Research

MOengineering

Peng Wu Laboratory: Chemical Glycobiology

The research in the Wu laboratory integrates synthetic chemistry with glycobiology to explore the relevance of protein glycosylation in human diseases and in host-symbiont interactions. The glycome, defined as the full complement of glycans that a cell produces, is involved in a variety of physiological processes, including angiogenesis, fertilization, stem cell development and neuronal development. Changes in the glycome have also been shown to mark the onset of cancer and inflammation. The wealth of biological information encoded in the glycome has motivated researchers to develop methods for its retrieval.

Produced by the secondary metabolism rather than encoded in the genome, glycans are assembled in a step-wise fashion by multiple enzymes and thus by multiple genes. Therefore, genetic and biochemical tools alone cannot be used to define all aspects of the glycome. Rather, many complementary approaches must be applied in parallel in order to assemble a picture of the glycome both from the “bottom up” and from the “top down”.

The major goal of our laboratory is to develop chemical biology platforms to image and characterize the glycome in tumors and in the immune system. The laboratory is also interested in chemical tools that enable selectively enrichment of glycoproteins in leukocytes for their molecular identification and functional studies. These new tools will facilitate the discovery of new biomarkers for diseases, and assist in the development of clinical diagnostics and therapeutics.

1. Development of new bioorthogonal chemistry for applications in living systems

In this project, our efforts are directed at discovering new bioorthogonal chemical reactions that can be utilized to probe protein glycosylation in living systems. Bioorthogonal reactions are non-native, non-perturbing chemical reactions that can incorporate an exogenously delivered probe to a target biomolecule in a highly selective manner in a cellular environment or in complex cell lysates.1 Therefore, these reactions are powerful tools for detection or isolation of the posttranslationally modified proteins that are glycosylated, lipidated and methylated among many other modifications.

Our laboratory pioneers the development of the first biocompatible Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC) reaction,2 a prototypical example of bioorthogonal click chemistry. This new biocompatible CuAAC allows rapid labeling of glycans in zebrafish embryos: three minutes vs. one hour required by copper-free click chemistry. The highly sensitive nature of this chemistry enabled us to visualize alterations in the biosynthesis of glycoproteins with exquisite temporal resolution exceeding the capabilities of previous techniques. It also enabled, for the first time, the implementation of imaging techniques to track dynamic glycan movement in live cells with single-molecule resolution.3 Currently, we are developing methods for labeling glycans on a specific membrane protein in situ.

2. Development of chemoenzymatic approaches for detecting cell-surface glycans

Metabolic labeling that utilizes unnatural monosaccharide derivatives enables the global remodeling of cell-surface glycans bearing that building block. However, higher order glycans, i.e. disaccharides or trisaccharides, of specific composition cannot be uniquely labeled by hijacking their biosynthetic pathways with unnatural monosaccharide building blocks.4 Because higher order glycans, rather than monosaccharides, encode information for specific cell-surface molecular recognition, methods are in urgent need for their detection. In 2011, our lab developed the first chemoenzymatic method for the specific labeling of Type II N-acetyllactosamine (LacNAc; Galβ1,4GlcNAc), an ubiquitous cell surface disaccharides, on cell surface glycoproteins of cultured cells and zebrafish embryos.5 The method utilizes a recombinant H. pylori α(1,3)fucosyltransferase to transfer a C-6 azide- or alkyne-tagged fucose residue to the 3-OH of N-acetylglucosamine (GlcNAc) of the LacNAc disaccharide. The tag may then be selectively derivatized with probes via bioorthogonal click chemistry.

This chemoenzymatic method has superior sensitivity and specificity as compared with antibody and lectin-based techniques, the current “gold standard” for glycan detection. We translated this method into a histological technique, which enabled high throughput screening of human tumor microarrays to correlate glycosylation patterns with lung adenocarcinoma progression. Lung cancer is the leading cause of cancer mortality with the five-year survival rate about 4.0%, and there is no reliable, early detection method. Using our chemoenzymatic method, we discovered a 13-fold decrease in LacNAc expression in grade one-lung adenocarcinoma patient samples as compared to that of healthy humans, suggesting that LacNAc can serve as an early detection marker for lung cancer.6

Based upon our success of identifying the chemoenzymatic method for the specific labeling of LacNAc, we will continue our effort on the development of a tool kit for the labeling of other sectors of higher order glycans. This tool kit will be used to characterize dynamic changes in cell-surface glycosylation in both living systems and in human tissue or serum samples. Given the strong correlation of altered glycosylation patterns with malignancy, we will combine glycosylation status mapping via the chemoenzymatic labeling with the antibody-based detection of protein-of-interest to develop new cancer diagnostic methods with high sensitivity and specificity.

3. Decipher the role of glycans in leukocyte activation and differentiation

After encountering an activation signal, resting leukocytes become activated and acquire distinct phenotypes and functions. For example, activated and quiescent T cells have distinct metabolic phenotypes.7 Activated T cells (effector T cells) have an anabolic metabolism, in which growth factor cytokines activate glycolysis, despite the presence of oxygen, to support cell proliferation. This process provides ATP for proliferating T cells while fatty acids and amino acids are incorporated into cellular components. By contrast, quiescent T cells (naïve and memory T cells) have a catabolic

metabolism, in which they use glucose, fatty acids, and amino acids for ATP generation through the TCA cycle and oxidative phosphorylation.

Although many studies have been performed to characterize changes in the genetic signatures during these processes, little is known about the accompanying changes in the cell-surface glycosylation, the mechanism governing these changes, as well as their functional roles. Combining the chemoenzymatic tools developed in our laboratory with genetic and biochemical techniques, we are poised to investigate these challenging questions. Given aberrant glycosylation is linked to the pathogenesis of autoimmune diseases and immunosuppressive strategies evolved by tumor, this study may point towards opportunities for new glycan-targeted immune therapies to counteract human disease.

4. Study of fucosylated glycoproteins in Bacteroidales species

Human symbionts of the Bacteroidales order are among the predominantly represented genera in the mammalian gut. Bacteroidales species impart numerous beneficial effects to their hosts: they participate in nutrient processing—hydrolyzing dietary polysaccharides and converting them into short chain fatty acids that can be utilized by the host.8 They are also involved in promoting the development of gut-associated lymphoid tissues and regulation of intestinal angiogenesis. New evidence suggests that some Bacteroidales species also play an important role in the maintenance of a healthy body weight.

L-Fucose, an abundant component of many host intestinal cell-surface glycoconjugates has risen as a key mediator of interactions between Bacteroidales and their mammalian hosts. Discovered by Gordon and coworkers, fucose serves as a principal carbon and energy source for B. thetaiotaomicron, a major member of the human intestinal microflora.9 Further, recent studies by Comstock and coworkers have shown that commensal Bacteroidales species B. fragilis and P. distasonis use mammalian-like pathways to decorate surface capsular polysaccharides and glycoproteins with fucosylated glycans, a process that has not been recognized for any other prokaryotes and is essential for persistence of these species within the competitive ecosystem of the gut.10 Taken together, these findings link fucose availability in the host intestine to the glycan constitution of Bacteroidales glycoconjugates.

Recently, our laboratory discovered that an alkyne-bearing fucose analog can be metabolized and incorporated into cell-surface glycoproteins in intestinal commensal Bacteroidales species, allowing subsequent detection via CuAAC.11 This chemical strategy allows us to enrich and identify fucosylated glycoproteins in these abundant intestinal symbionts with the ultimate goal of answering the following fundamental questions: How many Bacteroidales proteins are fucosylated? What are the functional roles of these fucosylated glycoproteins in intestinal colonization and in mediating a symbiotic relationship with the host?

Projects in our research group are highly multidisciplinary. Students joining our lab will gain expertise in biochemistry, organic synthesis, molecular and cell biology, immunology as well as proteomics.

References:

  1. Sletten EM, Bertozzi CR. Bioorthogonal chemistry: fishing for selectivity in a sea of functionality. Angew Chem Int Ed. 2009;48:6974-6998. PubMed PMID: 19714693.
  2. Soriano del Amo D, Wang W, Jiang H, Besanceney C, Yan AC, Levy M, Liu Y, Marlow FL, Wu P. Biocompatible copper(I) catalysts for in vivo imaging of glycans. J Am Chem Soc. 2010;132:16893-16899. PubMed PMID: 21062072.
  3. Jiang H, English BP, Hazan RB, Wu P, Ovryn B. Tracking surface glycans on live cancer cells with single-molecule sensitivity. Angew Chem Int Ed Engl. 2015;54:1765-1769. doi: 10.1002/anie.201407976. PubMed PMID: 25515330; PubMed Central PMCID: PMC4465920.
  4. Rouhanifard SH, Nordstrom LU, Zheng T, Wu P. Chemical probing of glycans in cells and organisms. Chem Soc Rev. 2013;42(10):4284-4296. doi: 10.1039/c2cs35416k. PubMed PMID: 23257905; PubMed Central PMCID: PMC3641795.
  5. Zheng T, Jiang H, Gros M, Soriano Del Amo D, Sundaram S, Lauvau G, Marlow F, Liu Y, Stanley P, Wu P. Tracking N-acetyllactosamine on cell-surface glycans in vivo. Angew Chem Int Ed. 2011;50:4113-4118. PubMed PMID: 21472942.
  6. Rouhanifard SH, López-Aguilar A, Wu P. CHoMP: A Chemoenzymatic Histology Method Using Clickable Probes. ChemBioChem. 2014;15:2667-2673.
  7. Pearce EL. Metabolism in T cell activation and differentiation. Curr Opin Immunol. 2010;22:314-320. doi: 10.1016/j.coi.2010.01.018. PubMed PMID: 20189791; PubMed Central PMCID: PMC4486663.
  8. Xu J, Gordon JI. Honor thy symbionts. Proc Natl Acad Sci U S A. 2003;100:10452-10459. doi: 10.1073/pnas.1734063100. PubMed PMID: 12923294; PubMed Central PMCID: PMC193582.
  9. Hooper LV, Xu J, Falk PG, Midtvedt T, Gordon JI. A molecular sensor that allows a gut commensal to control its nutrient foundation in a competitive ecosystem. Proc Natl Acad Sci U S A. 1999;96:9833-9838. PubMed PMID: 10449780.
  10. Coyne MJ, Reinap B, Lee MM, Comstock LE. Human symbionts use a host-like pathway for surface fucosylation. Science. 2005;307:1778-1781. PubMed PMID: 15774760.
  11. Besanceney-Webler C, Jiang H, Wang W, Baughn AD, Wu P. Metabolic labeling of fucosylated glycoproteins in Bacteroidales species. Bioorg Med Chem Lett. 2011;21:4989-4992. doi: 10.1016/j.bmcl.2011.05.038. PubMed PMID: 21676614; PubMed Central PMCID: PMC3156311.