Practical 3.. Analysis of N-linked glycans from the nat

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Practical 3.Analysis ofN-linked glycans from the native lactoferrin glycoproteinisolated from human and bovine milkGeneral BackgroundMany recombinant glycoproteins, including erythropoietin and tissue plasminogen activator(tPA), have been developed as therapeutics that are designed to mimic the native humanprotein. The oligosaccharides (glycans) attached to glycoproteins have been shown tovariously affect their physicochemical properties, thermal stability, activity, interaction withreceptors, immunogenicity and circulating half-life(Li and d'Anjou 2009). For the efficientand safe use of glycoprotein products as biopharmaceutical drugs, and for the monitoring oftheir production, it is necessary to determine the heterogeneous structures of the carbohydratemoieties on the glycoprotein. Since the oligosaccharide structures varies between species andin different expression systems, the change is often markedly different from the native humanstructures(Demain and Vaishnav 2009).In this practical we are using lactoferrin as a model glycoprotein to compare the glycosylationbetween two species; released oligosaccharides from the native protein isolated from humanand bovine milk will be compared(Legrand, Pierceet al. 2008).Lactoferrin is an iron-binding glycoprotein expressed in most biological fluids and because ofits iron binding capacity is a major component of the mammalian innate immune system(Legrand and Mazurier 2010).Lactoferrin is found in mucosal secretions, including tears,saliva, bile, urine and most highly in milk and colostrum (7 g/L) making it the second mostabundant protein in human milk. The protein consists of a single polypeptide chaincomprising 673 amino acid residues that form two homologous domains, the N- and C-lobes.Lactoferrin contains two potentialN-linked glycosylation sites; however, the degree ofglycosylation varies and as a result, the molecular mass of the protein varies from 76 to 80kDa(Legrand, Pierceet al. 2008).1
Fig. 1. Three-dimesional structure of biferric bovine lactoferrin. Source: Lactoferrin:structure, function and applications. International Journal of Antimicrobial Agents, 33:301(Gonzalez-Chavez, Arevalo-Gallegoset al. 2009).The aim of this experiment is to compare the protein glycosylation of native lactoferrinisolated from human and bovine milk. This will be done by analysing theN-linkedoligosaccharides released enzymatically from the glycoproteins.The oligosaccharides will be released in solution from the glycoproteins by the enzymeN-Glycosidase F (PNGase F) that releases allN-linked glycans from mammalian glycoproteins.The released glycans from the two samples will be analysed by capillary carbon liquidchromatography (LC) interfaced with an Electrospray Ionisation (ESI) mass spectrometer(Zaia 2010).References:Demain, A. L. and P. Vaishnav (2009). Production of recombinant proteins by microbes andhigher organisms. Biotechnol Adv 27: 297-306.Gonzalez-Chavez, S. A., S. Arevalo-Gallegos, Q. Rascon-Cruz. (2009). Lactoferrin:structure, function and applications. Int J Antimicrob Agents 33: 301 e301-308.Legrand, D. and J. Mazurier (2010). A critical review of the roles of host lactoferrin inimmunity. Biometals 23: 365-376.Legrand, D., A. Pierce, E. Elass, M Carpentier, C. Mariller, J Mazurier. (2008). Lactoferrinstructure and functions. Adv Exp Med Biol 606: 163-194.Li, H. and M. d'Anjou (2009). Pharmacological significance of glycosylation in therapeuticproteins. Curr Opin Biotechnol 20: 678-684.Zaia, J. (2010). Mass spectrometry and glycomics. OMICS 14: 401-418.2bound ironglycans
You will need per pair:50 μg lactoferrin purified from human and bovine milk samples (froma 5 mg/mL solution)0.1 M DTT (Dithiothreitol)0.5 M IodoacetamideMade Freshly2 Zip TipC18from Merck Millipore LtdRapid PNGase F(2 x 4μLRapid PNGaseF buffer + enzyme solution,#P0711S, New England Biolabs)For the class:100 % MethanolGlacial acetic acid1 M sodium borohydride (NaBH4in 50mM Potassium hydroxide(KOH)Made FreshlyCation exchange resin-Dowex AG 50W X81 M Hydrocholoric acid1 M Ammonium acetate (CH3COON H4),pH 5Eppendorf tubes (1.5ml)Glass Autosampler Vials (Waters)50°C heat blockRefrigerated vacuum concentrator3
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