Tianqing Zheng

Increasing the efficacy of bioorthogonal click reactions for bioconjugation: a comparative study.

Raising the bar: the efficacy of bioorthogonal reactions for bioconjugation has been thoroughly evaluated in four different biological settings. Powered by the development of new biocompatible ligands, the copper-catalyzed azide-alkyne cycloaddition has brought about unsurpassed bioconjugation efficiency, and thus it holds great promise as a highly potent and adaptive tool for a broader spectrum of biological applications.

Chemoenzymatic synthesis of GDP-l-fucose and the Lewis X glycan derivatives

Lewis X (Lex)-containing glycans play important roles in numerous cellular processes. However, the absence of robust, facile, and cost-effective methods for the synthesis of Lex and its structurally related analogs has severely hampered the elucidation of the specific functions of these glycan epitopes. Here we demonstrate that chemically defined guanidine 5′-diphosphate-β-l-fucose (GDP-fucose), the universal fucosyl donor, the Lex trisaccharide, and their C-5 substituted derivatives can be synthesized on preparative scales, using a chemoenzymatic approach. This method exploits l-fucokinase/GDP-fucose pyrophosphorylase (FKP), a bifunctional enzyme isolated from Bacteroides fragilis 9343, which converts l-fucose into GDP-fucose via a fucose-1-phosphate (Fuc-1-P) intermediate. Combining the activities of FKP and a Helicobacter pylori α1,3 fucosyltransferase, we prepared a library of Lex trisaccharide glycans bearing a wide variety of functional groups at the fucose C-5 position. These neoglycoconjugates will be invaluable tools for studying Lex-mediated biological processes.

Tracking N-Acetyllactosamine on Cell-Surface Glycans In Vivo†

The glycome, the totality of glycans produced by a cell, is a dynamic indicator of the cells physiology.[1] Changes in the glycome reflect a cells developmental stage and the transformation state of a cell. Recently, imaging glycans in vivo has been enabled using a bioorthogonal chemical reporter strategy by treating cells or organisms with azide- or alkyne-tagged monosaccharide precursors.[2, 3] The modified monosaccharides, when taken up by cells, are activated in the cytoplasm to form nucleotide sugars, substrates of glycosyltransferases that generate complex glycans in the endoplasmic reticulum and Golgi. Once incorporated into cell surface glycoconjugates, the bioorthogonal chemical tags allow covalent conjugation with fluorescent probes for visualization,[2] or with affinity probes for enrichment and glycomic analysis.[4] This approach has been successfully used for the detection and imaging of mucin O-linked glycans,[2] sialylated[2] and fucosylated glycans,[5] and cytosolic O-GlcNAcylated proteins.[2] However, only monosaccharides are tracked by this strategy, and each monosaccharide is usually found on a plethora of glycans.[6] Higher order glycans, i.e. disaccharides or trisaccharides, of specific composition cannot be uniquely labeled by hijacking their biosynthetic pathways with unnatural monosaccharides (Figure 1a). Here we report a rapid and highly specific chemoenzymatic method for labeling cell surface glycans bearing a ubiquitous disaccharide—N-acetyllactosamine (LacNAc, Galβ1,4GlcNAc)—with biophysical probes for imaging or glycomic analysis.

Chemical Probing of Glycans in Cells and Organisms

Among the four major building blocks of life, glycans play essential roles in numerous physiological and pathological processes. Due to their non-templated biosynthesis, advances towards elucidating the molecular details of glycan functions are relatively slow compared with the pace of protein and nucleic acid research. Over the past 30 years, chemical tools have emerged as powerful allies to genetics and molecular biology in the study of glycans in their native environment. This tutorial review will provide an overview of the recent technological developments in the field, as well as the progress in the application of these techniques to probe glycans in cells and organisms.

Single-Stranded DNA as a Cleavable Linker for Bioorthogonal Click Chemistry-Based Proteomics

In this communication, we report a new class of cleavable linker based on automatically synthesized, single-stranded DNAs. We incorporated a DNA oligo into an azide-functionalized biotin (biotin-DNA-N3) and used the probe to enrich for alkyne-tagged glycoproteins from mammalian cell lysates. Highly efficient and selective release of the captured proteins from streptavidin agarose resins was achieved using DNase treatment under very mild conditions. A total of 36 sialylated glycoproteins were identified from the lysates of HL60 cells, an acute human promyeloid leukemia cell line. These sialylated glycoproteins were involved in many different biological processes ranging from glycan biosynthesis to cell adhesion events.

Click Triazoles for Bioconjugation.

Click Chemistry is a set of rapid, selective and robust reactions that give near-quantitative yield of the desired product in aqueous solutions. The Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC) that forms 1,4-disubstituted triazoles is a prototypical example of click chemistry that features exquisite selectivity and bioorthogonality-that is, non-interacting with biological components while proceeding under physiological conditions. Over the past ten years, CuAAC has found extensive applications in the field of chemical biology. In this chapter, we describe the discovery of Cu(I) catalysts for this transformation and the recent development of the strain-promoted azide-alkyne cycloaddition that eliminate the use of copper. We also highlight several recent applications toward conjugating biomolecules, including proteins, nucleic acids, lipids and glycans, with biophysical probes for both in vitro and in vivo studies.

Chemoenzymatic synthesis of the sialyl Lewis X glycan and its derivatives

A combination of recombinant FKP and alpha-(1-->3)-fucosyltransferase allows the facile synthesis of the sialyl Lewis X tetrasaccharide glycan and its derivatives in excellent yield. In this system, the universal fucosyl donor, guanidine 5-diphosphate-beta-L-fucose (GDP-fucose), or its analogues can be generated in situ by cofactor recycling using pyruvate kinase.

Diversity of Functionally Permissive Sequences in the Receptor-Binding Site of Influenza Hemagglutinin.

Influenza A virus hemagglutinin (HA) initiates viral entry by engaging host receptor sialylated glycans via its receptor-binding site (RBS). The amino acid sequence of the RBS naturally varies across avian and human influenza virus subtypes and is also evolvable. However, functional sequence diversity in the RBS has not been fully explored. Here, we performed a large-scale mutational analysis of the RBS of A/WSN/33 (H1N1) and A/Hong Kong/1/1968 (H3N2) HAs. Many replication-competent mutants not yet observed in nature were identified, including some that could escape from an RBS-targeted broadly neutralizing antibody. This functional sequence diversity is made possible by pervasive epistasis in the RBS 220-loop and can be buffered by avidity in viral receptor binding. Overall, our study reveals that the HA RBS can accommodate a much greater range of sequence diversity than previously thought, which has significant implications for the complexxa0evolutionary interrelationships between receptor specificity and immune escape.

Interferon-γ is a master checkpoint regulator of cytokine-induced differentiation

Significance The understanding of the molecular mechanisms of activation and checkpoint processes has important therapeutic implications. Here, we show that interferon-γ is a master checkpoint regulator for many cytokines. It operates partially by activating STAT1 signaling. However, most important is the mechanism that allows it to assume master regulator status. To do this, it induces internalization of gp130, a common component of many heterodimeric cytokine receptors. Therefore, this cytokine checkpoint could open a whole new paradigm in cell biology. Cytokines are protein mediators that are known to be involved in many biological processes, including cell growth, survival, inflammation, and development. To study their regulation, we generated a library of 209 different cytokines. This was used in a combinatorial format to study the effects of cytokines on each other, with particular reference to the control of differentiation. This study showed that IFN-γ is a master checkpoint regulator for many cytokines. It operates via an autocrine mechanism to elevate STAT1 and induce internalization of gp130, a common component of many heterodimeric cytokine receptors. This targeting of a receptor subunit that is common to all members of an otherwise diverse family solves the problem of how a master regulator can control so many diverse receptors. When one adds an autocrine mechanism, fine control at the level of individual cells is achieved.

Immunochemical engineering of cell surfaces to generate virus resistance.

Significance A method that renders cells resistant to virus infection is reported. The method is based on anchoring antibodies to cell surface virus receptors to the plasma membrane. Because of their effective molarity, the antibodies are much more effective than if they were free in the circulation. The antibodies interact with the receptors and block virus attachment. In infections with viruses such as HIV, cells rendered resistant to infection will be selected over infected cells, thereby opening the possibility of curing the infection. Modern immunochemical engineering allows the creation of cells that either secrete antibodies or incorporate them into various cellular compartments, including the plasma membrane. Because the receptors for most viruses are known, if one can achieve the proper stoichiometry and geometry, plasma membrane-associated antibodies to these receptors should block viral infection. In this report, we test this concept for two different viruses, human rhinovirus and HIV. Plasma membrane-tethered antibodies efficiently rendered cells permanently nonpermissive for infection by both these viruses. Membrane-bound antibodies were much more efficient than free antibody in preventing infection, likely because of the effective molarity of membrane bound antibodies. Such resistant cells may restore immune-competence to otherwise compromised HIV patients.