ABSTRACT
Generation of defined glycoconjugates is necessary for the study of glycoprotein function, as well as the development of therapeutics. The biosynthesis of glycoproteins produces multiple glycoforms, proteins which differ only in the structure of the attached glycan. This inherent heterogeneity complicates the study of isolated glycans and, in particular, could obscure the role of individual glycan epitopes in biological function. We present a general strategy based on the Staudinger ligation to introduce specific glycan epitopes onto azide-containing proteins. The use of a phosphane-based Staudinger reagent allows for extremely mild reaction conditions which can be applied to aqueous proteins or cells. We demonstrate that multiple carbohydrate epitopes can be incorporated onto a protein backbone, and that the resulting glycans are competent for recognition by lectins. We propose that this general strategy will allow for testing the role of specific glycan epitopes in cellular and biochemical assays and increasing the stability of protein conjugates.
Subject(s)
Amino Sugars/chemistry , Azides/chemistry , Glycoproteins/chemistry , Amines/chemistry , Amino Sugars/chemical synthesis , Disaccharides/chemistry , Epitopes/chemistry , Epitopes/metabolism , Glycoproteins/metabolism , Indicators and Reagents/chemistry , Monosaccharides/chemistry , Phosphines/chemistry , Plant Lectins/metabolismABSTRACT
The function of cellular membranes remains a critical area of study. Advances in molecular biology and biochemistry have helped to define the plasma membrane as a dynamic and heterogeneous structure. Probes capable of resolving molecular interactions and biochemical changes within the membrane are becoming a necessary tool for cell biology studies. We review current examples that apply small molecule fluorophores to label either lipids or proteins to study the plasma membrane. We discuss probes of the lipid environment itself, as well as labeling strategies for membrane proteins and membrane receptors.
