The cellular microenvironment and cell adhesion: a role for O-glycosylation

Glycosylation is one of the most abundant protein modifications in Nature, having roles in protein stability, secretion and function. Alterations in mucin-type O-glycosylation are responsible for a number of human diseases and developmental defects, as well as associated with certain types of cancer. However, the mechanistic role of this form of glycosylation in many of these instances is unclear. Here we describe how one glycosyltransferase responsible for initiating mucin-type O-glycosylation (PGANT3), specifically modulates integrin-mediated cell adhesion by influencing the secretion and localization of an integrin ligand. The integrin ligand Tiggrin, is normally O-glycosylated and localized to the basal matrix, where adhesion of two opposing cell layers takes place. In pgant3 mutants, Tiggrin is no longer O-glycosylated and fails to be properly secreted to the basal cell layer interface, resulting in disruption of proper cell adhesion. pgant3-mediated effects are dependent on the enzymatic activity of PGANT3 and cannot be rescued by another pgant family member, indicating a unique role for this glycosyltransferase. These results provide in vivo evidence for the role of O-glycosylation in the secretion of specific extracellular matrix proteins, which thereby influences the composition of the cellular 'microenvironment' and modulates cell adhesion events. The studies described in this review provide insight into the long-standing association between aberrant O-glycosylation and tumorigenesis, as changes in tumour environment and cell adhesion are hallmarks of cancer progression.     

An O-Glycosyltransferase Promotes Cell Adhesion during Development by Influencing Secretion of an Extracellular Matrix Integrin Ligand

Protein secretion and localization are crucial during eukaryotic development, establishing local cell environments as well as mediating cell interactions, signaling, and adhesion. In this study, we demonstrate that the glycosyltransferase, pgant3, specifically modulates integrin-mediated cell adhesion by influencing the secretion and localization of the integrin ligand, Tiggrin. We demonstrate that Tiggrin is normally O-glycosylated and localized to the basal matrix where the dorsal and ventral cell layers adhere in wild type Drosophila wings. In pgant3 mutants, Tiggrin is no longer O-glycosylated and fails to be properly secreted to this basal cell layer interface, resulting in disruption of integrin-mediated cell adhesion in the wing. pgant3-mediated effects are dependent on enzymatic activity, as mutations that form a stable protein yet abrogate O-glycosyltransferase activity result in Tiggrin accumulation within the dorsal and ventral cells comprising the wing. Our results provide the first in vivo evidence for the role of O-glycosylation in the secretion of specific extracellular matrix proteins, thus altering the composition of the cellular “microenvironment” and thereby modulating developmentally regulated cell adhesion events. As alterations in cell adhesion are a hallmark of cancer progression, this work provides insight into the long-standing association between aberrant O-glycosylation and tumorigenesis.     

Multiple members of the UDP-GalNAc: polypeptide N-acetylgalactosaminyltransferase family are essential for viability in Drosophila

Mucin-type O-glycosylation represents a major form of post-translational modification that is conserved across most eukaryotic species. This type of glycosylation is initiated by a family of enzymes (GalNAc-Ts in mammals and PGANTs in Drosophila) whose members are expressed in distinct spatial and temporal patterns during development. Previous work from our group demonstrated that one member of this family is essential for viability and another member modulates extracellular matrix composition and integrin-mediated cell adhesion during development. To investigate whether other members of this family are essential, we employed RNA interference (RNAi) to each gene in vivo. Using this approach, we identified 4 additional pgant genes that are required for viability. Ubiquitous RNAi to pgant4, pgant5, pgant7, or the putative glycosyltransferase CG30463 resulted in lethality. Tissue-specific RNAi was also used to define the specific organ systems and tissues in which each essential family member is required. Interestingly, each essential pgant had a unique complement of tissues in which it was required. Additionally, certain tissues (mesoderm, digestive system, and tracheal system) required more than one pgant, suggesting unique functions for specific enzymes in these tissues. Expanding upon our RNAi results, we found that conventional mutations in pgant5 resulted in lethality and specific defects in specialized cells of the digestive tract, resulting in loss of proper digestive system acidification. In summary, our results highlight essential roles for O-glycosylation and specific members of the pgant family in many aspects of development and organogenesis.     

A UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase is required for epithelial tube formation

Epithelial tubes are essential for the proper function of a diverse array of eukaryotic organs. Here we present a novel class of genes required for maintaining epithelial cell shape, polarity, and paracellular barrier function in the Drosophila embryonic tracheal system. Mutations in one member of the UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase family (pgant35A) are recessive lethal and result in tracheal tubes that are irregular in diameter and morphology. Further analysis of the pgant35A mutants reveals diminished levels of the apical determinant Crbs and the luminal marker 2A12, concomitant with increased staining in cytoplasmic vesicles within tracheal cells. GalNAc-containing glycoproteins are severely diminished along the apical region of the tracheal system as well. Tracheal cells become irregular in size and shape, and septate junction proteins are mislocalized to a more apical position. Most notably, paracellular barrier function is lost in the tracheal system of the mutants. Overexpression of wild type pgant35A under control of the trachea-specific breathless (btl) promoter results in partial rescue of the lethality. We propose a model where pgant35A is required to establish proper apical composition of tracheal cells by influencing apical delivery of proteins/glycoproteins. Disruption of the normal apical content results in altered cell morphology and loss of paracellular barrier function. These studies demonstrate a previously unrecognized requirement for mucin-type O-glycosylation in epithelial tube integrity and have obvious implications for epithelial morphogenesis in higher eukaryotes, since a unique ortholog to pgant35A exists in mammals.     

An inhibitor of O-glycosylation induces apoptosis in NIH3T3 cells and developing mouse embryonic mandibular tissues

The family of UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferases (ppGaNTases) is responsible for initiating mucin-type O-linked glycosylation in higher eukaryotes. To begin to examine the biological role of O-linked glycosylation, mammalian cells were treated with a small molecule inhibitor (designated 1-68A, Ref. 15) of ppGaNTase activity. NIH3T3 cells exposed to the inhibitor were shown to undergo a significant reduction in cell surface O-glycosylation as detected by staining with jacalin and peanut agglutinin lectins after 30 min of treatment; no reduction in staining using antibodies to O-linked N-acetylglucosamine or the lectin concanavalin A was detected. Apoptosis was also observed in treated cells after 45 min of exposure, ostensibly following the O-glycosylation reduction. Overexpression of several different ppGaNTase isoforms restored cell surface O-glycosylation and rescued inhibitor-induced apoptosis. Additionally, mouse embryonic mandibular organ cultures exposed to 1-68A developed abnormally, presumably because of epithelial and mesenchymal apoptosis that followed a reduction in jacalin and peanut agglutinin staining. Our studies suggest that mucin-type O-linked glycosylation may be required for normal development and that ppGaNTases may play a role in the regulation of apoptosis.     

Dissecting the Biological Role of Mucin-type O-Glycosylation Using RNA Interference in Drosophila Cell Culture

Mucin type O-glycosylation is a highly conserved form of post-translational modification initiated by the family of enzymes known as the polypeptide α-N-acetylgalactosaminyltransferases (ppGalNAcTs in mammals and PGANTs in Drosophila). To address the cellular functions of the many PGANT family members, RNA interference (RNAi) to each pgant gene was performed in two independent Drosophila cell culture lines. We demonstrate that RNAi to individual pgant genes results in specific reduction in gene expression without affecting the expression of other family members. Cells with reduced expression of individual pgant genes were then examined for changes in viability, morphology, adhesion, and secretion to assess the contribution of each family member to these cellular functions. Here we find that RNAi to pgant3, pgant6, or pgant7 resulted in reduced secretion, further supporting a role for O-glycosylation in proper secretion. Additionally, RNAi to pgant3 or pgant6 resulted in altered Golgi organization, suggesting a role for each in establishing or maintaining proper secretory apparatus structure. Other subcellular effects observed included multinucleated cells seen after RNAi to either pgant2 or pgant35A, suggesting a role for these genes in the completion of cytokinesis. These studies demonstrate the efficient and specific knockdown of pgant gene expression in two Drosophila cell culture systems, resulting in specific morphological and functional effects. Our work provides new information regarding the biological roles of O-glycosylation and illustrates a new platform for interrogating the cellular and subcellular effects of this form of post-translational modification.     

Drosophila integrin-linked kinase is required at sites of integrin adhesion to link the cytoskeleton to the plasma membrane

Integrin-linked kinase (ILK) was identified by its interaction with the cytoplasmic tail of human beta1 integrin and previous data suggest that ILK is a component of diverse signaling pathways, including integrin, Wnt, and protein kinase B. Here we show that the absence of ILK function in Drosophila causes defects similar to loss of integrin adhesion, but not similar to loss of these signaling pathways. ILK mutations cause embryonic lethality and defects in muscle attachment, and clones of cells lacking ILK in the adult wing fail to adhere, forming wing blisters. Consistent with this, an ILK-green fluorescent protein fusion protein colocalizes with the position-specific integrins at sites of integrin function: muscle attachment sites and the basal junctions of the wing epithelium. Surprisingly, mutations in the kinase domain shown to inactivate the kinase activity of human ILK do not show any phenotype in Drosophila, suggesting a kinase-independent function for ILK. The muscle detachment in ILK mutants is associated with detachment of the actin filaments from the muscle ends, unlike integrin mutants, in which the primary defect is detachment of the plasma membrane from the extracellular matrix. Our data suggest that ILK is a component of the structure linking the cytoskeleton and the plasma membrane at sites of integrin-mediated adhesion.     

Early steps in O-linked glycosylation and clustered O-linked glycans of herpes simplex virus type 1 glycoprotein C: effects on glycoprotein properties

The pathogenesis of herpes simplex virus type 1 (HSV-1) implies the sequential infection of many cell types from mucosal cells to neurons, each having a unique pattern of protein glycosylation. The HSV-1 glycoprotein gC-1 is highly glycosylated and contains not only N-linked glycans but also a large number of O-linked glycans, some of which are clustered into two pronase-resistant arrays in the vicinity of the HSV-1 receptor-binding domain of gC-1. The aim of the present study was to characterize gC-1 signals for addition of clustered glycans, to determine the efficacy of synthetic peptides, representing putative O-glycosylation signals, as substrates for a panel of GalNAc transferases, and to identify possible effects of early O-linked glycosylation on the biological functions of gC-1. Gel filtration analysis of the pronase-resistant gC-1 O-glycan clusters from a glycoprotein mutant, lacking a site for N-linked glycosylation at Asn 73 in the vicinity of the O-glycosylation signal, suggested that one function of this N-linked glycan was to modulate the access for GalNAc transferases to one particular O-glycosylation peptide signal (aa 80-104). The ability of four GalNAc-transferase isoenzymes with different cell type expression patterns to initialize O-glycosylation of synthetic gC-1 derived peptides was analyzed. Two synthetic gC-1 peptides (aa 55-69 and aa 80-104) were excellent substrates for all four GalNAc-transferases, suggesting that cell types expressing less frequent GalNAc transferase species with unusual acceptor peptide sequence specificities may also produce a highly O-glycosylated gC-1 after HSV-1 infection. The O-linked glycans were not essential for cell surface expression of gC-1, but monoclonal antibody-assisted epitope analysis of N-acetylgalactosaminidase-treated gC-1 showed that the O-linked monosaccharide GalNAc contributed to expression of a three-dimensional epitope overlapping the heparan sulfate-binding domain of gC-1.     

Basic amino acids as modulators of an O-linked glycosylation signal of the herpes simplex virus type 1 glycoprotein gC: functional roles in viral infectivity

The herpes simplex virus type 1 (HSV-1) glycoprotein gC-1 is engaged both in viral attachment and viral immune evasion mechanisms in the infected host. Besides several N-linked glycans, gC-1 contains numerous O-linked glycans, mainly localized in two pronase-resistant clusters in the N-terminal domain of gC-1. In the present study we construct and characterize one gC-1 mutant virus, in which two basic amino acids (114K and 117R) in a putative O-glycosylation sequon were changed to alanine. We found that this modification did not modify the N-linked glycosylation but increased the content of O-linked glycans considerably. Analysis of the O-glycosylation capacity of wild-type and mutant gC-1 was performed by in vitro glycosylation assays with synthetic peptides derived from the mutant region predicted to present new O-glycosylation sites. Thus the mutant peptide region served as a better substrate for polypeptide GalNAc-transferase 2 than the wild-type peptide, resulting in increased rate and number of O-glycan attachment sites. The predicted increase in O-linked glycosylation resulted in two modifications of the biological properties of mutant virus-that is, an impaired binding to cells expressing chondroitin sulfate but not heparan sulfate on the cell surface and a significantly reduced plaque size in cultured cells. The results suggested that basic amino acids present within O-glycosylation signals may down-regulate the amount of O-linked glycans attached to a protein and that substitution of such amino acid residues may have functional consequences for a viral glycoprotein involving virus attachment to permissive cells as well as viral cell-to-cell spread.     

O-Glycosylation of the V2 vasopressin receptor.

The human V2 vasopressin receptor contains one consensus site for N-linked glycosylation at asparagine 22 in the predicted extracellular amino terminal segment of the protein. This segment also contains clusters of serines and threonines that are potential sites for O-glycosylation. Mutagenesis of asparagine 22 to glutamine abolished N-linked glycosylation of the V2 receptor (N22Q-V2R), without altering its function or level of expression. The N22Q-V2R expressed in transfected cells migrated in denaturing acrylamide gels as two protein bands with a difference of 7000 Da. Protein labeling experiments demonstrated that the faster band could be chase to the slower one suggesting the presence of O-linked sugars. Sialidase treatment of membranes from cells expressing the N22Q-V2R or of immunoprecipitated metabolically labeled V2R accelerated the migration of the protein in acrylamide gels demonstrating the existence of O-glycosylation, the first time this type of glycosylation has been found in a G protein coupled receptor. Synthesis of metabolically labeled receptor in the presence of 1 mM phenyl-N-acetyl-alpha-D-galactosaminide, a competitive inhibitor of N-acetyl-alpha-D-galactose and N-acetylneuraminic acid transferases, also produced a receptor that migrated faster in denaturing gels. Serines and threonines present in the amino terminus were analyzed by alanine scanning mutagenesis to identify the acceptor sites. O-glycosylation was found at most serines and threonines present in the amino terminus. Because the disappearance of a site opened the availability of others to the transferases, the exact identification of the acceptor sites was not feasible. The wild type V2R expressed in HEK 293, COS, or MDCK cells underwent N- and O-linked glycosylation. The mutant V2R bearing all serine/threonine substitutions by alanine at the amino terminus yielded a receptor functionally indistinguishable from the wild type protein, whose mobility in polyacrylamide gels was no longer affected by sialidase treatment.     

A modified method of islet isolation preserves the ability of pancreatic islets to increase protein O-glycosylation in response to glucose and streptozotocin

An important link has recently been shown in vivo between beta-cell O-linked protein glycosylation and beta-cell apoptosis, with hyperglycemia having been demonstrated to reversibly increase beta-cell O-linked protein glycosylation by providing substrate for the glucosamine pathway. In contrast, the same study showed that the administration of streptozotocin to rats prior to the induction of hyperglycemia results in irreversible increases in O-glycosylation and subsequent beta-cell apoptosis. In light of these data, we investigated beta-cell O-glycosylation in vitro by exposing isolated rat islets to high glucose, glucosamine, or streptozotocin and analyzing the pattern of O-glycosylated proteins present. All three compounds acutely increased O-glycosylation of a predominate 135-kDa protein (p135); however, their ability to stimulate p135 O-glycosylation was only consistently observed when islets were isolated in the presence of high glucose and 1 mM L-glutamine. Islets isolated in low glucose and no added L-glutamine demonstrated no consistent increase in p135 O-glycosylation in response to glucose, glucosamine, or streptozotocin. These data suggest that during islet isolation, beta-cell enzymes responsible for regulating p135 O-glycosylation may be adversely affected by the absence of high glucose and glutamine, which together are necessary for O-linked N-acetylglucosamine synthesis. We propose that the combination of high glucose and glutamine during islet isolation generates UDP-N-acetylglucosamine and O-linked N-acetylglucosamine, thus providing substrate protection for these enzymes and preserving the ability of isolated islets to O-glycosylate p135.     

O-linked protein glycosylation structure and function

There has been a recent resurgence of interest in the post-translational modification of serine and threonine hydroxyl groups by glycosylation, because the resulting O-linked oligosaccharide chains tend to be clustered over short stretches of peptide and hence they can present multivalent carbohydrate antigenic or functional determinants for antibody recognition, mammalian cell adhesion and microorganism binding. Co-operativity can greatly increase the affinity of interactions with antibodies or carbohydrate binding proteins. Thus, in addition to their known importance in bearing tumour associated antigens in the gastrointestinal and respiratory tracts, glycoproteins with O-linked chains have been implicated as ligands or co-receptors for selectins (mammalian carbohydrate binding proteins). Microorganisms may have adopted similar mechanisms for interactions with mammalian cells in infection, by having relatively low affinity ligands (adhesins) for carbohydrate binding, which may bind with higher affinity due to the multivalency of the host ligand and which are complemented by other virulence factors such as interactions with integrin-type molecules. In addition to specific adhesion signals from O-linked carbohydrate chains, multivalent O-glycosylation is involved in determining protein conformation and forming conjugate oligosaccharide-protein antigenic, and possible functional determinants.     

O-glycosylation of the mucin type

While only about ten percent of the databank entries are defined as glycoproteins, it has been estimated recently that more than half of all proteins are glycoproteins. Mucin-type O-glycosylation is a widespread post-translational modification of proteins found in the entire animal kingdom, but also in higher plants. The structural complexity of the chains initiated by O-linked GalNAc exceeds that of N-linked chains by far. The process during which serine and threonine residues of proteins become modified is confined to the cis to trans Golgi compartments. The initiation of this process by peptidyl GalNAc-transferases is ruled by the sequence context of putative O-glycosylation sites, but also by epigenetic regulatory mechanisms, which can be mediated by enzyme competition. The cellular repertoir of glycosyltransferases with their distinct donor sugar and acceptor sugar specificities, their sequential action at highly-ordered surfaces, and their localizations in subcompartments of the Golgi finally determine the cell-specific O-glycosylation profile. Dramatic alterations of the glycosylation machinery are observed in cancer cells, resulting in aberrantly O-glycosylated proteins that expose previously masked peptide motifs and new antigenic targets. The functional aspects of O-linked glycans, which comprise among many others their potential role in sorting and secretion of glycoproteins, their influence on protein conformation, and their multifarious involvement in cell adhesion and immunological processes, appear as complex as their structures.     

Potential roles of N-glycosylation in cell adhesion

The functional units of cell adhesion are typically multiprotein complexes made up of three general classes of proteins; the adhesion receptors, the cell-extracellular matrix (ECM) proteins, and the cytoplasmic plaque/peripheral membrane proteins. The cell adhesion receptors are usually transmembrane glycoproteins (for example E-cadherin and integrin) that mediate binding at the extracellular surface and determine the specificity of cell-cell and cell-ECM recognition. E-cadherin-mediated cell-cell adhesion can be both temporally and spatially regulated during development, and represents a key step in the acquisition of the invasive phenotype for many tumors. On the other hand, integrin-mediated cell-ECM interactions play important roles in cytoskeleton organization and in the transduction of intracellular signals to regulate various processes such as proliferation, differentiation and cell migration. ECM proteins are typically large glycoproteins, including the collagens, fibronectins, laminins, and proteoglycans that assemble into fibrils or other complex macromolecular arrays. The most of these adhesive proteins are glycosylated. Here, we focus mainly on the modification of N-glycans of integrins and laminin-332, and a mutual regulation between cell adhesion and bisected N-glycan expression, to address the important roles of N-glycans in cell adhesion.     

Integrins modulate the Egfr signaling pathway to regulate tendon cell differentiation in the Drosophila embryo

Changes in the extracellular matrix (ECM) govern the differentiation of many cell types during embryogenesis. Integrins are cell matrix receptors that play a major role in cell-ECM adhesion and in transmitting signals from the ECM inside the cell to regulate gene expression. In this paper, it is shown that the PS integrins are required at the muscle attachment sites of the Drosophila embryo to regulate tendon cell differentiation. The analysis of the requirements of the individual alpha subunits, alphaPS1 and alphaPS2, demonstrates that both PS1 and PS2 integrins are involved in this process. In the absence of PS integrin function, the expression of tendon cell-specific genes such as stripe and beta1 tubulin is not maintained. In addition, embryos lacking the PS integrins also exhibit reduced levels of activated MAPK. This reduction is probably due to a downregulation of the Epidermal Growth Factor receptor (Egfr) pathway, since an activated form of the Egfr can rescue the phenotype of embryos mutant for the PS integrins. Furthermore, the levels of the Egfr ligand Vein at the muscle attachment sites are reduced in PS mutant embryos. Altogether, these results lead to a model in which integrin-mediated adhesion plays a role in regulating tendon cell differentiation by modulating the activity of the Egfr pathway at the level of its ligand Vein.     

Inflated wings, tissue autolysis and early death in tissue inhibitor of metalloproteinases mutants of Drosophila

In vertebrates, tissue inhibitors of metalloproteinases (TIMPs) play key roles in extracellular matrix (ECM) homeostasis and growth control. Deletion of the recently cloned Timp gene of Drosophila results in a subviable phenotype. Adult flies display inflated wings similar to integrin mutants, suffer from a bloated gut and progressive dissolution of internal tissues, and die prematurely. Our results demonstrate that the Timp gene product controls selective aspects of ECM function in Drosophila, and suggest that it is involved in cell adhesion/cell signaling pathways. Hence, Drosophila Timp mutants may prove useful as a model system for a wide variety of pathological conditions related to ECM dysregulation.     

Agl22 and Agl23 are involved in the synthesis and utilization of the lipid-linked intermediates in the glycosylation pathways of the halophilic archaeaon Haloarcula hispanica

Like both eukaryotes and bacteria, archaea can decorate proteins with N- and O-linked glycans. Whereas pathways and roles of N-glycosylation have been studied in several model archaeal organisms, little is known of O-glycosylation. To explore commonalities and variations of these two versions of glycosylation, we used Haloarcula hispanica as a model. Our previous work showed that H. hispanica S-layer glycoproteins are modified by an N-linked glucose-α-(1, 2)-[sulfoquinovosamine-β-(1, 6)-]galactose trisaccharide and an O-linked glucose-α-(1, 4)-galactose disaccharide. Here, we found that H. hispanica membrane contains C60 dolichol phosphate (DolP) as a lipid carrier for glycosylation. As revealed by bioinformatics, gene deletion and phenotype analysis, gene HAH_1571, renamed agl22, encodes a predicted glucosyltransferase that transfers glucose from glucose-DolP onto galactose-DolP to form the glucose-α-(1, 4)-galactose-DolP precursor of the N-glycosylation. Gene HAH_2016, renamed agl23, encodes a putative flippase-associated protein responsible for flipping of hexose-DolPs across the membrane to face the exterior. Our results also suggested that the synthesis of the N- and O-linked glycans onto target protein occurs on the outer surface of the cell using hexose-DolPs as sugar donors. Deletion mutant showed that N- and O-glycosylation are required for growth in the defined medium mimicking the natural habitat of H. hispanica.     

Differential expression of Dystroglycan-spliceforms with and without the mucin-like domain during Drosophila embryogenesis

Dystroglycan (DG) is a widely expressed extracellular matrix (ECM) receptor required for muscle viability, synaptogenesis, basement-membrane formation and epithelial development. As an integral component of the Dystrophin-associated glycoprotein complex, DG plays a central role in linking the ECM and the cytoskeleton. Disruption of this linkage in skeletal muscle is the underlying cause in various types of muscular dystrophies (MD). One particular type of MD is caused by alterations of O-linked glycosylation in the mucin-like domain of DG, which is required for binding of the ECM molecules Laminin and Perlecan. In epithelial cells, reduced expression of DG is associated with increased invasiveness of cancer cells and loss of cell polarity. Drosophila Dg is, in contrast to vertebrate Dg, subjected to differential splicing of the mRNA. Interestingly, the shorter DG splice forms lack the mucin?like domain. Here, we describe the embryonic expression patterns of full-length DG and a short variant of DG. We find that differential splicing of Dg is developmentally regulated and tissue-specific. In some tissues, e.g., hindgut, midgut constrictions, gonads, both DG variants can be detected. For the long form, we detected specific expression at the blastoderm stage, in the epidermis and in the tracheal pits. The short form showed exclusive expression in dorsal vessel cells, chordotonal organs and dorsal median cells. In the nervous system, the long form is predominantly expressed on axons, while the short form is present on glial cells. Our findings further support the idea that DG forms lacking the mucin-like domain serve a specific function in Drosophila.