Neurociencias

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J Chem Educ. Author manuscript; available in PMC 2012 January 1.
Published in final edited form as: J Chem Educ. 2011 January 1; 88(1): 95–97. doi:10.1021/ed900054b.

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Kinetics of Neuraminidase Action on Glycoproteins by 1D and 2D NMR
Adam W. Barb, John N. Glushka, and James H.Prestegard* Complex Carbohydrate Research Center, University of Georgia, 315 Riverbend Road, Athens, GA 30602

Abstract
The surfaces of mammalian cells are coated with complex carbohydrates, many terminated with a negatively charged N-acetylneuraminic acid residue. This motif is specifically targeted by pathogens, including influenza viruses and many pathogenic bacteria, to gain entry into thecell. A necessary step in the influenza virus life cycle is the release of viral particles from the cell surface; this is achieved by cleaving N-acetylneuraminic acid from cell surface glycans with a virallyproduced neuraminidase. Here we present a laboratory exercise to model this process using a glycoprotein as a glycan carrier and using real time nuclear magnetic resonance (NMR) spectroscopy tomonitor N-acetylneuraminic acid release as catalyzed by neuraminidase. A timeresolved two dimensional data processing technique, statistical total correlation spectroscopy (STOCSY), enhances the resolution of the complicated 1D glycoprotein spectrum and isolates characteristic peaks corresponding to substrates and products. This exercise is relatively straightforward and leads students through awide range of biologically and chemically relevant procedures, including use of NMR spectroscopy, enzymology and data processing techniques.

Keywords Upper-Division Undergraduate; Biochemistry; Inquiry-Based/Discovery Learning; Bioanalytical Chemistry; Biotechnology; Biosynthesis; Carbohydrates; Drugs/Pharmaceuticals; Enzymes; NMR Spectroscopy

Lab Summary
Glycobiology is a rapidly-expandingand vibrant field buoyed by recent discoveries that highlight the critical nature of complex carbohydrates in cell-cell communication and pathology. Introductory biochemistry and biology courses often focus on molecular biology because of the great impact this field has had on our understanding of much of biology and the very logical route it portrays to the synthesis of proteins and the directroles they play in cellular processes. Because complex carbohydrates are not synthesized from templates like DNA, RNA, and proteins, they exhibit a higher level of primary structural diversity, and a basic understanding of their synthesis and biological roles has lagged behind that of their more-studied counterparts. They are, however, primary mediators of cellular communication and function, andtheir roles in these processes deserve more attention. Braced by the importance and diversity of the glycome, the inclusion of glycobiology into early undergraduate education will enhance introductory biochemistry and biology courses and may excite students to this field of biological research.

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Mammalian cell surfaces are a dynamicmixture of lipids, proteins and carbohydrates. The carbohydrate moieties presented on cell surfaces are highly variable, and are often anchored to proteins or lipids embedded in the cell membrane. Complex carbohydrate (glycan) termini on the cell surface are commonly modified by a sialic acid residue, N-acetylneuraminic acid in humans (Figure 1), which functions to distinguish self from non-selfand regulate a number of cellular processes (1). However, pathogens have evolved to recognize these motifs and use the sialylated glycans (a complex carbohydrate modified with Nacetylneuraminic acid residues on the non-reducing end) to gain entry into host cells. The influenza virus, for example, binds to the surface of epithelial cells in the upper respiratory mucosa by coordinating glycans...