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Methods in Enzymology

Barbara Imperiali
No abstract text is available yet for this article.
2017: Methods in Enzymology
Satoru Horiya, Jennifer K Bailey, Isaac J Krauss
Directed evolution is a useful method for the discovery of nucleic acids, peptides, or proteins that have desired binding abilities or functions. Because of the abundance and importance of glycosylation in nature, directed evolution of glycopeptides and glycoproteins is also highly desirable. However, common directed evolution platforms such as phage-, yeast-, or mammalian-cell display are limited for these applications by several factors. Glycan structure at each glycosylation site is not genetically encoded, and yeast and mammalian cells produce a heterogeneous mixture of glycoforms at each site on the protein...
2017: Methods in Enzymology
Thapakorn Jaroentomeechai, Xiaolu Zheng, Jasmine Hershewe, Jessica C Stark, Michael C Jewett, Matthew P DeLisa
Asparagine-linked (N-linked) protein glycosylation is one of the most abundant types of posttranslational modification, occurring in all domains of life. The central enzyme in N-linked glycosylation is the oligosaccharyltransferase (OST), which catalyzes the covalent attachment of preassembled glycans to specific asparagine residues in target proteins. Whereas in higher eukaryotes the OST is comprised of eight different membrane proteins, of which the catalytic subunit is STT3, in kinetoplastids and prokaryotes the OST is a monomeric enzyme bearing homology to STT3...
2017: Methods in Enzymology
Suttipun Sungsuwan, Xuanjun Wu, Xuefei Huang
Tumor-associated carbohydrate antigens (TACAs) are attractive targets for anticancer vaccine development. Due to the low immunogenicity of TACAs, a powerful carrier system is needed to boost immune responses. Virus-like particles (VLPs) are an exciting platform for delivering TACAs to the immune system. The high symmetry of VLPs enables the display of TACAs in an organized manner, which in turn can potently activate antibody secreting B cells, eliciting high titers of antiglycan IgG antibodies. In this chapter, the protocol for conjugating a prototypical TACA, the Tn antigen to a VLP, bacteriophage Qβ, is presented...
2017: Methods in Enzymology
Abirami Lakshminarayanan, Balakumar Vijayakrishnan, Hagan Bayley, Benjamin G Davis
The relative structural conservation of "internal glycans" in the cell walls of pathogens suggests that they might as target epitopes less prone to variation and hence with greater potential universality as vaccine targets. Examples of such glycans include the inner core sugars of lipopolysaccharides in Gram-negative bacteria. However, due to the buried nature of such internal epitopes, this approach has been rarely adopted. Here we briefly review and compare strategic approaches and outline practical methods associated with evaluating one synergistic strategy that combines (i) blocking of the display of "external glycans" with (ii) vaccination targeted at "internal glycans...
2017: Methods in Enzymology
Felix Broecker, Peter H Seeberger
Synthetic oligosaccharide-based vaccines are promising alternatives to conventional antibacterial carbohydrate vaccines prepared with isolated polysaccharides. Unlike polysaccharides, synthetic glycans are well defined, contaminant-free, and accessible even for pathogens that cannot be fermented or show limited carbohydrate biosynthesis in vitro. However, identifying synthetic glycan B cell epitopes that induce protective immunity has traditionally been a time-consuming trial-and-error process, as predicting the immunogenicity of an oligosaccharide by means of structure alone is not straightforward...
2017: Methods in Enzymology
Zach Armstrong, Peter Rahfeld, Stephen G Withers
Microorganisms routinely perform complex enzymatic transformations of natural material; thus, their enzymes have the potential to tackle medical and industrial challenges. However, a vast number of microbes are recalcitrant to cultivation. Functional screening of environmental DNA allows us to tap into the seemingly boundless diversity of enzymes encoded by microbial populations. In this chapter, we describe methods for the isolation of environmental DNA, generation of metagenomic libraries, and the functional screening of these libraries for new glycosidases...
2017: Methods in Enzymology
Ezequiel Valguarnera, Mario F Feldman
As we enter into the postantibiotic era, vaccines to prevent bacterial infections previously treatable with antibiotics are urgently needed. Most successful antibacterial vaccines are glycoconjugates, composed of cell surface carbohydrates chemically attached to a carrier protein. Glycoconjugate vaccines provide a safe and consistent strategy against polysaccharide-encapsulated pathogens. The best examples are the conjugate vaccines against Haemophilus influenzae type b, Streptococcus pneumoniae, and Neisseria meningitidis, all based on capsular polysaccharides...
2017: Methods in Enzymology
Qiang Yang, Lai-Xi Wang
N-glycosylation plays important roles in modulating the biological functions of glycoproteins, such as protein folding, stability, and immunogenicity. However, acquiring homogeneous glycoforms of glycoproteins has been a challenging task for functional studies and therapeutic applications. In this chapter, we describe an efficient chemoenzymatic glycan remodeling protocol for making homogeneous glycoproteins that involves enzymatic deglycosylation and subsequent reglycosylation procedures. Two therapeutic glycoproteins, Herceptin (trastuzumab, a therapeutic monoclonal antibody) and erythropoietin (EPO, a glycoprotein hormone), were chosen as the model systems...
2017: Methods in Enzymology
David H Kwan
Directed evolution is an incredibly powerful strategy for engineering enzyme function. Applying this approach to glycosidases offers enormous potential for the development of highly specialized tools in chemical glycobiology. Performing enzyme directed evolution requires the generation, by random mutagenesis, of mutant libraries from which large numbers of variant enzymes must be screened in high-throughput assays. A structure-guided "semirational" method for library creation allows researchers to target specific amino acid positions for mutagenesis, concentrating mutations where they might be most effective in order to produce mutant libraries of a manageable size, minimizing screening effort while maximizing the chances of finding improved mutants...
2017: Methods in Enzymology
Naoko Komura, Kenichi G N Suzuki, Hiromune Ando, Miku Konishi, Akihiro Imamura, Hideharu Ishida, Akihiro Kusumi, Makoto Kiso
Gangliosides, glycosphingolipids containing one or more sialic acids in the glycan chain, are involved in various important biological processes in cell plasma membranes (PMs). However, the behaviors and functions of gangliosides are poorly understood, primarily because of the lack of fluorescent analogs that are equivalent to native gangliosides that can be used as chemical and physical probes. In this study, we developed entirely chemical methods to synthesize fluorescent gangliosides (GM3, GM2, GM1, and GD1b) in which the glycan components are site-specifically labeled with various fluorescent dyes...
2017: Methods in Enzymology
Martin Rejzek, Lionel Hill, Edward S Hems, Sakonwan Kuhaudomlarp, Ben A Wagstaff, Robert A Field
Sugar nucleotides are essential building blocks for the glycobiology of all living organisms. Detailed information on the types of sugar nucleotides present in a particular cell and how they change as a function of metabolic, developmental, or disease status is vital. The extraction, identification, and quantification of sugar nucleotides in a given sample present formidable challenges. In this chapter, currently used techniques for sugar nucleotide extraction from cells, separation from complex biological matrices, and detection by optical and mass spectrometry methods are discussed...
2017: Methods in Enzymology
Ian C Schoenhofen, N Martin Young, Michel Gilbert
Legionaminic acids are analogs of sialic acid that occur in cell surface glycoconjugates of several bacteria. Because legionaminic acids share the same stereochemistry as sialic acid but differ at C7 and C9, they are interesting analogs to probe the impact of varying exocyclic moieties (C7-C9) on biological activities such as susceptibilities to sialidases, interactions with Siglecs and immunogenicity. There are currently no reports on the bacterial enzymes that transfer legionaminic acids to these cell surface glycoconjugates, but some mammalian and bacterial sialyltransferases display donor promiscuity and can use CMP-Leg5,7Ac2 efficiently enough to transfer Leg5,7Ac2 to their natural acceptor glycans...
2017: Methods in Enzymology
Cristina Y Zamora, Nathaniel S Schocker, Michelle M Chang, Barbara Imperiali
This method describes the chemoenzymatic synthesis of several nucleotide sugars, which are essential substrates in the biosynthesis of prokaryotic N- and O-linked glycoproteins. Protein glycosylation is now known to be widespread in prokaryotes and proceeds via sequential action of several enzymes, utilizing both common and modified prokaryote-specific sugar nucleotides. The latter, which include UDP-hexoses such as UDP-diNAc-bacillosamine (UDP-diNAcBac), UDP-diNAcAlt, and UDP-2,3-diNAcManA, are also important components of other bacterial and archaeal glycoconjugates...
2017: Methods in Enzymology
Michael E Harris, Vernon E Anderson
No abstract text is available yet for this article.
2017: Methods in Enzymology
Sam Hay
Enzyme reaction progress curves, or time course datasets, are often rich in information, yet their analysis typically reduces their information content to a single parameter, the initial velocity. An alternative approach is described here, where the time course is described by a model constructed from rate equations. In combination with global nonlinear regression, intrinsic rate and/or equilibrium constants can be directly obtained by fitting these data. This method can be greatly enhanced when combined with the measurement of (usually deuterium) isotope effects, which selectively perturb individual step(s) within the reaction, allowing better separation of fitted parameters and more robust model testing...
2017: Methods in Enzymology
Natalia Sannikova, Andrew R Lewis, Andrew J Bennet
Nuclear magnetic spectroscopic (NMR) methods are discussed for the measurement of heavy atom ((13)C, (18)O, (15)N) and secondary deuterium kinetic isotope effects. The discussion focuses primarily on the NMR methods that enable the measurement of quantitative spectra and not on methods to make labeled substrates. Two main techniques are considered: single-point determinations on natural abundance material and the continuous monitoring of isotopically enriched materials. The second method is described in more detail, and we include a discussion of the current state of instrumentation and computer programs for data acquisition and analysis...
2017: Methods in Enzymology
Govardhan R Veerareddygari, Eugene G Mueller
The synthesis of specifically deuterated uridine, its incorporation into an RNA oligonucleotide substrate, and the use of the labeled substrate to determine the deuterium kinetic isotope effect for the reaction catalyzed by the pseudouridine synthases (enzymes that isomerize uridine to pseudouridine in RNA) are described. Both enzymes-TruB and RluA-display a primary kinetic isotope effect, which indicates the formation of a glycal intermediate in the ribose ring during turnover. Although the details of the protocols are specific to these two enzymes, the general methodology is readily adaptable to the synthesis and incorporation of other labeled nucleosides into any RNA molecule by in vitro transcription...
2017: Methods in Enzymology
Scott O C Mundle, Barbara Sherwood Lollar, Ronald Kluger
Isotope ratio mass spectrometry (IRMS) provides accurate measurements of relative abundance of isotopes of heavy atoms for reactions that are subject to kinetic isotope effects (KIEs). The recent development of compound-specific isotope analysis (CSIA) allows the use of multiple time points that provide data for a rate plot as well as isotope ratios. Utilizing CSIA in enzymology presents opportunities for obtaining heavy atom KIEs in diverse areas.
2017: Methods in Enzymology
Mark W Ruszczycky, Hung-Wen Liu
Steady-state kinetic isotope effects on enzyme-catalyzed reactions are often interpreted in terms of the microscopic rate constants associated with the elementary reactions of interest. Unfortunately, this approach can lead to confusion, especially when more than one elementary reaction is isotopically sensitive, because it forces one to consider the full catalytic cycle one step at a time rather than as a complete whole. Herein we argue that shifting focus from intrinsic effects to net rate constants and enzyme intermediate concentrations provides a more natural and holistic interpretation by which the effects of partial rate limitation are more easily understood...
2017: Methods in Enzymology
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