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Mammalian Protein Glycosylation in Cellular Mechanisms of Health and Disease
Summary: Jamey Marth is investigating the biological functions of protein glycosylation and the cellular mechanisms by which this type of post-translational modification regulates mammalian physiology and participates in disease pathogenesis.
Structural modifications of proteins are essential for the normal activities of living cells among all organisms, and yet such modifications when aberrantly regulated are often the basis of disease. Glycosylation is a major form of protein modification that involves the addition of monosaccharides that for the most part occurs in the secretory pathway, especially in the Golgi apparatus, resulting in glycoproteins that are then typically trafficked to the cell surface and reside among extracellular compartments. These saccharide linkages, often termed glycans, may be the most abundant and diverse of nature's biopolymers, contributing a significant amount of structural variation in biologic systems. Glycans constitute one of the four basic components of cells, along with nucleic acids (DNA and RNA), lipids, and proteins, and their repertoire at the cell surface composes what is termed the glycome.
The mammalian genome has conserved approximately 1–2 percent of genes that encode enzymes to produce and modify glycan structures (Figure 1). Most of these enzymes are glycosyltransferases that are responsible for producing glycan linkages in the process of glycosylation. Glycans bearing multiple saccharide linkages may exist as linear as well as branched structures, resulting in a structural repertoire that is estimated in the thousands and may be similar to or exceed the diversity of the proteome (Figure 2). During embryogenesis, normal adult physiology, and in many disease states, the appearance of different glycan linkages occurs by altered gene expression and other forms of regulation involving glycosyltransferases and glycan-modifying enzymes.
We have found that genetic approaches and intact model organisms can be used to establish the purpose of glycosylation and its regulation. The mystery of what glycans do and the mechanisms by which they function to control health and disease are the focus of research in this laboratory. Enforcing or inhibiting the activity of specific glycosyltransferases and glycosidases can protect against disease, as well as intervene in disease pathogenesis and provide therapeutic benefits in models of inflammation, autoimmune disease, hematologic disorders, and neurologic activity. Examples of fundamental and potentially translational discoveries involving protein glycosylation are provided below.
Defects in Protein Glycosylation That Affect Pre- and Postnatal Development
The inherited human genetic diseases termed congenital disorders of glycosylation (CDGs) include many separate defects in the formation of asparagine (N)-linked glycans. The frequency of CDG cases in the human population is not known, as the clinical diagnosis of CDG is inexact and infrequently employed. An example is human CDG type IIa, a severe childhood disorder resulting from the absence of the Golgi-localized enzyme GlcNAcT-II produced by the MGAT2 gene. Inherited loss of MGAT2 gene function results in a block in the production of complex N-glycan structures and thus has a broad and severe impact on formation of the cell's glycome (Figure 3).
To determine whether the CDGs can be modeled in the mouse to provide insights into glycan-dependent disease mechanisms and reveal possible therapeutic approaches, we compared two mammalian species with the same defect in glycan formation. We recapitulated in the mouse an inherited mutation of the Mgat2 gene resulting in the biochemical loss of GlcNAcT-II enzyme activity. Disease signs in the mouse were similar to those reported in the literature among humans with CDG-IIa, indicating significant conservation of N-glycan structure-function relationships through the evolutionary time that separates these two mammalian species. These findings suggested the potential for misdiagnosis and underdiagnosis among humans and a possible contribution of CDG-IIa in some cases of gestational and perinatal death.
Glycan Control of Protein Function in Neural Connectivity and Brain Morphogenesis
The developing neural system and adult brain regions associated with neurogenesis express a glycan structure known as polysialic acid (PSA). Polysialyltransferases are glycosyltransferases responsible for placing this modification on the neural cell adhesion molecule (NCAM), the predominant carrier of PSA glycans. We recently found that polysialyltransferase ST8Sia-II produces distinct modifications to neuronal axonal trafficking that correlate with anxiety and fear behavior (Figure 4). This is associated with morphologic alterations in the brain involving mossy fiber tracts and synapse formation in the hippocampus. Similar topographic alterations in mossy fiber axonal projections have been correlated with emotional cognition and fear behavior among different mouse strains and in response to environmental stimuli. By comparisons to studies published by others involving mice lacking the second polysialyltransferase, ST8Sia-IV, and animals lacking NCAM, it was possible to infer the contribution of PSA to NCAM function. We observed that these two polysialyltransferases promote distinct neurologic activities and that the majority of NCAM function is attributable to the attached PSA glycan structure. In further collaborative studies, we found that loss of PSA is lethal, while the additional loss of NCAM restores viability. Therefore, PSA is the predominant carrier of biological information in this system, controlling the function of the NCAM glycoprotein in directing neuronal connectivity and suppressing the otherwise lethal effect of NCAM expression during early brain development.
The High-Fat Diet and Glycan-Dependent Pathogenesis of Type 2 Diabetes
Type 2 diabetes, which is increasing to epidemic proportions worldwide, has been associated with obesity and a high-fat diet. The initial disease process is diagnosed by high blood glucose levels, and pancreatic beta cell failure occurs with deregulation of normal insulin secretion mechanisms, followed by the development of insulin resistance. We have recently discovered a dietary and genetic mechanism of pancreatic beta cell failure that results in the pathogenesis of type 2 diabetes. The mechanism involves insufficient expression of the Mgat4a-encoded GnT-4a glycosyltransferase. Remarkably, reduced Mgat4a expression occurs among mice fed a high-fat diet, as well as type 2 diabetic human islet cells. Reduced levels of GnT-4a activity lead to loss of pancreatic beta cell surface glucose transporter expression, by what appears to be the disruption of a lectin-based cell surface retention mechanism (Figure 5). The resulting hyperglycemia and insulinopenia appear to drive early disease progression, leading to insulin resistance. We are investigating whether this pathogenic mechanism also exists in the human population and whether constitutively high levels of Mgat4a gene expression provide resistance to the diabetogenic effects of the high-fat diet.
Glycans as Signal Modulators in Adaptive Immunity
Glycan structures often contain a sialic acid linkage at the distal tip of a glycan branch, placing this negatively charged monosaccharide in an appropriate location to form or mask biological ligands of endogenous lectins. The ST6Gal-I sialyltransferase participates by adding sialic acid to underlying galactose on N-glycan branches, thereby producing the endogenous ligand of the B lymphocyte–specific lectin and immune signal attenuator CD22. We recently published findings that show ST6Gal-I suppresses CD22-dependent B cell antigen receptor endocytosis and maintains the normal induction of protein-tyrosine phosphorylation among key molecules that transmit immune activation signals from the B lymphocyte antigen receptor. ST6Gal-I protein glycosylation controls the degree of B lymphocyte receptor colocalization with CD22 and the amount of intracellular Shp-1 recruited to CD22. These findings provide a mechanistic explanation for how the threshold of immune activation is established in B lymphocytes. Our studies revealed that glycans produced by ST6Gal-I control trafficking and colocalization involving specific B lymphocyte cell surface glycoproteins, thereby modulating intracellular signal transduction (Figure 6). Remarkably, loss of ST6Gal-I function alleviates autoimmune pathogenesis in a mouse model of severe systemic lupus erythematosus due to Lyn tyrosine kinase deficiency, suggesting a novel therapeutic approach to autoimmune diseases.
Aberrant Protein Glycosylation in Autoimmune Disease
Loss of alpha-mannosidase II function results in a dyserythropoietic anemia similar to human congenital dyserythropoietic anemia type II. Unexpectedly, nonerythroid cells among mice lacking alpha-mannosidase II continue to produce complex N-glycans to varying degrees by the expression of a second alpha-mannosidase activity (Figure 3). This bypass mechanism does not fully compensate, however, and as these mice age they develop autoimmune disease signs that are diagnostic of systemic lupus erythematosus. We are identifying the cell types and mechanisms by which altered protein glycosylation causes this autoimmune disease in mice. Our recent findings implicate a novel mechanism of autoimmune disease pathogenesis mediated by chronic stimulation of the innate immune system.
Further Development of Conditional Gene Mutagenesis and Its Application to Cytoplasmic Protein Glycosylation
This laboratory has continued to develop techniques to enhance the use of gene targeting by transgenic applications of the Cre-loxP recombination system. We have recently shown that this approach to conditional gene mutagenesis (Figure 7) includes a method to produce heterozygous female animals bearing mutations on X chromosome–linked genes that are necessary for embryogenesis. By separately manipulating maternal and paternal X-linked gene function in the mouse germline, we were able to generate heterozygous female mice bearing mutations of the X chromosome–linked Ogt gene, which is otherwise necessary for embryo development and viability. The encoded Ogt glycosyltransferase is responsible for the formation of a single glycan linkage on cytoplasmic and nuclear proteins at sites often otherwise phosphorylated. Our findings imply that all somatic cell types require Ogt activity for survival, and support the view that this form of protein glycosylation modulates essential and conserved signaling pathways in mammalian cells.
Research encompassing glycobiology provides insights into cell function that can explain how extracellular signals are composed and how cell-cell communication is established among multicellular organisms. Discoveries in this field often integrate knowledge presently relegated to distinct scientific disciplines and will contribute to the advances needed to decode the biologic systems that comprise living organisms.
Grants from the National Institutes of Health have provided support for some of these projects.
Last updated: August 7, 2008
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