Moremen Lab Research Interests
Research in the Moremen lab focuses on the structure, regulation, and localization of enzymes involved in the biosynthesis, recognition, and catabolism of mammalian glycoproteins. Carbohydrate structures on glycoproteins contribute to many biological recognition events during development, oncogenic transformation, and cell adhesion. In addition, protein-linked glycans can influence bioactivity, folding, localization, and immunogenicity of the attached polypeptide. Large numbers of intracellular and extracellular proteins contain covalently bound oligosaccharides, including enzymes, cell-surface receptors, hormones, immunoglobulins, and viral antigens. Alterations in the synthesis and degradation of these structures can also occur in human genetic diseases and cancer. Despite the ubiquity of glycoprotein structures, much is still unknown about the regulation of the glycosylation pathway and aspects of structure and function of the processing enzymes. Many questions also remain regarding oligosaccharide catabolism in different intracellular compartments as well as the specificity and regulation of protein-carbohydrate interactions during development. Work in the Moremen lab is focused on four main areas: (1) expression, purification, and structural characterization of mammalian glycosyltransferases and glycosidases and their corresponding substrate interactions, (2) Characterization of protein carbohydrate interactions with a focus on interactions between proteoglycans (PGs) and their cognate binding proteins, (3) developing technologies for measuring transcript abundance and regulation for glycan-related genes, and (4) characterization of the roles of mammalian Asn-linked glycan trimming enzymeson the biosynthesis and catabolism of nascent glycoproteins by the expression and characterization of the enzymes involved in these processes. Each of these research programs is described below and is supported by grant funding by the National Institutes of Health. The first three projects are collaborative projects with other members of the Complex Carbohydrate Research Center.

1. Determination of the structural and molecular basis for the interaction between glycosidases and glycosyltransferases and their corresponding substrates: One of the major research areas in the Moremen lab is the characterization of mamalian glycosylation enzymes. This work, which is supported by the NIH Resource Center for Biomedical Complex Carbohydrates, is an integrated study on the structure and interactions of glycosyltransferases and glycosidases and the corresponding substrates. The project takes advantage of the unique expertise of several research programs within the CCRC. The program in the Moremen lab is foused on the production of all human glycosyltransferases and glycoside hydrolases by recombinant expression in mammalian cells followed by purification, enzymatic characterization, and structure determination. The first of the structures of a glycosyltransferase derived from this expression platform is rat ST6GAL1 and several more are in the production and characterization pipeline. The goal of these studies are to provide a biochemical and structural uderstanding of glycan biosynthesis and catabolism as well as providing enzymatic catalysys for chemoenzymatic synthesis. This project is funded by an NIH P01 grant (P01GM107012, Mammalian Glycosyltransferases for Use in Chemistry and Biology). For further information please see: glycoenzymes.ccrc.uga.edu.

2. Protein-carbohydrate interactions: Cell-surface and extracellular glycoproteins and glycolipids play unique and critical roles in mammalian physiology. One of the major glycan classes at the cell surface and within the extracellular matrix (ECM) are the proteoglycans (PGs), with extended linear disaccharide repeat polymers usually comprised of alternating amino sugar and uronic acid residues attached to a secreted or cell surface core protein. PGs play diverse roles as co-receptors for cell surface signaling, scaffolds for cell-matrix interactions, ligands that create morphogen or chemokine gradients in development and inflammation, and numerous other contributions to signaling and cell surface structure. Despite their critical importance in diverse biological processes, very little is known about the details of PG interactions with binding partners or their mechanisms of biological function. The Moremen lab directs and is involved in a multi-investigator project (Resource for Integrated Glycotechnology) with goals to develop a novel, integrated technology toolkit that will address the challenges of PG structures, interactions, and biological functions by leveraging advances in analytical, synthetic, structural, biochemical and biological tools. The role of the Moremen lab in the project is focused on recombinant, genetic and biochemical approaches to assess PG functions. Large-scale expression strategies have been developed for glycoprotein production in mammalian cells for structural and functional studies. Sparse labeling (13C/15N) of amino acids in recombinant products as well as development of strategies for incorporation of paramagnetic tags are the focus of ongoing studies to provide reagents for NMR structural studies, particularly beneficial for studying large systems. In addition, recombinant expression of PG core proteins has been achieved in the last funding period providing the opportunity to generate altered polymer structures by metabolic engineering. These molecules can be produced in large quantities and will act as alternative PG sources for interaction studies, as well as ligands for array binding analysis and biological studies on the influence of higher order oligomerization. Recombinant binding proteins and the isolated PGs derived from recombinant expression will be employed by all of the Technology Projects in the Resources as well as numerous Driving Biomedical Projects as key reagents to probe biological functions. This project is funded by an NIH P41 grant (P41GM103390, Resource for Integrated Glycotechnology). For further information please see: glycotech.uga.edu.

3. Development of methods for determining the levels of transcripts for glycan-related genes in animal systems: The third area of focus in the Moremen lab is a collaborative project focused on determining the structures and regulation of glycans associated with glycoproteins and glycolipids in animal systems. The overall aims of the program are to examine the changes glycan structures and proteome during development in a mammalian system, and in particular embryonic stem cell differentiation. The aims of the Moremen lab project are to apply our custom real-time RT-PCR strategy and RNA-Seq for measuring transcript abundance for all glycan-related genes in mouse ES cells. The technology employs a medium-throughput robotic real-time RT-PCR strategy to measure mRNA transcript levels over 7-orders-of-magnitude. Integration of the transcript abundance data with the glycan structure analysis developed by other research groups in the CCRC will be accomplished through the bioinformatics group associated with the research program. This project is funded by an NIH P41 grant (P41GM103490,National Center for Biomedical Glycomics). For further information please see: glycomics.ccrc.uga.edu

4. Structure, function, and biochemistry of enzymes involved in glycoprotein biosynthesis and catabolism: The third area of focus in the moremen lab is on the enzymes involved in glycan trimming in the secretory pathway that are essential for N-glycan maturation as well as playing a role in the catabolism of misfolded nascent glycoprotiens in the endoplasmic reticulum (ER-associated degradation). The major accomplishments of this project have been the identification and isolation of the structural genes and cDNAs encoding several of the enzymes involved in glycoprotein biosynthesis and catabolism and the protein structure determination of members of one of the families in the presence and absence of competitive inhibitors, substrate analogs , and intact substrates.

a. Class 1 (CAZy GH47) mannosidases in glycoprotein processing and quality control: The class 1 (CAZy GH47) mannosidases act as the committed steps in the synthesis of complex oligosaccharides by determining the extent of mannose trimming, and in some cases appear to control the rate of unfolded glycoproteins in the endoplasmic reticulum. The cloning of several of mammalian class 1 mannosidases by the lab has allowed a more detailed characterization of the roles of these enzymes in glycoprotein maturation and quality control degradation. The lab employs approaches of structure determination, enzymatic and biochemical characterization, cloning and expression of novel family members, and cell biology studies in mammalian and yeast model systems to investigate the roles, functions, and mechanisms of the individual family members in glycoprotein maturation and ER associated degradation of unfolded glycoproteins.The lab has determined the structure of wild type and mutant processing mannosidases that they have expressed and purified in multi-milligram quantities.

b. Class 2 (CAZy GH 38) processing mannosdiases in glycoprotein maturation: The projects on the processing mannosidases also include studies on the later Golgi processing mannosidases, including Golgi mannosidase II and Golgi mannosidase IIx. These studies are directed at the identification of the individual roles of the processing mannosidases in glycoprotein maturation and the consequences of genetic alterations in human disease.