Profiling of isomer-specific IgG N-glycosylation in cohort of Chinese colorectal cancer patients

BackgroudGiven the increasing morbidity and mortality of colorectal cancer (CRC), it is urgent to develop a noninvasive screening strategy for early diagnosis of CRC. Altered IgG glycosylation is associated with CRC progression, whereas the association of IgG isomeric glycosylation with CRC were not investigated.MethodsMethylamidation of IgG N-glycans was conducted prior to PGC-based nanoLC-ESI-MS/MS analysis. Data processing was operated by a self-developed application based on MATLAB solution. Statistical analysis including K–S test, t-test, ROC curve and OPLS-DA were successively performed. Additionally, an independent set was utilized to validate the results.ResultsTotal 28 IgG glycans and 79 compositional isomers were identified, over half of which are firstly identified so far. Statistical analysis showed that CRC associates with increase in IgG agalactosylation, decrease in IgG sialylation and fucosylation of sialylated glycans. Additionally, it was found that three compositional isomers (H3N4F1-a, H3N4F1-b and H4N3S1F1-e) could distinguish CRC and early stages from controls with an accurate area under the receiver operating characteristic curve. Significantly, these results were validated in an independent set by multivariate statistical analysis.ConclusionsThis is the first comprehensively profiling of isomer-specific IgG N-glycosylation, which could differentiate normal controls from colorectal disease patients. The candidate IgG glyco-biomarkers provide important screening indicators for early diagnosis of CRC.General significanceColorectal cancer progression is strongly associated with isomer-specific IgG N-glycosylation.     

Immunoglobulin G Fc N-glycan profiling in patients with gastric cancer by LC-ESI-MS: relation to tumor progression and survival

The IgG Fc glycans strongly influence the Fcγ receptor interactions and Fc-mediated effector mechanisms. Changes in the structure of IgG glycans are associated with various diseases, such as infections and autoimmunity. However, the possible role of Fc glycans in tumor immunity is not yet fully understood. The aim of this study was to profile the Fc N-glycans of IgG samples from patients with gastric cancer (n = 80) and controls (n = 51) using LC-ESI-MS method to correlate the findings with stage of cancer and patients survival. Analysis of 32 different IgG N-glycans revealed significant increase of agalactosylated (GnGnF, GnGn(bi)F), and decrease of galactosylated (AGn(bi), AGn(bi)F, AA(bi), AAF) and monosialylated IgG glycoforms (NaAF, NaA(bi)) in cancer patients. A statistically significant increase of Fc fucosylation was observed in tumor stage II and III whereas reverse changes were found for the presence of bisecting GlcNAc. Higher level of fully sialylated glycans and elevated expression of glycans with bisecting GlcNAc were associated with better survival rate. Our findings provide the first evidence that the changes in Fc glycan profile may predict the survival of patients with gastric cancer. Cancer stage-dependent changes in Fc fucosylation and the bisecting N-acteylglucosamine expression as well as an association of several IgG glycoforms with the survival suggest that IgG glycosylation is related to pathogenesis of cancer and progression of the disease.     

Activation of murine CD4+ and CD8+ T lymphocytes leads to dramatic remodeling of N-linked glycans

Differentiation and activation of lymphocytes are documented to result in changes in glycosylation associated with biologically important consequences. In this report, we have systematically examined global changes in N-linked glycosylation following activation of murine CD4 T cells, CD8 T cells, and B cells by MALDI-TOF mass spectrometry profiling, and investigated the molecular basis for those changes by assessing alterations in the expression of glycan transferase genes. Surprisingly, the major change observed in activated CD4 and CD8 T cells was a dramatic reduction of sialylated biantennary N-glycans carrying the terminal NeuGcalpha2-6Gal sequence, and a corresponding increase in glycans carrying the Galalpha1-3Gal sequence. This change was accounted for by a decrease in the expression of the sialyltransferase ST6Gal I, and an increase in the expression of the galactosyltransferase, alpha1-3GalT. Conversely, in B cells no change in terminal sialylation of N-linked glycans was evident, and the expression of the same two glycosyltransferases was increased and decreased, respectively. The results have implications for differential recognition of activated and unactivated T cells by dendritic cells and B cells expressing glycan-binding proteins that recognize terminal sequences of N-linked glycans.     

Region-specific upregulation of HNK-1 glycan in the PRMT1-deficient brain

BackgroundBrains express structurally unique glycans, including human natural killer-1 (HNK-1), which participate in development and high-order functions. However, the regulatory mechanisms of expression of these brain-specific glycans are largely unknown. We examined whether arginine methylation, another type of protein modification essential for neural development, impacts the expression of various glycans in the developing brain.MethodsWe analyzed several types of glycans, including the HNK-1 epitope, in the cerebellum and cerebral cortex from mice with nervous system-specific knockout of protein arginine methyltransferase 1 (PRMT1). We also analyzed the expression levels of glycosyltransferases responsible for HNK-1 and of HNK-1 carrier glycoproteins by quantitative RT-PCR and western blotting.ResultsAmong several glycans, expression of HNK-1 glycan was strikingly upregulated in the PRMT1-deficient cerebellum. Furthermore, such upregulation was found in the cerebellum but not in the cerebral cortex. Regarding the mechanisms, we demonstrated that the mRNA level and activity of the responsible glycosyltransferase (B3gat1) were elevated in the knockout cerebellum. We also showed that the expression of HNK-1 carrier glycoproteins such as neural cell adhesion molecule (NCAM), L1 and AMPA receptor subunit GluA2 were also increased in the PRMT1-deficient cerebellum.ConclusionsLoss of arginine methylation leads to an increase in HNK-1 glycan in the developing cerebellum but not in the cerebral cortex via upregulation of the biosynthetic enzyme and carrier glycoproteins.General significancePRMT1 is a novel regulator of HNK-1 glycan production in the cerebellum. Mechanisms involving crosstalk between glycosylation and arginine methylation are suggested to occur.     

In vitro galactosylation of human IgG at 1 kg scale using recombinant galactosyltransferase

The Fc effector functions of immunoglobulin G (IgG) antibodies are in part determined by structural features of carbohydrates linked to each of the paired gamma heavy chains in the antibody constant domain (C(H)2). One glycoform that has been shown to be advantageous is G2, where both arms of complex bi-antennary N-glycans terminate in galactose. In vitro treatment with glycosyltransferases can remodel heterogeneous IgG glycoforms, enabling preparation of IgG molecules with homogeneous glycan chains. Here we describe optimization of conditions for use of a soluble recombinant galactosyltransferase in vitro to remodel glycans of human serum IgG, and we demonstrate a scaled-up reaction in which >98% of neutral glycans attached to 1 kg IgG are converted to the G2 glycoform. Removal of glycosylation reagents from the product is achieved in one step by affinity chromatography on immobilized Protein A.     

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.     

miR-10b对MCF10A细胞表达N-糖链 及O-糖链的影响

蛋白质糖基化是蛋白质翻译后修饰之一,对蛋白质功能有重要的调节作用,而异常糖基化在肿瘤的发生、发展以及癌细胞转移过程中起到关键作用.MiRNAs在癌症的发生发展过程中同样起到非常关键的作用,但其如何影响糖基化进而在肿瘤恶性转化过程中发挥生物学功能的研究甚少.本文将miR-10b在人正常乳腺上皮细胞MCF10A中过表达,利用糖类相关基因芯片系统筛选了发生显著变化的糖基转移酶;随后利用本实验室建立的N-糖链及O-糖链测定方法,分析糖链水平的表达差异;最后对关键糖基转移酶基因Fut8、MGAT3及OGT通过荧光定量PCR、蛋白质免疫印迹和凝集素免疫印迹进行了验证,为研究miR-10b在乳腺癌中的作用提供更多糖组学方面的理论基础.     

Glycosylation potential of human prostate cancer cell lines

Altered glycosylation is a universal feature of cancer cells and altered glycans can help cancer cells escape immune surveillance, facilitate tumor invasion, and increase malignancy. The goal of this study was to identify specific glycoenzymes, which could distinguish prostate cancer cells from normal prostatic cells. We investigated enzymatic activities and gene expression levels of key glycosyl- and sulfotransferases responsible for the assembly of O- and N-glycans in several prostatic cells. These cells included immortalized RWPE-1 cells derived from normal prostatic tissues, and prostate cancer cells derived from metastasis in bone (PC-3), brain (DU145), lymph node (LNCaP), and vertebra (VCaP). We found that all cells were capable of synthesizing complex N-glycans and O-glycans with the core 1 structure, and each cell line had characteristic biosynthetic pathways to modify these structures. The in vitro measured activities corresponded well to the mRNA levels of glycosyltransferases and sulfotransferases. Lectin and antibody binding to whole cells supported these results, which form the basis for the development of tumor cell-specific targeting strategies.     

IgG and IgM glycosylation patterns in patients undergoing image-guided tumor ablation

BackgroundImage-guided tumor ablation is a technique whereby needle-like applicators are placed directly into solid tumors under guidance typically with computed tomography or ultrasound. Changes in IgG and IgM antibody glycosylation were studied during ablation-induced immune response to cancer, and the use of glycosylation as a biomarker for diagnosis, prognosis and disease treatment was examined.MethodsPlasma from 27 tumor patients was collected immediately before, after and for 6 months following ablation. IgG and IgM antibodies were isolated by use high-throughput chromatography, and analyzed by hydrophilic liquid chromatography. Thorough identification of glycan structures in each chromatography peak was performed by nano-liquid chromatography electrospray ionization mass spectrometry.ResultsAlthough antibody glycosylation was found to vary with cancer type, discernable patterns of change based on the successful treatment of tumors by ablation were not identified. One patient with renal clear cell carcinoma and poor disease outcome had unexpectedly high amount of oligomannose IgG glycans during the whole period of monitoring. In contrast, IgM antibodies did not follow the same pattern.ConclusionsThese findings suggest that glycosylation patterns are indicative of an immune system that is unable to prevent different types of cancer, rather than products of the immunostimulatory response to the ablation of tumor itself. Analyses of the outcome effect suggested that IgG glycosylation and IgM glycosylation are not associated with tumor ablation.General significancePresent work opens a new way for parallel determination of glycosylation changes of both IgG and IgM antibodies by use of high-throughput methods, and their future use as biomarkers for disease diagnosis and prognosis. This article is part of a Special Issue entitled "Glycans in personalised medicine" Guest Editor: Professor Gordan Lauc.     

Glycosylation of site-specific glycans of α1-acid glycoprotein and alterations in acute and chronic inflammation

Backgroundα₁-Acid glycoprotein (AGP), an acute phase reactant, is extensively glycosylated at five Asn-linked glycosylation sites. In a number of pathophysiological states, including inflammation, rheumatoid arthritis, and cancer, alterations of Asn-linked glycans (N-glycans) have been reported. We investigated alteration of N-glycans at each of glycosylation sites of AGP in the sera of patients with acute and chronic inflammation.MethodsAGP purified from sera was digested with Glu-C and the liberated glycopeptides were isolated by reverse phase HPLC. N-glycans released with peptide N-glycosidase F and followed by neuraminidase treatment were analyzed by matrix-assisted laser desorption ionization-time of flight mass spectrometry.ResultsSite-specific differences in branching structures were observed among N-glycosylation sites 1, 3, 4 and 5. Within the sera of patients with acute inflammation, increases in bi-antennary and decreases in tri- and tetra-antennary structures were observed, as well as increases in α1,3-fucosylation, at most glycosylation sites. In the sera of patients with chronic inflammation, increased rates of tri-antennary α1,3-fucosylation at sites 3 and 4 and tetra-antennary α1,3-fucosylation at sites 3, 4 and 5 were detected. Although there were no significant differences between acute and chronic sera in site directed branching structures, significant differences of α1,3-fucosylation were detected in tri-antennary at sites 2, 4 and 5 and in tetra-antennary at sites 3 and 4.ConclusionLittle variation in the N-glycan composition of the glycosylation sites of AGP was observed among healthy individuals, while the sera of patients with acute inflammation demonstrated increased numbers of bi-antennary and α1,3-fucosylated N-glycan structures at each glycosylation site.     

High-mannose glycans are elevated during breast cancer progression

Alteration in glycosylation has been observed in cancer. However, monitoring glycosylation changes during breast cancer progression is difficult in humans. In this study, we used a well-characterized transplantable breast tumor mouse model, the mouse mammary tumor virus-polyoma middle T antigen, to observe early changes in glycosylation. We have previously used the said mouse model to look at O-linked glycosylation changes with breast cancer. In this glycan biomarker discovery study, we examined N-linked glycan variations during breast cancer progression of the mouse model but this time doubling the number of mice and blood draw points. N-glycans from total mouse serum glycoproteins were profiled using matrix-assisted laser desorption/ionization Fourier transform-ion cyclotron resonance mass spectrometry at the onset, progression, and removal of mammary tumors. We observed four N-linked glycans, m/z 1339.480 (Hex(3)HexNAc), 1485.530 (Hex(3)HexNAc(4)Fuc), 1809.639 (Hex(5)HexNAc(4)Fuc), and 1905.630 (Man(9)), change in intensity in the cancer group but not in the control group. In a separate study, N-glycans from total human serum glycoproteins of breast cancer patients and controls were also profiled. Analysis of human sera using an internal standard showed the alteration of the low-abundant high-mannose glycans, m/z 1419.475, 1581.528, 1743.581, 1905.634 (Man(6-9)), in breast cancer patients. A key observation was the elevation of a high-mannose type glycan containing nine mannoses, Man(9), m/z 1905.630 in both mouse and human sera in the presence of breast cancer, suggesting an incompletion of the glycosylation process that normally trims back Man(9) to produce complex and hybrid type oligosaccharides.     

N-glycosylation patterns of HSA/CD24 from different cell lines and brain homogenates: a comparison

N-glycans of the mouse glycoprotein HSA and its human analogue CD24 from lymphoblastoma, neuroblastoma and astrocytoma cell lines as well as from mouse brain homogenate were analysed and compared to each other and to the N-glycosylation pattern of total glycoproteins from mouse and human brain. The N-glycans were released from PVDF-blotted HSA or CD24 and separated on Carbograph SPE into neutral and acid glycans. The naturally neutral glycan fraction and the fraction of glycans rendered neutral after neuraminidase treatment were analysed without further purification by MALDI-MS. In each fraction, about 25 molecular ions with an intensity >10% of the base peak were identified which corresponded to glycans with distinct isobaric monosaccharide compositions. Comparison of the neutral and desialylated glycans revealed some similarities between the samples analysed, but also clear differences. HSA and CD24 from all cell lines express almost no neutral N-glycans with two or more fucose in contrast to brain HSA and glycoproteins from mouse and human brain. The lack of extensive fucosylation was also observed for desialylated glycans of HSA and CD24 from all cell lines analysed except for CD24 from a human neuroblastoma cell line which exhibits like total human and mouse brain glycoproteins a large variety of highly fucosylated, higher branched N-glycans. HSA from mouse brain carries in addition desialylated non-fucosylated glycans of high abundance which were detected, if at all, only at low intensity in all other samples analysed suggesting that they may be implicated in specific functions of mouse brain HSA. Therefore, a rapid assessment of similarities or differences between glycosylation patterns of a glycoprotein isolated from different sources is possible using methods as described here.     

High throughput isolation and glycosylation analysis of IgG-variability and heritability of the IgG glycome in three isolated human populations

All immunoglobulin G molecules carry N-glycans, which modulate their biological activity. Changes in N-glycosylation of IgG associate with various diseases and affect the activity of therapeutic antibodies and intravenous immunoglobulins. We have developed a novel 96-well protein G monolithic plate and used it to rapidly isolate IgG from plasma of 2298 individuals from three isolated human populations. N-glycans were released by PNGase F, labeled with 2-aminobenzamide and analyzed by hydrophilic interaction chromatography with fluorescence detection. The majority of the structural features of the IgG glycome were consistent with previous studies, but sialylation was somewhat higher than reported previously. Sialylation was particularly prominent in core fucosylated glycans containing two galactose residues and bisecting GlcNAc where median sialylation level was nearly 80%. Very high variability between individuals was observed, approximately three times higher than in the total plasma glycome. For example, neutral IgG glycans without core fucose varied between 1.3 and 19%, a difference that significantly affects the effector functions of natural antibodies, predisposing or protecting individuals from particular diseases. Heritability of IgG glycans was generally between 30 and 50%. The individual's age was associated with a significant decrease in galactose and increase of bisecting GlcNAc, whereas other functional elements of IgG glycosylation did not change much with age. Gender was not an important predictor for any IgG glycan. An important observation is that competition between glycosyltransferases, which occurs in vitro, did not appear to be relevant in vivo, indicating that the final glycan structures are not a simple result of competing enzymatic activities, but a carefully regulated outcome designed to meet the prevailing physiological needs.     

N-Glycosylation of a mouse IgG expressed in transgenic tobacco plants

Since plants are emerging as an important system for the expression of recombinant glycoproteins, especially those intended for therapeutic purposes, it is important to scrutinize to what extent glycans harbored by mammalian glycoproteins produced in transgenic plants differ from their natural counterpart. We report here the first detailed analysis of the glycosylation of a functional mammalian glycoprotein expressed in a transgenic plant. The structures of the N-linked glycans attached to the heavy chains of the monoclonal antibody Guy's 13 produced in transgenic tobacco plants (plantibody Guy's 13) were identified and compared to those found in the corresponding IgG1 of murine origin. Both N-glycosylation sites located on the heavy chain of the plantibody Guy's 13 are N-glycosylated as in mouse. However, the number of Guy's 13 glycoforms is higher in the plant than in the mammalian expression system. Despite the high structural diversity of the plantibody N-glycans, glycosylation appears to be sufficient for the production of a soluble and biologically active IgG in the plant system. In addition to high-mannose-type N-glycans, 60% of the oligosaccharides N-linked to the plantibody have beta(1, 2)-xylose and alpha(1, 3)-fucose residues linked to the core Man3GlcNAc2. These plant-specific oligosaccharide structures are not a limitation to the use of plantibody Guy's 13 for topical immunotherapy. However, their immunogenicity may raise concerns for systemic applications of plantibodies in human.     

Alterations of serum protein N-glycosylation in two mouse models of chronic liver disease are hepatocyte and not B cell driven

N-glycosylation of immunoglobulin G (IgG) has an important impact on the modification of the total serum N-glycome in chronic liver patients. Our aim was to determine the role and magnitude of the alterations in which hepatocytes and B cells are involved in two mouse models of chronic liver disease. Common bile duct ligation (CBDL) and subcutaneous injections with CCl(4) were induced in B cell-deficient and wild-type (WT) mice. IgG depletion was performed with beads covered with protein A/G and the depletions were evaluated by SDS-PAGE and Western blot analysis. N-glycan analysis was performed by improved DSA-FACE technology. Structural analysis of the mouse serum N-glycans was performed by exoglycosidase digests and MALDI-TOF mass spectrometry of permethylated glycans. The alterations seen in B cell-deficient mice closely resembled the alterations in WT mice, in both the CBDL and the CCl(4) models. N-glycan analysis of the IgG fraction in both mouse models revealed different changes compared with humans. Overall, the impact of IgG glycosylation on total serum glycosylation was marginal. Interestingly, the amount of fibrosis present in CBDL B cell-deficient mice was significantly increased compared with CBDL WT mice, whereas the opposite was true for the CCl(4) model as determined by Sirius red staining. However, this had no major effect on the alteration of N-glycosylation of serum proteins. Alterations of total serum N-glycome in mouse models of chronic liver disease are hepatocyte-driven. Undergalactosylation of IgG is not present in mouse models of chronic liver disease.     

Short O-GalNAc glycans: regulation and role in tumor development and clinical perspectives

BackgroundWhile the underlying causes of cancer are genetic modifications, changes in cellular states mediate cancer development. Tumor cells display markedly changed glycosylation states, of which the O-GalNAc glycans called the Tn and TF antigens are particularly common. How these antigens get over-expressed is not clear. The expression levels of glycosylation enzymes fail to explain it.Scope of ReviewWe describe the regulation of O-GalNAc glycosylation initiation and extension with emphasis on the initiating enzymes ppGalNAcTs (GALNTs), and introduce the GALA pathway — a change in GALNTs compartmentation within the secretory pathway that regulates Tn levels. We discuss the roles of O-GalNAc glycans and GALNTs in tumorigenic processes and finally consider diagnostic and therapeutic perspectives.Major conclusionsContrary to a common hypothesis, short O-glycans in tumors are not the result of an incomplete glycosylation process but rather reveal the activation of regulatory pathways. Surprisingly, high Tn levels reveal a major shift in the O-glycoproteome rather than a shortening of O-glycans. These changes are driven by membrane trafficking events.General SignificanceMany attempts to use O-glycans for biomarker, antibody and therapeutic vaccine development have been made, but suffer limitations including poor sensitivity and/or specificity that may in part derive from lack of a mechanistic understanding. Deciphering how short O-GalNAc glycans are regulated would open new perspectives to exploit this biology for therapeutic usage. This article is part of a Special Issue entitled "Glycans in personalised medicine" Guest Editor: Professor Gordan Lauc.     

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;