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LF f(21-31) modified [id=LFH0048]

Synonym: HLP6

Producer Organism : Native Protein : Production Method :
Human Lactoferrin (LF) Synthetic
Activity : Antibacterial
Target Organisms :

Gram-positive: Staphylococcus aureus NCTC 10571 (MIC=250 然).

Gram-negative: Acinetobacter sp. (MIC=10 然), Escherichia coli NCTC 10418 (MIC=750 然).

NOTE: No activity against Escherichia coli O111 NCTC 8007, Enterobacter aerogenes, Klebsiella sp. strain 3105, Providencia stuartii, Proteus mirabilis (>1000 然) .

Description :
Production method: Synthetic.

Seventh residue replaced with proline.
The difference between the activities of LFH0054 and LFH0045 against S. aureus could be explained by differences in peptide size, allowing uptake of HLP 2 rather than HLP 1, but it is more likely to be due to differences in flexibility and conformation if the peptide binds to the peptidoglycan. This would also explain why LFH0048 still has activity against S. aureus (Citation: 1).
In the presence of LPS, LFH0045 and LFH0048 were found to bind and adopt a beta-strand conformation rather than an alpha-helix. Furthermore, there is a time-dependent association of peptide that results in an ordered formation of peptide aggregates. The rate of interpeptide association was far greater in LFH0045-LPS than in LFH0048-LPS, which was consistent with the lag phase observed on the killing curves. These results allow us to propose a mechanism by which LFH0045 folds and self-assembles at the outer membrane surface before exerting its activity (Citation: 2).
Length : 11 Mass (Da): 1 515.38 Common Amino Acids : R
Isolectric Point : 12.81 Net Charge : 4 Absent Amino Acids : ACDEGHILMSTY
Basic Residues : 4 Acidic Residues : 0 Hydrophobic Residues : 3
Polar Residues : 1 Boman Index : -58.68 Hydropathy Index : -2.127
Aliphatic Index : 26.36 Instability Index : 0 Extinction Coefficient : 5500
Absorbance 280nm : 550

Wheel representation

Hydrophobicity plot

Red solid plot : values according to the hydrophobicity scale of Kyte and Doolittle (reference paper).
Yellow dashed plot : Experimentally determined hydrophobicity scale for proteins at membrane interfaces(reference paper).
Green dotted-dashed plot : prediction of transmembrane helices (reference paper). In this scale (unlike the others), more negative values reflect greater hydrophobicity.

Multiple Sequence Alignment (MSA)

 1 LFH0010  100.0%  GRRRRSVQWCAVSQPEATKCFQWQRNMRKVRGPPVSCIKRDSPIQCIQA 
 2 LFH0016  100.0%  -----------------TKCFQWQRN----------------------- 
 3 LFH0020   88.9%  -----------------TKCFQWQGN----------------------- 
 4 LFH0017   88.9%  -----------------TKCGQWQRN----------------------- 
 5 LFH0018   77.8%  -----------------TKCFGWGRN----------------------- 
 6 LFH0019   88.9%  -----------------TGCFQWQRN----------------------- 
 7 LFH0011  100.0%  -------------QPEATKCFQWQRNMRKVR------------------ 
 8 LFH0012  100.0%  --------------PEATKCFQWQRNMRKVR------------------ 
 9 LFH0013  100.0%  ---------------EATKCFQWQRNMRKVR------------------ 
10 LFH0014  100.0%  ----------------ATKCFQWQRNMRKVR------------------ 
11 LFH0041   91.7%  -------------------CFQWQRNMRKVA------------------ 
12 LFH0043  100.0%  --------------------FQWQRNMRK-------------------- 
13 LFH0040   91.7%  -------------------CFQWQRNMRKAR------------------ 
14 LFH0015  100.0%  -----------------TKCFQWQRNMRKVR------------------ 
15 LFH0038   91.7%  -------------------CFQWQRNMAKVR------------------ 
16 LFH0039   91.7%  -------------------CFQWQRNMRAVR------------------ 
17 LFH0021  100.0%  -----------------TKCFQWQRNMRKVR------------------ 
18 LFH0027  100.0%  ------------------KCFQWQRNMRKVR------------------ 
19 LFH0030   91.7%  -------------------AFQWQRNMRKVR------------------ 
20 LFH0044  100.0%  --------------------FQWQRNMRKV------------------- 
21 LFH0049  100.0%  --------------------FQWQRNMRKVR------------------ 
22 LFH0045  100.0%  --------------------FQWQRNMRKVR------------------ 
23 LFH0031   91.7%  -------------------CAQWQRNMRKVR------------------ 
24 LFH0055  100.0%  ---------------------QWQRNMRKVR------------------ 
25 LFH0022  100.0%  -----------------TKCFQWQRNMRKVRG----------------- 
26 LFH0029  100.0%  -------------------CFQWQRNMRKVR------------------ 
27 LFH0033   91.7%  -------------------CFQAQRNMRKVR------------------ 
28 LFH0056  100.0%  ------------------------RNMRKVR------------------ 
29 LFH0032   91.7%  -------------------CFAWQRNMRKVR------------------ 
30 LFH0034   91.7%  -------------------CFQWARNMRKVR------------------ 
31 LFH0023   93.3%  -----------------TKCFQWQWNMRKVRG----------------- 
32 LFH0057  100.0%  ---------------------------RKVR------------------ 
33 LFH0035   91.7%  -------------------CFQWQANMRKVR------------------ 
34 LFH0036   91.7%  -------------------CFQWQRAMRKVR------------------ 
35 LFH0046   90.9%  --------------------FQWQRNIRKVR------------------ 
36 LFH0047   90.9%  --------------------FQWQRNIRKVR------------------ 
37 LFH0048   90.9%  --------------------FQWQRNPRKVR------------------ 
38 LFH0037   91.7%  -------------------CFQWQRNARKVR------------------ 
39 LFH0024  100.0%  -----------------TKCFQWQRNMRKVRGPPVSCIKR--------- 
40 LFH0026  100.0%  -----------------TKCFQWQRNMRKVRGPPVSCIKRDS------- 
41 LFH0025  100.0%  -----------------TKCFQWQRNMRKVRGPPVSCIKRDS------- 
42 LFH0042  100.0%  -------------------CFQWQRNMRKVRGPPVSCI----------- 
43 LFH0054  100.0%  --------------------FQWQRNMRKVRGPPVS------------- 
44 LFH0028  100.0%  ------------------KCFQWQRNMRKVRGPPVSCI----------- 
45 LFH0007  100.0%  GRRRRSVQWCAVSQPEATKCFQWQRNMRKVRGPPVSCIKRDSPIQCI-- 
46 LFH0058  100.0%  ---------------------------RKVRGPPVSCIKRDSP------ 
47 LFH0059  100.0%  ------------------------------------CIKRDSP------ 
48 LFH0009A 100.0%  GRRRRSVQWCA-------------------------------------- 
49 LFH0009B 100.0%  -----------VSQPEATKCFQWQRNMRKVRGPPVSCIKRDSPIQCI-- 
50 LFH0005  100.0%  GRRRRSVQWCAVSQPEATKCFQWQRNMRKVRGPPVSCIKRDSPIQCI-- 
51 LFH0004  100.0%  GRRRRSVQWCA-------------------------------------- 
52 LFH0003  100.0%  GRRRRSVQW---------------------------------------- 
53 LFH0002  100.0%  GRRRRS------------------------------------------- 
54 LFH0006   97.9%  GRRRRSVQWCAVSQPEATKCFQWQRNMRRVRGPPVSCIKRDSPIQCI-- 

Citation: 1

Structure-function relationship of antibacterial synthetic peptides homologous to a helical surface region on human lactoferrin against Escherichia coli Serotype O111

Cited Entries: LFH0045, LFH0048, LFH0049, LFH0054

Authors:Chapple, D.S., Mason, D.J., Joannou, C.L., Odell, E.W., Gant, V., Evans, R.W.
Journal: Infection and Immunity 1998, 66(6).
Abstract: Lactoferricin includes an 11-amino-acid amphipathic alpha-helical region which is exhibited on the outer surface of the amino-terminal lobe of lactoferrin. Synthetic peptides homologous to this region exhibited potent antibacterial activity against a selected range of both gram-negative and gram-positive bacteria. An analog synthesized with methionine substituted for proline at position 26, which is predicted to disrupt the helical region, abolished antibacterial activity against Escherichia coli and considerably reduced antibacterial activity against Staphylococcus aureus and an Acinetobacter strain. The mode of action of human lactoferrin peptide (HLP) 2 against E. coli serotype O111 (NCTC 8007) was established by using flow cytometry, surface plasmon resonance, and transmission electron microscopy. Flow cytometry was used to monitor membrane potential, membrane integrity, and metabolic processes by using the fluorescent probes bis-1,3-(dibutylbarbituric acid)-trimethine oxonol, propidium iodide, and carbonyl cyanide m-chlorophenylhydrazone, respectively. HLP 2 was found to act at the cell membrane, causing complete loss of membrane potential after 10 min and of membrane integrity within 30 min, with irreversible damage to the cell as shown by rapid loss of viability. The number of particles, measured by light scatter on the flow cytometer, dropped significantly, showing that bacterial lysis resulted. The peptide was shown to bind to E. coli O111 lipopolysaccharide by using surface plasmon resonance. Transmission electron microscopy revealed bacterial distortion, with the outer membrane becoming detached from the inner cytoplasmic membrane. We conclude that HLP 2 causes membrane disruption of the outer membrane, resulting in lysis, and that structural considerations are important for antibacterial activity.
Citation: 2

Structure and association of human lactoferrin peptides with Escherichia coli lipopolysaccharide

Cited Entries: LFH0045, LFH0048

Authors:Chapple, D.S., Hussain, R., Joannou, C.L., Hancock, R.E.W., Odell, E., Evans, R.W., Siligardi, G.
Journal: Antimicrobial Agents and Chemotherapy 2004, 48(6).
Abstract: An 11-amino-acid amphipathic synthetic peptide homologous to a helical region on helix 1 of human lactoferrin HLP-2 exhibited bactericidal activity against Escherichia coli serotype O111, whereas an analogue synthesized with Pro substituted for Met, HLP-6, had greatly reduced antimicrobial activity. The bactericidal activity of HLP-2 was 10-fold greater than that of HLP-6 in both buffer and growth medium by time-kill assays. These assays also showed a pronounced lag phase that was both concentration and time dependent and that was far greater for HLP-2 than for HLP-6. Both peptides, however, were shown to be equally efficient in destabilizing the outer membrane when the hydrophobic probe 1-N-phenylnaphthylamine was used and to have the same lipopolysaccharide (LPS) binding affinity, as shown by polymyxin B displacement. Circular dichroism (CD) spectroscopy was used to study the structure and the organization of the peptides in solution and upon interaction with E. coli LPS. In the presence of LPS, HLP-2 and HLP-6 were found to bind and adopt a {beta}-strand conformation rather than an {alpha}-helix, as shown by nonimmobilized ligand interaction assay-CD spectroscopy. Furthermore, this assay was used to show that there is a time-dependent association of peptide that results in an ordered formation of peptide aggregates. The rate of interpeptide association was far greater in HLP-2 LPS than in HLP-6 LPS, which was consistent with the lag phase observed on the killing curves. These results allow us to propose a mechanism by which HLP-2 folds and self-assembles at the outer membrane surface before exerting its activity.

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