Characterization of b-lactoglobulin from buffalo (Bubalus bubalis) colostrum and its possible interaction with erythrocyte lipocalin-interacting membrane receptor Received February 18, 2011; accepted April 1, 2011; published online May 4, 2011 Rohit A. Chougule and Huligerepura S. Aparna* Department of Studies in Biotechnology, University of Mysore, Manasagangotri, Mysore 570 006, Karnataka, India *Dr H.S. Aparna, Department of Studies in Biotechnology, University of Mysore, Mysore- 570 006, Karnataka, India. Tel: 91-821-2419883, Fax: 91-821-2419363, email: hsa.uom@gmail.com Lipocalins form a widespread class of proteins involved in the transport of weakly soluble vitamins, hormones or hydrophobic molecules. b-lactoglobulin (BLG-col), a major lipocalin present in whey was purified and char- acterized from buffalo colostrum. The molecular weight of BLG-col as determined by Liquid chromatography electrospray ionization mass spectrometry (LC-ESI- MS) was 18.257 kDa and the peptide mass fingerprint of the purified protein revealed 67% sequence hom- ology to buffalo milk b-lg. The N-terminal-IIVTQ and LCESI-collision-induced dissociationElectron transfer dissociation mass spectrometry/mass spectro- metry analyses of doubly (m/z 1156 þ2 ) and triply (m/z 546 þ3 ) charged ion pairs corresponding to VYVEELKPTPEGDLEILLQK (4160) and TPEV DDEALEKFDK (125138) sequences confirmed the identity of BLG-col. Using these peptide sequences, the location of a gene encoding for BLG-col was iden- tified on chromosome 11 at 11q28 loci of bovine genome. The unique property of the BLG-col isolated from buffalo colostrum was its strong and specific hae- magglutinating activity with ‘O’ blood of human erythrocytes with 10,309 HAU/mg protein. The cell surface localization of BLG-col on human erythrocytes was confirmed by immunocytochemistry and the speci- ficity of interaction was established by immunoblot ana- lysis of human erythrocyte membrane proteins. Based on these observations, we suggest the presence of lipo- calin receptor (70 kDa) on human erythrocyte mem- brane and the multiple sequence alignment supported structural diversity among lipocalin receptors. Keywords: Bubalus bubalis/colostrum/b-lactoglobulin/ immunoblot/lipocalin/mass spectrometry. Abbreviations: DHB, 2, 5-Dihydroxybenzoic acid; FITC, fluorescein isothiocyanate; PMF, peptide mass fingerprint; PVDF, polyvinylidene fluoride; LCESI-CID/ETD MS/MS, Liquid ChromatographyElectro Spray IonizationCollision Induced Dissociation/Electron Transfer Dissociation Mass Spectrometry/Mass Spectrometry. Colostrum, nature’s specially designed food for neonates, is a complex biological fluid containing glycoconjugates, growth and tissue repair factors, phagocytes and array of bacteriostatic and antiviral defence factors along with normal components of mature milk (1). The immune-boosting property of colostrum stems from various bioactive molecules in conjunction with cellu- lar components protects not only the maternal mam- mary gland but also the recipient infant from various infections. The macromolecules are often resistant to enzymatic digestion in the gastrointestinal tract and inhibit the localized adherence of enteropathogens to the digestive tract of neonates (2). Thus, glycoconju- gates and bioactive peptides isolated from bovine col- ostrum are extensively used as neutraceuticals in infant food and formula (3). On a global scale, buffalo is a major milking mam- mal of Asia. Buffalo milk has almost 11.42% more pro- tein content compared with bovine milk (4), and the protein content is even higher in the colostrum (5). During our previous investigations on buffalo col- ostrum glycoconjugates, we have identified antigenic glycopeptides (6) and macrophage activating oligosac- charides (7) similar to those present in bovine and human colostrum. Further, presence of bifidogenic glycoproteins in colostral whey (8) in analogy to human colostrum whey proteins demonstrated the immunoprotective role of the colostrum glycoconju- gates. Hence, both buffalo milk and colostrum can be used as alternatives to bovine milk products for the isolation of probiotics. The colostrum whey proteins comprise 35% of the total milk proteins and are rich in b-lactoglobulin, a-lactalbumin, glycomacropeptides, albumin, proteose peptone-3 and immunoglobulins and their immunore- gulatory activities have been well documented by both in vivo and in vitro studies (9, 10). b-lactoglobulin is the most abundant whey protein, which belongs to lipocalin family of proteins (11). Lipocalins are a large group of extracellular proteins that show great diversity in their sequences and mo- lecular recognition properties. The members of the lipocalin family of proteins play pivotal roles in the transport of small hydrophobic molecules, colour- ation, olfaction, prostaglandin synthesis, cell homoeo- stasis and immunomodulation (12). Recent evidences suggest the functional advantage of b-lg in the trans- port of vitamin D and indicated that supplementation of milk with vitamin D effectively enhanced its uptake (13). The peptides derived from b-lactoglobulin have attracted considerable attention due to their J. Biochem. 2011;150(3):279–288 doi:10.1093/jb/mvr054 ß The Authors 2011. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved 279 Downloaded from https://academic.oup.com/jb/article-abstract/150/3/279/865923 by University of Mysore user on 08 August 2019