title: Buffalo colostrum β-lactoglobulin inhibits VEGF-induced angiogenesis by interacting with G protein-coupled receptor kinase.
abstract: β-lactoglobulin (β-lg), a major whey protein was purified and characterised from buffalo colostrum. The in silico analysis of the tryptic peptides based on LC-CID-MS/MS facilitated the identification of protein as β-lg. The sequences IIVTQ f[1-5] and LSFNPTQLEEQCHV f(149-162) of m/z 933(+) and 851(2+) were found to match N- and C-extreme of β-lg while IDALNENK f(84-91) and TPEVDDEALEKFDK f(125-138) sequences deduced for m/z 916(+) and 818(2+) were in compliance to buffalo milk β-lg. Considering the sequence similarity of β-lg to glycodelin, a proven angiogenic protein, similar role for β-lg from buffalo colostrum (BLG-col) was examined. Interestingly, BLG-col exhibited anti-angiogenic activity by potently inhibiting cell proliferation, micro-vessel sprouting, cell migration and tube formation of human umbilical vein endothelial cells (HUVECs) in a dose-dependent manner but having varied effect on Ehrlich ascites tumor cells, MCF-7, MDA-MB 435 and MDA-MB 231 cell lines. The anti-angiogenic potential of BLG-col was found to be vascular endothelial growth factor mediated. The immunolocalisation of BLG-col on the cell surface of HUVECs evidenced using FITC-labelled β-lg antibody indicated its extra-cellular binding. Furthermore, BLG-col interacting HUVEC membrane protein (64 kDa) was detected by immunoblot and its identity was established by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry analysis, which showed peptide sequence homology to G protein-coupled receptor kinase 4.
authors.affiliation: Department of Studies in Biotechnology, Manasagangotri, University of Mysore, Mysore 570 006, India.
authors.forename: Rohit A
authors.initials: RA
authors.lastname: Chougule
authors.forename: Shilpa
authors.initials: S
authors.lastname: P
authors.forename: Bharathi P
authors.initials: BP
authors.lastname: Salimath
authors.forename: Aparna H
authors.initials: AH
authors.lastname: Sosalegowda
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002020:Buffaloes; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D002465:Cell Movement / Q000187:drug effects; D049109:Cell Proliferation / Q000187:drug effects; D003126:Colostrum / Q000737:chemistry*; D054771:G-Protein-Coupled Receptor Kinase 4 / Q000378:metabolism*; D061307:Human Umbilical Vein Endothelial Cells / Q000166:cytology / Q000187:drug effects; D006801:Humans; D007782:Lactoglobulins / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology*; D008297:Male; D018919:Neovascularization, Physiologic / Q000187:drug effects*; D011485:Protein Binding; D040901:Proteomics; D051381:Rats; D042461:Vascular Endothelial Growth Factor A / Q000494:pharmacology*
chemical_list: D007782:Lactoglobulins; D042461:Vascular Endothelial Growth Factor A; D054771:G-Protein-Coupled Receptor Kinase 4