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Apo-H is a plasma glycoprotein. Nearly 19% of the molecular weight of this protein is composed of glycans. Up- and down-regulation and structural changes in protein glycans provide diagnostic value for disease detection. Here, an efficient, sensitive, and optimized method is developed for Apo-H N-glycans analysis by MALDI-TOF-MS in positive mode. This bioanalytical method includes sample preparation, sample purification, and detection. An Apo-H enrichment method is developed using standard proteins by anti-Apo-H beads followed by enrichment from plasma samples. SDS-PAGE confirms the Apo-H protein enrichment, which is further verified by LC-MS/MS analysis. The lower ionization efficiency of sialylated glycan hampers their analysis by MALDI-MS. For this, stabilization of sialic acids is done by selective derivatization of carboxyl groups to differentiate between α(2,3)- and α(2,6)-linked sialic acids. Glycans are further purified by HILIC-SPE and analyzed by MALDI-MS. Several branched bi- and tri-antennary glycans with fucosylation and sialylation are identified. The reproducibility of the developed method is tested by analyzing multiple replicates of human plasma, where the same glycans are consistently identified. This method could be applied for the Apo-H glycan profiling of large clinical cohorts for diagnostic purposes.

This study elucidated the regulatory roles of milk glycoprotein core fucosylation on the gut microbiota of breast-fed infants. In order to investigate the effects of core fucosylation of maternal milk N-glycan on the intestinal functions in newborn mice, Fut8+/+ → Fut8+/+ and Fut8± → Fut8+/+ models were established. The results indicated that the gut microbiota diversity was altered and production of Bifidobacterium and Lactobacillus was reduced in the intestine of Fut8± → Fut8+/+mice. Furthermore, the levels of SCFAs were decreased in the gut of Fut8± → Fut8+/+infant. To further investigate the effects of core fucosylation of breast milk N-glycan on the intestinal inflammation in newborns, NEC models were established, and Fut8+/+ off-springs with experimentally induced NEC were fed with the breast milk of Fut8+/+ and Fut8± maternal mice. Our data revealed that reduced core fucosylation of breast milk N-glycan could be responsible for the intestinal inflammation in NEC offspring, as the production of pro-inflammatory cytokines including IL-1b, IL-6, IL-17A and TNF-a was significantly upregulated in Fut8± → Fut8+/+NEC group, while the levels of anti-inflammatory cytokine IL-10 were remarkably reduced. Furthermore, Treg/Th17 balance in the intestine was disturbed in Fut8± → Fut8+/+NEC newborn, suggesting the impaired immune development caused by reduced core fucosylation of breast milk. These findings indicated that core fucosylation of maternal milk N-glycan could assist the colonization of gut Lactobacillus and Bifidobacterium and regulate the Treg/Th17 balance, subsequently preventing intestinal inflammation in breast-fed newborns.

Caveolin-1 (Cav-1) is a constitutive protein within caveolar membranes. Previous studies from our group and others indicated that Cav-1 could mediate N-glycosylation, α2,6-sialylation, and fucosylation in mouse hepatocarcinoma cells in vitro. However, little is known about the effect of Cav-1 expression on glycosylation modifications in vivo. In this study, the N-glycan profiles in serum from Cav-1-/- mice were investigated by lectin microarray and mass spectrometric analysis approaches. The results showed that levels of multi-antennary branched, α2,6-sialylated, and galactosylated N-glycans increased, while high-mannose typed and fucosylated N-glycans decreased in the serum of Cav-1-/- mice, compared with that of wild-type mice. Furthermore, the real-time quantitative PCR analysis indicated that α2,6-sialyltransferase gene expression decreased significantly in Cav-1-/- mouse organ tissues, but α2,3- and α2,8-sialyltransferase did not. Of them, both mRNA and protein expression levels of the β-galactoside α2,6-sialyltransferase 1 (ST6Gal-I) had dramatically reduced in Cav-1-/- mice organ tissues, which was consistent with the α2,6-sialyl Gal/GalNAc level reduced significantly in tissues instead of serum from Cav-1-/- mice. These results provide for the first time the N-glycans profile of Cav-1-/- mice serum, which will facilitate understanding the function of Cav-1 from the perspective of glycosylation.

Background: Haptoglobin (Hp), an acute-phase protein, is known as a potential diagnostic biomarker in human diseases. Two alleles of Hp (Hp1 and Hp2 ) exist in humans allowing three phenotypes (Hp1-1, Hp2-1, and Hp2-2), which influence the biophysical and biological properties of Hp. Objective: This work aimed to investigate the variation of serum level and fucosylation change among Hp phenotypes in patients with lung cancer compared to healthy donors. Methods: 44 patients with lung cancer and 26 healthy blood donors who lived in the Northern-Thailand region were investigated by the glycoproteomic procedure. Results: The phenotypic distribution of the Hp (Hp1-1:Hp2-1:Hp2-2) in healthy donors was 0.04:0.38:0.58, while the patient group was 0.09:0.52:0.39. The Hp1 allele frequency of patients with lung cancer (0.34) was higher than the healthy donor (0.23). Glycoprotein blotting technique represented that the level of serum Hp and its fucosylation were significantly higher among lung cancer patients compared to those of the healthy donors. However, a downward trend in the fucosylation level from Hp1-1 to Hp2-1, Hp2-2, was seen in the patient group, but varying in the serum Hp level. An N-linked glycan was enzymatically released from discrete Hp multimers of Hp2-1 and Hp2-2 samples. Analysis of glycan profiling by MALDI-TOF-MS showed that reduction of the fucosylated glycan was associated with the size of Hp multimers, resulting in the lower level of fucosylation in Hp2-1 and Hp2-2, respectively. Conclusion: Our finding demonstrates that the Hp phenotype is a dependent risk factor for lung cancer and should be incorporated into further clinical and biochemical investigations of diseases, including lung cancer.

In metazoans, protein O-fucosylation of Ser/Thr residues was only found in secreted or cell surface proteins, and this post-translational modification is catalyzed by ER-localized protein O-fucosyltransferases (POFUTs) in the GT65 family. Recently, a novel nucleocytoplasmic POFUT, SPINDLY (SPY), was identified in the reference plant Arabidopsis thaliana to modify nuclear transcription regulators DELLAs, revealing a new regulatory mechanism for gene expression. The paralog of AtSPY, SECRET AGENT (SEC), is an O-link-N-acetylglucosamine (GlcNAc) transferase (OGT), which O-GlcNAcylates Ser/Thr residues of target proteins. Both AtSPY and AtSEC are tetratricopeptide repeat-domain-containing glycosyltransferases in the GT41 family. The discovery that AtSPY is a POFUT clarified decades of miss-classification of AtSPY as an OGT. SPY and SEC play pleiotropic roles in plant development, and the interactions between SPY and SEC are complex. SPY-like genes are conserved in diverse organisms, except in fungi and metazoans, suggesting that O-fucosylation is a common mechanism in modulating intracellular protein functions.

Fucosylation alteration is involved in several steps of human cancer pathogenesis. Dysregulated long non-coding RNA (lncRNA) often leads to malignancy in colorectal cancer (CRC). Differential levels of LEF1-AS1, LEF1 and FUT8 are analyzed by qRT-PCR and western blot. Chip, RIP, EMSA and luciferase reporter assay confirm the direct interaction among LEF1-AS1, MLL1, H3K4me3, LEF1 and FUT8. Functionally, CRC cell proliferation, migration and invasion are analyzed by CCK8 assay, colony formation assay, transwell assay and flow cytometry. The xenografts nude mice models, lung metastasis and liver metastasis are established to determine the effect of LEF1-AS1/LEF1/FUT8 axis on CRC progression in vivo. Here, we identify that LEF1-AS1 and LEF1 are higher in CRC tissues than that in adjacent tissues, as well as upregulated in CRC cell lines than that in normal colorectal cells. Altered levels of LEF1-AS1 modulate LEF1 expression, while altered LEF1 could not regulate LEF1-AS1. LEF1-AS1 recruits MLL1 to the promoter region of LEF1, induces H3K4me3 methylation modification and mediates LEF1 transcription. Furthermore, α1-6 fucosyltransferase FUT8 is overexpressed in CRC tissues and positively correlated to LEF1. FUT8 is a direct target of transcription factor LEF1, which regulates FUT8 level. Altered FUT8 also regulates the core fucosylation of CRC cells, and LEF1-AS1 mediates FUT8 level through activation of Wnt/β-catenin/LEF1 pathway, thereby resulting in β-catenin nuclear translocation. In addition, LEF1-AS1 mediates the proliferation, migration and invasion of CRC cells in vitro. LEF1-AS1 silence hinders the tumorigenesis, liver and lung metastasis of SW620 cells in vivo, while overexpressed FUT8 abolishes the suppressive impact of LEF1-AS1 repression on the biological behavior of SW620 cells. Our studies uncovered a novel mechanism for constitutive LEF1-AS1/LEF1/FUT8 axis in CRC progression by regulating α1, 6-fucosylation via Wnt/β-catenin pathway, and consequently, as a potential therapeutic target in CRC.

Objective Anti-β-2 glycoprotein I (anti-β2GPI) antibodies, defined as primary pathogenic antibody in antiphospholipid syndrome (APS). It has been reported that IgG Fc N-glycosylation affects IgG effector, we aim to investigate the association of Fc glycosylation profiles of purified anti-β2GP1 IgG with clinical features of APS. Methods We purify anti-β2GPI IgG and total IgG from 82 APS patients including nine catastrophic antiphospholipid syndrome (CAPS) patients, as well as total IgG from 103 healthy controls to quantitatively analyse all detectable Fc N-glycanforms of all IgG subclasses with Multiple Reaction Monitoring (MRM) method based on UPLC-ESI-QqQ mass spectrometry. Results Both purified anti-β2GPI IgG and APS total IgG showed altered N-glycan profiles when compared with healthy control (HC) IgG. Anti-β2GPI IgG presented with lower galactosylation, increased bisection and core fucosylation compared with APS total IgG and HC IgG. We found higher galactosylation of aβ2GPI IgG2 in thrombotic APS compared with the obstetric APS, and lower galactosylation of aβ2GPI IgG2 associated with late pregnancy morbidity. Moreover, low galactosylation of all anti-β2GPI IgG subclasses, increased bisection and core fucosylation of anti-β2GPI IgG1/2 were strongly associated with CAPS and triple positivity of antiphospholipid antibodies (aPLs). Conclusion We comprehensively characterize the N-Glycans landscape of both anti-β2GP1 and total IgG in APS. Altered N-glycan profiles of anti-β2GPI IgG enables enabled the antibodies with proinflammatory properties. Furthermore, we associated levels of IgG Fc-glycosylation with clinical features antiphospholipid syndrome. These findings could increase our understanding of anti-β2GPI antibody mediated mechanisms in APS and be used to develop diagnostics and new target treatments.

Fc glycosylation profoundly impacts the effector functions of antibodies and often dictates an antibody’s pro- or anti-inflammatory activities. It is well established that core fucosylation of the Fc domain N-glycans of an antibody significantly reduces its affinity for FcγRIIIa receptors and antibody-dependent cellular cytotoxicity (ADCC). Previous structural studies have suggested that the presence of a core fucose remarkably decreases the unique and favorable carbohydrate–carbohydrate interactions between the Fc and the receptor N-glycans, leading to reduced affinity. We report here that in contrast to natural core fucose, special site-specific modification on the core fucose could dramatically enhance the affinity of an antibody for FcγRIIIa. The site-selective modification was achieved through an enzymatic transfucosylation with a novel fucosidase mutant, which was shown to be able to use modified α-fucosyl fluoride as the donor substrate. We found that replacement of the core l-fucose with 6-azide- or 6-hydroxy-l-fucose (l-galactose) significantly enhanced the antibody’s affinity for FcγRIIIa receptors and substantially increased the ADCC activity. To understand the mechanism of the modified fucose-mediated affinity enhancement, we performed molecular dynamics simulations. Our data revealed that the number of glycan contacts between the Fc and the Fc receptor was increased by the selective core-fucose modifications, showing the importance of unique carbohydrate–carbohydrate interactions in achieving high FcγRIIIa affinity and ADCC activity of antibodies. Thus, the direct site-selective modification turns the adverse effect of the core fucose into a favorable force to promote the carbohydrate–carbohydrate interactions.

Glycosylation changes are key molecular events in tumorigenesis, progression and glycosyltransferases play a vital role in the this process. FUT8 belongs to the fucosyltransferase family and is the key enzyme involved in N-glycan core fucosylation. FUT8 and/or core fucosylated proteins are frequently upregulated in liver, lung, colorectal, pancreas, prostate,breast, oral cavity, oesophagus, and thyroid tumours, diffuse large B-cell lymphoma, ependymoma, medulloblastoma and glioblastoma multiforme and downregulated in gastric cancer. They can be used as markers of cancer diagnosis, occurrence, progression and prognosis. Core fucosylated EGFR, TGFBR, E-cadherin, PD1/PD-L1 and α3β1 integrin are potential targets for tumour therapy. In addition, IGg1 antibody defucosylation can improve antibody affinity, which is another aspect of FUT8 that could be applied to tumour therapy.

Rationale: Epithelial-mesenchymal transition (EMT) has been recognized as an important step toward high invasion and metastasis of many cancers including hepatocellular carcinoma (HCC), while the mechanism for EMT promotion is still ambiguous. Methods: The dynamic alterations of site-specific glycosylation during HGF/TGF-β1-induced EMT process of three HCC cell lines were systematically investigated using precision glycoproteomic methods. The possible roles of EMT-related glycoproteins and site-specific glycans were further confirmed by various molecular biological approaches. Results: Using mass spectrometry-based glycoproteomic methods, we totally identified 2306 unique intact glycopeptides from SMMC-7721 and HepG2 cell lines, and found that core-fucosylated glycans were accounted for the largest proportion of complex N-glycans. Through quantification analysis of intact glycopeptides, we found that the majority of core-fucosylated intact glycopeptides from folate receptor α (FOLR1) were up-regulated in the three HGF-treated cell lines. Similarly, core-fucosylation of FOLR1 were up-regulated in SMMC-7721 and Hep3B cells with TGF-β1 treatment. Using molecular approaches, we further demonstrated that FUT8 was a driver for HGF/TGF-β1-induced EMT. The silencing of FUT8 reduced core-fucosylation and partially blocked the progress of HGF-induced EMT. Finally, we confirmed that the level of core-fucosylation on FOLR1 especially at the glycosite Asn-201 positively regulated the cellular uptake capacity of folates, and enhanced uptake of folates could promote the EMT of HCC cells. Conclusions: Based on the results, we proposed a potential pathway for HGF or TGF-β1-induced EMT of HCC cells: HGF or TGF-β1 treatment of HCC cells can increase the expression of glycosyltransferase FUT8 to up-regulate the core-fucosylation of N-glycans on glycoproteins including the FOLR1; core-fucosylation on FOLR1 can then enhance the folate uptake capacity to finally promote the EMT progress of HCC cells.

The α1,6-fucosyltransferase, FUT8, is the sole enzyme catalyzing the core-fucosylation of N-glycoproteins in mammalian systems. Previous studies using free N-glycans as acceptor substrates indicated that a terminal β1,2-GlcNAc moiety on the Man-α1,3-Man arm of N-glycan substrates is required for efficient FUT8-catalyzed core-fucosylation. In contrast, we recently demonstrated that, in a proper protein context, FUT8 could also fucosylate Man5GlcNAc2 without a GlcNAc at the non-reducing end. We describe here a further study of the substrate specificity of FUT8 using a range of N-glycans containing different aglycones. We found that FUT8 could fucosylate most of high-mannose and complex-type N-glycans, including highly branched N-glycans from chicken ovalbumin, when the aglycone moiety is modified with a 9-fluorenylmethyloxycarbonyl (Fmoc) moiety or in a suitable peptide/protein context, even if they lack the terminal GlcNAc moiety on the Man-α1,3-Man arm. FUT8 could also fucosylate paucimannose structures when they are on glycoprotein substrates. Such core-fucosylated paucimannosylation is a prominent feature of lysosomal proteins of human neutrophils and several types of cancers. We also found that sialylation of N-glycans significantly reduced their activity as a substrate of FUT8. Kinetic analysis demonstrated that Fmoc aglycone modification could either improve the turnover rate or decrease the KM value depending on the nature of the substrates, thus significantly enhancing the overall efficiency of FUT8 catalyzed fucosylation. Our results indicate that an appropriate aglycone context of N-glycans could significantly broaden the acceptor substrate specificity of FUT8 beyond what has previously been thought.

Chinese hamster ovary (CHO) cell is the predominant cell factory to produce biopharmaceuticals such as immunoglobulin G (IgG), but in CHO cells lactate accumulation and cell death compromise IgG production while fucosylation mitigates the antibody-dependent cellular cytotoxicity (ADCC) of IgG. To tackle these problems, we harnessed a burgeoning gene silencing system, CRISPR-Cas13d, to effectively suppress the endogenous genes governing lactate production (LDHA), fucosylation (GFT) and cell death (DDIT3), hence repressing lactate accumulation and core fucosylation. We further exploited the Sleeping Beauty system to integrate the CRISPR-Cas13d module co-targeting these 3 genes and generated a CHO cell platform for IgG production. The new platform exhibited simultaneous knockdown of LDHA, GFT and DDIT3, accumulated less lactate, had prolonged longevity, produced 59 ± 6% more IgG with less fucosylation and stronger ADCC efficacy. These data collectively warrant the potentials of CRISPR-Cas13d for CHO cell engineering and improving antibody production with regard to quantity and quality.

Intervertebral disc (IVD) degeneration causes low-back pain through disc compression, prolapse and herniation. Inflammation of the IVD and subsequent degeneration produce altered glycosylation profiles in several animal models of IVD injury and ageing, although the function of this altered glycosylation pattern in a human is unknown. Altered N-glycome, specifically sialylated and fucosylated N-glycosylation motif expression, might play a role in inflammation and disease progression. Healthy (foetal and adolescent idiopathic scoliosis) and degenerated (lumbar degeneration) human IVD glycosylation patterns were studied using lectin histochemistry. Small-molecule fluorinated sugar analogues (3Fax-Peracetyl Neu5Ac; 2F-Peracetyl-Fucose) were used to inhibit sialylation and fucosylation in an in vitro model of inflammation, to investigate their effects on the glycosignature, cell metabolism, extracellular matrix synthesis and cell migration. The effects of interleukin (IL)-1β, tumour necrosis factor (TNF)-α and IL-6 on glycosylation in human nucleus pulposus cells were investigated by lectin histochemistry, PCR and enzyme-linked immunosorbent assay (ELISA). In the in vitro model of IVD degeneration, cytokine-induced inflammation-induced hypersialylation was observed, as indicated by Sambucus nigra I binding. However, this modification was inhibited by the sialyltransferase inhibitor. Inhibition of sialylation and fucosylation modulates cell migration and protein translation of catabolic enzymes in response to inflammation. The altered patterns of glycosylation in human tissue in degeneration was consistent with previous IVD studies in murine, bovine and ovine models. The present study was the first functional investigation of glycosylation in human degenerated IVD, elucidating the role of the glycome in disease progression and identified potential therapeutic targets for future regenerative therapies.

In this study, we developed an efficient fluorous tagging strategy for site-selective enzymatic fucosylation/sialylation in which appending a readily removable auxiliary tag (SF17) to the reducing ...

The deoxyhexose sugar L-fucose is important for many biological processes within the human body and the associated microbiota. This carbohydrate is abundant in host gut mucosal surfaces, numerous microbial cell surface structures, and some dietary carbohydrates. Fucosylated oligosaccharides facilitate the establishment of a healthy microbiota and provide protection from infection. However, there are instances where pathogens can also exploit these fucosylated structures to cause infection. Furthermore, deficiencies in host fucosylation are associated with specific disease outcomes. This review focuses on our current understanding of the impact of fucosylation within the mucosal environment of the gastrointestinal tract with a specific emphasis on the mediatory effects in host-microbe interactions.

Immunoglobulin G (IgG) functionality can drastically change from anti- to proinflammatory by alterations in the IgG N-glycan patterns. Our previous studies have demonstrated that IgG N-glycans associated with the risk factors of dementia, such as aging, dyslipidemia, type 2 diabetes mellitus, hypertension, and ischemic stroke. Therefore, the aim is to investigate whether the effects of IgG N-glycan profiles on dementia exists in a Chinese Han population. A case–control study, including 81 patients with dementia, 81 age- and gender-matched controls with normal cognitive functioning (NC) and 108 non-matched controls with mild cognitive impairment (MCI) was performed. Plasma IgG N-glycans were separated by ultra-performance liquid chromatography. Fourteen glycan peaks reflecting decreased of sialylation and core fucosylation, and increased bisecting N-acetylglucosamine (GlcNAc) N-glycan structures were of statistically significant differences between dementia and NC groups after controlling for confounders (p q p q N-glycans might contribute to be potential novel biomarkers for the neurodegenerative process risk assessment of dementia.

Immunoglobulin G is the abundant antibody present in the colostrum and milk of major dairy animals. In the present study, buffalo milk IgG was characterized for its amino acid sequence and glycan diversity using reverse phase liquid chromatography coupled to ESI-Q-TOF MS in tandem mode. Amino acid sequence analysis of heavy chain constant region revealed the presence of two IgG subtypes namely IgG1 and IgG3, with IgG1 being the abundant. The complete light chain constant region sequence was also determined. N-glycan sequence analysis at a highly conserved site Asn-Ser-Thr revealed the presence of mainly biantennary complex type with core fucosylation (34%), bisecting GlcNAc (19%) and sialylation with both Neu5Ac and Neu5Gc (14%). The observed glycan diversity in buffalo milk IgG is in part comparable with bovine colostrum as well as human, bovine, goat serum counterparts.

BACKGROUND Inflammatory bowel disease (IBD) is a chronic, relapsing inflammation of the digestive tract. Although fecal and serum biomarkers have been extremely important and supportive for monitoring of IBD, their low sensitivity and high variability characteristics limit clinical efficacy. Thus, the establishment of better biomarkers is expected. Fucosylation is one of the most important glycosylation modifications of proteins. Fucosylated haptoglobin (Fuc-Hpt) is used as a biomarker for several cancers and inflammation-related diseases. We recently established a novel glycan monoclonal antibody (mAb), designated 10-7G, which recognizes Fuc-Hpt. We developed an enzyme-linked immunosorbent assay (ELISA) to measure serum levels of Fuc-Hpt (10-7G values). AIM To investigate the usefulness of the serum 10-7G values as a potential biomarker for monitoring disease activity in IBD. METHODS This was a case control study. Intestinal tissues of IBD patients (n = 10) were examined immunohistochemically using the 10-7G mAb. We determined 10-7G values using serum from patients with ulcerative colitis (UC, n = 110), Crohn’s disease (n = 45), acute enteritis (AE, n = 11), and healthy volunteers (HVs) who exhibited normal (n = 20) or high (n = 79) C-reactive protein (CRP) levels at medical check-up. We investigated the correlation between the 10-7G value and various clinical parameters of IBD patients by correlation analysis. Receiver operating characteristic (ROC) curve analysis was performed to evaluate the usefulness of the 10-7G values as a biomarker for clinical and endoscopic remission of UC compared to conventional serum biomarkers. RESULTS In the immunohistochemical analysis, positive 10-7G mAb staining was observed in lymphocytes infiltrating into inflammatory sites of the mucosal layer and lymphoid follicles. The 10-7G values were significantly higher in patients with IBD (P < 0.001) and AE (P < 0.05) compared with HVs. In addition, 10-7G values were correlated with clinical examination parameters related to inflammation in patients with UC, particularly the CRP level (rs = 0.525, P = 0.003) and clinical activity index score (rs = 0.435, P = 0.038). However, there was no correlation between 10-7G values and CRP in HVs with high CRP levels, suggesting that the 10-7G values is not the same as a general inflammation biomarker. ROC curve analysis showed that area under the curve (AUC) value of 10-7G values for the diagnosis of endoscopic remission was higher than other biomarkers (AUC value = 0.699). CONCLUSION The serum 10-7G value is a novel biomarker for evaluating intestinal inflammation and endoscopic mucosal healing in UC.

Fucosylation of glycans impacts a myriad of physiological and pathological processes. Inhibition of fucose expression emerges as a potential therapeutic avenue for example in cancer, inflammation, and infection. In this study, we found that protected 2‐fluorofucose 1‐phosphate efficiently inhibits cellular fucosylation with a four to seven times higher potency than known inhibitor 2FF, independently of the anomeric stereochemistry. Nucleotide sugar analysis revealed that both the α‐ and β‐GDP‐2FF anomers are formed inside the cell. In conclusion, we developed A2FF1P and B2FF1P as potent new tools for studying the role of fucosylation in health and disease and they are potential therapeutic candidates., Fucosylation of glycans impacts a myriad of physiological of pathological activities. In this study, new fucosylation inhibitors based on fluorination of fucose‐1‐phosphates were developed. Inhibitors A2FF1P and B2FF1P are potent, specific, non‐toxic, inhibit fucosylation for multiple days and have a known mechanism of action. These new tools will help study the role of fucosylation in health and disease and are potential therapeutic candidates.

CD3-bispecific antibodies represent an important therapeutic strategy in oncology. These molecules work by redirecting cytotoxic T cells to antigen-bearing tumor cells. Although CD3-bispecific antibodies have been developed for several clinical indications, cases of cancer-derived resistance are an emerging limitation to the more generalized application of these molecules. Here, we devised whole-genome CRISPR screens to identify cancer resistance mechanisms to CD3-bispecific antibodies across multiple targets and cancer types. By validating the screen hits, we found that deficiency in IFNγ signaling has a prominent role in cancer resistance. IFNγ functioned by stimulating the expression of T-cell killing–related molecules in a cell type–specific manner. By assessing resistance to the clinical CD3-bispecific antibody flotetuzumab, we identified core fucosylation as a critical pathway to regulate flotetuzumab binding to the CD123 antigen. Disruption of this pathway resulted in significant resistance to flotetuzumab treatment. Proper fucosylation of CD123 was required for its normal biological functions. In order to treat the resistance associated with fucosylation loss, flotetuzumab in combination with an alternative targeting CD3-bispecific antibody demonstrated superior efficacy. Together, our study reveals multiple mechanisms that can be targeted to enhance the clinical potential of current and future T-cell–engaging CD3-bispecific antibody therapies.

B-cell precursor acute lymphoblastic leukemia (BCP-ALL) with mixed-lineage leukemia gene rearrangement (MLL-r) is a poor-prognosis subtype for which additional therapeutic targets are urgently needed. Currently no multi-omics data set for primary MLL r patient cells exists that integrates transcriptomics, proteomics and glycomics to gain an inclusive picture of theranostic targets. Methods: We have integrated transcriptomics, proteomics and glycomics to i) obtain the first inclusive picture of primary patient BCP-ALL cells and identify molecular signatures that distinguish leukemic from normal precursor B-cells and ii) better understand the benefits and limitations of the applied technologies to deliver deep molecular sequence data across major cellular biopolymers. Results: MLL-r cells feature an extensive remodeling of their glycocalyx, with increased levels of Core 2-type O-glycans and complex N-glycans as well as significant changes in sialylation and fucosylation. Notably, glycosaminoglycan remodeling from chondroitin sulfate to heparan sulfate was observed. A survival screen, to determine if glycan remodeling enzymes are redundant, identified MGAT1 and NGLY1, essential components of the N-glycosylation/degradation pathway, as highly relevant within this in vitro screening. OGT and OGA, unique enzymes that regulate intracellular O-GlcNAcylation, were also indispensable. Transcriptomics and proteomics further identified Fes and GALNT7-mediated glycosylation as possible therapeutic targets. While there is overall good correlation between transcriptomics and proteomics data, we demonstrate that a systematic combined multi-omics approach delivers important diagnostic information that is missed when applying a single omics technology. Conclusions: Apart from confirming well-known MLL-r BCP-ALL glycoprotein markers, our integrated multi-omics workflow discovered previously unidentified diagnostic/therapeutic protein targets.

The effector functions of the IgGs are modulated by the N-glycosylation of their Fc region. Particularly, the absence of core fucosylation is known to increase the affinity of IgG1s for the Fcγ receptor IIIa expressed by immune cells, in turn translating in an improvement in the antibody-dependent cellular cytotoxicity. However, the impact of galactosylation and sialylation is still debated in the literature. In this study, we have investigated the influence of high and low levels of core fucosylation, terminal galactosylation and terminal α2,6-sialylation of the Fc N-glycans of trastuzumab on its affinity for the FcγRIIIa. A large panel of antibody glycoforms (i.e., highly α2,6-sialylated or galactosylated IgG1s, with high or low levels of core fucosylation) were generated and characterized, while their interactions with the FcγRs were analysed by a robust surface plasmon resonance-based assay as well as in a cell-based reporter bioassay. Overall, IgG1 glycoforms with reduced fucosylation display a stronger affinity for the FcγRIIIa. In addition, fucosylation, and the presence of terminal galactose and sialic acids are shown to increase the affinity for the FcγRIIIa as compared to the agalactosylated forms. These observations perfectly translate in the response observed in our reporter bioassay.

Inter-tumor heterogeneity is a result of genomic, transcriptional, translational, and post-translational molecular features. To investigate the roles of protein glycosylation in the heterogeneity of high-grade serous ovarian carcinoma (HGSC), we perform mass spectrometry-based glycoproteomic characterization of 119 TCGA HGSC tissues. Cluster analysis of intact glycoproteomic profiles delineates 3 major tumor clusters and 5 groups of intact glycopeptides. It also shows a strong relationship between N-glycan structures and tumor molecular subtypes, one example of which being the association of fucosylation with mesenchymal subtype. Further survival analysis reveals that intact glycopeptide signatures of mesenchymal subtype are associated with a poor clinical outcome of HGSC. In addition, we study the expression of mRNAs, proteins, glycosites, and intact glycopeptides, as well as the expression levels of glycosylation enzymes involved in glycoprotein biosynthesis pathways in each tumor. The results show that glycoprotein levels are mainly controlled by the expression of their individual proteins, and, furthermore, that the glycoprotein-modifying glycans correspond to the protein levels of glycosylation enzymes. The variation in glycan types further shows coordination to the tumor heterogeneity. Deeper understanding of the glycosylation process and glycosylation production in different subtypes of HGSC may provide important clues for precision medicine and tumor-targeted therapy., Altered protein glycosylation is increasingly recognized as a hallmark of cancer. Here, the authors profile the glycoproteome of 119 high-grade serous ovarian carcinoma tissues, showing that glycosylation patterns correlate with tumor molecular subtypes and clinical outcomes.

Human Milk Oligosaccharides (HMOs) are abundant carbohydrates fundamental to infant health and development. Although these oligosaccharides were discovered more than half a century ago, their biosynthesis in the mammary gland remains largely uncharacterized. Here, we used a systems biology framework that integrated glycan and RNA expression data to construct an HMO biosynthetic network and predict glycosyltransferases involved. To accomplish this, we constructed models describing the most likely pathways for the synthesis of the oligosaccharides accounting for >95% of the HMO content in human milk. Through our models, we propose candidate genes for elongation, branching, fucosylation, and sialylation of HMOs. We further explored selected enzyme activities through kinetic assay and their co-regulation through transcription factor analysis. These results provide the molecular basis of HMO biosynthesis necessary to guide progress in HMO research and application with the ultimate goal of understanding and improving infant health and development.Significance statementWith the HMO biosynthesis network resolved, we can begin to connect genotypes with milk types and thereby connect clinical infant, child and even adult outcomes to specific HMOs and HMO modifications. Knowledge of these pathways can simplify the work of synthetic reproduction of these HMOs providing a roadmap for improving infant, child, and overall human health with the specific application of a newly limitless source of nutraceuticals for infants and people of all ages.

Protein-based therapeutics such as mAbs have become emerging drugs in modern medicine. Most of the approved therapeutic proteins are glycoproteins. Glycosylation is an essential critical quality attribute (CQA) due to the influence that glycoforms have on the safety, efficacy, and pharmacokinetics/pharmacodynamics (PK/PD) of biotherapeutics. Here, we applied an LC-MS/MS-based glycoproteomics approach to characterize Fc glycans of an NISTmAb reference material (RM) 8671 (sample B) and a β-1,4-galactosidase-treated NISTmAb (sample A). Overall, 48 glycan compositions were identified and quantified. The glycan structure with the highest abundance was FA2, with a relative abundance of 52% in sample A and 38% in sample B. Over 50% of the identified glycans presented at levels smaller than 0.1%. Important glycan attributes were further derived using the quantitative results. The galactosylation level of modified NISTmAb was found to decrease by ∼10% when compared to the galactosylation level of NISTmAb. There was no significant difference between the two samples in the levels of sialylation, fucosylation, and high mannose. Moreover, unglycosylated peptides were also observed at a level of 1-2%.

Drug resistance represents an obstacle in colorectal cancer (CRC) treatment because of its association with poor prognosis. rBC2LCN is a lectin isolated from Burkholderia that binds cell surface glycans that have fucose moieties. Because fucosylation is enhanced in many types of cancers, this lectin could be an efficient drug carrier if CRC cells specifically present such glycans. Therefore, we examined the therapeutic efficacy and toxicity of lectin drug conjugate therapy in CRC mouse xenograft models. The affinity of rBC2LCN for human CRC cell lines HT‐29, LoVo, LS174T, and DLD‐1 was assessed in vitro. The cytocidal efficacy of a lectin drug conjugate, rBC2LCN‐38 kDa domain of pseudomonas exotoxin A (PE38) was evaluated by MTT assay. The therapeutic effects and toxicity for each CRC cell line‐derived mouse xenograft model were compared between the intervention and control groups. LS174T and DLD‐1 cell lines showed a strong affinity for rBC2LCN. In the xenograft model, the tumor volume in the rBC2LCN‐PE38 group was significantly reduced compared with that using control treatment alone. However, the HT‐29 cell line showed weak affinity and poor therapeutic efficacy. No significant toxicities or adverse responses were observed. In conclusion, we demonstrated that rBC2LCN lectin binds CRC cells and that rBC2LCN‐PE38 significantly suppresses tumor growth in vivo. In addition, the efficacy of the drug conjugate correlated with its binding affinity for each CRC cell line. These results suggest that lectin drug conjugate therapy has potential as a novel targeted therapy for CRC cell surface glycans., rBC2LCN lectin showed affinity for colorectal cancer (CRC) cell surface. rBC2LCN‐PE38 suppressed subcutaneous CRC tumor growth rates in mouse models. Lectin drug conjugate therapy could be a novel therapeutic option for CRC.

The α(1,6)fucose residue attached to the N-glycoprotein core is suspected to play an essential role in the progression of several types of cancer. Lectins remain the first choice for probing glycan modifications, although they may lack specificity. Thus, efforts have been made to identify new lectins with a narrower core fucose (CF) detection profile. Here, we present a comparison of the classical Aleuria aurantia lectin (AAL), Lens culinaris agglutinin (LCA) and Aspergillus oryzae lectin (AOL) with the newer Pholiota squarrosa lectin (PhoSL), which has been described as being specific for core fucosylated N-glycans. To this end, we studied the binding profiles of the four lectins using mammalian glycan arrays from the Consortium of Functional Glycomics. To validate their glycan specificity, we probed AOL, LCA and PhoSL in western-blot assays using protein extracts from eight common colorectal cancer (CRC) lines and colorectal biopsies from a small cohort of patients with CRC. The results showed that (i) LCA and PhoSL were the most specific lectins for detecting the presence of CF in a concentration-dependent manner; (ii) PhoSL exhibited the highest N-glycan sequence restriction, with preferential binding to core fucosylated paucimannosidic-type N-glycans, (iii) the recognition ability of PhoSL was highly influenced by the presence of terminal N-acetyl-lactosamine; (iv) LCA bound to paucimannosidic, bi-antennary and tri-antennary core fucosylated N-glycans and (v) AOL and AAL exhibited broader specificity towards fucosylation. Together, our results support the choice of LCA as the most appropriate lectin for CF detection, as validated in protein extracts from CRC cell lines and tissue specimens from patients with CRC.

In a cohort of infants, we found that lack of the Lewis histo-blood group antigen was associated with increased susceptibility to shigellosis. Broadly inhibiting fucosylation in epithelial cells in vitro decreased invasion by Shigella flexneri. These results support a role for fucosylated glycans in susceptibility to shigellosis.

N-glycans on the cell surface provide distinct signatures that are recognized by different glycan-binding proteins (GBPs) and pathogens. Most glycans in humans are asymmetric and isomeric, yet their biological functions are not well understood due to their lack of availability for studies. In this work, we have developed an improved strategy for asymmetric N-glycan assembly and diversification using designed common core substrates prepared chemically for selective enzymatic fucosylation and sialylation. The resulting 26 well-defined glycans that carry the sialic acid residue on different antennae were used in a microarray as a representative application to profile the binding specificity of hemagglutinin (HA) from the avian influenza virus (H5N2). We found distinct binding affinity for the Neu5Ac-Gal epitope linked to the N-acetylglucosamine (GlcNAc) of different branches and only a minor effect in binding for the terminal galactose on different branches. Overall, the microarray analysis showed branch-biased and context-based recognition patterns.

N-glycan alterations in the nervous system can result in different neuropathological symptoms such as mental retardation, seizures, and epilepsy. Studies have reported the characterization of N-glycans in rodent brains, but there is a lack of spatial resolution as either the tissue samples were homogenized or specific proteins were selected for analysis of glycosylation. We hypothesize that region-specific resolution of N-glycans isolated from the striatum and substantia nigra (SN) can give an insight into the establishment and pathophysiological degeneration of neural circuitry in Parkinson’s disease. Specific objectives of the study include isolation of N-glycans from the rat striatum and SN; reproducibility, resolution, and relative quantitation of N-glycome using ultra-performance liquid chromatography (UPLC), weak anion exchange-UPLC, and lectin histochemistry. The total N-glycomes from the striatum and SN were characterized using database mining (GlycoStore), exoglycosidase digestions, and liquid chromatography-mass spectrometry. It revealed significant differences in complex and oligomannose type N-glycans, sialylation (mono-, di-, and tetra-), fucosylation (tri-, core, and outer arm), and galactosylation (di-, tri-, and tetra-) between striatum and SN N-glycans with the detection of phosphorylated N-glycans in SN which were not detected in the striatum. This study presents the most comprehensive comparative analysis of relative abundances of N-glycans in the striatum and SN of rodent brains, serving as a foundation for identifying “brain-type” glycans as biomarkers or therapeutic targets and their modulation in neurodegenerative disorders.

The N-linked glycoprofile of bovine whey is the combined result of individual protein glycoprofiles. In this work, we provide in-depth structural information on the glycan structures of known whey glycoproteins, namely lactoferrin, lactoperoxidase, α-lactalbumin, immunoglobulin-G (IgG) and glycosylation dependent cellular adhesion molecule 1 (GlyCAM-1, PP3). The majority (~95%) of N-glycans present in the overall whey glycoprofile were attributed to three proteins; Lactoferrin, IgG and GlyCAM-1. We identified specific signature glycans for these main proteins; Lactoferrin contributes oligomannose-type glycans, while IgG carries fucosylated di-antennary glycans with Gal-β(1,4)GlcNAc (LacNAc) motifs. GlyCAM-1 is the sole whey glycoprotein carrying tri- and tetra-antennary structures, with a high degree of fucosylation and sialylation. Signature glycans can be used to recognize individual proteins in the overall whey glycoprofile, as well as for protein concentration estimations. Application of the whey glycoprofile analysis to colostrum samples revealed dynamic protein concentration changes for IgG, lactoferrin and GlyCAM-1 over time.

To study the Chinese human milk N-glycome over lactation, N-glycans were released and separated from serum proteins, purified by solid-phase extraction, and analyzed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS). In total, 66 different putative N-glycans were found in the colostrum (week 1) and mature milk (week 4) of seven Chinese mothers. A clear difference was observed between milk of five secretor and two nonsecretor mothers, based on the type and relative amounts of the individual N-glycans. The relative levels of the total neutral nonfucosylated and the fucosylated N-glycans in milk of five secretor mothers increased and decreased over lactation, respectively. This pattern could not be observed for the milk from the two nonsecretor mothers. Overall, this was the first study that provided detailed information on individual N-glycans in milk among mothers and over time as well as that fucosylation of N-glycans in milk was associated with the mother's secretor status.

Myocardial infarction (MI) is one of the leading causes of deaths worldwide. Because of the incapability of regeneration, the cardiomyocyte loss with MI is replaced by fibrotic scar tissue, which eventually leads to heart failure. Reconstructing regeneration of an adult human heart has been recognized as a promising strategy for cardiac therapeutics. A neonatal mouse heart, which possesses transient regenerative capacity at the first week after birth, represents an ideal model to investigate processes associated with cardiac regeneration. In this work, an integrated glycoproteomic and proteomic analysis was performed to investigate the differences in glycoprotein abundances and site-specific glycosylation between postneonatal day 1 (P1) and day 7 (P7) of mouse hearts. By large-scale profiling and quantifying more than 2900 intact N-glycopeptides in neonatal mouse hearts, we identified 227 altered N-glycopeptides between P1 and P7 hearts. By extracting protein changes from the global proteome data, the normalized glycosylation changes for site-specific glycans were obtained, which showed heterogeneity on glycosites and glycoproteins. Systematic analysis of the glycosylation changes demonstrated an overall upregulation of sialylation and core fucosylation in P7 mice. Notably, the upregulated sialylation was a comprehensive result of increased sialylated glycans with Neu5Gc, with both Neu5Gc and core fucose, and decreased sialylated glycans with Neu5Ac. The upregulated core fucosylation resulted from the increase of glycans containing both core fucose and Neu5Gc but not glycans containing sole core fucose. These data provide a valuable resource for future functional and mechanism studies on heart regeneration and discovery of novel therapeutic targets. All mass spectrometry proteomic data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the data set identifier PXD017139.

Like sialylation, fucose usually locates at the nonreducing ends of various glycans on glycoproteins and constitutes important glycan epitopes. Detecting the substrate glycans of fucosyltransferases is important for understanding how these glycan epitopes are regulated in response to different growth conditions and external stimuli. Here we report the detection of these glycans on glycoproteins as well as in their free forms via enzymatic incorporation of fluorophore-conjugated fucose using FUT2, FUT6, FUT7, FUT8 and FUT9. Specifically, we describe the detection of the substrate glycans of these enzymes on fetal bovine fetuin, recombinant H1N1 viral neuraminidase and therapeutic antibodies. The detected glycans include complex and high-mannose N-glycans. By establishing a series of precursors for the synthesis of Lewis X and sialyl Lewis X structures, we not only provide convenient electrophoresis methods for studying glycosylation but also demonstrate the substrate specificities and some kinetic features of these enzymes. Our results support the notion that fucosyltransferases are key targets for regulating the synthesis of Lewis X and sialyl Lewis X structures.

Fucosylation of the inner-most N-acetyl-glucosamine (GlcNAc) of N-glycans by fucosyltransferase 8 (FUT8) is an important step in the maturation of complex and hybrid N-glycans. This simple modification can have a dramatic impact on the activity and half-life of glycoproteins. These effects are relevant to understanding the invasiveness of some cancers, the development of monoclonal antibody therapeutics, and to a congenital disorder of glycosylation. The acceptor substrate preferences of FUT8 are well characterised and provide a framework for understanding N-glycan maturation in the Golgi, however the structural basis for these substrate preferences and the mechanism through which catalysis is achieved remains unknown. Here, we describe several structures of mouse and human FUT8 in the apo state and in complex with guanosine diphosphate (GDP), a mimic of the donor substrate, and a glycopeptide acceptor substrate. These structures provide insights into: a unique conformational change associated with donor substrate binding; common strategies employed by fucosyltransferases to coordinate GDP; features that define acceptor substrate preferences; and a likely mechanism for enzyme catalysis. Together with molecular dynamics simulations, the structures also reveal how FUT8 dimerisation plays an important role in defining the acceptor substrate binding site. Collectively, this information significantly builds on our understanding of the core-fucosylation process.

Lectins are the primary recognition macromolecules for various types of fucosylation, a common eukaryotic post-translational modification. In this study, we report the heterologous expression and molecular binding properties of a fucose-specific lectin, AofleA, isolated from Arthrobotrys oligospora. This is the first reported fucose-specific lectin found in nematophagous fungi. The recombinant AofleA (r-AofleA) was expressed in Escherichia coli with high efficiency, yielding at least 500 mg of soluble and functional r-AofleA per liter of broth. Using hemagglutination inhibition assay and glycan microarray analysis, r-AofleA was found to be broadly specific for fucosylated glycans or oligosaccharides including Fucα(1–2), Fucα(1–3), Fucα(1–4) and Fucα(1–6) linkages, similar to Aleuria aurantia lectin (AAL). Frontal affinity chromatography showed that r-AofleA has high affinity towards PA-L-fucose with an average Kd value of 15 nM. These findings provide a basis for improved understanding of the structure and functions of AofleA during recognition and capture of prey nematodes by nematophagous fungus A. oligospora.

Fucosyltransferase 8 (FUT8) and β-galactoside α-2,6-sialyltransferase 1 (ST6GAL1) are glycosyltransferases that catalyze α1,6-fucosylation and α2,6-sialylation, respectively, in the mammalian N-glycosylation pathway. They are aberrantly expressed in various human diseases. FUT8 is non-glycosylated but is responsible for the fucosylation of ST6GAL1. However, the mechanism for the interaction between these two enzymes is unknown. In this study, we show that serum levels of α2,6-sialylated N-glycans are increased in Fut8−/− mice, whereas the mRNA and protein levels of ST6GAL1 are unchanged in mouse live tissues. The level of α2,6-sialylation on IgG was also enhanced in Fut8−/− mice along with ST6GAL1 catalytic activity increase in both serum and liver. Moreover, it was observed that ST6GAL1 prefers non-fucosylated substrates. Interestingly, increased core fucosylation accompanied by a reduction in α2,6-sialylation, was detected in rheumatoid arthritis patient serum. These findings provide new insight into the interactions between FUT8 and ST6GAL1.

Protein glycosylation impacts the development and function of innate immune cells. The glycophenotypes and the glycan remodelling associated with the maturation of macrophages from monocytic precursor populations remain incompletely described. Herein, label-free porous graphitised carbon–liquid chromatography–tandem mass spectrometry (PGC-LC-MS/MS) was employed to profile with high resolution the N- and O-glycome associated with human monocyte-to-macrophage transition. Primary blood-derived CD14+ monocytes were differentiated ex vivo in the absence of strong anti- and proinflammatory stimuli using a conventional 7-day granulocyte-macrophage colony-stimulating factor differentiation protocol with longitudinal sampling. Morphology and protein expression monitored by light microscopy and proteomics validated the maturation process. Glycomics demonstrated that monocytes and macrophages display similar N-glycome profiles, comprising predominantly paucimannosidic (Man1-3GlcNAc2Fuc0–1, 22.1–30.8%), oligomannosidic (Man5-9GlcNAc2, 29.8–35.7%) and α2,3/6-sialylated complex-type N-glycans with variable core fucosylation (27.6–39.1%). Glycopeptide analysis validated conjugation of these glycans to human proteins, while quantitative proteomics monitored the glycoenzyme expression levels during macrophage differentiation. Significant interperson glycome variations were observed suggesting a considerable physiology-dependent or heritable heterogeneity of CD14+ monocytes. Only few N-glycome changes correlated with the monocyte-to-macrophage transition across donors including decreased core fucosylation and reduced expression of mannose-terminating (paucimannosidic-/oligomannosidic-type) N-glycans in macrophages, while lectin flow cytometry indicated that more dramatic cell surface glycan remodelling occurs during maturation. The less heterogeneous core 1-rich O-glycome showed a minor decrease in core 2-type O-glycosylation but otherwise remained unchanged with macrophage maturation. This high-resolution glycome map underpinning normal monocyte-to-macrophage transition, the most detailed to date, aids our understanding of the molecular makeup pertaining to two vital innate immune cell types and forms an important reference for future glycoimmunological studies.

Background Exosomes are vesicles of endocytic origin released by various cell types and emerging as important mediators in tumor cells. Human metastases-associated lung adenocarcinoma transcript 1 (MALAT1) is a long non-coding RNA known to promote cell proliferation, metastasis, and invasion in colorectal cancer (CRC). Methods The expression of MALAT1 was analyzed in CRC using qRT-PCR. FUT4 and fucosylation levels were detected in CRC clinical samples and CRC cell lines by immunofluorescent staining, western blot and lectin blot analysis. CRC derived exosomes were isolated and used to examine their tumor-promoting effects in vitro and in vivo. Results The invasive and metastatic abilities of primary CRC cells were enhanced after exposure to exosomes derived from highly metastatic CRC cells, which increased the fucosyltransferase 4 (FUT4) levels and fucosylation not by directly transmitting FUT4 mRNA. Exosomal MALAT1 increased FUT4 expresssion via sponging miR-26a/26b. Furthermore, MALAT1/miR-26a/26b/FUT4 axis played an important role in exosome-mediated CRC progression. Exosomal MALAT1 also mediated FUT4-associated fucosylation and activated the PI3K/AKT/mTOR pathway. Conclusions These data indicated that exosomal MALAT1 promoted the malignant behavior of CRC cells by sponging miR-26a/26b via regulating FUT4 and activating PI3K/Akt/mTOR pathway.

The complex enzyme network responsible for glycan synthesis suffers significant changes during the first steps of tumor development, leading to the early formation of tumor-associated glycan signatures. Among the glycosylation pathways, changes in fucosylation emerged as one of most important features in cancer. Αlpha-1,3/4-fucosyltransferase (FUT3) has been linked to pro-tumor and anti-tumor pathways depending on the cancer type. The present study aimed to understand the gene and protein expression profiles of FUT3 in three different and independent cohorts composed by invasive breast cancer patients: Local Brazilian population, METABRIC and TCGA. FUT3 transcripts and protein were measured in the Brazilian population by real-time PCR and Western blotting, respectively. Clinical records and FUT3 levels from public METABRIC and TCGA cohorts were accessed through CBioPortal database. FUT3 expression was analyzed in each cohort using the appropriated statistic tools. Survival meta-analysis in triple negative patients was performed using five independent cohorts including GSE41119, GSE47994 and GSE86945, data obtained from GEO repository available at NCBI database, and METABRIC and TCGA. Our analysis showed that high FUT3 levels were consistently associated to reduced invasive breast cancer patients overall survival. This finding is particularly significant in triple negative patients. These results together with the previously knowledge regarding the involvement of FUT3 in pro-tumor and anti-tumor mechanisms led us to purpose a model for FUT3 expression regulation throughout breast cancer establishment and progression.

Glycosylation of cell receptors influences their function and development of tumour induces changes in glycosylation. Cell growth depends on the activation of receptors which bind growth factors and the insulin-like growth factor (IGF) receptors are among the most important ones. Usually, only small quantities of isolated receptors are available thus there is a need of suitable assay to study receptors glycosylation. Therefore, we developed a lectin-based reverse-phase protein microarray method for screening the glycosylation pattern of receptors in picomolar (pM) concentrations. The method was applied to glycoprofile IGF1 and IGF2 receptors and the solubilised membrane proteins isolated from tumour and non-tumour colon tissue of patients with colorectal cancer. We found that common to both receptors was partial overlapping of the major glycan structures with those present in the entire glycome of membrane proteins. In contrast, receptors possess higher level of α2,3 sialic acid residues and lower level of tri-/tetra-antennary complex type N-glycans and terminal mannose in high-mannose structures. Increased levels of fucosylation and branched mannose structures were observed in both receptors derived from tumour tissue compared to non-tumour tissue. The described method enabling glycan analysis of receptors has a big application potential in e.g. biomarker research, biology and diagnostics.

FcγRIIIa (CD16a) and FcγRIIa (CD32a) on monocytes are essential for proper effector functions including antibody dependent cellular cytotoxicity (ADCC) and phagocytosis (ADCP). Indeed, therapeutic monoclonal antibodies (mAbs) that bind FcγRs with greater affinity exhibit greater efficacy. Furthermore, post-translational modification impacts antibody binding affinity, most notably the composition of the asparagine(N)-linked glycan at N162 of CD16a. CD16a is widely recognized as the key receptor for the monocyte response, however the post-translational modifications of CD16a from endogenous monocytes are not described. Here we isolated monocytes from individual donors and characterized the composition of CD16a and CD32a N-glycans from all modified sites. The composition of CD16a N-glycans varied by glycosylation site and donor. CD16a displayed primarily complex-type biantennary N-glycans at N162, however some individuals expressed CD16a V158 with ∼20% hybrid and oligomannose types which increased affinity for IgG1 Fc according to surface plasmon resonance binding analyses. The CD16a N45-glycans contain markedly less processing than other sites with >75% hybrid and oligomannose forms. N38 and N74 of CD16a both contain highly processed complex-type N-glycans with N-acetyllactosamine repeats and complex-type biantennary N-glycans dominate at N169. The composition of CD16a N-glycans isolated from monocytes included a higher proportion of oligomannose-type N-glycans at N45 and less sialylation plus greater branch fucosylation than we observed in a recent analysis of NK cell CD16a. The additional analysis of CD32a from monocytes revealed different features than observed for CD16a including the presence of a predominantly biantennary complex-type N-glycans with two sialic acids at both sites (N64 and N145).

Core fucosylation (CF) is a special form of N-glycosylation and plays an important role in pathological and biological processes. Increasing efforts in this area are focused on the identification of CF glycosites, whereas evidence showed that the stoichiometry of CF occupancy is functionally important. Here, an integrated strategy based on "Glycan-Simplification and Paired-Peaks-extraction" (GSPPE) for detecting large-scale stoichiometries of CF was developed. After HILIC enrichment of intact glycopeptides, sequential cleavages by endoglycosidases H and endoglycosidases F3 were performed to generate simplified glycopeptide forms (SGFs), i.e., peptide-GlcNAc (pep-HN) and peptide-GlcNAc-Fucose (pep-CF). These paired SGFs were found to be eluted consecutively on a reversed-phase chromatography column, which allowed us to obtain peak areas of SGF pairs, even if only one of the peaks was captured by the mass spectrometer (MS), by introducing the Paired-Peaks-Extraction algorithm. Thus, the missing value dilemma of random data-dependent MS/MS acquisition was reduced and the stoichiometry of site-specific CF could be calculated. We systematically evaluated the feasibility of this strategy using standard glycoproteins and then explored urinary samples from healthy individuals and hepatocellular carcinoma (HCC) patients. In total, 1449 highly reliable core fucose glycosites and their corresponding CF stoichiometries were obtained. Dozens of glycosites that differed significantly in the urine of healthy individuals and HCC patients were disclosed. The developed approach and program presented here may promote studies on core fucosylation and lead to a deeper understanding of their dysregulation in physiological- or pathological processes.

The canonical Wnt signalling pathway is a critical pathway involved in the proliferation of cells. It has been well‐established that it plays the central role during colorectal carcinogenesis and development. Yet the exact molecular mechanism of how the canonical Wnt pathway is fine‐tuned remains elusive. We found that SLC35C1, a GDP‐fucose transporter, negatively regulates the Wnt signalling pathway. We show here that SLC35C1 is reduced in all colon cancer by both immunohistochemistry images and TCGA data, whereas β‐catenin is increased. Down‐regulation of SLC35C1 is also detected by real‐time PCR in stage 3 and stage 4 colorectal cancer tissues. Moreover, analysing the TCGA database with cBioPortal reveals the negative correlation of SLC35C1 mRNA level to the expression of β‐catenin. Reduced SLC35C1 significantly promotes cell proliferation and colony formation of HEK293 cells. Meanwhile, in HEK293 cells silencing SLC35C1 activates canonical Wnt pathway, whereas overexpressing SLC35C1 inhibits it. Consistently, the reduction of SLC35C1 in HEK293 cells also elevated the mRNA level of Wnt target genes C‐myc, Axin2 and Cyclin D1, as well as the secretion of Wnt3a. In conclusion, we identified SLC35C1 as a negative regulator of the Wnt signalling pathway in colon cancer. Decreased SLC35C1 may cause over‐activation of Wnt signalling in colorectal cancer.

Pancreatic ductal adenocarcinoma (PDAC) is the most common type of pancreatic cancer. It is an aggressive malignancy associated with poor prognosis because of recurrence, metastasis, and treatment resistance. Aberrant glycosylation of cancer cells triggers their migration and invasion and is considered one of the most important prognostic cancer biomarkers. The current study aimed to identify glycan alterations and their relationship with the malignant potential of PDAC. Using a lectin microarray, we evaluated glycan expression in 62 PDAC samples. Expression of fucosyltransferase 8 (FUT8), the only enzyme catalyzing core fucosylation, was investigated by immunohistochemistry. The role of FUT8 in PDAC invasion and metastasis was confirmed using an in vitro assay and a xenograft peritoneal metastasis mouse model. The microarray data demonstrated that core fucose-binding lectins were significantly higher in carcinoma than in normal pancreatic duct tissues. Similarly, FUT8 protein expression was significantly higher in carcinoma than in normal pancreatic duct tissues. High FUT8 protein expression was significantly associated with lymph-node metastases and relapse-free survival. FUT8 knockdown significantly reduced the invasion in PDAC cell lines and impaired peritoneal metastasis in the xenograft model. The findings of this study provide evidence that FUT8 plays a pivotal role in PDAC invasion and metastasis and might be a therapeutic target for this disease.

Epidermal growth factor receptor (EGFR) is a major driver of head and neck cancer, a devastating malignancy with a major sub-site in the oral cavity manifesting as oral squamous cell carcinoma (OSCC). EGFR is a glycoprotein receptor tyrosine kinase (RTK) whose activity is upregulated in >80% OSCC. Current anti-EGFR therapy relies on the use of cetuximab, a monoclonal antibody against EGFR, although it has had only a limited response in patients. Here, we uncover a novel mechanism regulating EGFR activity, identifying a role of the nuclear branch of the Wnt/β-catenin signaling pathway, the β-catenin/CBP axis, in control of post-translational modification of N-glycans on the EGFR. Genomic and structural analyses reveal that β-catenin/CBP signaling represses fucosylation on the antennae of N-linked glycans on EGFR. By employing nUPLC-MS/MS, we determined that malignant human OSCC cells harbor EGFR with a paucity of N-glycan antennary fucosylation, while indolent cells display higher levels of fucosylation at sites N420 and N579. Additionally, treatment with either ICG-001 or E7386, which are both small molecule inhibitors of β-catenin/CBP signaling, leads to increased transcriptional expression of fucosyltransferases FUT2 and FUT3, with a concomitant increase in EGFR N-glycan antennary fucosylation. In order to discover which fucosylated glycan epitopes are involved in the observed effect, we performed in-depth characterization of multiply-fucosylated N-glycans via tandem mass spectrometry analysis of the EGFR tryptic glycopeptides. Data are available via ProteomeXchange with identifier PXD017060. We propose that β-catenin/CBP signaling promotes EGFR oncogenic activity in OSCC by inhibiting its N-glycan antennary fucosylation through transcriptional repression of FUT2 and FUT3.

A facile enzymatic modular assembly strategy for the preparative-scale synthesis of poly-N-acetyllactosamine (poly-LacNAc) glycans with varied lengths and designed sialylation and/or fucosylation patterns is described. These glycans were printed as a microarray to investigate their interactions with a panel of glycan binding proteins (GBPs). Binding affinities revealed that the avidity of GBPs could be largely affected by the length and the patterns of sialylation and fucosylation.

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.

Aberrant immunoglobulin G (IgG) N-glycosylation offers new prospects to detect changes in cell metabolism and by extension, for biomarker discovery in type 2 diabetes mellitus (T2DM). However, past studies did not analyze the individual IgG subclasses in relation to T2DM pathophysiology. We report here original findings through a comparison of the IgG subclass-specific fragment crystallizable (Fc) glycan biosignatures in 115 T2DM patients with 122 healthy controls within the Uyghur population in China. IgG Fc glycosylation profiles were analyzed using nano- liquid chromatography–mass spectrometry to exclude changes attributed to fragment antigen binding N- glycosylation. After correction for clinical covariates, 27 directly measured and 4 derived glycan traits of the IgG subclass-specific N- glycopeptides were significantly associated with T2DM. Furthermore, we observed in T2DM a decrease in bisecting N-acetylglucosamine of IgG2 and agalactosylation of IgG4, and an increase in sialylation of IgG4 and digalactosylation of IgG2. Classification model based on IgG subclass- specific N-glycan traits was able to distinguish patients with T2DM from controls with an area under the receiver operating characteristic curve of 0.927 (95% confidence interval 0.894–0.960, p < 0.001). In conclusion, a robust association between the IgG subclass-specific Fc N-glycomes and T2DM was observed in the Uyghur population sample in China, suggesting a potential for the IgG Fc glycosylation as a biomarker candidate for type 2 diabetes. The integration of glycomics with other system science biomarkers might offer further hope for innovation in diagnosis and treatment of T2DM in the future. Finally, it is noteworthy that “Population Glycomics” is an emerging approach to biomarker discovery for common complex diseases.