Levels Of Sialylation Research Articles

Immunoglobulin G Subclass-Specific Glycosylation Changes in Primary Epithelial Ovarian Cancer.

Epithelial ovarian cancer (EOC) was previously shown to be associated with glycosylation changes of total serum and total IgG proteins. However, as a majority of previous studies analyzed released glycan profiles, still little is known about IgG subclass-specific alterations in ovarian cancer. Hence, in this study, we investigated EOC-related glycosylation changes of the three most abundant IgG subclasses, namely, IgG1, IgG2 and IgG3 isolated from sera of 87 EOC patients and 74 age-matched healthy controls. In order to separate IgG2 and IgG3, we performed a two-step affinity purification employing Protein A and Protein G Sepharose. After tryptic digestion, IgG glycopeptides were enriched and measured by MALDI-TOF-MS. Finally, EOC-related glycosylation changes were monitored at the level of total agalactosylation, monogalactosylation, digalactosylation, sialylation, bisection and fucosylation, which were calculated separately for each IgG subclass. Interestingly, aside from an EOC-related increase in agalactosylation/decrease in monogalactosylation and digalactosylation observed in all IgG subclasses, some subclass-specific trends were detected. Glycosylation of IgG1 was found to be most strongly affected in EOC, as it exhibited the highest number of significant differences between healthy controls and EOC patients. Specifically, IgG1 was the only subclass that showed a significant decrease in sialylation and a significant increase in fucosylation in EOC patients. Interestingly, IgG2 and IgG3 that were often investigated collectively in previous studies, were found to have distinct glycosylation patterns. IgG3 displayed stronger EOC-related increase in agalactosylation/decrease in digalactosylation and was characterized by notably higher sialylation, which consequently decreased in EOC patients. In conclusion, our study indicates that IgG subclasses exhibit subtly distinct glycosylation patterns of EOC-related alterations and that IgG1 and IgG3 agalactosylation show the strongest association with CA125, the routine diagnostic marker. Additionally, our results show that simultaneous analyses of IgG2 and IgG3 might lead to wrong conclusions as these two subclasses exhibit noticeably different glycosylation phenotypes.

Abberant Immunoglobulin G Glycosylation in Rheumatoid Arthritis by LTQ-ESI-MS.

Aberrant glycosylation has been observed in many autoimmune diseases. For example, aberrant glycosylation of immunoglobulin G (IgG) has been implicated in rheumatoid arthritis (RA) pathogenesis. The aim of this study is to investigate IgG glycosylation and whether there is an association with rheumatoid factor levels in the serum of RA patients. We detected permethylated N-glycans of the IgG obtained in serum from 44 RA patients and 30 healthy controls using linear ion-trap electrospray ionization mass spectrometry (LTQ-ESI-MS), a highly sensitive and efficient approach in the detection and identification of N-glycans profiles. IgG N-glycosylation and rheumatoid factor levels were compared in healthy controls and RA patients. Our results suggested that total IgG purified from serum of RA patients shows significantly lower galactosylation (p = 0.0012), lower sialylation (p < 0.0001) and higher fucosylation (p = 0.0063) levels compared with healthy controls. We observed a positive correlation between aberrant N-glycosylation and rheumatoid factor level in the RA patients. In conclusion, we identified aberrant glycosylation of IgG in the serum of RA patients and its association with elevated levels of rheumatoid factor.

Defining the human kidney N-glycome in normal and cancer tissues using MALDI imaging mass spectrometry.

Clear‐cell renal cell carcinoma (ccRCC) presents challenges to clinical management because of late‐stage detection, treatment resistance, and frequent disease recurrence. Metabolically, ccRCC has a well‐described Warburg effect utilization of glucose, but how this affects complex carbohydrate synthesis and alterations to protein and cell surface glycosylation is poorly defined. Using an imaging mass spectrometry approach, N‐glycosylation patterns and compositional differences were assessed between tumor and nontumor regions of formalin‐fixed clinical ccRCC specimens and tissue microarrays. Regions of normal kidney tissue samples were also evaluated for N‐linked glycan‐based distinctions between cortex, medullar, glomeruli, and proximal tubule features. Most notable was the proximal tubule localized detection of abundant multiantennary N‐glycans with bisecting N‐acetylglucosamine and multziple fucose residues. These glycans are absent in ccRCC tissues, while multiple tumor‐specific N‐glycans were detected with tri‐ and tetra‐antennary structures and varying levels of fucosylation and sialylation. A polycystic kidney disease tissue was also characterized for N‐glycan composition, with specific nonfucosylated glycans detected in the cyst fluid regions. Complementary to the imaging mass spectrometry analyses was an assessment of transcriptomic gene array data focused on the fucosyltransferase gene family and other glycosyltransferase genes. The transcript levels of the FUT3 and FUT6 genes responsible for the enzymes that add fucose to N‐glycan antennae were significantly decreased in all ccRCC tissues relative to matching nontumor tissues. These striking differences in glycosylation associated with ccRCC could lead to new mechanistic insight into the glycobiology underpinning kidney malignancies and suggest the potential for new therapeutic interventions and diagnostic markers.

High levels of fucosylation and sialylation of milk N‐glycans from mothers with gestational diabetes mellitus alter the offspring gut microbiome and immune balance in mice

Gestational diabetes mellitus (GDM) is significantly associated with allergen sensitization in early childhood, and this may influence the gut microbiome and immune system of the children. In addition to mother-to-child transmission of microbes, milk glycans play a pivotal role in shaping the gut microbiome of infants. A previous study has demonstrated alterations in the major milk N-glycans of mothers with GDM. However, the impact of these changes on the gut microbiome and immune response of the neonates has yet to be studied. Here, we aimed to compare the glycosylation levels of various milk glycans between normal and GDM mice, and to characterize the intestinal microbiome and immune responses of the offspring after weaning. We found that GDM mouse milk contained significantly higher concentrations of fucosylated and sialylated N-glycans than control mice, but there was no difference in the concentration of milk oligosaccharides between the groups. The differences in milk N-glycans had direct effects on the intestinal microbiome of the offspring, which in turn affected their immune response upon challenge with ovalbumin (OVA), with disruptions in the Th1/Th2 and Th17/Treg cell balances. This study lays the foundation for further research and development of specific nutritional care for the offspring of GDM mothers.

Comparative Membrane N-Glycomics of Different Breast Cancer Cell Lines To Understand Breast Cancer Brain Metastasis.

The mechanism of brain metastatic breast cancer has gained attention because of its increased incidence rate and its low survival rate. Aberrant protein glycosylation is thought to be a contributing factor in this metastatic mechanism, in which metastatic cancer cells can pass through the blood-brain barrier (BBB). The cell membrane is the outermost layer of a cell and in direct contact with the environment and with other cells, making membrane glycans especially important in many biological processes that include mediating cell-cell adhesion, cell signaling, and interactions. Thus, membrane glycomics has attracted more interest for a variety of disease studies in recent years. To reveal the role that membrane N-glycans play in breast cancer brain metastasis, in this study, membrane enrichment was achieved by ultracentrifugation. Liquid chromatography-tandem mass spectrometry was employed to analyze enriched membrane N-glycomes from five breast cancer cell lines and one brain cancer cell line. Relative quantitative glycomic data from each cell line were compared to MDA-MB-231BR, which is the brain-seeking cell line. The higher sialylation level observed in MDA-MB-231BR suggested the importance of sialylation as it might assist with cell invasion and the penetration of the BBB. Some highly sialylated N-glycans, such as HexNAc5Hex6DeoxyHex1NeuAc3 and HexNAc6Hex7DeoxyHex1NeuAc3, exhibited higher abundances in 231BR, indicating their possible contributions to breast cancer brain metastasis as well as their potential to be indicators for the breast cancer brain metastasis.

Surface Plasmon Resonance-Based Method for Rapid Product Sialylation Assessment in Cell Culture.

To streamline cell culture process development, surface plasmon resonance (SPR) biosensors offer a versatile platform for the rapid quantification and quality analysis of recombinant proteins. As a representative case study, the present chapter details a procedure employing a SPR biosensor for determining the differential sialylation levels of recombinant interferon α2b contained in cell culture samples, using immobilized Sambucus nigra lectin. Of interest, this semiquantitative approach can be adapted to work with other lectins with unique carbohydrate-binding specificities, enabling a wide range of product characterization analysis.

Comprehensive analysis of human IgG Fc N-glycopeptides and construction of a screening model for colorectal cancer

Currently, analyzing intact glycopeptides remains a challengeable task. Considerable progress has been achieved in the knowledge of immunoglobulin G (IgG) glycans in patients with colorectal cancer (CRC), whereas data on IgG Fc N-glycopeptides are scarce in the literature. To fill this gap in knowledge, we developed a rapid and effective method to obtain and analyze IgG Fc N-glycopeptides in the plasma from 46 CRC patients and 67 healthy individuals using chitosan@poly (glycidyl methacrylate) @iminodiacetic acid (CS@PGMA@IDA) nanomaterial in combination with matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF-MS). A total of 29 N-glycopeptides were detected and analyzed. Compared with healthy individuals, CRC patients had increased levels of N-acteylglucosamine, yet decreased levels of galactosylation, fucosylation and sialylation. Further, a multivariate logistic regression model was developed using the levels of IgG Fc N-glycopeptides to distinguish CRC patients from healthy individuals, and the prediction performance was good, with an average AUC of the ROC curves of 0.893. SignificanceIn this study, we proposed a strategy for obtaining and analyzing IgG glycopeptides using CS@PGMA@IDA nanomaterial in combination with MALDI-TOF-MS. Using this strategy, IgG Fc N-glycopeptides were analyzed in the plasma of CRC patients, and our findings indicated that glycosylation levels in the IgG Fc region were closely related to CRC. By using the IgG N-glycopeptide enrichment method and screening model designed in this study, early large-scale colorectal cancer screening can be implemented easily and fast.

Hypersialylation promotes tumor immune escape

Sialylation is one type of glycosylation that adds sialic acid residues to the tip of glycans in glycoprotein or glycolipid on cell membrane and participates in many critical cellular processes via cell-cell contact. The level of sialylation is usually elevated during carcinogenesis. Increased sialic acid could interact with Siglecs on the surface of immune cells, and thus induce immunosuppressive tumor microenvironment and tumor immune escape. It is believed that sialic acid-Siglecs can be served as novel immune checkpoints and bring hope to the development of immunotherapy in the future. Key words: Sialic acid; Cancer; Immune escape; Siglec

Knockout of sialidase and pro-apoptotic genes in Chinese hamster ovary cells enables the production of recombinant human erythropoietin in fed-batch cultures

Sialic acid, a terminal monosaccharide present in N-glycans, plays an important role in determining both the in vivo half-life and the therapeutic efficacy of recombinant glycoproteins. Low sialylation levels of recombinant human erythropoietin (rhEPO) in recombinant Chinese hamster ovary (rCHO) cell cultures are considered a major obstacle to the production of rhEPO in fed-batch mode. This is mainly due to the accumulation of extracellular sialidases released from the cells. To overcome this hurdle, three sialidase genes (Neu1, 2, and 3) were initially knocked-out using the CRISPR/Cas9-mediated large deletion method in the rhEPO-producing rCHO cell line. Unlike wild type cells, sialidase knockout (KO) clones maintained the sialic acid content and proportion of tetra-sialylated rhEPO throughout fed-batch cultures without exhibiting a detrimental effect with respect to cell growth and rhEPO production. Additional KO of two pro-apoptotic genes, BAK and BAX, in sialidase KO clones (5X KO clones) further improved rhEPO production without any detrimental effect on sialylation. On day 10 in fed-batch cultures, the 5X KO clones had 1.4-times higher rhEPO concentration and 3.0-times higher sialic acid content than wild type cells. Furthermore, the proportion of tetra-sialylated rhEPO on day 10 in fed-batch cultures was 42.2–44.3% for 5X KO clones while it was only 2.2% for wild type cells. Taken together, KO of sialidase and pro-apoptotic genes in rCHO cells is a useful tool for producing heavily sialylated glycoproteins such as rhEPO in fed-batch mode.

Platelet Desialylation As a Predictive Marker in Childhood Immune Thrombocytopenia (ITP)

Background and aim: Immune thrombocytopenia (ITP) is the most common bleeding condition in children. Its prognosis is mostly superior, however, severe refractory disease remains diagnostic and therapeutic challenge. Low platelet counts (&amp;lt;100× 109/L) are associated with increased platelet clearance by two parallel mechanisms: classical antibody-mediated pathway and a novel lectin-carbohydrate mediated pathway. The latter is based on platelet desialylation, where terminal sialic acids are cleaved from glycoconjugates, mainly glycoproteins (GPs), on the platelet surface. The loss of sialic acid enhances bond of the penultimate β-galactose to asialoglycoprotein receptors (ASGPRs, also called Ashwell-Morell receptors) on hepatocytes. Desialylated platelets are then captured and phagocytosed by ASGPR-expressing hepatocytes. Desialylation has been shown to be responsible for platelet destruction in many contexts, e.g., infection-related thrombocytopenia or clearance of senescent platelets. Loss of T-cell tolerance is another underlying mechanism in ITP; CD8+ regulatory T cells (Tregs) are able to inhibit overactive immune response and maintain immune homeostasis. Forkhead box P3 (FOXP3) and GATA3 are transcription factors crucial for development and proper function of Tregs limiting the Th2-type inflammatory response. Our aims were to distinguish contribution of the mentioned processes to ITP development and to characterize immune response in children with ITP during the course of disease (diagnosis, ongoing therapy, remission, refractory/persistent ITP). Patients and Methods: We examined 30 samples from 20 children with ITP (12 males, 8 females, age 3-17 years; 3 acute ITP, 17 chronic ITP) and 10 healthy controls (age 4-15). The degree of desialylation was determined by flow cytometry using FITC-labeled Ricinus communis agglutinin (RCA-I) specific for terminal galactose or N-acetylgalactosamine. Expression of platelet surface markers was given quantitatively as mean fluorescence intensity (MFI). Presence of platelet surface-bond antibodies (IgG, IgA and IgM) was examined by flow cytometry. Subpopulations of CD4+ and CD8+ T-cells were characterized based on intracellular expression of transcription factors T-bet (Th1 cells), GATA3 (Th2 cells), ROR gamma T (Th17 cells) and FOXP3 (for Tregs) using multicolor flow cytometry. Results: Patients with ITP showed significant increase in RCA-I reactivity in comparison with healthy controls (p&amp;lt;0.001). Patients with newly diagnosed ITP showed the most aberrant sialylation (i.e., maximum desialylation) of platelet surface proteins. A decrease in desialylation intensity was noticeable as soon as at three days after therapy initiation. Sialylation levels returned to normal after one month of successful treatment and were similar to healthy controls in children with ITP remission. Platelet surface-bond immunoglobulins were increased in 10 (50%) patients independently on their sialylation level. We observed significant changes in T-cell subpopulations in ITP: T lymphocytes producing T-bet were decreased within both CD4+ and CD8+ populations. Percentage of CD4+ cells expressing ROR gamma T was also reduced. Proportions of cells expressing FOXP3 and GATA3 were decreased within the CD8+ but not within the CD4+ population. Conclusion: Our results highlight the importance of Fc-independent hepatic platelet clearance in ITP. Interindividual differences in ITP pathophysiology are reflected by treatment response and may improve therapeutic management and prognostication. E.g., intravenous immunoglobulins or splenectomy will be ineffective in patients with prevalent Fc-independent mechanisms, and contrarily, possibilities for novel targeted treatment (neuraminidase inhibitors) arise. Better understanding of immune-mediated processes involved in ITP pathogenesis may reduce adverse effects of immunosuppressive therapy and considerably improve quality of life in patients with ITP. Supported by: MH CZ - DRO (FNOl, 00098892), Project ENOCH (No. CZ.02.1.01/0.0/0.0/16_019/0000868) and Ministry of Education, Youth and Sports OPVVV CEREBIT CZ.02.1.01/0.0/0.0/16_025/0007397. Disclosures No relevant conflicts of interest to declare.

Production of α2,6-sialylated and non-fucosylated recombinant alpha-1-antitrypsin in CHO cells

Alpha-1-antitrypsin (A1AT) is an abundant serum inhibitor of serine proteases. A1AT deficiency is a common genetic disorder which is currently treated with augmentation therapies. These treatments involve weekly injections of patients with purified plasma-derived A1AT. Such therapies can be extremely expensive and rely on plasma donors. Hence, large-scale production of recombinant A1AT (rA1AT) could greatly benefit these patients, as it could decrease the cost of treatments, reduce biosafety concerns and ensure quantitative and qualitative controls of the protein. In this report, we sought to produce α2,6-sialylated rA1AT with our cumate-inducible stable CHO pool expression system. Our different CHO pools could reach volumetric productivities of 1,2 g/L. The human α2,6-sialyltransferase was stably expressed in these cells in order to mimic elevated α2,6-sialylation levels of native A1AT protein. Sialylation of the recombinant protein was stable over the duration of the fed-batch production phase and was higher in a pool where cells were sorted and enriched by FACS based on cell-surface α2,6-sialylation. Addition of ManNAc to the cell culture media during production enhanced both α2,3 and α2,6 A1AT sialylation levels whereas addition of 2F-peracetylfucose potently inhibited fucosylation of the protein. Finally, we demonstrated that rA1AT proteins exhibited human neutrophil elastase inhibitory activities similar to the commercial human plasma-derived A1AT.

The impact of sialylation linkage-type on the pharmacokinetics of recombinant butyrylcholinesterases.

Chinese hamster ovary (CHO) cells typically produce glycoproteins with N-glycans terminating in α-2,3 sialylation. Human cells produce glycoproteins that include α-2,3 and α-2,6 sialic acids. To examine the impact of altering protein sialylation on pharmacokinetic properties, recombinant human butyrylcholinesterase (BChE) was produced in CHO cells by knocking out the α-2,3 sialyltransferase genes followed by overexpression of the α-2,6 sialyltransferase (26BChE) enzyme. The N-glycan composition of 26BChE was compared to BChE with α-2,3 sialylation (23BChE) derived from wild-type CHO cells. Both 23BChE and 26BChE exhibited comparable antennarity distributions with bi-antennary di-sialylated glycans representing the most abundant glycoform. CD-1 mice were intravenously injected with the 23BChE or 26BChE, and residual BChE activities from blood collected at various time points for pharmacokinetic analyses. Although 23BChE contained a slightly lower initial sialylation level compared to 26BChE, the molecule exhibited higher residual activity between 5 and 24 hr postinjection. Pharmacokinetic analyses indicated that 23BChE exhibited an increase in area under the curve and a lower volume of distribution at steady state than that of 26BChE. These findings suggest that the type of sialylation linkage may play a significant role in the pharmacokinetic behavior of a biotherapeutic when tested in in vivo animal models.

Exploiting the Disialyl Galactose Activity of α2,6-Sialyltransferase from Photobacterium damselae To Generate a Highly Sialylated Recombinant α-1-Antitrypsin.

Sialic acids are sugars present in many animal glycoproteins and are of particular interest in biopharmaceuticals, where a lack of sialylation can reduce bioactivity. Here, we describe how α-2,6-sialyltransferase from Photobacterium damselae can be used to markedly increase the level of sialylation of CHO-produced α-1-antitrypsin. Detailed analysis of the sialylation products showed that in addition to the expected α-2,6-sialylation of galactose, a second disialyl galactose motif Neu5Ac-α2,3(Neu5Ac-α2,6)Gal was produced, which, to our knowledge, had never been detected on a mammalian glycoprotein. We exploited this disialyl galactose activity of the P. damselae in a multienzyme reaction to produce a highly sialylated α-1-antitrypsin. The influence of this unique disialylation on the in vitro activity of α-1-antitrypsin was studied, and a toolkit of mass spectrometry methods for identifying this new disialyl galactose motif in complex mixtures was developed.

Revealing the Biological Attributes of N-Glycan Isomers in Breast Cancer Brain Metastasis Using Porous Graphitic Carbon (PGC) Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS).

Breast cancer is a leading cancer in women and is considered to be the second-most common metastatic cancer following lung cancer. An estimated 10-16% of breast cancer patients are suffering from brain metastasis, and the diagnostic cases of breast cancer brain metastasis are increasing. Nevertheless, the mechanisms behind this process are still unclear. Aberrant glycosylation has been proved to be related to many diseases and cancer metastasis. However, studies of N-glycan isomer function in breast cancer brain metastasis are limited. In this study, the expressions of N-glycan isomers derived from five breast cancer cell lines and one brain cancer cell line were investigated and compared to a brain-seeking cell line, 231BR, to acquire a better understanding of the role glycan isomers play in breast cancer brain metastasis. The high temperature nanoPGC-LC-MS/MS achieved an efficient isomeric separation and permitted the identification and quantitation of 144 isomers from 50 N-glycan compositions. There were significant expression alterations of these glycan isomers among the different breast cancer cell lines. The increase of total glycan abundance and sialylation level were observed to be associated with breast cancer invasion. With regard to individual isomers, the greatest number of sialylated isomers was observed along with significant expression alterations in 231BR, suggesting a relationship between glycan sialylation and breast cancer brain metastasis. Furthermore, the increase of the α2,6-sialylation level in 231BR likely contributes to the passage of breast cancer cells through the blood-brain barrier, thus facilitating breast cancer brain metastasis. Meanwhile, the upregulation of highly sialylated glycan isomers with α2,6-linked sialic acids were found to be associated with breast cancer metastasis. This investigation of glycan isomer expressions, especially the unique isomeric expression in brain-seeking cell line 231BR, provides new information toward understanding the potential roles glycan isomers play during breast cancer metastasis and more clues for a deeper insight of this bioprocess.

&lt;p&gt;The β-galactoside α2,6-sialyltranferase 1 (ST6GAL1) inhibits the colorectal cancer metastasis by stabilizing intercellular adhesion molecule-1 via sialylation&lt;/p&gt;

Background: Colorectal cancer (CRC) is one of the most frequent malignancies of the digestive system. Elevated expression of β-galactoside α2,6-sialyltranferase 1 (ST6GAL1) has been observed in multiple cancers. But the mechanism of how ST6GAL1 might affect cancer cells remains to be clarified. Our previous study recognized intercellular adhesion molecule-1(ICAM-1) as a probable substrate of ST6GAL1 through mass spectrometry (MS) analysis. ICAM-1 is related to tumor metastasis in various cancers.Methods: First, ST6GAL1 was overexpressed and knocked down to perform transwell and wound healing assays, and the results were further confirmed in vivo. Based on the results of MS, GO and KEGG analysis were applied to reveal the connection between ST6GAL1 and ICAM-1. Immunoblot and tissue microarrays were administered to investigate the expression of ICAM-1 in different stages of CRC. Next, PCR, lectin precipitation and cycloheximide (CHX) were used to demonstrate the mechanism of ST6GAL1 on ICAM-1. Moreover, we investigated the sialylation on soluble ICAM in serum and its connection to tumor staging.Results: Overexpression of ST6GAL1 inhibited the migratory ability, while knockdown of ST6GAL1 cells had the reverse effect. Moreover, nude mice injected with ST6GAL1-knockdown cells harvested more liver metastases. Based on the GO and KEGG analysis, data from TCGA database showed a positive correlation between ST6GAL1 and ICAM-1. ICAM-1 also demonstrated a significant decrease in stage III/IV compared with stage I/II tumors. Our results revealed that ST6GAL1 could increase the stability of ICAM-1 through sialylation but had little influence on transcriptional level. Additionally, results of serum lectin precipitation revealed a correlation between the level of sialylation on soluble ICAM and CRC staging.Conclusion: This study illustrated that ST6GAL1 inhibited the metastatic ability of CRC by stabilizing ICAM-1 via sialylation and demonstrated a correlation between CRC staging and the sialylation on soluble ICAM-1 in serum.

Insights into the loss of protein sialylation in an fc-fusion protein-producing CHO cell bioprocess.

Sialylation affects circulating half-life, charge distribution, and other biochemical properties of therapeutic glycoproteins. Loss of protein sialylation during glycoprotein-producing bioprocesses could lead to a low final protein sialylation level and bring negative effects on subsequent clinical efficacy. In this work, an Fc-fusion protein-producing Chinese hamster ovary cell fed-batch culture process was studied and insights into the loss of protein sialylation during the Fc-fusion protein production phase (days 5 to 13) were presented. The results showed that the decreased total sialic acid content was 13.84 μg/mg during the production phase, which accounted for 24% of the total sialic acid content on day 5. The lost sialic acids were predominantly from α 2-3 sialylation on N- and O-glycans. Through cell-free incubation and kinetics studies, it was found that the decreased sialic acid content caused by extracellular sialic acid degradation and incomplete glycan biosynthesis were 7.79 μg/mg and 6.05 μg/mg, respectively. The two processes had a nearly equal contribution to the loss of final product sialylation. Detailed characterizations revealed that decreases in sialic acid content were due either to extracellular sialic acid degradation via hydrolysis of α 2-3 sialic acids probably by released cytosolic sialidase or to a lack of galactosylated glycan availability for sialylation during late-stage glycosylation. Our work provides a better understanding of losses in protein sialylation during glycoprotein manufacturing.

N-Glycomic and Transcriptomic Changes Associated with CDX1 mRNA Expression in Colorectal Cancer Cell Lines.

The caudal-related homeobox protein 1 (CDX1) is a transcription factor, which is important in the development, differentiation, and homeostasis of the gut. Although the involvement of CDX genes in the regulation of the expression levels of a few glycosyltransferases has been shown, associations between glycosylation phenotypes and CDX1 mRNA expression have hitherto not been well studied. Triggered by our previous study, we here characterized the N-glycomic phenotype of 16 colon cancer cell lines, selected for their differential CDX1 mRNA expression levels. We found that high CDX1 mRNA expression associated with a higher degree of multi-fucosylation on N-glycans, which is in line with our previous results and was supported by up-regulated gene expression of fucosyltransferases involved in antenna fucosylation. Interestingly, hepatocyte nuclear factors (HNF)4A and HNF1A were, among others, positively associated with high CDX1 mRNA expression and have been previously proven to regulate antenna fucosylation. Besides fucosylation, we found that high CDX1 mRNA expression in cancer cell lines also associated with low levels of sialylation and galactosylation and high levels of bisection on N-glycans. Altogether, our data highlight a possible role of CDX1 in altering the N-glycosylation of colorectal cancer cells, which is a hallmark of tumor development.

Highly Variable Sialylation Status of Donor-Specific Antibodies Does Not Impact Humoral Rejection Outcomes.

Clinical outcome in antibody-mediated rejection (AMR) shows high inter-individual heterogeneity. Sialylation status of the Fc fragment of IgGs is variable, which could modulate their ability to bind to C1q and/or Fc receptors. In this translational study, we evaluated whether DSA sialylation influence AMR outcomes. Among 938 kidney transplant recipients for whom a graft biopsy was performed between 2004 and 2012 at Lyon University Hospitals, 69 fulfilled the diagnosis criteria for AMR and were enrolled. Sera banked at the time of the biopsy were screened for the presence of DSA by Luminex. The sialylation status of total IgG and DSA was quantified using Sambucus nigra agglutinin-based chromatography. All patients had similar levels of sialylation of serum IgGs (~2%). In contrast, the proportion of sialylated DSA were highly variable (median = 9%; range = 0–100%), allowing to distribute the patients in two groups: high DSA sialylation (n = 44; 64%) and low DSA sialylation (n = 25; 36%). The two groups differed neither on the intensity of rejection lesions (C4d, ptc, and g; p > 0.05) nor on graft survival rates (Log rank test, p = 0.99). in vitro models confirmed the lack of impact of Fc sialylation on the ability of a monoclonal antibody to trigger classical complement cascade and activate NK cells. We conclude that DSA sialylation status is highly variable but has not impact on DSA pathogenicity and AMR outcome.

Insertion of N-Terminal Hinge Glycosylation Enhances Interactions of the Fc Region of Human IgG1 Monomers with Glycan-Dependent Receptors and Blocks Hemagglutination by the Influenza Virus.

In therapeutic applications in which the Fc of IgG is critically important, the receptor binding and functional properties of the Fc are lost after deglycosylation or removal of the unique Asn297 N-X-(T/S) sequon. A population of Fcs bearing sialylated glycans has been identified as contributing to this functionality, and high levels of sialylation also lead to longer serum retention times advantageous for therapy. The efficacy of sialylated Fc has generated an incentive to modify the unique N-linked glycosylation site at Asn297, either through chemical and enzymatic methods or by mutagenesis of the Fc, that disrupts the protein–Asn297 carbohydrate interface. In this study, we took an alternative approach by inserting or deleting N-linked attachment sites into the body of the Fc to generate a portfolio of mutants with tailored effector functions. For example, we describe mutants with enhanced binding to low-affinity inhibitory human Fcγ and glycan receptors that may be usefully incorporated into existing Ab engineering approaches to treat or vaccinate against disease. The IgG1 Fc fragments containing complex sialylated glycans attached to the N-terminal Asn221 sequon bound influenza virus hemagglutinin and disrupted influenza A–mediated agglutination of human erythrocytes.

Qualitative and quantitative characterization of sialylated N-glycans using three fluorophores, two columns, and two instrumentations

Sialylation can influence the stability, half-life, and immunogenicity of glycoproteins, but sialylated N-glycans are known to be difficult to analyze. Human alpha1-acid glycoprotein (AGP) is reported to have glycans that consist of sialylated N-glycans. The N-glycan profiling of AGP is qualitatively and quantitatively investigated here by UPLC and LC-ESI-MS/MS. Three fluorescent tags (AB, AA, and ProA) and two separation columns (HILIC and AEX-HILIC) were adopted to confirm and compare each analytical characteristic. The results of AA were comparable to those of the well-established AB. The qualification of ProA was notable due to its superior fluorescence intensity and ionization efficiency, and ProA showed smaller quantitative or larger-sized fragments in LC-ESI-MS/MS compared to AB and AA. However, the MS quantification of ProA was distorted because the increased sialylation level decreased the LC-ESI-MS/MS ionization efficiency. HILIC had better peak separability, AEX-HILIC had an advantage in UPLC sialylation profiling, and each isomeric glycan could be identified by both columns in LC-ESI-MS/MS. In conclusion, ProA is favored for UPLC and LC-ESI-MS/MS detection but not reliable for MS quantification. This study firstly demonstrates the qualification and quantification of sialylated N-glycans by comparing the commonly used analytical conditions with different fluorescent tags, columns, and instruments.