Charles E. Warren

Glycoprotein glycosylation and cancer progression.

Glycosylation of glycoproteins and glycolipids is one of many molecular changes that accompany malignant transformation. GlcNAc-branched N-glycans and terminal Lewis antigen sequences have been observed to increase in some cancers, and to correlate with poor prognosis. Herein, we review evidence that beta1, 6GlcNAc-branching of N-glycans contributes directly to cancer progression, and we consider possible functions for the glycans. Mgat5 encodes N-acetylglucosaminyltransferase V (GlcNAc-TV), the Golgi enzyme required in the biosynthesis of beta1,6GlcNAc-branched N-glycans. Mgat5 expression is regulated by RAS-RAF-MAPK, a signaling pathway commonly activated in tumor cells. Ectopic expression of GlcNAc-TV in epithelial cells results in morphological transformation and tumor growth in mice, and over expression in carcinoma cells has been shown to induce metastatic spread. Ectopic expression of GlcNAc-TIII, an enzyme that competes with GlcNAc-TV for acceptor, suppresses metastasis in B16 melanoma cells. Furthermore, breast cancer progression and metastasis induced by a viral oncogene expressed in transgenic mice is markedly suppressed in a GlcNAc-TV-deficient background. Mgat5 gene expression and beta1, 6GlcNAc-branching of N-glycans are associated with cell motility, a required phenotype of malignant cells.

Protein glycosylation in development and disease

N‐ and O‐linked glycan structures of cell surface and secreted glycoproteins serve a variety of functions related to cell–cell communication in systems affecting development and disease. The more sophisticated N‐glycan biosynthesis pathway of metazoans diverges from that of yeast with the appearance of the medial‐Golgi β‐N‐acetylglucosaminyltransferases (GlcNAc‐Ts). Tissue‐specific regulation of medial‐ and trans‐Golgi glycosyltransferases contribute structural diversity to glycoproteins in metazoans, and this can affect their molecular properties including localization, half‐life, and biological activity. Null mutations in glycosyltransferase genes positioned later in the biosynthetic pathway disrupt expression of smaller subsets of glycan structures and are progressively milder in phenotype. In this review, we examine data on targeted mutations affecting glycosylation in mice and congenital mutations in man, with a view to understanding the molecular functions of glycan structures as modulators of glycoprotein activity. Finally, pathology associated with the expression of GlcNAc‐Ts in cancer and diabetes‐induced cardiac hypertrophy suggest that inhibitors of these enzymes may have therapeutic value. BioEssays 21:412–421, 1999.

Identification and Characterization of a Gene Regulating Enzymatic Glycosylation which is Induced by Diabetes and Hyperglycemia Specifically in Rat Cardiac Tissue

Primary cardiac abnormalities have been frequently reported in patients with diabetes probably due to metabolic consequences of the disease. Approximately 2,000 mRNA species from the heart of streptozotocin-induced diabetic and control rats were compared by the mRNA differential display method, two of eight candidate clones thus isolated (DH1 and 13) were confirmed by Northern blot analysis. The expression of clone 13 was increased in the heart by 3.5-fold (P < 0.05) and decreased in the aorta by twofold (P < 0.05) in diabetes as compared to control. Sequence analysis showed that clone 13 is a rat mitochondrial gene. DH1 was predominantly expressed in the heart with an expression level 6.8-fold higher in the diabetic rats than in control (P < 0.001). Insulin treatment significantly (P < 0.001) normalized the expression of DH1 in the hearts of diabetic rats. DH1 expression was observed in cultured rat cardiomyocytes, but not in aortic smooth muscle cells or in cardiac derived fibroblasts. The expression in cardiomyocytes was regulated by insulin and glucose concentration of culture media. The full length cDNA of DH1 had a single open-reading frame with 85 and 92% amino acid identity to human and mouse UDP-GlcNAc:Gal beta 1-3GalNAc alpha R beta 1-6 N-acetylglucosaminyltransferase (core 2 GlcNAc-T), respectively, a key enzyme determining the structure of O-linked glycosylation. Transient transfection of DH1 cDNA into Cos7 cells conferred core 2 GlcNAc-T enzyme activity. In vivo, core 2 GlcNAc-T activity was increased by 82% (P < 0.05) in diabetic hearts vs controls, while the enzymes GlcNAc-TI and GlcNAc-TV responsible for N-linked glycosylation were unchanged. These results suggest that core 2 GlcNAc-T is specifically induced in the heart by diabetes or hyperglycemia. The induction of this enzyme may be responsible for the increase in the deposition of glycoconjugates and the abnormal functions found in the hearts of diabetic rats.

Overexpression of core 2 N-acetylglycosaminyltransferase enhances cytokine actions and induces hypertrophic myocardium in transgenic mice

Elevated levels of glycocojugates, commonly observed in the myocardium of diabetic animals and patients, are postulated to contribute to the myocardial dysfunction in diabetes. Previously, we reported that UDP‐GlcNAc: Galβ 1–3GalNAcαRβ 1–6‐N‐acetylglucosaminyltransferase (core 2 GlcNAc‐T), a developmentally regulated enzyme of O‐linked glycans biosynthesis pathway, is specifically increased in the heart of diabetic animals and is regulated by hyperglycemia and insulin. In this study, transgenic mice overexpressing core 2 GlcNAc‐T with severe increase in cardiac core 2 GlcNAc‐T activities were normal at birth but showed progressive and significant cardiac hypertrophy at 6 months of age. The heart of transgenic mice showed elevation of sialylated O‐glycan and increases of c‐fos gene expression and AP‐1 activity, which are characteristics of cardiac stress. Furthermore, transfection of PC12 cells with core 2 GlcNAc‐T also induced c‐fos promoter activation, mitogen activated‐protein kinase (MAPK) phosphorylation, Trk receptor glyco‐sylation, and cell differentiation. These results suggested a novel role for core 2 GlcNAc‐T in the development of diabetic cardiomyopathy and modulation of the MAP kinase pathway in the heart.—Koya, D., Dennis, J. W., Warren, C. E., Takahara, N., Schoen, F. J., Nishio, Y., Nakajima, T., Lipes, M. A., King, G. L. Overexpression of core 2 N‐acetylglyco‐saminyltransferase enhances cytokine actions and induces hypertrophic myocardium in transgenic mice. FASEB J. 13, 2329–2337 (1999)

Genetic defects in N-glycosylation and cellular diversity in mammals

Glycoproteins in mammalian cells are modified with complex-type aspargine-linked glycans of variable chain lengths and composition. Observations of mice carrying mutations in glycosyltransferase genes imply that N-glycan structures regulate T-cell receptor clustering and hence sensitivity to agonists. We argue that the heterogeneity inherent in N-glycosylation contributes to cellular diversity and, thereby, to adaptability in the immune system.

Modeling a congenital disorder of glycosylation type I in C. elegans: A genome-wide RNAi screen for N-glycosylation-dependent loci

Inefficient glycosylation caused by defective synthesis of lipid-linked oligosaccharide donor results in multi-systemic syndromes known as congenital disorders of glycosylation type I (CDG-I). Strong loss of function mutations are embryonic lethal, patients with partial losses of function are occasionally born but are very ill, presenting with defects in virtually every tissue. CDG-I clinical expression varies considerably and ranges from very mild to severe, and the underlying cause of the variable clinical features is not yet understood. We postulate that accompanying defects in an individuals genetic background enhance the severity of CDG-I clinical phenotypes. Since so many protein structures and functions are compromised in CDG-I illnesses, the gene products that are dependent on N-linked glycosylation which cause lethality or particular symptoms are difficult to resolve. The power of genetic silencing that is a characteristic of C. elegans has allowed us to systematically dissect the complex glycosylation phenotype observed in CDG-I patients into specific glycan-dependent gene products. To accomplish this, we inhibited glycosylation with a sub-phenotypic dose of tunicamycin, reduced single genes by RNA interference, and then sought loci where the combination caused a synthetic or dramatically enhanced phenotype. This screen has identified genes in C. elegans that require N-linked glycans to function properly as well as candidate gene homologues that may enhance the clinical severity of CDG-I disorders in humans.