Tissue and serum α2-3- and α2-6-linkage specific sialylation changes in oral carcinogenesis

Increased sialylation of cell surface glycoconjugates is among the key molecular changes associated with malignant transformation and cancer progression. We investigated significance of linkage-specific sialylation changes in oral carcinogenesis. Tissue and serum levels of total sialic acid (TSA), linkage-specific sialyltransferases (ST) and sialoproteins were analyzed from patients with oral precancerous conditions (OPC) and oral cancer as well as the post-treatment follow-up blood samples of oral cancer patients. TSA levels were measured using a spectrophotometric method. The linkage-specific lectins, Sambusus nigra (SNA) and Maackia amurensis (MAM) detects α2-6- and α2-3-linked sialic acid, respectively, were used to analyze ST activity and sialoproteins. Malignant tissues showed significantly higher levels of TSA, reactivity of SNA and MAM, and α2,3-ST activity compared to the adjacent normal tissues. α2,6-ST was also higher in malignant tissues. Similarly, the marker levels were higher in precancerous tissues than their adjacent normal tissues. Serum levels of TSA, TSA/ total proteins, α2-6-sialoproteins and α2,6-ST were markedly increased in untreated oral cancer patients compared to the controls and OPC as well as responder (CR) patients. Serum levels of the markers were higher or comparable between untreated oral cancer patients and non-responders (NR). Serum levels of α2-3-sialylation were elevated in non-responders compared with the responders. Further, the observed sialylation changes in tissue and serum were found to be associated with various clinicopathological features and disease progression. Thus, the data suggest potential utility of sialylation markers in early detection, prognostication and treatment monitoring of oral cancer.     

Fluorescent CMP-sialic acids as a tool to study the specificity of the CMP-sialic acid carrier and the glycoconjugate sialylation in permeabilized cells.

The specificity of the Golgi carrier for CMP-sialic-acids and the lumenal sialylation of glycoconjugates in mechanically permeabilized cells (semi-intact CHO 15B cells) was studied with CMP-activated fluorescent sialic acids as sensitive markers. Semi-intact cells represent a well-established cellular model for studies on the constitutive secretion pathway because the perforated plasma membrane allows membrane-impermeable CMP-sialic-acids to gain access to cellular organelles. The subcellular structures of semi-intact cells remain morphologically intact and hence synthetic CMP-sialic-acids can be assayed as substrates for the corresponding Golgi sugar-nucleotide transporter. The results prove that the CMP-sialic-acid carrier is able to translocate fluorescent CMP-glycosides, despite the bulky fluoresceinyl residue located at position C5 or C9 of the sialic-acid moiety; the data suggest a slightly higher affinity of the carrier for the C9-substituted CMP-glycoside, whereas the affinity of cellular sialyltransferases is fourfold higher for CMP-5-N-fluoresceinylaminoacetylneuraminic acid (5-FTIUNeuAc; 5-N-fluoresceinylaminoneuraminic acid). Using CMP-9-fluoresceinylthioureido-N-acetylneuraminic acid (CMP-9-FTIUNeuAc), an easy and sensitive fluorometric assay was established for the lumenal sialylation in semi-intact cells. Cellular proteins and gangliosides are both labelled by covalent incorporation of the fluorescent N-acetylneuraminic acid analogue. The assay allows rapid screening for small biomolecules or proteins that influence cellular sialyl transport and sialyl transfer; the lumenal fluorescence incorporation does not require ATP or cytosolic compounds. The suitability of fluorescent CMP-glycosides as markers for intracellular sialylation, proven in this paper, introduces the use of synthetic sialic acids for visualisation of cellular sialic acid pathways by fluorescence microscopy. Based on the data presented here, specific CMP-N-acetylneuraminic-acid analogues can be produced and used for the characterization of the Golgi CMP-sialic-acid carrier.     

Changes in antigen-specific IgG1 Fc N-glycosylation upon influenza and tetanus vaccination

Antibody effector functions have been shown to be influenced by the structure of the Fc N-glycans. Here we studied the changes in plasma or serum IgG Fc N-glycosylation upon vaccination of 10 Caucasian adults and 10 African children. Serum/plasma IgG was purified by affinity chromatography prior to and at two time points after vaccination. Fc N-glycosylation profiles of individual IgG subclasses were determined for both total IgG and affinity-purified anti-vaccine IgG using a recently developed fast nanoliquid chromatography-electrospray ionization MS (LC-ESI-MS) method. While vaccination had no effect on the glycosylation of total IgG, anti-vaccine IgG showed increased levels of galactosylation and sialylation upon active immunization. Interestingly, the number of sialic acids per galactose increased during the vaccination time course, suggesting a distinct regulation of galactosylation and sialylation. In addition we observed a decrease in the level of IgG1 bisecting N-acetylglucosamine whereas no significant changes were observed for the level of fucosylation. Our data indicate that dependent on the vaccination time point the infectious agent will encounter IgGs with different glycosylation profiles, which are expected to influence the antibody effector functions relevant in immunity.     

Increase in Sialylation and Branching in the Mouse Serum N-glycome Correlates with Inflammation and Ovarian Tumour Progression

Ovarian cancer is the most lethal gynaecological cancer and is often diagnosed in late stage, often as the result of the unavailability of sufficiently sensitive biomarkers for early detection, tumour progression and tumour-associated inflammation. Glycosylation is the most common posttranslational modification of proteins; it is altered in cancer and therefore is a potential source of biomarkers. We investigated the quantitative and qualitative effects of anti-inflammatory (acetylsalicylic acid) and pro-inflammatory (thioglycolate and chlorite-oxidized oxyamylose) drugs on glycosylation in mouse cancer serum. A significant increase in sialylation and branching of glycans in mice treated with an inflammation-inducing compound was observed. Moreover, the increases in sialylation correlated with increased tumour sizes. Increases in sialylation and branching were consistent with increased expression of sialyltransferases and the branching enzyme MGAT5. Because the sialyltransferases are highly conserved among species, the described changes in the ovarian cancer mouse model are relevant to humans and serum N-glycome analysis for monitoring disease treatment and progression might be a useful biomarker.     

Antipsychotic treatment of acute paranoid schizophrenia patients with olanzapine results in altered glycosylation of serum glycoproteins

Atypical antipsychotic drugs, such as olanzapine, have been shown to alleviate the positive, negative and, to a lesser degree, the cognitive symptoms of schizophrenia in many patients. However, the detailed mechanisms of action of these drugs have yet to be elucidated. We have carried out the first investigation aimed at evaluating the effects of olanzapine treatment on the glycosylation of serum proteins in schizophrenia patients. Olanzapine treatment resulted in increased levels of a disialylated biantennary glycan and reduced levels of a number of disialylated bi- and triantennary glycans on whole serum glycoproteins. These changes were not observed on a low-abundance serum protein fraction. α1 acid glycoprotein was identified as a carrier of some of the detected altered oligosaccharides. In addition, glycan analysis of haptoglobin, transferrin, and α1 antitrypsin reported similar findings, although these changes did not reach significance. Exoglycosidase digestion analysis showed that olanzapine treatment increased galactosylation and sialylation of whole serum proteins, suggesting increased activity of specific galactosyltransferases and increased availability of galactose residues for sialylation. Taken together, these findings indicate that olanzapine treatment results in altered glycosylation of serum proteins.     

Sialylation enhancement of CTLA4‐Ig fusion protein in Chinese hamster ovary cells by dexamethasone

The importance of glycoprotein sialic acid levels is well known, as increased levels have been shown to increase in vivo serum half‐life profiles. Here we demonstrate for the first time that dexamethasone (DEX) was capable of improving the sialylation of a CTLA4‐Ig fusion protein produced by Chinese hamster ovary (CHO) cells. DEX was shown to enhance the intracellular addition of sialic acid by sialyltransferases as well as reduce extracellular removal of sialic acid by sialidase cleavage. We illustrated that DEX addition resulted in increased expression of the glycosyltransferases α2,3‐sialyltransferase (α2,3‐ST) and β1,4‐galactosyltransferase (β1,4‐GT) in CHO cells. Based upon our previous results showing DEX addition increased culture cell viability, we confirmed here that cultures treated with DEX also resulted in decreased sialidase activity. Addition of the glucocorticoid receptor (GR) antagonist mifepristone (RU‐486) was capable of blocking the increase in sialylation by DEX which further supports that DEX affected sialylation as well as provides evidence that the sialylation enhancement effects of DEX on recombinant CHO cells occurred through the GR. Finally, the effects of DEX on increasing sialylation were then confirmed in 5‐L controlled bioreactors. Addition of 1 µM DEX to the bioreactors on day 2 resulted in harvests with average increases of 16.2% for total sialic acid content and 15.8% in the protein fraction with N‐linked sialylation. DEX was found to be a simple and effective method for increasing sialylation of this CTLA4‐Ig fusion protein expressed in CHO cells. Biotechnol. Bioeng. 2010;107: 488–496. © 2010 Wiley Periodicals, Inc.     

Highly Efficient Identification of O‐GalNAc Glycosylation by an Acid‐Assisted Glycoform Simplification Approach

Compared with N‐linked glycosylation, the analysis of O‐GalNAc glycosylation is extremely challenging due to the high structure diversity of glycans and lack of glycosidases to release O‐GalNAc glycans. In this work, a glycoform simplification strategy by combining HILIC enrichment with chemical de‐sialylation to characterize O‐GalNAc glycosylation of human serum is presented. This method is first validated by using the bovine fetuin as the test sample. It is found that more than 90% of the sialic acid residues can be removed from bovine fetuin by the acid‐assisted de‐sialylation method, which significantly simplifies the glycan structure and improves identification sensitivity. Indeed, the number of identified peptide backbones increases nearly one fold when this strategy is used. This method is further applied to analyze the human serum sample, where 185 O‐GalNAc modified peptide sequences corresponding to 94 proteins with high confidence (FDR (false detection rate) <1%) are identified. This straight forward strategy can significantly reduce the variations of glycan structures, and is applicable to analysis of other biological samples with high complexity.     

The adhesive properties of recombinant soluble L-selectin are modulated by its glycosylation

The leukocyte adhesion molecule L-selectin, which mediates the initial steps of leukocyte attachment to vascular endothelium, is intensely glycosylated. Different glycoforms of L-selectin are expressed on different leukocyte subsets and differences in L-selectin glycosylation appear to be correlated with the leukocyte's ability to attach to different endothelial targets. In the present study we addressed the question whether glycosylation of L-selectin influences L-selectin-ligand interactions. To obtain different glycoforms of L-selectin, recombinant proteins were expressed both in the baby hamster kidney (BHK) cell line and in the human myelogenous cell line K562, resulting in sL-sel[BHK] or sL-sel[K562], respectively. The glycosylation characteristics of the purified proteins were determined. The most striking differences in glycosylation were seen in the terminal sialylation. Each of the two proteins carried sialic acids in the alpha 2-3 position, while alpha 2-6-bound sialic acids were found exclusively on sL-sel[K562]. To investigate their adhesive properties, both recombinant sL-selectins were used in cell adhesion assays and interactions with the ligands present on various hematopoietic cell lines or activated human cardiac microvascular endothelial cells were examined. The binding capacity of sL-sel[K562] was about 1.6 fold higher compared to sL-sel[BHK] under static as well as under flow conditions. These findings indicate that the terminal sialylation pattern of L-selectin modulates its binding characteristics.     

Acute phase inflammation is characterized by rapid changes in plasma/peritoneal fluid <Emphasis Type="Italic">N</Emphasis>-glycosylation in mice

Murine zymosan-induced peritonitis is a widely used model for studying the molecular and cellular events responsible for the initiation, persistence and/or resolution of inflammation. Among these events, it is becoming increasingly evident that changes in glycosylation of proteins, especially in the plasma and at the site of inflammation, play an important role in the inflammatory response. Using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS)-based glycosylation profiling, we investigated the qualitative and quantitative effect of zymosan-induced peritonitis on N-glycosylation in mouse plasma and peritoneal fluid. Our results show that both N-glycomes exhibit highly similar glycosylation patterns, consisting mainly of diantennary and triantennary complex type N-glycans with high levels (>95 %) of galactosylation and sialylation (mostly NeuGc) and a medium degree of core fucosylation (30 %). Moreover, MS/MS structural analysis, assisted by linkage-specific derivatization of sialic acids, revealed the presence of O-acetylated sialic acids as well as disialylated antennae (“branching sialylation”) characterized by the presence of α2-6-linked NeuGc on the GlcNAc of the NeuGcα2-3-Galβ1-3-GlcNAc terminal motif. A significant decrease of (core) fucosylation together with an increase of both α2-3-linked NeuGc and “branching sialylation” were observed in N-glycomes of mice challenged with zymosan, but not in control mice injected with PBS. Importantly, substantial changes in glycosylation were already observed 12 h after induction of peritonitis, thereby demonstrating an unexpected velocity of the biological mechanisms involved.     

Sialic acid metabolism's dual function in Haemophilus influenzae

Many bacterial commensals and pathogens use the sialic acids as carbon and nitrogen sources. In Escherichia coli, the breakdown of these sugars is catalysed by gene products of the nan (Nacylneuraminate) operon; other microorganisms may use a similar catabolic strategy. Despite the known ligand and antirecognition functions of the sialic acids, the contribution of their catabolism to infection or host colonization has never been directly investigated. We addressed these questions with Haemophilus influenzae type b, which metabolizes relatively few carbohydrates, using the infant-rat infection model. The predicted H. influenzae homologue (HI0142) of the E. coli sialic acid aldolase structural gene, nanA, was subcloned and mutagenized by insertion of a kanamycin resistance cassette. Phenotypic investigation of the resulting H. influenzae aldolase mutants showed that: (i) HI0142 is essential for sialic acid degradation; (ii) the products of the open reading frames (ORFs) flanking HI0142 (HI0140, 41, 44 and 45) are likely to have the same functions as those of their counterparts in E. coli; (iii) sialylation of the lipooligosaccharide (LOS) epitope recognized by monoclonal antibody 3F11 is dependent on an environmental source of sialic acid; (iv) a nanA mutant hypersialylates its LOS sialyl acceptor, corresponding to an apparent increased fitness of the mutant in the infant-rat model; and (v) expression of the LOS sialyl acceptor is altered in cells grown without exogenous sialic acid, indicating the direct or indirect effect of sialic acid metabolism on LOS antigenicity. Taken together the data show the dual role of sialic acid catabolism in nutrition and cell surface modulation.     

Reversible sialylation: synthesis of cytidine 5'-monophospho-N-acetylneuraminic acid from cytidine 5'-monophosphate with alpha2,3-sialyl O-glycan-, glycolipid-, and macromolecule-based donors yields diverse sialylated products

Sialyltransferases transfer sialic acid from cytidine 5'-monophospho-N-acetylneuraminic acid (CMP-NeuAc) to an acceptor molecule. Trans-sialidases of parasites transfer alpha2,3-linked sialic acid from one molecule to another without the involvement of CMP-NeuAc. Here we report another type of sialylation, termed reverse sialylation, catalyzed by mammalian sialyltransferase ST3Gal-II. This enzyme synthesizes CMP-NeuAc by transferring NeuAc from the NeuAcalpha2,3Galbeta1,3GalNAcalpha unit of O-glycans, 3-sialyl globo unit of glycolipids, and sialylated macromolecules to 5'-CMP. CMP-NeuAc produced in situ is utilized by the same enzyme to sialylate other O-glycans and by other sialyltransferases such as ST6Gal-I and ST6GalNAc-I, forming alpha2,6-sialylated compounds. ST3Gal-II also catalyzed the conversion of 5'-uridine monophosphate (UMP) to UMP-NeuAc, which was found to be an inactive sialyl donor. Reverse sialylation proceeded without the need for free sialic acid, divalent metal ions, or energy. Direct sialylation with CMP-NeuAc as well as the formation of CMP-NeuAc from 5'-CMP had a wide optimum range (pH 5.2-7.2 and 4.8-6.4, respectively), whereas the entire reaction comprising in situ production of CMP-NeuAc and sialylation of acceptor had a sharp optimum at pH 5.6 (activity level 50% at pH 5.2 and 6.8, 25% at pH 4.8 and 7.2). Several properties distinguish forward/conventional versus reverse sialylation: (i) sodium citrate inhibited forward sialylation but not reverse sialylation; (ii) 5'-CDP, a potent forward sialyltransferase inhibitor, did not inhibit the conversion of 5'-CMP to CMP-NeuAc; and (iii) the mucin core 2 compound 3-O-sulfoGalbeta1,4GlcNAcbeta1,6(Galbeta1,3)GalNAcalpha-O-benzyl, an efficient acceptor for ST3Gal-II, inhibited the conversion of 5'-CMP to CMP-NeuAc. A significant level of reverse sialylation activity is noted in human prostate cancer cell lines LNCaP and PC3. Overall, the study demonstrates that the sialyltransferase reaction is readily reversible in the case of ST3Gal-II and can be exploited for the enzymatic synthesis of diverse sialyl products.     

An investigation of intracellular glycosylation activities in CHO cells: Effects of nucleotide sugar precursor feeding

Controlling glycosylation of recombinant proteins produced by CHO cells is highly desired as it can be directed towards maintaining or increasing product quality. To further our understanding of the different factors influencing glycosylation, a glycosylation sub‐array of 79 genes and a capillary electrophoresis method which simultaneously analyzes 12 nucleotides and 7 nucleotide sugars; were used to generate intracellular N‐glycosylation profiles. Specifically, the effects of nucleotide sugar precursor feeding on intracellular glycosylation activities were analyzed in CHO cells producing recombinant human interferon‐γ (IFN‐γ). Galactose (±uridine), glucosamine (±uridine), and N‐acetylmannosamine (ManNAc) (±cytidine) feeding resulted in 12%, 28%, and 32% increase in IFN‐γ sialylation as compared to the untreated control cultures. This could be directly attributed to increases in nucleotide sugar substrates, UDP‐Hex (～20‐fold), UDP‐HexNAc (6‐ to 15‐fold) and CMP‐sialic acid (30‐ to 120‐fold), respectively. Up‐regulation of B4gal and St3gal could also have enhanced glycan addition onto the proteins, leading to more complete glycosylation (sialylation). Combined feeding of glucosamine + uridine and ManNAc + cytidine increased UDP‐HexNAc and CMP‐sialic acid by another two‐ to fourfold as compared to feeding sugar precursors alone. However, it did not lead to a synergistic increase in IFN‐γ sialylation. Other factors such as glycosyltransferase or glycan substrate levels could have become limiting. In addition, uridine feeding increased the levels of uridine‐ and cytidine‐activated nucleotide sugars simultaneously, which could imply that uridine is one of the limiting substrates for nucleotide sugar synthesis in the study. Hence, the characterization of intracellular glycosylation activities has increased our understanding of how nucleotide sugar precursor feeding influence glycosylation of recombinant proteins produced in CHO cells. It has also led to the optimization of more effective strategies for manipulating glycan quality. Biotechnol. Bioeng. 2010;107: 321–336. © 2010 Wiley Periodicals, Inc.     

“Lost sugars” — reality of their biological and medical applications

The glycan chains attached to cell surfaces or to single proteins are highly dynamic structures with various functions. The glycan chains of mammals and of some microorganisms often terminate in sialic acids or α-1,3-galactose. Although these two sugars are completely distinct, there are several similarities in their biological and medical importance. First, one type of sialic acid, N-glycolylneuraminic acid, and the galactose bound by an α-1,3-linkage to LacNAc, that forms an α-gal epitope, were both eliminated in human evolution, resulting in the production of antibodies to these sugars. Both of these evolutionary events have consequences connected with the consumption of foods of mammalian origin, causing medical complications of varying severity. In terms of ageing, sialic acids prevent the clearance of glycoproteins and circulating blood cells, whereas cryptic α-gal epitopes on senescent red blood cells contribute to their removal from circulation. The efficiency of therapeutic proteins can be increased by sialylation. Another common feature is the connection with microorganisms since sialic acids and α-gal epitopes serve as receptors on host cells and can also be expressed on the surfaces of some microorganisms. Whereas, the sialylation of IgG antibodies may help to treat inflammation, the expression of the α-gal epitope on microbial antigens increases the immunogenicity of the corresponding vaccines. Finally, sialic acids and the α-gal epitope have applications in cancer immunotherapy. N-glycolylneuraminic acid is a powerful target for cancer immunotherapy, and the α-gal epitope increases the efficiency of cancer vaccines. The final section of this article contains a brief overview of the methods for oligosaccharide chain synthesis and the characteristics of sialyltransferases and α-1,3-galactosyltransferase.     

Generation of Biologically Active Multi-Sialylated Recombinant Human EPOFc in Plants

Hyperglycosylated proteins are more stable, show increased serum half-life and less sensitivity to proteolysis compared to non-sialylated forms. This applies particularly to recombinant human erythropoietin (rhEPO). Recent progress in N-glycoengineering of non-mammalian expression hosts resulted in in vivo protein sialylation at great homogeneity. However the synthesis of multi-sialylated N-glycans is so far restricted to mammalian cells. Here we used a plant based expression system to accomplish multi-antennary protein sialylation. A human erythropoietin fusion protein (EPOFc) was transiently expressed in Nicotiana benthamiana ΔXTFT, a glycosylation mutant that lacks plant specific N-glycan residues. cDNA of the hormone was co-delivered into plants with the necessary genes for (i) branching (ii) β1,4-galactosylation as well as for the (iii) synthesis, transport and transfer of sialic acid. This resulted in the production of recombinant EPOFc carrying bi- tri- and tetra-sialylated complex N-glycans. The formation of this highly complex oligosaccharide structure required the coordinated expression of 11 human proteins acting in different subcellular compartments at different stages of the glycosylation pathway. In vitro receptor binding assays demonstrate the generation of biologically active molecules. We demonstrate the in planta synthesis of one of the most complex mammalian glycoforms pointing to an outstanding high degree of tolerance to changes in the glycosylation pathway in plants.     

The O-linked glycosylation of secretory/shed MUC1 from an advanced breast cancer patient's serum

MUC1 is a high molecular weight glycoprotein that is overexpressed in breast cancer. Aberrant O-linked glycosylation of MUC1 in cancer has been implicated in disease progression. We investigated the O-linked glycosylation of MUC1 purified from the serum of an advanced breast cancer patient. O-Glycans were released by hydrazinolysis and analyzed by liquid chromatography-electrospray ionization-mass spectrometry and by high performance liquid chromatography coupled with sequential exoglycosidase digestions. Core 1 type glycans (83%) dominated the profile which also confirmed high levels of sialylation: 80% of the glycans were mono-, di- or trisialylated. Core 2 type structures contributed approximately 17% of the assigned glycans and the oncofoetal Thomsen-Friedenreich (TF) antigen (Galbeta1-3GalNAc) accounted for 14% of the total glycans. Interestingly, two core 1 type glycans were identified that had sialic acid alpha2-8 linked to sialylated core 1 type structures (9% of the total glycan pool). This is the first O-glycan analysis of MUC1 from the serum of a breast cancer patient; the results suggest that amongst the cell lines commonly used to express recombinant MUC1 the T47D cell line processes glycans that are most similar to patient-derived material.