Role of E-cadherin N-glycosylation profile in a mammary tumor model

Modifications in cell surface glycosylation affecting cell adhesion are common characteristics of transformed cells. This study characterizes the N-glycosylation profile of E-cadherin in models of canine mammary gland adenoma and carcinoma evaluating the importance of these glycosylation modifications in the malignant phenotype.Our results show that the pattern of E-cadherin N-glycosylation in mammary carcinoma is characterized by highly branched N-glycans, increase in sialylation and an expression of few high mannose structures. Detailed mass spectrometry analysis demonstrated a new N-glycosylation site containing a potential complex type N-glycan in E-cadherin from a mammary carcinoma cell line.Our study demonstrates the importance of E-cadherin N-glycans in the process of tumor development and in the transformation to the malignant phenotype.     

The N-glycosylation of classical swine fever virus E2 glycoprotein extracellular domain expressed in the milk of goat

Classical swine fever virus (CSFV) outer surface E2 glycoprotein represents an important target to induce protective immunization during infection but the influence of N-glycosylation pattern in antigenicity is yet unclear. In the present work, the N-glycosylation of the E2-CSFV extracellular domain expressed in goat milk was determined. Enzymatic N-glycans releasing, 2-aminobenzamide (2AB) labeling, weak anion-exchange and normal-phase HPLC combined with exoglycosidase digestions and mass spectrometry of 2AB-labeled and unlabeled N-glycans showed a heterogenic population of oligomannoside, hybrid and complex-type structures. The detection of two Man₈GlcNAc₂ isomers indicates an alternative active pathway in addition to the classical endoplasmic reticulum processing. N-acetyl or N-glycolyl monosialylated species predominate over neutral complex-type N-glycans. Asn207 site-specific micro-heterogeneity of the E2 most relevant antigenic and virulence site was determined by HPLC-mass spectrometry of glycopeptides. The differences in N-glycosylation with respect to the native E2 may not disturb the main antigenic domains when expressed in goat milk.     

Plant N-glycan profiling of minute amounts of material

Development of convenient strategies for identification of plant N-glycan profiles has been driven by the emergence of plants as an expression system for therapeutic proteins. In this article, we reinvestigated qualitative and quantitative aspects of plant N-glycan profiling. The extraction of plant proteins through a phenol/ammonium acetate procedure followed by deglycosylation with peptide N-glycosidase A (PNGase A) and coupling to 2-aminobenzamide provides an oligosaccharide preparation containing reduced amounts of contaminants from plant cell wall polysaccharides. Such a preparation was also suitable for accurate qualitative and quantitative evaluation of the N-glycan content by mass spectrometry. Combining these approaches allows the profiling to be carried out from as low as 500 mg of fresh leaf material. We also demonstrated that collision-induced dissociation (CID) mass spectrometry in negative mode of N-glycans harboring α(1,3)- or α(1,6)-fucose residue on the proximal GlcNAc leads to specific fragmentation patterns, thereby allowing the discrimination of plant N-glycans from those arising from mammalian contamination.     

ABH blood group antigens in N-glycan of human glycophorin A

We previously showed that a small proportion of the O-linked oligosaccharide chains of human glycophorin A (GPA) contains blood group A, B or H antigens, relevant to the ABO phenotype of the donor. The structures of these minor O-glycans have been established (Podbielska et al. (2004) [20]). By the use of immunochemical methods we obtained results indicating that ABH blood group epitopes are also present in N-glycan of human GPA (Podbielska and Krotkiewski (2000) [22]). In the present paper we report a detailed analysis of GPA N-glycans using nanoflow electrospray ionization tandem mass spectrometry. N-glycans containing A-, B- and H-related sequences were identified in GPA preparations obtained from erythrocytes of blood group A, B and O donors, respectively. The ABH blood group epitopes are present on one antenna of the N-glycan, whereas a known sialylated sequence NeuAcα2-6Galβ1-4GlcNAc- occurs on the other antenna and other details are in agreement with the known major structure of the GPA N-glycan. In the bulk of the biantennary sialylated N-glycans released from GPA preparations, the blood group ABH epitopes-containing N-glycans, similarly O-glycans, constituted only a minor part. The amount relative to other N-glycans was estimated to 2-6% of blood group H epitope-containing glycans released from GPA-O preparations and 1-2% of blood group A and B epitope-containing glycans, released from GPA-A and GPA-B, respectively.     

Accumulation of free Neu5Ac-containing complex-type N-glycans in human pancreatic cancers

We have analyzed the structures of glycosphingolipids and intracellular free glycans in human cancers. In our previous study, trace amounts of free N-acetylneuraminic acid (Neu5Ac)-containing complex-type N-glycans with a single GlcNAc at each reducing terminus (Gn1 type) was found to accumulate intracellularly in colorectal cancers, but were undetectable in most normal colorectal epithelial cells. Here, we used cancer glycomic analyses to reveal that substantial amounts of free Neu5Ac-containing complex-type N-glycans, almost all of which were α2,6-Neu5Ac-linked, accumulated in the pancreatic cancer cells from three out of five patients, but were undetectable in normal pancreatic cells from all five cases. These molecular species were mostly composed of five kinds of glycans having a sequence Neu5Ac-Gal-GlcNAc-Man-Man-GlcNAc and one with the following sequence Neu5Ac-Gal-GlcNAc-Man-(Man-)Man-GlcNAc. The most abundant glycan was Neu5Acα2-6Galβ1-4GlcNAcβ1-2Manα1-3Manβ1-4GlcNAc, followed by Neu5Acα2-6Galβ1-4GlcNAcβ1-2Manα1-6Manβ1-4GlcNAc. This is the first study to show unequivocal evidence for the occurrence of free Neu5Ac-linked N-glycans in human cancer tissues. Our findings suggest that free Neu5Ac-linked glycans may serve as a useful tumor marker.     

N-acetylglucosamine drives myelination by triggering oligodendrocyte precursor cell differentiation

Myelination plays an important role in cognitive development and in demyelinating diseases like multiple sclerosis (MS), where failure of remyelination promotes permanent neuro-axonal damage. Modification of cell surface receptors with branched N-glycans coordinates cell growth and differentiation by controlling glycoprotein clustering, signaling, and endocytosis. GlcNAc is a rate-limiting metabolite for N-glycan branching. Here we report that GlcNAc and N-glycan branching trigger oligodendrogenesis from precursor cells by inhibiting platelet-derived growth factor receptor-α cell endocytosis. Supplying oral GlcNAc to lactating mice drives primary myelination in newborn pups via secretion in breast milk, whereas genetically blocking N-glycan branching markedly inhibits primary myelination. In adult mice with toxin (cuprizone)-induced demyelination, oral GlcNAc prevents neuro-axonal damage by driving myelin repair. In MS patients, endogenous serum GlcNAc levels inversely correlated with imaging measures of demyelination and microstructural damage. Our data identify N-glycan branching and GlcNAc as critical regulators of primary myelination and myelin repair and suggest that oral GlcNAc may be neuroprotective in demyelinating diseases like MS.     

A new insect cell line engineered to produce recombinant glycoproteins with cleavable N-glycans

Glycoproteins are difficult to crystallize because they have heterogeneous glycans composed of multiple monosaccharides with considerable rotational freedom about their O-glycosidic linkages. Crystallographers studying N-glycoproteins often circumvent this problem by using β1,2-N-acetylglucosaminyltransferase I (MGAT1)–deficient mammalian cell lines, which produce recombinant glycoproteins with immature N-glycans. These glycans support protein folding and quality control but can be removed using endo-β-N-acetylglucosaminidase H (Endo H). Many crystallographers also use the baculovirus-insect cell system (BICS) to produce recombinant proteins for their work but have no access to an MGAT1-deficient insect cell line to facilitate glycoprotein crystallization in this system. Thus, we used BICS-specific CRISPR–Cas9 vectors to edit the Mgat1 gene of a rhabdovirus-negative Spodoptera frugiperda cell line (Sf-RVN) and isolated a subclone with multiple Mgat1 deletions, which we named Sf-RVN^Lec1. We found that Sf-RVN and Sf-RVN^Lec1 cells had identical growth properties and served equally well as hosts for baculovirus-mediated recombinant glycoprotein production. N-glycan profiling showed that a total endogenous glycoprotein fraction isolated from Sf-RVN^Lec1 cells had only immature and high mannose-type N-glycans. Finally, N-glycan profiling and endoglycosidase analyses showed that the vast majority of the N-glycans on three recombinant glycoproteins produced by Sf-RVN^Lec1 cells were Endo H-cleavable Man₅GlcNAc₂ structures. Thus, this study yielded a new insect cell line for the BICS that can be used to produce recombinant glycoproteins with Endo H-cleavable N-glycans. This will enable researchers to combine the high productivity of the BICS with the ability to deglycosylate recombinant glycoproteins, which will facilitate efforts to determine glycoprotein structures by X-ray crystallography.     

N-Glycosylation and N-Glycan Moieties of CTB Expressed in Rice Seeds

Cholera toxin B subunit (CTB) is widely used as a carrier molecule and mucosal adjuvant and for the expression of fusion proteins of interest. CTB-fusion proteins are also expressed in plants, but the N-glycan structures of CTB have not been clarified. To gain insights into the N-glycosylation and N-glycans of CTB expressed in plants, we expressed CTB in rice seeds with an N-terminal glutelin signal and a C-terminal KDEL sequence and analyzed its N-glycosylation and N-glycan structures. CTB was successfully expressed in rice seeds in two forms: a form with N-glycosylation at Asn32 that included both plant-specific N-glycans and small oligomannosidic N-glycans and a non-N-glycosylated form. N-Glycan analysis of CTB showed that approximately 50 % of the N-glycans had plant-specific M3FX structures and that almost none of the N-glycans was of high-mannose-type N-glycan even though the CTB expressed in rice seeds contains a C-terminal KDEL sequence. These results suggest that the CTB expressed in rice was N-glycosylated through the endoplasmic reticulum (ER) and Golgi N-glycosylation machinery without the ER retrieval.     

Site-specific and linkage analyses of fucosylated <Emphasis Type="Italic">N</Emphasis>-glycans on haptoglobin in sera of patients with various types of cancer: possible implication for the differential diagnosis of cancer

Fucosylation is an important type of glycosylation involved in cancer, and fucosylated proteins could be employed as cancer biomarkers. Previously, we reported that fucosylated N-glycans on haptoglobin in the sera of patients with pancreatic cancer were increased by lectin-ELISA and mass spectrometry analyses. However, an increase in fucosylated haptoglobin has been reported in various types of cancer. To ascertain if characteristic fucosylation is observed in each cancer type, we undertook site-specific analyses of N-glycans on haptoglobin in the sera of patients with five types of operable gastroenterological cancer (esophageal, gastric, colon, gallbladder, pancreatic), a non-gastroenterological cancer (prostate cancer) and normal controls using ODS column LC-ESI MS. Haptoglobin has four potential glycosylation sites (Asn184, Asn207, Asn211, Asn241). In all cancer samples, monofucosylated N-glycans were significantly increased at all glycosylation sites. Moreover, difucosylated N-glycans were detected at Asn 184, Asn207 and Asn241 only in cancer samples. Remarkable differences in N-glycan structure among cancer types were not observed. We next analyzed N-glycan alditols released from haptoglobin using graphitized carbon column LC-ESI MS to identify the linkage of fucosylation. Lewis-type and core-type fucosylated N-glycans were increased in gastroenterological cancer samples, but only core-type fucosylated N-glycan was relatively increased in prostate cancer samples. In metastatic prostate cancer, Lewis-type fucosylated N-glycan was also increased. These data suggest that the original tissue/cell producing fucosylated haptoglobin is different in each cancer type and linkage of fucosylation might be a clue of primary lesion, thereby enabling a differential diagnosis between gastroenterological cancers and non-gastroenterological cancers.     

Multiple N-Glycans Cooperate in the Subcellular Targeting and Functioning of Arabidopsis KORRIGAN1

Arabidopsis thaliana KORRIGAN1 (KOR1) is an integral membrane endo-β1,4-glucanase in the trans-Golgi network and plasma membrane that is essential for cellulose biosynthesis. The extracellular domain of KOR1 contains eight N-glycosylation sites, N1 to N8, of which only N3 to N7 are highly conserved. Genetic evidence indicated that cellular defects in attachment and maturation of these N-glycans affect KOR1 function in vivo, whereas the manner by which N-glycans modulate KOR1 function remained obscure. Site-directed mutagenesis analysis of green fluorescent protein (GFP)-KOR1 expressed from its native regulatory sequences established that all eight N-glycosylation sites (N1 to N8) are used in the wild type, whereas stt3a-2 cells could only inefficiently add N-glycans to less conserved sites. GFP-KOR1 variants with a single N-glycan at nonconserved sites were less effective than those with one at a highly conserved site in rescuing the root growth phenotype of rsw2-1 (kor1 allele). When functionally compromised, GFP-KOR1 tended to accumulate at the tonoplast. GFP-KOR1Δall (without any N-glycan) exhibited partial complementation of rsw2-1; however, root growth of this line was still negatively affected by the absence of complex-type N-glycan modifications in the host plants. These results suggest that one or several additional factor(s) carrying complex N-glycans cooperate(s) with KOR1 in trans to grant proper targeting/functioning in plant cells.     

Sequences from the First Fibronectin Type III Repeat of the Neural Cell Adhesion Molecule Allow O-Glycan Polysialylation of an Adhesion Molecule Chimera

Polysialic acid is a developmentally regulated, anti-adhesive polymer that is added to N-glycans on the fifth immunoglobulin domain (Ig5) of the neural cell adhesion molecule (NCAM). We found that the first fibronectin type III repeat (FN1) of NCAM is required for the polysialylation of N-glycans on the adjacent Ig5 domain, and we proposed that the polysialyltransferases recognize specific sequences in FN1 to position themselves for Ig5 N-glycan polysialylation. Other studies identified a novel FN1 acidic surface patch and α-helix that play roles in NCAM polysialylation. Here, we characterize the contribution of two additional FN1 sequences, Pro⁵¹⁰-Tyr⁵¹¹-Ser⁵¹² (PYS) and Gln⁵¹⁶-Val⁵¹⁷-Gln⁵¹⁸ (QVQ). Replacing PYS or the acidic patch dramatically decreases the O-glycan polysialylation of a truncated NCAM protein, and replacing the α-helix or QVQ shifts polysialic acid to FN1 O-glycans in full-length NCAM. We also found that the FN1 domain of the olfactory cell adhesion molecule, a homologous but unpolysialylated protein, could partially replace NCAM FN1. Inserting Pro⁵¹⁰-Tyr⁵¹¹ eliminated N-glycan polysialylation and enhanced O-glycosylation of an NCAM- olfactory cell adhesion molecule chimera, and inserting other FN1 sequences unique to NCAM, predominantly the acidic patch, created a new polysialyltransferase recognition site. Taken together, our results highlight the role of the FN1 α-helix and QVQ sequences in N-glycan polysialylation and demonstrate that the acidic patch primarily functions in O-glycan polysialylation.     

Glycan analysis of glycoprotein pharmaceuticals: Evaluation of analytical approaches to Z number determination in pharmaceutical erythropoietin products

N-Glycosylation of many glycoprotein drugs is important for biological activity and should therefore be the target of specific and quantitative analytical methods. In this study, we focus on the two N-glycan mapping approaches that are used in pharmacopoeial monograph to analyse N-glycans released from fifteen preparations of recombinant human erythropoietin supplied by ten Chinese manufacturers. Underivatised N-glycans were analysed by high performance anion-exchange chromatography with pulsed amperometric detection and fluorophore-labelled N-glycans were analysed by weak anion-exchange and normal-phase high performance liquid chromatography. N-glycans were also analysed by matrix assisted laser desorption ionisation mass spectrometry. The release of N-glycans by PNGase F was shown to be consistent. Z number, a mathematical expression of the total negatively charged N-glycans composition has provided a convenient way to summarise the complex dataset and it might be suitable for product consistency monitoring. However, this Z number reduces the information of individual acidic N-glycan structure and is also found to be method dependent. Therefore, its use requires clear specification and validation. In this study, we only found weak but positive correlation between the Z number and its bioactivity. Wide range of N-glycans yields were obtained from the fifteen preparations but the significance of their differences is unclear.     

The ammonia-catalyzed release of glycoprotein <Emphasis Type="Italic">N</Emphasis>-glycans

Despite the great significance of release and analysis of glycans from glycoproteins, the existing N-glycan release methods are undermined by some limitations and deficiencies. The traditional enzymatic protocols feature high N-glycan release specificity but are generally costly and inefficient for some types of N-glycans. The existing chemical methods require harsh reaction conditions or are accompanied by the remarkable formation of by-products. Herein, we describe a versatile chemical method for the release and analysis of N-glycans from glycoproteins. This method differs from the existing methods as only aqueous ammonia is used to catalyze the N-glycan release reactions. Optimization of reaction conditions was performed using RNase B as a model glycoprotein and the obtained results indicated a highest N-glycan yield in ammonia at 60 °C for 16 h. Comparison of this method with traditional enzymatic protocols and recently reported NaClO methods confirmed the good reliability and efficiency of the novel approach. We also successfully applied this method to some complex biological samples, such as Ginkgo seed protein, fetal bovine serum (FBS) and hen egg white, and demonstrated its great compatibility with various neutral N-glycans, core α-1,3-fucosylated N-glycans and sialylated N-glycans. This method is very simple and cost-effective, enabling convenient analysis and large-scale preparation of released reducing N-glycans from various biological samples for structural and functional glycomics studies.     

Discovery and characterization of a novel extremely acidic bacterial N-glycanase with combined advantages of PNGase F and A

Peptide-N4-(N-acetyl-β-glucosaminyl) asparagine amidases [PNGases (peptide N-glycosidases), N-glycanases, EC 3.5.1.52] are essential tools in the release of N-glycans from glycoproteins. We hereby report the discovery and characterization of a novel bacterial N-glycanase from Terriglobus roseus with an extremely low pH optimum of 2.6, and annotated it therefore as PNGase H⁺. The gene of PNGase H⁺ was cloned and the recombinant protein was successfully expressed in Escherichia coli. The recombinant PNGase H⁺ could liberate high mannose-, hybrid- and complex-type N-glycans including core α1,3-fucosylated oligosaccharides from both glycoproteins and glycopeptides. In addition, PNGase H⁺ exhibited better release efficiency over N-glycans without core α1,3-fucose compared with PNGase A. The facile expression, non-glycosylated nature, unusual pH optimum and broad substrate specificity of this novel type of N-glycanase makes recombinant PNGase H⁺ a versatile tool in N-glycan analysis.     

Type and branched pattern of N-glycans and their structural effect on the chicken egg allergen ovotransferrin: a comparison with ovomucoid

Ovotransferrin (OT), a multifunctional glycoprotein with defensive and protective activities, accounts for approximately 13 % of chicken egg white proteins and is known as a major egg-associated allergen along with ovomucoid (OM). In contrast to the well-characterized N-glycans of OM, the N-glycan structure of OT has not been reported. Here, using HPLC equipped with a fluorescence detector and mass spectrometric analysis in combination with exoglycosidase digestion, we investigated the N-glycan type and branched pattern of OT, and compared them with those of OM. The HPLC peak area was used to calculate the relative quantity (%) of each glycan. Seventeen N-glycans, including 11 glycans (1 core structure and 10 complex-type oligosaccharides), that commonly exist in ovotransferrin and ovomucoid were identified. Six characteristic glycans (2 truncated structures, 1 complex-type, and 3 hybrid-type oligosaccharides) in OT and eight characteristic glycans in OM were classified. OT contains the following branched complex-type structures: mono-(13.2 %), bi-(23.9 %), tri-(9.0 %), tetra-(2.7 %), and penta-(2.8 %) antennary oligosaccharides. However, OM contained mostly tri-(33.5 %) and penta-(31.2 %) antennary oligosaccharides. The N-glycan–containing bisecting N-acetylglucosamine comprised 43.4 % and 79.8 % of the total glycans in OT and OM, respectively. Moreover, using circular dichroism analysis, we observed that the secondary structure of the deglycosylated OT is quite different from that of the intact protein. To our knowledge, this is the first study to analyze N-glycans in OT in comparison with those of OM.     

Malonic acid suppresses mucin-type O-glycan degradation during hydrazine treatment of glycoproteins

Hydrazine treatment is frequently used for releasing mucin-type O-glycans (O-glycans) from glycoproteins because the method provides O-glycans that retain a reducible GalNAc at their reducing end, which is available for fluorescent labeling. However, many O-glycans are degraded by “peeling” during this treatment. In the current study, it was found that malonic acid suppressed O-glycan degradation during hydrazine treatment of bovine fetuin or porcine gastric mucin in both the gas and liquid phases. This is paradoxical because the release of O-glycans from glycoproteins occurs under alkaline conditions. However, malonic acid seems to prevent the degradation through its acidic property given that other weak acids also prevented the degradation. Accordingly, disodium malonate did not suppress O-glycan degradation. Application of this method to rat gastric mucin demonstrated that the majority of the major O-glycans obtained in the presence of malonic acid were intact, whereas those obtained in the absence of malonic acid were degraded. These results suggest that hydrazine treatment in the presence of malonic acid would allow glycomic analysis of native mucin glycoproteins.     

Jack bean α-mannosidase digestion profile of hybrid-type N-glycans: effect of reaction pH on substrate preference

Jack bean α-mannosidase (JBM) is a well-studied plant vacuolar α-mannosidase, and is widely used as a tool for the enzymatic analysis of sugar chains of glycoproteins. In this study, the JBM digestion profile of hybrid-type N-glycans was examined using pyridylamino (PA-) sugar chains. The digestion efficiencies of the PA-labeled hybrid-type N-glycans Manα1,6(Manα1,3)Manα1,6(GlcNAcβ1,2Manα1,3)Manβ1,4GlcNAcβ1,4GlcNAc-PA (GNM5-PA) and Manα1,6(Manα1,3)Manα1,6(Galβ1,4GlcNAcβ1,2Manα1,3)Manβ1,4GlcNAcβ1,4GlcNAc-PA (GalGNM5-PA) were significantly lower than that of the oligomannose-type N-glycan Manα1,6(Manα1,3)Manα1,6Manβ1,4GlcNAcβ1,4GlcNAc-PA (M4-PA), and the trimming pathways of GNM5-PA and GalGNM5-PA were different from that of M4-PA, suggesting a steric hindrance to the JBM activity caused by GlcNAcβ1-2Man(α) residues of the hybrid-type N-glycans. We also found that the substrate preference of JBM for the terminal Manα1-6Man(α) and Manα1-3Man(α) linkages in the hybrid-type N-glycans was altered by the change in reaction pH, suggesting a pH-dependent change in the enzyme-substrate interaction.     

N-glycan of ErbB family plays a crucial role in dimer formation and tumor promotion

More and more evidence indicates that N-glycan regulates signal transduction by modulating receptor functions. Previous studies suggested that glycosylation of EGFR is involved in dimerization and endocytosis. We further determined the role of N-glycosylation of ErbB family. A series of human ErbB3 mutants that lack each of the 10 N-glycosylation sites were prepared and transfected to Flp-In-CHO cells for stable expression. A crosslinking study showed that Asn 418 to Gln mutant (N418Q) of ErbB3 underwent autodimerization without its ligand, heregulin, and the heterodimer formation with ErbB2 was also increased. The N418Q mutant of ErbB3 co-expressed with ErbB2 promoted downstream signaling, anchorage-independent cell growth and the tumor growth in athymic mice. These findings suggest that the specific N-glycan in domain III of ErbB family plays an essential role in regulating receptor dimerization and transforming activity. We assume that the N-glycans affect the conformation of ErbB family, which is crucial for their activity. Together with findings from other laboratories, it is suggested that N-glycosylation controls ErbB signaling by various mechanisms.