Review
Glycoprotein glycosylation and cancer progression

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Abstract

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 β1,6GlcNAc-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 β1,6GlcNAc-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 β1,6GlcNAc-branching of N-glycans are associated with cell motility, a required phenotype of malignant cells.

Introduction

The N- and O-glycans are structurally diverse and widely distributed on cell surface and secreted glycoproteins where they are thought to function in cell–cell communication. With the recent sequencing of the Caenorhabditis elegans genome [1], it is apparent that the number of genes dedicated to glycoconjugate biosynthesis and the conservation of many of these genes with mammals is very substantial. Conserved glycan functions may include cellular processes and signaling pathways common to all metazoans. Glycans are structural components of glycoprotein receptors that transmit information between cells and the environment to control fundamental aspects of cell behavior. Indeed, many receptors, cytokines and signaling pathways are conserved between mammals and C. elegans [2]. Recent studies on targeted mutations of glycosyltransferase genes in mice and ectopic expression of glycosyltransferases in cell lines suggest that tissue-specific and disease-associated programs of glycosylation may regulate the activity of certain receptors, and thereby cell behavior.

The structural variability of glycans is dictated by tissue-specific regulation of glycosyltransferase genes, the availability of sugar nucleotides, and competition between enzymes for acceptor intermediates during glycan elongation. A number of substrate–product relationships govern N-glycan biosynthesis, and many of these were described Schachter’s group [3]. Briefly, the GlcNAc-TI product is required for substrate recognition by α-mannosidase II and GlcNAc-TII, and the subsequent biosynthesis of the complex-type N-glycans. GlcNAc-TV initiates a specific branch that is variably elongation in the trans-Golgi generating structural diversity in the mature N-glycans. The GlcNAc-TV product is the preferred intermediate for extension with polylactosamine chains (i.e. Galβ1,4GlcNAcβ1,3 repeating units of 2 to >10 in length) [4]. GlcNAc-TV also appears to be a rate-limiting enzyme for polylactosamine addition to N-glycans in tumor cells. In this regard, GlcNAc-TV-deficient BW5147-PHAR2.1 lymphoma cells [5], and MDAY-D2 tumor cell mutants [6] are depleted in N-glycan polylactosmine, while O-glycan was unaffected. Polylactosamine adds considerable heterogeneity in the form of both polymer length and capping with various sequences including the Lewis antigens. Polylactosamine synthesis is affected by β1,3GlcNAc-T(i) activity [7], glycoprotein transit time in the trans-Golgi [8], and competition by chain-terminating enzymes including α1,2Fuc-T and α2,6SA-T [9]. Thus control of polylactosamine and Lewis antigen expression appears to be, in large measure, a consequence of substrate preference, enzyme competition and tissue-specific regulation of glycosyltransferases.

A range of biosynthetically related glycan structures may be present at any particular glycosylation site of a mature glycoprotein [10]. In this manner, heterogeneous glycosylation creates structural diversity within individual glycoprotein populations, and this may be the most significant feature of glycan function. Each ‘glycoform’ may have different molecular properties affecting half-life or their intrinsic biological activity. The incidence of different glycoforms in the molecular population can thereby affect overall potency; and may be viewed as producing a gradient of biological activity. For example, certain glycan sequences on serum glycoproteins bind to hepatic lectins, which mediate their clearance. The glycopeptide hormones are produced in the pituitary with glycans bearing either terminal N-acetylgalactosamine-4 sulfate or sialic acid, and these glycoforms differ in liver clearance rates and biological activity [11]. Similarly, relative levels of sialylated and branched N-glycans on erythropoietin affect the stability and activity of the glycoprotein in vivo [12]. Other examples include the glycoform heterogeneity observed for CD44 [13], ICAM-1 [14] and CD43 [15] cell surface receptors, which is suggested to modify their cell adhesion activities.

N-Glycan biosynthesis can be viewed in four distinct phases, each associated with different compartments of the secretory pathway [16]: (1) transfer of Glc3Man9GlcNAc2 oligosaccharide from a dolichol-linked donor to nascent glycoproteins in the lumen of the rough ER; (2) glycosidase-mediated trimming in the rough ER and Golgi; (3) substitution by GlcNAc-Ts in the medial-Golgi; and (4) elongation in the trans-Golgi network to complete the glycan structures.

These phases may reflect the evolution of the biosynthetic pathway, as well as an increased demand for the structural and functional complexity of N-glycans in metazoans. Bacteria do not N-glycosylate via the dolichol pathway, and yeast make only oligomannose-type N-glycans. A critical evolutionary event may have been the appearance of GlcNAc-TI, and the specialization of the medial-Golgi compartment. GlcNAc-TI is found in all metazoans and plants examined to date, but not in protozoa. This correlates with an evolutionary boundary between uni- and multi-cellular organisms, and suggests that complex-type N-glycans may well have arisen originally in response to a need for mechanisms of intercellular interaction. Certainly, glycosylation of proteins occurs in the ER–Golgi complex, an organelle dedicated to the production of cell surface and secreted materials. However, plant and animal kingdoms may have diverged in their exploitation of complex-type N-glycans, as suggested by the observation that GlcNAc-TI is dispensable for plant growth [17], but required for embryogenesis in mice [18], [19].

Glycan binding to lectins is a common means of adhesion between multicellular organisms and parasitic or symbiotic organisms. Different terminal glycan sequences on glycoproteins can also mask lectin-binding sites for parasite infection [20]. The influenza virus surface hemagglutinin-neuraminidase recognizes sialic acids present in bronchial epithelium, and strain variation affects binding specificity, cell surface attachment and virulence [21]. Structural diversification of glycans in metazoans may have enhanced population fitness as a tactic for evading parasitic organisms. However, newly evolved glycan structures were likely co-opted into functions unrelated to pathogen evasion. With their predominantly extracellular location, these functions appear to be associated with cell–cell interactions of metazoans. Conserved glycan structures appear to have become acceptors, intermediates for newly evolved glycosyltransferases, expanding the pathway and glycan diversity. The earlier the biosynthetic capacity for an oligosaccharide structure arose, the more likely that the glycans have acquired functional niches within the biology of the organism. Thus, mutations in upstream, and evolutionarily ancient, parts of the glycosylation pathway will affect many downstream oligosaccharide structures, many of which will have been co-opted into developmental functions. Indeed, the phenotypes of null mutations in glycosyltransferase genes in mice appear to be progressively milder for enzymes operating later in the biosynthetic pathway [22]. GlcNAc-TI-deficient mouse embryos (Mgat1−/−) lack all complex-type N-glycans, and they die at around E9.5 due to a failure of multiple organ systems [18], [19], whereas mutations affecting subsets of complex-type N-glycans are less severe. GalT1−/− [23], Mgat3−/− [24] and Mgat5−/− mice are viable (Granovsky et al., submitted). Characterization of these mutant embryos and mice suggests that specific subsets of glycan structures have cell-type specific functions that can affect cell differentiation, growth and migration. These cellular processes are central to cancer development and metastasis.

Section snippets

GlcNAc-TV and tumor progression

Rat GlcNAc-TV protein is a 740 amino acid type II transmembrane glycoprotein, the characteristic organization for Golgi enzymes [25]. The C-terminal portion of rat GlcNAc-TV comprising S213–740 is essential for the catalytic activity [26]. A region required for Golgi localization is defined by a point mutation in the GlcNAc-TV gene of Lec4A mutant cells. This missense mutation changes Leu to Arg at position 188, and causes mis-localization of the enzyme without affecting catalytic activity [27]

Ectopic expression of GlcNAc-TV and GlcNAc-III

To test for transforming activity of GlcNAc-TV, an immortalized lung epithelial cell line (Mv1Lu) was transfected with a GlcNAc-TV expression vector [56]. The GlcNAc-TV expressing transfectants formed tumors when injected into nude mice, while control cell lines were non-tumorigenic. Tumor growth showed an initial slow phase and at 4–6 months a rapid growth phase suggesting the occurrence in vivo of an additional genetic event. In cell culture, the GlcNAc-TV-transfected cells showed loss of

T-cell receptor function in GlcNAc-TV-deficient mice

As suggested by the studies discussed above, the kinetics of receptor aggregation might be regulated by glycosylation, and thereby influence intracellular signaling. Mgat5-dependent glycosylation in T-cells affects the kinetics of agonist-induced T-cell receptor (TCR) aggregation and signaling (Granovsky et al., submitted). Rates of TCR internalization immediately following addition of agonist were increased for Mgat5−/− T-cells compared to wild-type cells. This results in a marked increase in

Tumor growth and metastasis is suppressed in GlcNAc-TV-deficient mice

Mutant MDAY-D2 tumor cell lines selected resistance to L-PHA and deficient in GlcNAc-TV produced 95% fewer spontaneous metastases in liver, and solid tumor growth rate was ∼50% slower compared to wild-type cells [33]. Similarly, GlcNAc-TV-deficient mutants of 168.1 mammary tumor cell line cells formed 2–10 times fewer colonies in the lungs following intravenous injection [67]. However, as the nature of the mutations in these cell lines remains uncharacterized, it may be argued that GlcNAc-TV

Polylactosamine

Polylactosamine is found on O- and N-glycans, and both classes of glycans may affect malignant cell behavior. O-Glycans in cancer cells show increased β1,6GlcNAc branching and polylactosamine due to increased expression of core 2 GlcNAc-T [6], [77]. Inhibition of GalNAc-Ser/Thr elongation by treating tumor cells with benzyl-α-GalNAc reduces organ colonization [78]. Furthermore, MDAY-D2 mutants deficient in UDP-Gal transport lack Gal in both O- and N-glycans, and of all mutants examined, show

Sialylation and Lewis antigens

Terminal N- and O-glycan sequences contribute to malignancy, as suggested by studies on tumor cell glycosylation mutations. An MDAY-D2 mutant over-expressing α2,6SA-T by 40-fold, showed 3–10-fold fewer metastases and 60% slower tumor growth. The mutant cells had predominantly α2-6SA rather than the wild-type α2,3SA on the cell surface [86], and the mutation is due to a retroviral insertion into the ST6Gal gene promoter (Lo et al., in prep.). Loss of sialylation in mutant of B16 melanoma

Carbohydrate processing inhibitors as anti-cancer agents

The alkaloids swainsonine and castanospermine block tumor cell metastasis and invasion through extracellular matrix in vitro [100]. Swainsonine is a competitive inhibitor of Golgi α-mannosidaseII, which blocks the N-glycan biosynthetic pathway prior to β1,6GlcNAc-branching. Swainsonine-treated cells, as well as GlcNAc-TV-deficient mutant cells, showed increased transcription rates for tissue inhibitor of metalloproteinases (TIMP-1) [101]. Swainsonine also suppresses MMP-2 expression in human

Future directions

The Magt5 null mutation antagonizes tumor progression and metastasis in PyMT transgenic mice. In addition, leukocyte migration into sites of inflammation in the Magt5 null mice was reduced in vivo. Focal-adhesion formation by embryonic fibroblasts and tumor cells in cell culture was impaired in Magt5 null cells. These observations suggest a role for GlcNAc-TV glycosylation in the regulation of cell motility. Cell motility propagates intracellular signals that intersect with growth factor

Acknowledgments

The authors thank the National Cancer Institute of Canada, the Mizutani Foundation, the National Science and Engineering Research Council of Canada and GlycoDesign Inc., Toronto for research grants awarded to J.W.D.

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