-mediated activation of monocytes. Due to its inhibitory activity for various LPS, BPI has therapeutic potential in endotoxic shock. To be efficient in vivo, BPI should overcome the action of LPS-binding protein (LBP), a serum molecule that increases the expression of LPS-inducible genes via CD14 of monocytes, rBPI23, a recombinant fragment of BPI, prevented in a dose-dependent manner the binding and the internalization of LPS mediated by LBP. Consequently, rBPI23 also inhibited LPS-induced tumor necrosis factor (TNF alpha) synthesis from monocytes. LPS- and LBP-mediated activation of monocytes was totally inhibited when LPS was preincubated with rBPI23. Adding rBPI23 at the same time as LBP resulted in an important but partial inhibition of TNF alpha release, but this inhibition vanished with delaying the time of addition of rBPI23. These studies suggest that the inhibitory activity of BPI is related to its ability to compete with LBP for LPS.

• Marra MN, Wilde CG, Griffith JE, Snable JL, Scott RW
• Bactericidal/permeability-increasing protein has endotoxin-neutralizing activity.
• J Immunol. 1990; 144: 662-6
• Display abstract

• Neutrophil granules contain proteins important in host defense against bacterial pathogens. Granule proteins released from activated neutrophils facilitate opsonization, phagocytosis, tissue digestion, and antimicrobial activity. Three similar, if not identical, neutrophil proteins, bactericidal/permeability-increasing protein (BPI), 57,000 m.w. cationic antimicrobial protein, and bactericidal protein have been described that specifically kill gram negative bacteria. Since LPS is a structure common to all gram-negative bacteria, we investigated whether the microbicidal protein BPI affects biologic activity of LPS in vitro. Human neutrophils can be activated both in vitro and in vivo by LPS. Upon stimulation, surface expression of CR1 and CR3 increases markedly. Using flow microfluorimetry, we analyzed surface expression of CR1 and CR3 as a measure of neutrophil stimulation in response to LPS. CR up-regulation on neutrophils was TNF independent, suggesting direct LPS stimulation of neutrophils in this system. Purified BPI completely inhibited CR up-regulation on neutrophils stimulated with both rough and smooth LPS chemotypes at 1.8 to 3.6 nM (100 to 200 ng/ml). By comparison, the polypeptide antibiotic polymyxin B completely inhibited the same dose of LPS at 0.4 nM. The inhibitory activity of BPI appeared to be specific for LPS because neutrophil stimulation by formylated peptide or TNF was unaffected. The specificity of BPI for LPS was further demonstrated by inhibition of LPS activity in the limulus amebocyte lysate assay. Therefore, the role of BPI in infection may not be limited to its microbicidal activity, but it may also regulate the neutrophil response to LPS.

• Tobias PS, Mathison JC, Ulevitch RJ
• A family of lipopolysaccharide binding proteins involved in responses to gram-negative sepsis.
• J Biol Chem. 1988; 263: 13479-81
• Display abstract

• The lipopolysaccharides (LPS) of Gram-negative bacteria initiate potentially fatal processes in many host organisms. Recently published amino acid sequence data suggest that there is a family of LPS binding proteins that may participate in the host response to Gram-negative bacteremia. The first two members of the family to be identified are an LPS binding protein present in serum after an acute phase response in humans, mice, rabbits, and rats and a bactericidal/permeability increasing protein present in the primary granules of human and rabbit neutrophils. LPS binding protein and bactericidal/permeability increasing protein share an ability to bind to LPS, have homologous NH2-terminal amino acid sequences, and are immunologically cross-reactive. Nevertheless, these two molecules differ in their effects on LPS and Gram-negative bacteria, in their sites of biosynthesis, and localization in vivo.

© 2021 EMBL | Privacy Policy