Work in the Moremen lab is focused on four main areas: (1) characterization of mammalian glycoprotein biosynthesis and catabolism by the cloning and characterization of the enzymes involved in these processes, (2) identification of inhibitors of glycoprotein processing and maturation as potential anti-metastatic therapeutic agents, (3) determination of the structural and molecular basis for the interaction between glycosidases and glycosyltransferases and their corresponding substrates and (4) developing technologies for measuring transcript abundance for glycan-related genes. Each of these research programs is described below and is supported by grant funding by the National Institutes of Health. The latter three projects are collaborative projects with other members of the Complex Carbohydrate Research Center.

1. Structure, function, and biochemistry of enzymes involved in glycoprotein biosynthesis and catabolism. This research program is the most comprehensive of the projects in the lab and is supported by an NIH grant. 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. The group's determination that these enzymes are members of larger multi-gene families that are differentially expressed in a tissue- and cell-specific manner has allowed them to use the conserved elements among the family members to isolate additional novel family members. Several projects have been undertaken as an extension of the initial cloning work.

A. Class 1 mannosidases in glycoprotein processing and quality control: The class 1 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 members of 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. In collaboration with Dr. Lynne Howell (Hospital for Sick Children, Toronto) and Dr. B.C. Wang (University of Georgia) the lab has determined the structure of wild type and mutant processing mannosidases that they have expressed and purified in multi-milligram quantities (see web report and Chime files for a summary of the studies on these enzymes).

B. Class 2 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 include ongoing collaborations with Dr. Michiko Fukuda in the cloning, expression, and characterization of the genetic basis of the naturally ocurring human deficiency in processing mannosidases that result in congenital dyserythropoetic anemia type II (HEMPAS), as well as characterization of the enzymology of glycoprotein maturation in mice deficient in Golgi mannosidase II in collaboration with Jamey Marth (UCSD). 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.

C. Lysosomal a-mannosidase: The recent cloning of the human and mouse cDNAs and genes encoding the lysosomal a-mannosidase by members of the lab has allowed the identification of the molecular basis of the human genetic defects in this enzyme. Human patients with this enzymatic defect have classical symptoms of a lysosomal storage disease: severe neural, skeletal, and immune system defects; proliferation of lysosomes in most cell types resulting from the accumulation of undegraded oligosaccharides, and elevated tissue, serum, and urinary oligosaccharide levels. The Moremen group has expressed large quantities of the recombinant human lysosomal a-mannosidase in Pichia pastoris and mammalian cells. These enzymes are presently being characterized in biochemical, enzymatic, and structural studies for potential use in enzyme replacement therapies for the lysosomal storage disease. In addition members of the lab have characterized the organization of the human and mouse lysosomal a-mannosidase genes for potential use in gene replacement therapy. Recombinant expression of the mouse lysosomal a-mannosidase has been accomplished as a prelude to testing its efficacy a mouse model for the enzymatic deficiency by enzyme replacement approaches.

2. Identification of inhibitors of glycoprotein processing and maturation as potential anti-metastatic therapeutic agents: At the cellular level, N- and O-glycan structures have been shown to contribute to several aspects of biological recognition, including cell adhesion events during immune surveillance, inflammatory reactions, hormone action, arthritis, and viral infections. Although some of the roles of N-linked glycans in cell adhesion have been identified, many of the details relating to the dynamics of cell adhesion events and changes in cell surface carbohydrate structures remain unresolved. The cell- and tissue-specific changes in cell surface oligosaccharides during development have indicated that these structures may be involved in cell adhesion and migration events during embryogenesis. Alterations in the branching and extension of N-glycans have also been found on the surfaces of cells that have undergone oncogenic transformation and these changes correlate with alterations in cell adhesion that contribute to the invasiveness and metastatic potential of malignant cells. A model has been developed over the last decade linking oncogene activation to the induction of oligosaccharide branching and extension at the cell surface. These changes in oligosaccharide structure have a direct influence on the cell adhesion characteristics of the transformed cells and contribute to the development of the metastatic phenotype. a-Mannosidase inhibitors that act late in the N-glycan processing pathway provide one route to blocking the oncogene-induced changes in cell surface oligosaccharide structures. By inducing the formation of altered structures, the acceptor for oligosaccharide branching is no longer available. Although these compounds have been shown to exhibit potent anti-tumor and anti-metastatic activities they also lead to the serious complications as result of their activity toward the catabolic lysosomal a-mannosidases, causing a phenocopy of the lysosomal storage disease, a-mannosidosis. A collaborative group in the Complex Carbohydrate Research Center including Drs. Moremen, Pierce, Boons, Siriwardena, have taken a biochemical and organic synthetic approach for the development of selective inhibitors for N-glycan maturation that should inhibit tumor progression. Members of the Moremen lab have cloned, expressed, and characterized both the processing and catabolic a-mannosidases from a variety of mammalian sources and have established a strategy for the screening of inhibitors for selectivity toward the N-glycan processing enzyme. The Boons/Siriwardena group has established a chemical approach whereby glycosidase lead inhibitors can be efficiently modified with a view to accentuating their potency and selectivity for their target enzymes. The joint project will result in the synthesis of large numbers of diverse analogs derived from lead compounds and to test them in vitro against the enzymatic targets (Moremen group) as well as in tissue culture (Pierce group) in an effort to identify selective, high-affinity, cell permeable anti-metastatic agents.
 

3. Determination of the structural and molecular basis for the interaction between glycosidases and glycosyltransferases and their corresponding substrates: The third area of research is a collaborative project with three research groups in the Complex Carbohydrate Research Center. 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 three research programs within the CCRC: a program in the Moremen lab to express large quantities of the protein by recombinant methods, a program in the Pierce lab to synthesize large quantities of the oligosaccharide ligands in an isotope-enriched form, and the analysis of the interactions of the isotope-enriched oligosaccharides with the isotope-enriched binding protein by high-field NMR in the Prestegard lab.
 

4. Development of methods for determining the levels of transcripts for glycan-related genes in animal systems: The fourth area is a collaborative project focused on determining the structurea and regulation of glycan structures associated with glycoproteins and glycolipids in animal systems. The overall proposed studies 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 develop a real-time RT-PCR strategy for measuring transcript abundance for all glycan-related genes in mouse ES cells. The technology being developed employs a medium-throughput robotic real-time RT-PCR strategy to measure mRNA transcript levels over 7-orders-of-magnitude. Present technologies commonly employ microarray or SAGE methods for large-scale transcript analysis, but several limitations inherent in these technologies suggest that the real-time RT-PCR strategy developed in this project will be superior for quantifying the low-abundance glycosidase and glycosyltransferase transcripts and determining their alterations during development. 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. For further information on the CCRC initiative on the Integrated Technology Resource for Biomedical Glycomics please see the web site HERE.