Overview
Glycosylation is crucial to a myriad of biological processes including bacterial pathogenesis, cell signaling, tumor cell metastasis and neuronal development. Research in my laboratory is focused on the creation and application of chemical, biological and systems-based methods to the study of glycosylation. We work on three main areas of interest:1. Understanding the role of glycosylation in development, cancer and bacterial pathogenesis using systems-based approaches.
Mammalian and bacterial cell surfaces are coated with a wealth of carbohydrate epitopes. In nature, pattern recognition of glycans plays an important role in innate immunity against pathogens and may be involved in tumor metastasis and embryogenesis. This argues that the overall composition of the glycan coat, rather than any specific sugar epitope, is important to recognition. To obtain systems-level data on glycosylation, we must develop new technologies for glycomics research (ACA, 2008). Our laboratory has recently established lectin microarrays as a method for the high throughput analysis of carbohydrates on glycoproteins, bacteria and mammalian cells (ChemBioChem , 2005 , 6 , 985-989 and Nat. Chemical Biology, 2006, 2, 153-7 ). Lectins are carbohydrate-binding proteins that can discriminate between structural isomers and epimers. Lectin microarrays consist of a collection of lectins immobilized onto a solid support at a high spatial density. Over 100 lectins are known and >60 are commercially available. By utilizing a microarray format, we can obtain comprehensive lectin-binding profiles of fluorescently labeled samples. The resulting pattern of spots (glycopattern) yields information on the glycosylation of the sample based on cross-correlating the glycan-binding specificities of the lectins. We have demonstrated the use of our arrays for bacterial glycomics, observing distinct glycan fingerprints for closely related Escherichia coli strains. Recently, we have also applied lectin microarrays to the study of mammalian glycosylation, observing subtle differences in carbohydrate epitope expression (PNAS, 2007 ).
We are currently utilizing this technology to study three critical and fundamental questions in glycobiology. 1. What role does glycosylation play in defining cell state and type and how does this inform our ideas about cell differentiation and cancer metastasis? The glycocode hypothesis states that each cell type has a unique coat of glycans that defines them. Although this hypothesis has been around for decades, we have yet to determine the validity of this idea. If glycans are an absolute determinant of cell type and function, this has profound implications for tissue targeting, determination of secondary metastatic sites and for biomarkers of differentiation. Our laboratory is studying this question from many angles including the study of stem cell differentiation ( in collaboration with Dr. Stephen Dalton at University of Georgia), haemopoetic cell differentiation and cancer. 2. How does the genome encode the glycome? Our current understanding of the glycome and how it is encoded at the genomic level is cursory. To help shed light on this issue, we are obtaining multifaceted integratable datasets including genomic, proteomic and glycomic information. These datasets will allow us to model how glycosylation is controlled within the cell. 3. Does the glycosylation of microbial pathogens reflect the host and to what extent do dynamic changes in glycosylation enhance pathogenicity? We are currently collaborating with several virology and microbiology laboratories to address these questions.2. Creation and Identification of Novel Microbial Lectins.
We are constantly expanding the utility of our lectin microarrays by increasing the diversity of lectins on the array (Mol. BioSyst., 2008). At the present time, we are actively involved in creating more specific lectins using novel evolutionary approaches and in identifying lectins via a combination of bioinformatics and high-throughput screening.
3. Dissecting the function of b -O-N-acetyl-D-glucosamine (O-GlcNAc) in cell signaling.
O-GlcNAc is a simple sugar modification of serine and threonine residues on cytosolic and nuclear proteins that may play an important role in the etiologies of diabetes, Alzheimer's disease and cancer. O-GlcNAc is a dynamic modification and is controlled by two enzymes, O-GlcNAc transferase (OGT), which catalyzes the addition of O-GlcNAc onto the polypeptide, and O-GlcNAcase, which removes the modification. O-GlcNAc competes with phosphate for the same serine/threonine sites utilized by the kinases in some proteins. In addition, global reciprocity between O-GlcNAc and phosphate has been observed at the cellular levels, ie. ↓O-GlcNAc = ↑ phosphorylation and vice versa. This alters the transcription and activation of a multitude of proteins critical to normal cell function and profoundly challenges the current idea that cell signaling through serine/threonine modification is controlled solely by the kinases. Little is known about either the regulation and spatial localization of O-GlcNAc or the dynamic reciprocity between O-GlcNAc and phosphorylation in signal transduction pathways in living systems. To study O-GlcNAc within the living cells we are developing new tools and methods. One such tool is a genetically encoded FRET sensor which enables us to directly study O-GlcNAc dynamicsl in live cells (JACS, 2006). To see a movie of HeLa cells expressing our FRET sensor, treated with PuGNAc, an O-GlcNAcase inhibitor, and glucosamine click here. An increase in FRET = an increase in RED.
