N-terminal stretch Arg2, Arg3, Arg4 and Arg5 of human lactoferrin is essential for binding to heparin, bacterial lipopolysaccharide, human lysozyme and DNA

Berkel, Patrick H.C. van; Geerts, M.E.J.; Veen, Harrie A. van; Mericskay, Mathias; Boer, H. H.; Nuijens, Jan H.

doi:10.1042/bj3280145

Cited by 184 publications

(132 citation statements)

References 34 publications

(51 reference statements)

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“…Like nAzu, lactoferrin, an antimicrobial protein found in the secondary granules of human neutrophils, binds several ligands including heparin and LPS (31). The N-terminal region of hLF contains two basic clusters that act synergistically in the binding of the protein to heparin (26) as well as an antimicrobial domain (32)(33)(34).…”

Section: Discussionmentioning

confidence: 99%

Basic Residues in Azurocidin/HBP Contribute to Both Heparin Binding and Antimicrobial Activity

McCabe

Cukierman

Gabay

2002

Journal of Biological Chemistry

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Azurocidin/CAP37/HBP is an antimicrobial and chemotactic protein that is part of the innate defenses of human neutrophils. In addition, azurocidin is an inactive serine protease homolog with binding sites for diverse ligands including heparin and the bovine pancreatic trypsin inhibitor (BPTI). The structure of the protein reveals a highly cationic domain concentrated on one side of the molecule and responsible for its strong polarity. To investigate the role of this highly basic region, we produced three recombinant azurocidin mutant proteins that were altered in either one or both of two clusters of 4 basic residues located symmetrically on each side of a central cleft in the cationic domain. Two of the mutant proteins (Loop 3: R5Q, K6Q, R8Q, and R10Q; Loop 4: R61Q, R62Q, R63Q, and R65Q) exhibited little or no change in heparin and BPTI binding or in antimicrobial function. In contrast, the Loop 3/Loop 4 mutant (R5Q, K6Q, R8Q, R10Q, R61Q, R62Q, R63Q, and R65Q) in which all 8 basic residues were replaced showed greatly decreased ability to bind heparin and to kill Escherichia coli and Candida albicans. Thus, we report that the 8 basic residues that were altered in the Loop 3/Loop 4 mutant contribute to the ability of the wild-type azurocidin molecule to bind heparin and to kill E. coli and C. albicans. Because BPTI binding was comparable in wild-type and Loop 3/Loop 4 mutant protein, we conclude that the same 8 basic residues are not involved in the binding of BPTI to azurocidin, supporting the notion that the binding site for BPTI is distinct from the site involved in heparin binding and antimicrobial activity. Finally, we show that removal of all 4 positively charged amino acids in the 20 -44 azurocidin sequence (DMC1: R23Q,H24S,H32S,R34Q), a region previously thought to contain an antimicrobial domain, does not affect the activity of the protein against E. coli, Streptococcus faecalis, and C. albicans.Azurocidin/CAP37/HBP is an antimicrobial and chemotactic protein (1-7) that forms part of the innate defenses of human neutrophils (8 -11). It has also been reported to play a role in neutrophil-induced increase in vascular permeability (12). The mechanism for these functions is largely unknown. Structurally, azurocidin belongs to the serprocidin family, a group of serine proteases with antimicrobial function but lacking enzymatic activity (13-16). Its three-dimensional structure has been elucidated and shows a highly cationic region made of 16 basic amino acid residues and located at one pole of the molecule (17,18). In addition to its high density of positive charges, the cationic region also contains potential heparin binding consensus sequences that are located in loop areas of azurocidin tertiary structure (4, 17).Several ligands have been shown to bind to azurocidin, including heparin and the serine protease inhibitor BPTI 1 (4,19). In this report, we show that heparin and BPTI bind to distinct regions of the molecule and that these two ligands differentially affect antimicrobial activity. Indeed, binding ...

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Section: Discussionmentioning

confidence: 99%

Basic Residues in Azurocidin/HBP Contribute to Both Heparin Binding and Antimicrobial Activity

McCabe

Cukierman

Gabay

2002

Journal of Biological Chemistry

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“…Each lobe has two domains (N1 and N2, C1 and C2) and can bind a single ferric ion concomitantly with one bicarbonate ion very tightly [2]. There are striking conservation between these two lobes in respect of their iron retention ability (C-lobe bind iron more tightly) [3] and biological functions (some functions of Lf are thought to be involved in the N-lobe) [4,5]. N-and C-lobes also possess unique binding regions for microbial membranes [6].…”

Section: Introductionmentioning

confidence: 99%

Bovine lactoferrin region responsible for binding to bifidobacterial cell surface proteins

et al. 2009

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Bovine lactoferrin (bLf) is a multifunctional iron-binding glycoprotein secreted mainly in milk and other secretory fluids. Bovine lactoferrin is reported to promote the growth of bifidobacteria and binding of bLf to bifidobacteria cell is thought to be involved. After separation of bLf half molecule and extraction of surface proteins from bifidobacteria, binding profiles were observed by immunoblotting. No binding was appeared when bLf C-lobe was being reacted with cell surface proteins on PVDF membrane. Conversely, a 50-kDa band was appeared when reacted with either intact bLf or nicked bLf. This result strongly suggests that binding region could be N-lobe.Moreover, blot, probed with nicked bLf, reacted with anti-lactoferricin antibody also produced a 50-kDa band that indicates the binding occurred at lactoferricin region of bLf molecule. Interestingly, despite absence of binding, bLf C-lobe can stimulate the growth of bifidobacteria.

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“…The anti-bacterial activity of LF originates from the Nterminal Arg-rich region in hLF (Arg 2 , Arg 3 , Arg 4 , Arg 5 , and Arg 28 -31 ), and this region is also involved in binding heparin, LPS, and lysozyme (2). We examined the anti-bacterial activity of hLF using E. coli BL21 in the presence of the LFpep-1 peptide (1-500 M).…”

Section: Discussionmentioning

confidence: 99%

“…The antibacterial activity of LF is due to its N-terminal Arg-rich region, which binds to lipopolysaccharides (LPS) on Gram-negative bacteria, and this region is also responsible for binding lysozyme, heparin, and DNA (2,3). With respect to its antiviral activity, LF acts as an inhibitor for viral entry to target cells but does not repress the intracellular replication of viruses (4).…”

Section: Lactoferrin (Lf)mentioning

confidence: 99%

Interaction of Human Lactoferrin with Cell Adhesion Molecules through RGD Motif Elucidated by Lactoferrin-binding Epitopes

Sakamoto

Ito

Mori

et al. 2006

Journal of Biological Chemistry

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Lactoferrin (LF) is an iron-binding secretory protein, which is distributed in the secondary granules of polynuclear lymphocytes as well as in the milk produced by female mammals. Although it has multiple functions, for example antimicrobial, immunomodulatory, antiviral, and anti-tumor metastasis activities, the receptors responsible for these activities are not fully understood. In this study, the binding epitopes for human LF were first isolated from a hexameric random peptide library displayed on T7 phage. Interestingly, two of the four isolated peptides had a representative cell adhesion motif, Arg-Gly-Asp (RGD), implying that human LF interacts with proteins with the RGD motif. We found that human LF bound to the RGD-containing human extracellular matrix proteins, fibronectin and vitronectin. Furthermore, human LF inhibited cell adhesion to these matrix proteins in a concentration-dependent manner but not to the RGD-independent cell adhesion molecule like laminin or collagen. These results indicate that a function of human LF is to block the various interactions between the cell surface and adhesion molecules. This may explain the multifunctionality of LF.

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N-terminal stretch Arg2, Arg3, Arg4 and Arg5 of human lactoferrin is essential for binding to heparin, bacterial lipopolysaccharide, human lysozyme and DNA

Cited by 184 publications

References 34 publications

Basic Residues in Azurocidin/HBP Contribute to Both Heparin Binding and Antimicrobial Activity

Basic Residues in Azurocidin/HBP Contribute to Both Heparin Binding and Antimicrobial Activity

Bovine lactoferrin region responsible for binding to bifidobacterial cell surface proteins

Interaction of Human Lactoferrin with Cell Adhesion Molecules through RGD Motif Elucidated by Lactoferrin-binding Epitopes

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