O-linked glycan expression during Drosophila development

Mucin-type O-linked glycosylation is an evolutionarily conserved protein modification that is essential for viability in Drosophila melanogaster. However, the exact role of O-glycans and the identity of the crucial apoproteins modified with O-linked N-acetylgalactosamine (O-GalNAc) remain unknown. In an effort to elucidate the O-linked glycans expressed during Drosophila development, we have employed fluorescent confocal microscopy using a battery of lectins and an antibody specific for the GalNAcalpha-Ser/Thr structure (Tn antigen). Confocal microscopy provides high-resolution images of the diversity of glycans expressed in many developing organ systems. In particular, O-glycans are highly expressed on a number of ectodermally derived tissues such as the salivary glands, developing gut, and the tracheal system, suggesting a role for O-glycans in cell polarity and tube formation common to these organs. Additionally, O-glycans are found in the developing nervous system and within subregions of developing tissues known to be active in cell signaling events. This study provides us with temporal and spatial information regarding O-glycan expression as well as a set of reagents for the isolation of glycoproteins from specific developmental stages and organ systems. This information will aid us in identifying the in vivo substrates of the UDP-GalNAc: polypeptide N-acetylgalactosaminyltranferases, in a continuing effort to define the biological role of O-linked glycoproteins during development.     

黏蛋白型O-聚糖：结构、功能及与肿瘤的相关性

黏蛋白是细胞表面的或分泌的、具有高度O-糖基化修饰的糖蛋白。黏蛋白型O-聚糖是由多肽：N-乙酰氨基半乳糖转移酶（ppGalNAc-T）家族催化起始合成的,在肿瘤中常常伴随着黏蛋白型O-聚糖结构和数量上的改变,形成肿瘤特异聚糖结构（cancer-associated glycans）,如肿瘤Tn和T抗原等。肿瘤特异聚糖使肿瘤细胞的抗原性和黏附能力发生改变,促进肿瘤细胞的恶性增生与转移。而这些肿瘤特异聚糖结构,也为肿瘤的诊断与抗肿瘤药物或疫苗开发提供了理论基础。     

Pathways of O-glycan biosynthesis in cancer cells

Glycoproteins with O-glycosidically linked carbohydrate chains of complex structures and functions are found in secretions and on the cell surfaces of cancer cells. The structures of O-glycans are often unusual or abnormal in cancer, and greatly contribute to the phenotype and biology of cancer cells. Some of the mechanisms of changes in O-glycosylation pathways have been determined in cancer model systems. However, O-glycan biosynthesis is a complex process that is still poorly understood. The glycosyltransferases and sulfotransferases that synthesize O-glycans appear to exist as families of related enzymes of which individual members are expressed in a tissue- and growth-specific fashion. Studies of their regulation in cancer may reveal the connection between cancerous transformation and glycosylation which may help to understand and control the abnormal biology of tumor cells. Cancer diagnosis may be based on the appearance of certain glycosylated epitopes, and therapeutic avenues have been designed to attack cancer cells via their glycans.     

Diverse glycosylation of MUC1 and MUC2: potential significance in tumor immunity

Mucins are major epithelial luminal surface proteins and function as a physical and biological barrier protecting mucous epithelia. Diverse glycosylation of mucins potentially provides a basis for tissue-specific interaction with the milieu. When mucins are associated with malignant epithelial cells, they not only protect these cells from a host environment during metastatic dissemination but also generate immunogenic epitopes which are used by the host in the detection and immunological elimination of carcinoma cells potentially depending upon their status of glycosylation. Diverse mucin structures are generated by the combination of different core peptides, of which 10 have been reported so far, multiple types of UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferases (pp-GalNAc-Ts), and the consequences of stepwise glycosylation events. For example, the mucin 1 (MUC1) associated with malignant cells was previously believed to exhibit unique features with a lower percentage of threonine and serine residues attached to N-acetylgalactosamine and/or without extension through core 2 structures. Some of MUC1-specific monoclonal antibodies and cytotoxic lymphocytes recognize the peptide sequences PDTR within the tandem repeat portion exposed by decreased degree of glycosylation. The specific arrangement of N-acetylgalactosamine residues is shown to be generated by a combination of pp-GalNAc-Ts with different specificities. The role of core 2 branching is somewhat confusing because well-known carcinoma-associated carbohydrate epitopes such as sialyl-Le(X), sialyl-Le(a), Le(Y), and others are often expressed when O-glycans are extended through core 2 branching. The other series of well-known carcinoma-associated carbohydrate structures are truncated O-glycans, conventionally called Tn and sialyl-Tn antigens. Interestingly, these are often found to be aligned on core polypeptides, resulting in three or more consecutive truncated O-glycans. MUC2 and other mucins, but not MUC1, have unique tandem repeats containing three or more consecutive serine or threonine residues, which potentially serve as a scaffold for trimeric Tn and sialyl-Tn epitopes. We recently found, using the MUC2 tandem repeat, that trimeric Tn is a high-affinity receptor for a calcium-type lectin expressed on the surface of histiocytic macrophages. The biosynthesis of trimeric Tn was strictly regulated by the acceptor specificity of pp-GalNAc-Ts. These results strongly suggest that variation in both glycan structures and distribution of glycans on the core polypeptides give mucins unique and diverse biological functions that play essential roles in carcinoma-host and other cellular interactions.     

Fermentation Monitoring and Control: A Perspective

Abstract

One of the fastest growing fields in the pharmaceutical industry is the market for therapeutic glycoproteins. Today, these molecules play a major role in the treatment of various diseases, and include several protein classes, i.e., clotting factors, hormones, cytokines, antisera, enzymes, enzyme inhibitors, Ig-Fc-Fusion proteins, and monoclonal antibodies. Optimal glycosylation is critical for therapeutic glycoproteins, as glycans can influence their yield, immunogenicity and efficacy, which impact the costs and success of such treatments. While several mammalian cell expression systems currently used can produce therapeutic glycoproteins that are mostly decorated with human-like glycans, they can differ from human glycans by presenting two structures at the terminal and therefore most exposed position. First, natural human N-glycans are lacking the terminal Gal? 1–3Gal (alpha-Gal) modification;     

Regulation of dolichol-linked glycosylation

In the majority of congenital disorders of glycosylation, the assembly of the glycan precursor GlcNAc₂Man₉Glc₃ on the polyprenol carrier dolichyl-pyrophosphate is compromised. Because N-linked glycosylation is essential to life, most types of congenital disorders of glycosylation represent partial losses of enzymatic activity. Consequently, increased availability of substrates along the glycosylation pathway can be beneficial to increase product formation by the compromised enzymes. Recently, we showed that increased dolichol availability and improved N-linked glycosylation can be achieved by inhibition of squalene biosynthesis. This review summarizes the current knowledge on the biosynthesis of dolichol-linked glycans with respect to deficiencies in N-linked glycosylation. Additionally, perspectives on therapeutic treatments targeting dolichol and dolichol-linked glycan biosynthesis are examined.     

Evaluation of analogues of GalNAc as substrates for enzymes of the mammalian GalNAc salvage pathway

Changes in glycosylation are correlated to disease and associated with differentiation processes. Experimental tools are needed to investigate the physiological implications of these changes either by labeling of the modified glycans or by blocking their biosynthesis. N-Acetylgalactosamine (GalNAc) is a monosaccharide widely encountered in glycolipids, proteoglycans, and glycoproteins; once taken up by cells it can be converted through a salvage pathway to UDP-GalNAc, which is further used by glycosyltransferases to build glycans. In order to find new reporter molecules able to integrate into cellular glycans, synthetic analogues of GalNAc were prepared and tested as substrates of both enzymes acting sequentially in the GalNAc salvage pathway, galactokinase 2 (GK2) and uridylpyrophosphorylase AGX1. Detailed in vitro assays identified the GalNAc analogues that can be transformed into sugar nucleotides and revealed several bottlenecks in the pathway: a modification on C6 is not tolerated by GK2; AGX1 can use all products of GK2 although with various efficiencies; and all analogues transformed into UDP-GalNAc analogues except those with alterations on C4 are substrates for the polypeptide GalNAc transferase T1. Besides, all analogues that could be incorporated in vitro into O-glycans were also integrated into cellular O-glycans as attested by their detection on the cell surface of CHO-ldlD cells. Altogether our results show that GalNAc analogues can help to better define structural requirements of the donor substrates for the enzymes involved in GalNAc metabolism, and those that are incorporated into cells will prove valuable for the development of novel diagnostic and therapeutic tools.     

Identification and characterization of protein glycosylation using specific endo- and exoglycosidases

Glycosylation, the addition of covalently linked sugars, is a major post-translational modification of proteins that can significantly affect processes such as cell adhesion, molecular trafficking, clearance, and signal transduction. In eukaryotes, the most common glycosylation modifications in the secretory pathway are additions at consensus asparagine residues (N-linked); or at serine or threonine residues (O-linked) (Figure 1). Initiation of N-glycan synthesis is highly conserved in eukaryotes, while the end products can vary greatly among different species, tissues, or proteins. Some glycans remain unmodified ("high mannose N-glycans") or are further processed in the Golgi ("complex N-glycans"). Greater diversity is found for O-glycans, which start with a common N-Acetylgalactosamine (GalNAc) residue in animal cells but differ in lower organisms. The detailed analysis of the glycosylation of proteins is a field unto itself and requires extensive resources and expertise to execute properly. However a variety of available enzymes that remove sugars (glycosidases) makes possible to have a general idea of the glycosylation status of a protein in a standard laboratory setting. Here we illustrate the use of glycosidases for the analysis of a model glycoprotein: recombinant human chorionic gonadotropin beta (hCGβ), which carries two N-glycans and four O-glycans. The technique requires only simple instrumentation and typical consumables, and it can be readily adapted to the analysis of multiple glycoprotein samples. Several enzymes can be used in parallel to study a glycoprotein. PNGase F is able to remove almost all types of N-linked glycans. For O-glycans, there is no available enzyme that can cleave an intact oligosaccharide from the protein backbone. Instead, O-glycans are trimmed by exoglycosidases to a short core, which is then easily removed by O-Glycosidase. The Protein Deglycosylation Mix contains PNGase F, O-Glycosidase, Neuraminidase (sialidase), β1-4 Galactosidase, and β-N-Acetylglucosaminidase. It is used to simultaneously remove N-glycans and some O-glycans. Finally, the Deglycosylation Mix was supplemented with a mixture of other exoglycosidases (α-N-Acetylgalactosaminidase, α1-2 Fucosidase, α1-3,6 Galactosidase, and β1-3 Galactosidase), which help remove otherwise resistant monosaccharides that could be present in certain O-glycans. SDS-PAGE/Coomasie blue is used to visualize differences in protein migration before and after glycosidase treatment. In addition, a sugar-specific staining method, ProQ Emerald-300, shows diminished signal as glycans are successively removed. This protocol is designed for the analysis of small amounts of glycoprotein (0.5 to 2 μg), although enzymatic deglycosylation can be scaled up to accommodate larger quantities of protein as needed.     

Glycomic studies of Drosophila melanogaster embryos

With the complete genome sequence of Drosophila melanogaster defined a systematic approach towards understanding the function of glycosylation has become possible. Structural assignment of the entire Drosophila glycome during specific developmental stages could provide information that would shed further light on the specific roles of different glycans during development and pinpoint the activity of certain glycosyltransferases and other glycan biosynthetic genes that otherwise might be missed through genetic analyses. In this paper the major glycoprotein N- and O-glycans of Drosophila embryos are described as part of our initial undertaking to characterize the glycome of Drosophila melanogaster. The N-glycans are dominated by high mannose and paucimannose structures. Minor amounts of mono-, bi- and tri-antennary complex glycans were observed with GlcNAc and Galβ1–4GlcNAc non-reducing end termini. O-glycans were restricted to the mucin-type core 1 Galβ1-3GalNAc sequence.     

A combined defect in the biosynthesis of N- and O-glycans in patients with cutis laxa and neurological involvement: the biochemical characteristics

Based on our preliminary observation of abnormal glycosylation in a cutis laxa patient, nine cutis laxa patients were analyzed for congenital defects of glycosylation (CDG). Isoelectric focusing of plasma transferrin and apolipoproteinC-III showed that three out of nine patients had a defect in the biosynthesis of N-glycans and core 1 mucin type O-glycans, respectively. Mass spectrometric N-glycan analyses revealed a relative increase of glycans lacking sialic acid and glycans lacking sialic acid and galactose residues. Mutation analysis of the fibulin-5 gene (FBLN5), which has been reported in cases of autosomal recessive cutis laxa, revealed no mutations in the patients' DNA. Evidence is presented that extracellular matrix (ECM) proteins of skin are likely to be highly glycosylated with N- and/or mucin type O-glycans by using algorithms for predicting glycosylation. The conclusions in this study were that the clinical phenotype of autosomal recessive cutis laxa seen in three patients is not caused by mutations in the FBLN5 gene. Our findings define a novel form of CDG with cutis laxa and neurological involvement due to a defect in the sialylation and/or galactosylation of N- and O-glycans. Improper glycosylation of ECM proteins of skin may form the pathophysiological basis for the cutis laxa phenotype.     

Mass spectrometric characterization of N- and O-glycans of plasma-derived coagulation factor VII

Factor VII (FVII) is a vitamin K-dependent glycoprotein which, in its activated form (FVIIa), participates in the coagulation process by activating factor X and factor IX. FVII is secreted as single peptide chain of 406 residues. Plasma-derived FVII undergoes many post-translational modifications such as γ-carboxylation, N- and O-glycosylation, β-hydroxylation. Despite glycosylation of recombinant FVIIa has been fully characterized, nothing is reported on the N- and O-glycans of plasma-derived FVII (pd-FVII) and on their structural heterogeneity at each glycosylation site. N- and O-glycosylation sites and site specific heterogeneity of pd-FVII were studied by various complementary qualitative and quantitative techniques. A MALDI-MS analysis of the native protein indicated that FVII is a 50.1 kDa glycoprotein modified on two sites by diantennary, disialylated non-fucosylated (A2S2) glycans. LC–ESIMS/MS analysis revealed that both light chain and heavy chain were N-glycosylated mainly by A2S2 but also by triantennary sialylated glycans. Nevertheless, lower amounts of triantennary structures were found on Asn₃₂₂ compared to Asn₁₄₅. Moreover, the triantennary glycans were shown to be fucosylated. In parallel, quantitative analysis of the isolated glycans by capillary electrophoresis indicated that the diantennary structures represented about 50% of the total glycan content. Glycan sequencing using different glycanases led to the identification of triantennary difucosylated structures. Last, MS and MS/MS analysis revealed that FVII is O-glycosylated on the light chain at position Ser₆₀ and Ser₅₂ which are modified by oligosaccharide structures such as fucose and Glc(Xyl)_0–1–2, respectively. These latter three O-glycans coexist in equal amounts in plasma-derived FVII.     

Almost all human gastric mucin O-glycans harbor blood group A, B or H antigens and are potential binding sites for Helicobacter pylori

Helicobacter pylori infects more than half of the world's population. Although most patients are asymptomatic, persistent infection may cause chronic gastritis and gastric cancer. Adhesion of the bacteria to the gastric mucosa is a necessary prerequisite for the pathogenesis of H. pylori-related diseases and is mediated by mucin O-glycans. In order to define which glycans may be implicated in the binding of the bacteria to the gastric mucosa in humans, we have characterized the exact pattern of glycosylation of gastric mucins. We have identified that the major component was always a core 2-based glycan carrying two blood group H antigens, whatever was the blood group of individuals. We have also demonstrated that around 80% of O-glycans carried blood group A, B or H antigens, suggesting that the variation of gastric mucin glycosylation between individuals is partly due to the blood group status. This study will help better understanding the role of O-glycans in the physiology and homeostasis of gastric mucosa. Overall, the results reported here give us the necessary background information to begin studies to determine whether individuals who express certain carbohydrate epitopes on specific mucins are predisposed to certain gastric diseases.     

Large induces functional glycans in an O-mannosylation dependent manner and targets GlcNAc terminals on alpha-dystroglycan

Alpha-dystroglycan (α-DG) is a ubiquitously expressed receptor for extracellular matrix proteins and some viruses, and plays a pivotal role in a number of pathological events, including cancer progression, muscular dystrophies, and viral infection. The O-glycans on α-DG are essential for its ligand binding, but the biosynthesis of the functional O-glycans remains obscure. The fact that transient overexpression of LARGE, a putative glycosyltransferase, up-regulates the functional glycans on α-DG to mediate its ligand binding implied that overexpression of LARGE may be a novel strategy to treat disorders with hypoglycosylation of α-DG. In this study, we focus on the effects of stable overexpression of Large on α-DG glycosylation in Chinese hamster ovary (CHO) cell and its glycosylation deficient mutants. Surprisingly, stable overexpression of Large in an O-mannosylation null deficient Lec15.2 CHO cells failed to induce the functional glycans on α-DG. Introducing the wild-type DPM2 cDNA, the deficient gene in the Lec15.2 cells, fully restored the Large-induced functional glycosylation, suggesting that Large induces the functional glycans in a DPM2/O-mannosylation dependent manner. Furthermore, stable overexpression of Large can effectively induce functional glycans on N-linked glycans in the Lec8 cells and ldlD cells growing in Gal deficient media, in both of which circumstances galactosylation are deficient. In addition, supplement of Gal to the ldlD cell culture media significantly reduces the amount of functional glycans induced by Large, suggested that galactosylation suppresses Large to induce the functional glycans. Thus our results revealed a mechanism by which Large competes with galactosyltransferase to target GlcNAc terminals to induce the functional glycans on α-DG.     

Glycosylation of α-dystroglycan: O-mannosylation influences the subsequent addition of GalNAc by UDP-GalNAc polypeptide N-acetylgalactosaminyltransferases

O-Linked glycosylation is a functionally and structurally diverse type of protein modification present in many tissues and across many species. α-Dystroglycan (α-DG), a protein linked to the extracellular matrix, whose glycosylation status is associated with human muscular dystrophies, displays two predominant types of O-glycosylation, O-linked mannose (O-Man) and O-linked N-acetylgalactosamine (O-GalNAc), in its highly conserved mucin-like domain. The O-Man is installed by an enzyme complex present in the endoplasmic reticulum. O-GalNAc modifications are initiated subsequently in the Golgi apparatus by the UDP-GalNAc polypeptide N-acetylgalactosaminyltransferase (ppGalNAc-T) enzymes. How the presence and position of O-Man influences the action of the ppGalNAc-Ts on α-DG and the distribution of the two forms of glycosylation in this domain is not known. Here, we investigated the interplay between O-Man and the addition of O-GalNAc by examining the activity of the ppGalNAc-Ts on peptides and O-Man-containing glycopeptides mimicking those found in native α-DG. These synthetic glycopeptides emulate intermediate structures, not otherwise readily available from natural sources. Through enzymatic and mass spectrometric methods, we demonstrate that the presence and specific location of O-Man can impact either the regional exclusion or the site of O-GalNAc addition on α-DG, elucidating the factors contributing to the glycosylation patterns observed in vivo. These results provide evidence that one form of glycosylation can influence another form of glycosylation in α-DG and suggest that in the absence of proper O-mannosylation, as is associated with certain forms of muscular dystrophy, aberrant O-GalNAc modifications may occur and could play a role in disease presentation.     

The action of TNFalpha and TGFbeta include specific alterations of the glycosylation of bovine and human chondrocytes

Joint destruction in arthritis is often associated with high levels of inflammatory cytokines. Previous work has shown that inflammatory conditions can alter the activities of glycosyltransferases that synthesize the glycan chains of glycoproteins, and that these changes in turn can influence the functions of glycoproteins. We therefore examined glycosyltransferases involved in glycoprotein biosynthesis in primary cultures of bovine articular chondrocytes and human chondrocytes isolated from knee cartilage of osteoarthritis patients. Bovine chondrocytes exhibited enzyme activities involved in the synthesis of bi-antennary complex Asn-linked N-glycans, as well as the enzymes involved in the synthesis of GalNAc-Ser/Thr-linked O-glycans with the core 1 structure. Human chondrocytes, in addition, were able to synthesize more complex O-glycans with core 2 structures. TNFalpha was found to induce apoptosis in chondrocytes, and this process was associated with significant changes in lectin binding to chondrocyte cell surface glycans. TGFbeta increased cell proliferation, and had significant effects on cell surface glycosylation in bovine but not in human cells. These cytokine-specific effects were partially correlated with changes in glycosyltransferase activities. Thus, chondrocytes have many of the enzymes necessary for the synthesis of N- and O-glycan chains of glycoproteins. The O-glycosylation pathways and the effects of TNFalpha and TGFbeta on glycosylation differed between bovine and human chondrocytes. These alterations are of potential importance for the regulation of the functions of cell surface receptors on chondrocytes, and for an understanding of the pathophysiology of arthritis.     

Glycosylation of human pancreatic ribonuclease: differences between normal and tumor states

Characterization of the N-glycans from human pancreatic ribonuclease (RNase 1) isolated from healthy pancreas and from pancreatic adenocarcinoma tumor cells (Capan-1 and MDAPanc-3) revealed completely different glycosylation patterns. RNase 1 from healthy cells contained neutral complex biantennary structures, with smaller amounts of tri- and tetraantennary compounds, and glycans with poly-N-acetyllactosamine extensions, all extensively fucosylated. In contrast, RNase 1 glycans from tumor cells (Capan-1) were fucosylated hybrid and complex biantennary glycans with GalNAc-GlcNAc antennae. RNase 1 glycans from Capan-1 and MDAPanc-3 cells also contained sialylated structures completely absent in the healthy pancreas. Some of these features provide distinct epitopes that were clearly detected using monoclonal antibodies against carbohydrate antigens. Thus monoclonal antibodies to Lewis(y) reacted only with normal pancreatic RNase 1, whereas, in contrast, monoclonal antibodies to sialyl-Lewis(x) and sialyl-Lewis(a) reacted only with RNase 1 secreted from the tumor cells. These glycosylation changes in a tumor-secreted protein, which reflect fundamental changes in the enzymes involved in the glycosylation pathway, open up the possibility of using serum RNase 1 as a tumor marker of pancreatic adenocarcinoma.     

Glycan-Dependent Immunogenicity of Recombinant Soluble Trimeric Hemagglutinin

Recombinant soluble trimeric influenza A virus (IAV) hemagglutinin (sHA₃) has proven an effective vaccine antigen against IAV. Here, we investigate to what extent the glycosylation status of the sHA₃ glycoprotein affects its immunogenicity. Different glycosylation forms of subtype H5 trimeric HA protein (sH5₃) were produced by expression in insect cells and different mammalian cells in the absence and presence of inhibitors of N-glycan-modifying enzymes or by enzymatic removal of the oligosaccharides. The following sH5₃ preparations were evaluated: (i) HA proteins carrying complex glycans produced in HEK293T cells; (ii) HA proteins carrying Man₉GlcNAc₂ moieties, expressed in HEK293T cells treated with kifunensine; (iii) HA proteins containing Man₅GlcNAc₂ moieties derived from HEK293S GnTI(−) cells; (iv) insect cell-produced HA proteins carrying paucimannosidic N-glycans; and (v) HEK293S GnTI(−) cell-produced HA proteins treated with endoglycosidase H, thus carrying side chains composed of only a single N-acetylglucosamine each. The different HA glycosylation states were confirmed by comparative electrophoretic analysis and by mass spectrometric analysis of released glycans. The immunogenicity of the HA preparations was studied in chickens and mice. The results demonstrate that HA proteins carrying terminal mannose moieties induce significantly lower hemagglutination inhibition antibody titers than HA proteins carrying complex glycans or single N-acetylglucosamine side chains. However, the glycosylation state of the HA proteins did not affect the breadth of the antibody response as measured by an HA1 antigen microarray. We conclude that the glycosylation state of recombinant antigens is a factor of significant importance when developing glycoprotein-based vaccines, such as recombinant HA proteins.     

Genetic defects in the hexosamine and sialic acid biosynthesis pathway

BackgroundCongenital disorders of glycosylation are caused by defects in the glycosylation of proteins and lipids. Classically, gene defects with multisystem disease have been identified in the ubiquitously expressed glycosyltransferases required for protein N-glycosylation. An increasing number of defects are being described in sugar supply pathways for protein glycosylation with tissue-restricted clinical symptoms.Scope of reviewIn this review, we address the hexosamine and sialic acid biosynthesis pathways in sugar metabolism. GFPT1, PGM3 and GNE are essential for synthesis of nucleotide sugars uridine diphosphate N-acetylglucosamine (UDP-GlcNAc) and cytidine-5′-monophospho-N-acetylneuraminic acid (CMP-sialic acid) as precursors for various glycosylation pathways. Defects in these enzymes result in contrasting clinical phenotypes of congenital myasthenia, immunodeficiency or adult-onset myopathy, respectively. We therefore discuss the biochemical mechanisms of known genetic defects in the hexosamine and CMP-sialic acid synthesis pathway in relation to the clinical phenotypes.Major conclusionsBoth UDP-GlcNAc and CMP-sialic acid are important precursors for diverse protein glycosylation reactions and for conversion into other nucleotide-sugars. Defects in the synthesis of these nucleotide sugars might affect a wide range of protein glycosylation reactions. Involvement of multiple glycosylation pathways might contribute to disease phenotype, but the currently available biochemical information on sugar metabolism is insufficient to understand why defects in these pathways present with tissue-specific phenotypes.General significanceFuture research on the interplay between sugar metabolism and different glycosylation pathways in a tissue- and cell-specific manner will contribute to elucidation of disease mechanisms and will create new opportunities for therapeutic intervention. This article is part of a Special Issue entitled "Glycans in personalised medicine" Guest Editor: Professor Gordan Lauc.