Properties of a Heparin-binding Peptide Derived from Bovine Lactoferrin

Abstract

A heparin-binding peptide was isolated from a proteolytic hydrolysate of bovine lactoferrin by affinity chromatography using an immobilized heparin column. Analysis of amino acid sequences at the N-terminus showed that this heparin-binding peptide is derived from the region beginning at the 17th amino acid residue of the bovine lactoferrin sequence. The molecular mass of this peptide was 3195.5 as measured by matrix-assisted laser desorption-time of flight mass spectrometry. This peptide is the same as the bactericidal peptide lactoferricin B. In an aqueous environment, this peptide displays mainly a beta-sheet structure and an unordered structure as assessed by measurements of circular dichroism spectra. When this peptide was mixed with heparin, a distinct spectral change was induced because of conformational alteration of the peptide. This spectral change was reversible. Analysis of data from peptide synthesis indicated that binding by the sequence Arg28-Met29-Lys30-Lys31 of bovine lactoferrin is significant and that there is a synergistic contribution from Lys18-Cys19-Arg20-Arg21, and Arg38-Arg39.

Bovine Lactoferricin Inhibits Basic Fibroblast Growth Factor- and Vascular Endothelial Growth Factor165-Induced Angiogenesis by Competing for Heparin-Like Binding Sites on Endothelial Cells

“…We also observed that LfcinB bound to immobilized heparin, consistent with earlier published findings. 30 However, LfcinB did not bind to immobilized bFGF or VEGF 165 , suggesting that a direct interaction between LfcinB and bFGF or VEGF 165 was not responsible for the antiangiogenic effect of LfcinB. Importantly, LfcinB binding to heparin was reduced in the presence of bFGF or VEGF 165 , suggesting that LfcinB, bFGF, and VEGF 165 might all interact with the same heparin-like binding sites on cell-surface heparan sulfate proteoglycans.…”

“…44 LfcinB did not exhibit any cytotoxic activity against HUVECs, excluding the possibility that LfcinB simply caused HUVECs to undergo apoptosis, as occurs when various cancer cell lines are exposed to LfcinB. 24,27 Because both bovine lactoferrin and LfcinB are known to bind heparin, 29,30 our findings led us to hypothesize that LfcinB competed with bFGF and VEGF 165 for heparin-like binding sites on heparan sulfate proteoglycans on the surface of HUVECs. Heparan sulfate proteoglycans are required for bFGF and VEGF 165 binding and signaling through their respective cell-surface receptors.…”

“…29,30 Figure 5 demonstrates that , bFGF alone (1 g/ml), VEGF 165 alone (5 g/ml), nonheparin-binding EGF alone (2 g/ml), or LfcinB (200 g/ml) in combination with bFGF (1 g/ml), VEGF 165 (5 g/ml), or nonheparin-binding EGF (2 g/ml) was implanted in mice by subcutaneous injection. After 6 days, mice were sacrificed, and Matrigel plugs were surgically excised, sectioned, and stained with H&E. a: Representative sections of Matrigel plugs containing bFGF or VEGF 165 alone or in combination with LfcinB.…”

“…Endothelial cell proliferation and migration induced by proangiogenic factors are crucial steps in the development of tumor vasculature. 5 Because LfcinB is derived from bovine lactoferrin, and both molecules exhibit heparin-binding activity, 29,30 we also determined the ability of LfcinB to bind heparinlike molecules that are involved in bFGF and VEGF 165 interactions with their respective receptors. 15,16 …”

Inhibitory Effect of Bovine Milk Lactoferrin on the Interaction between a Streptococcal Surface Protein Antigen and Human Salivary Agglutinin

“…The lactoferrin molecule is proposed to consist of two lobes (N-lobe and C-lobe) (40). The N-lobe contains the active domains for bactericidal action and heparin binding (31,41), whereas the C-lobe contains a functional domain for hepatocyte binding and internalization (42). In these previous studies, lactoferrin fragments were prepared by tryptic cleavage of lactoferrin and isolated by high performance liquid chromatography.…”