Glucosidase II and N-glycan mannose content regulate the half-lives of monoglucosylated species in vivo

Glucosidase II (GII) sequentially removes the two innermost glucose residues from the glycan (Glc(3)Man(9)GlcNAc(2)) transferred to proteins. GII also participates in cycles involving the lectin/chaperones calnexin (CNX) and calreticulin (CRT) as it removes the single glucose unit added to folding intermediates and misfolded glycoproteins by the UDP-Glc:glycoprotein glucosyltransferase (UGGT). GII is a heterodimer in which the α subunit (GIIα) bears the active site, and the β subunit (GIIβ) modulates GIIα activity through its C-terminal mannose 6-phosphate receptor homologous (MRH) domain. Here we report that, as already described in cell-free assays, in live Schizosaccharomyces pombe cells a decrease in the number of mannoses in the glycan results in decreased GII activity. Contrary to previously reported cell-free experiments, however, no such effect was observed in vivo for UGGT. We propose that endoplasmic reticulum α-mannosidase-mediated N-glycan demannosylation of misfolded/slow-folding glycoproteins may favor their interaction with the lectin/chaperone CNX present in S. pombe by prolonging the half-lives of the monoglucosylated glycans (S. pombe lacks CRT). Moreover, we show that even N-glycans bearing five mannoses may interact in vivo with the GIIβ MRH domain and that the N-terminal GIIβ G2B domain is involved in the GIIα-GIIβ interaction. Finally, we report that protists that transfer glycans with low mannose content to proteins have nevertheless conserved the possibility of displaying relatively long-lived monoglucosylated glycans by expressing GIIβ MRH domains with a higher specificity for glycans with high mannose content.     

The Rate of Phaseolin Assembly Is Controlled by the Glucosylation State of Its N-Linked Oligosaccharide Chains

Many of the proteins that are translocated into the endoplasmic reticulum are glycosylated with the addition of a 14-saccharide core unit (Glc3Man9GlcNAc2) to specific asparagine residues of the nascent polypeptide. Glucose residues are then removed by endoplasmic reticulum-located glucosidases, with diglucosylated and monoglucosylated intermediates being formed. In this study, we used a cell-free system constituted of wheat germ extract and bean microsomes to examine the role of glucose trimming in the structural maturation of phaseolin, a trimeric glycoprotein that accumulates in the protein storage vacuoles of bean seeds. Removal of glucose residues from the N-linked chains of phaseolin was blocked by the glucosidase inhibitors castanospermine and N-methyldeoxynojirimycin. If glucose trimming was not allowed to occur, the assembly of phaseolin was accelerated. Conversely, polypeptides bearing partially trimmed glycans were unable to form trimers. The effect of castanospermine on the rate of assembly was much more pronounced for phaseolin polypeptides that have two glycans but was also evident when a single glycan chain was present, indicating that glycan clustering can modulate the effect of glucose trimming on the rate of trimer formation. Therefore, the position of glycan chains and their accessibility to the action of glucosidases can be fundamental elements in the control of the structural maturation of plant glycoproteins.     

Folding of Insulin Receptor Monomers Is Facilitated by the Molecular Chaperones Calnexin and Calreticulin and Impaired by Rapid Dimerization

Many complex membrane proteins undergo subunit folding and assembly in the ER before transport to the cell surface. Receptors for insulin and insulin-like growth factor I, both integral membrane proteins and members of the family of receptor tyrosine kinases (RTKs), are unusual in that they require homodimerization before export from the ER. To better understand chaperone mechanisms in endogenous membrane protein assembly in living cells, we have examined the folding, assembly, and transport of the human insulin receptor (HIR), a dimeric RTK. Using pulse-chase labeling and nonreducing SDS-PAGE analysis, we have explored the molecular basis of several sequential maturation steps during receptor biosynthesis. Under normal growth conditions, newly synthesized receptor monomers undergo disulfide bond formation while associated with the homologous chaperones calnexin (Cnx) and calreticulin (Crt). An inhibitor of glucose trimming, castanospermine (CST), abolished binding to Cnx/Crt but also unexpectedly accelerated receptor homodimerization resulting in misfolded oligomeric proreceptors whose processing was delayed and cell surface expression was also decreased by ~30%. Prematurely-dimerized receptors were retained in the ER and more avidly associated with the heat shock protein of 70 kD homologue binding protein. In CST-treated cells, receptor misfolding followed disordered oligomerization. Together, these studies demonstrate a chaperone function for Cnx/Crt in HIR folding in vivo and also provide evidence that folding efficiency and homodimerization are counterbalanced.     

Glycopeptide specificity of the secretory protein folding sensor UDP-glucose glycoprotein:glucosyltransferase

Secretory and membrane N-linked glycoproteins undergo folding and oligomeric assembly in the endoplasmic reticulum with the aid of a folding mechanism known as the calnexin cycle. UDP–glucose glycoprotein:glucosyltransferase (UGGT) is the sensor component of the calnexin cycle, which recognizes these glycoproteins when they are incompletely folded, and transfers a glucose residue from UDP–glucose to N-linked Man9-GlcNAc2 glycans. To determine how UGGT recognizes incompletely folded glycoproteins, we used purified enzyme to glucosylate a set of Man9-GlcNAc2 glycopeptide substrates in vitro, and determined quantitatively the glucose incorporation into each glycan by mass spectrometry. A ranked order of glycopeptide specificity was found that provides the criteria for the recognition of substrates by UGGT. The preference for amino-acid residues close to N-linked glycans provides criteria for the recognition of glycopeptide substrates by UGGT.     

Glucosidase II β Subunit Modulates N-Glycan Trimming in Fission Yeasts and Mammals

Glucosidase II (GII) plays a key role in glycoprotein biogenesis in the endoplasmic reticulum (ER). It is responsible for the sequential removal of the two innermost glucose residues from the glycan (Glc₃Man₉GlcNAc₂) transferred to Asn residues in proteins. GII participates in the calnexin/calreticulin cycle; it removes the single glucose unit added to folding intermediates and misfolded glycoproteins by the UDP-Glc:glycoprotein glucosyltransferase. GII is a heterodimer whose α subunit (GIIα) bears the glycosyl hydrolase active site, whereas its β subunit (GIIβ) role is controversial and has been reported to be involved in GIIα ER retention and folding. Here, we report that in the absence of GIIβ, the catalytic subunit GIIα of the fission yeast Schizosaccharomyces pombe (an organism displaying a glycoprotein folding quality control mechanism similar to that occurring in mammalian cells) folds to an active conformation able to hydrolyze p-nitrophenyl α-d-glucopyranoside. However, the heterodimer is required to efficiently deglucosylate the physiological substrates Glc₂Man₉GlcNAc₂ (G2M9) and Glc₁Man₉GlcNAc₂ (G1M9). The interaction of the mannose 6-phosphate receptor homologous domain present in GIIβ and mannoses in the B and/or C arms of the glycans mediates glycan hydrolysis enhancement. We present evidence that also in mammalian cells GIIβ modulates G2M9 and G1M9 trimming.     

Interaction mode between catalytic and regulatory subunits in glucosidase II involved in ER glycoprotein quality control

The glycoside hydrolase family 31 (GH31) α‐glucosidases play vital roles in catabolic and regulated degradation, including the α‐subunit of glucosidase II (GIIα), which catalyzes trimming of the terminal glucose residues of N‐glycan in glycoprotein processing coupled with quality control in the endoplasmic reticulum (ER). Among the known GH31 enzymes, only GIIα functions with its binding partner, regulatory β‐subunit (GIIβ), which harbors a lectin domain for substrate recognition. Although the structural data have been reported for GIIα and the GIIβ lectin domain, the interaction mode between GIIα and GIIβ remains unknown. Here, we determined the structure of a complex formed between GIIα and the GIIα‐binding domain of GIIβ, thereby providing a structural basis underlying the functional extension of this unique GH31 enzyme.     

Structure of the Lectin Mannose 6-Phosphate Receptor Homology (MRH) Domain of Glucosidase II,an Enzyme That Regulates Glycoprotein Folding Quality Control in the Endoplasmic Reticulum

Here we report for the first time the three-dimensional structure of a mannose 6-phosphate receptor homology (MRH) domain present in a protein with enzymatic activity, glucosidase II (GII). GII is involved in glycoprotein folding in the endoplasmic reticulum. GII removes the two innermost glucose residues from the Glc₃Man₉GlcNAc₂ transferred to nascent proteins and the glucose added by UDP-Glc:glycoprotein glucosyltransferase. GII is composed of a catalytic GIIα subunit and a regulatory GIIβ subunit. GIIβ participates in the endoplasmic reticulum localization of GIIα and mediates in vivo enhancement of N-glycan trimming by GII through its C-terminal MRH domain. We determined the structure of a functional GIIβ MRH domain by NMR spectroscopy. It adopts a β-barrel fold similar to that of other MRH domains, but its binding pocket is the most shallow known to date as it accommodates a single mannose residue. In addition, we identified a conserved residue outside the binding pocket (Trp-409) present in GIIβ but not in other MRHs that influences GII glucose trimming activity.     

1H and 13C NMR assignments for the glycans in glycoproteins by using 2H/13C-labeled glucose as a metabolic precursor

In order to understand the role of the glycans in glycoproteins in solution, structural information obtained by NMR spectroscopy is obviously required. However, the assignment of the NMR signals from the glycans in larger glycoproteins is still difficult, mainly due to the lack of appropriate methods for the assignment of the resonances originating from the glycans. By using [U-¹³C₆,²H₇]glucose as a metabolic precursor, we have successfully prepared a glycoprotein whose glycan is uniformly labeled with ¹³C and partially with D at the sugar residues. The D to H exchange ratios at the C1-C6 positions of the sugar residues have been proven to provide useful information for the spectral assignments of the glycan in the glycoprotein. This is the first report on the residue-specific assignment of the anomeric resonances originating from a glycan attached to a glycoprotein by using the metabolic incorporation of hydrogen from the medium into a glycan labeled with [U-¹³C₆,²H₇]glucose.     

A two-dimensional array for simultaneous sequencing of N- and o-glycans and their glycoforms on specific glycosylation sites

Glycans play major roles in living organisms. Thus, essential information is required on diverse glycans, their location, and moieties in proteins, as well as for technology in a high-throughput manner, for improving functional glycomics. In the present study, we describe a new approach involving a 2-D array, which has the potential to fulfill both requirements. The first dimension of the array is composed of various lectins immobilized to a MALDI plate. The second dimension consists of initial proteolysis, then sequential exoglycosidase digestion using highly specific enzymes. The products of such digestions are peptide/glycopeptide mixtures conjugating different glycan fragments from which the exoglycosidase has removed specific terminal residues. Consequently, a series of spectra are obtained when lectin-attached products are analyzed by MALDI-TOF MS. By using well-known glycoproteins and NKp46D2-Ig, a recombinant fusion natural killer receptor with unknown glycans produced in CHO cells, we proved the usefulness of the method, demonstrating rapid and simultaneous determination of N- and O-glycan sequences, their glycan moieties, and subtypes on each of the determined glycosylation sites. This strategy provides a tool that can rapidly explore glycan structures and might contribute to a better understanding of process- and disease-related glycoproteins.     

N-Glycosylation in Chrysosporium lucknowense enzymes

Twenty-eight enzymes, encoded by different genes and secreted by different mutant strains of Chrysosporium lucknowense, were subjected to MALDI-TOF MS peptide fingerprinting followed by analysis of the MS data using the GlycoMod tool from the ExPASy proteomic site. Various N-linked glycan structures were discriminated in the C. lucknowense proteins as a result of the analysis. N-Glycosylated peptides with modifications matching the oligosaccharide compositions contained in the GlycoSuiteDB were found in 12 proteins. The most frequently encountered N-linked glycan, found in 9 peptides from 7 proteins, was (Man)(3)(GlcNAc)(2), that is, the core pentasaccharide structure forming mammalian-type high-mannose and hybrid/complex glycans in glycoproteins from different organisms. Nine out of 12 enzymes represented variably N-glycosylated proteins carrying common (Hex)(0-4)(HexNAc)(0-6)+(Man)(3)(GlcNAc)(2) structures, most of them being hybrid/complex glycans. Various glycan structures were likely formed as a result of the enzymatic trimming of a 'parent' oligosaccharide with different glycosidases. The N-glycosylation patterns found in C. lucknowense proteins differ from those reported for the extensively studied enzymes from Aspergilli and Trichoderma species, where high-mannose glycans of variable structure have been detected.     

Release and preparation of intact and unreduced N-linked oligosaccharides from Sf-9 insect cells

Glycosylation, the addition of carbohydrates to a peptide backbone, is the most extensive cotranslational and posttranslational modification made to proteins by eukaryotic cells. The glycosylation profile of a recombinant glycoprotein can significantly affect its biological activity, which is particularly important when being used in human therapeutic applications. Therefore, defining glycan structures to ensure consistency of recombinant glycoproteins among different batches is critical. In this study we describe a method to prepare N-linked glycans derived from insect cell glycoproteins for structural analysis by capillary electrophoresis. Briefly, glycoproteins obtained from uninfected Spodoptera frugiperda Sf-9 insect cells were precipitated with ammonium sulfate and the glycans were chemically cleaved by hydrazinolysis. Following the regeneration of the glycan reducing terminal residue and the removal of contaminating proteins and peptides, the glycans were fluorescently labeled by reductive amination. Fluorescent labeling greatly enhanced the detection limit of the glycan structures determined by capillary electrophoresis. Five major glycan structures were found that migrated between tetra-mannosylated hexasaccharide and nonamannosylated undecasaccharide standards. Upon alpha-mannosidase digestion the number of glycan structures was reduced to two major structures with shorter migration times than the undigested glycans. None of the glycans were susceptible to hexosaminidase or galactosidase treatment. These results are consistent with the majority of previous results demonstrating hypermannosylated glycan structures in Sf-9 insect cells.     

Online nanoliquid chromatography-mass spectrometry and nanofluorescence detection for high-resolution quantitative N-glycan analysis

The characterization of the repertoire of glycans at the quantitative and qualitative levels on cells and glycoproteins is a necessary step to the understanding of glycan functions in biology. In addition, there is an increasing demand in the field of biotechnology for the monitoring of glycosylation of recombinant glycoproteins, an important issue with regard to their safety and biological activity. The enzymatic release followed by fluorescent derivatization of glycans and separation by normal phase high-performance liquid chromatography (HPLC) has proven for many years to be a powerful approach to the quantification of glycans. Characterization of glycans has classically been performed by mass spectrometry (MS) with external standardization. Here, we report a new method for the simultaneous quantification and characterization of the N-glycans on glycoproteins without the need for external standardization. This method, which we call glycan nanoprofiling, uses nanoLC-coupled electrospray ionization (ESI)-MS with an intercalated nanofluorescence reader and provides effective single glycan separation with subpicomolar sensitivity. The method relies on the isolation and coumaric derivatization of enzymatically released glycans collected by solid phase extraction with porous graphitized carbon and their separation over polyamide-based nanoHPLC prior to serial nanofluorescence and nanoelectrospray mass spectrometric analysis. Glycan nanoprofiling is a broadly applicable and powerful approach that is sufficient to identify and quantify many glycan oligomers in a single run. Glycan nanoprofiling was successfully applied to resolve the glycans of monoclonal antibodies, showing that this method is a fast and sensitive alternative to available methods.     

N-linked glycosylation is required for nicotinic receptor assembly but not for subunit associations with calnexin

We investigated how asparagine (N)-linked glycosylation affects assembly of acetylcholine receptors (AChRs) in the endoplasmic reticulum (ER). Block of N-linked glycosylation inhibited AChR assembly whereas block of glucose trimming partially blocked assembly at the late stages. Removal of each of seven glycans had a distinct effect on AChR assembly, ranging from no effect to total loss of assembly. Because the chaperone calnexin (CN) associates with N-linked glycans, we examined CN interactions with AChR subunits. CN rapidly associates with 50% or more of newly synthesized AChR subunits, but not with subunits after maturation. Block of N-linked glycosylation or trimming did not alter CN-AChR subunit associations nor did subunit mutations prevent N-linked glycosylation. Additionally, CN associations with subunits lacking N-linked glycans occurred without subunit aggregation or misfolding. Our data indicate that CN associates with AChR subunits without N-linked glycan interactions. Furthermore, CN-subunit associations only occur early in AChR assembly and have no role in events later that require N-linked glycosylation.     

Unusual β1-4-galactosidase activity of an α1-6-mannosidase from Xanthomonas manihotis in the processing of branched hybrid and complex glycans

Mannosidases are a diverse group of glycoside hydrolases that play crucial roles in mannose trimming of oligomannose glycans, glycoconjugates, and glycoproteins involved in numerous cellular processes, such as glycan biosynthesis and metabolism, structure regulation, cellular recognition, and cell–pathogen interactions. Exomannosidases and endomannosidases cleave specific glycosidic bonds of mannoside linkages in glycans and can be used in enzyme-based methods for sequencing of isomeric glycan structures. α1-6-mannosidase from Xanthomonas manihotis is known as a highly specific exoglycosidase that removes unbranched α1-6 linked mannose residues from oligosaccharides. However, we discovered that this α1-6-mannosidase also possesses an unexpected β1-4-galactosidase activity in the processing of branched hybrid and complex glycans through our use of enzymatic reactions, high performance anion-exchange chromatography, and liquid chromatography mass spectrometric sequencing. Our docking simulation of the α1-6-mannosidase with glycan substrates reveals potential interacting residues in a relatively shallow pocket slightly differing from its homologous enzymes in the glycoside hydrolase 125 family, which may be responsible for the observed higher promiscuity in substrate binding and subsequent terminal glycan hydrolysis. This observation of novel β1-4-galactosidase activity of the α1-6-mannosidase provides unique insights into its bifunctional activity on the substrate structure-dependent processing of terminal α1-6-mannose of unbranched glycans and terminal β1-4-galactose of hybrid and complex glycans. The finding thus suggests the dual glycosidase specificity of this α1-6-mannosidase and the need for careful consideration when used for the structural elucidation of glycan isomers.     

Swainsonine is a useful tool to monitor the intracellular traffic of N-linked glycoproteins as a function of the state of enterocytic differentiation of HT-29 cells.

After treatment with swainsonine, an inhibitor of both lysosomal alpha-mannosidase and Golgi alpha-mannosidase-II activities, analysis of [3H]mannose-labeled glycans showed that HT-29 cells, derived from a human colonic adenocarcinoma, displayed distinct patterns of N-glycan expression, depending upon their state of enterocytic differentiation. In differentiated HT-29 cells hybrid-type chains were detected, whereas undifferentiated HT-29 cells accumulated high-mannose-type oligosaccharide, despite our demonstration of Golgi alpha-mannosidase-II activity in both cell populations. Pulse/chase experiments carried out in the presence of swainsonine revealed that the persistence of high-mannose-type chains in undifferentiated HT-29 cells was the result of the stabilization of glycoproteins substituted with these glycans. These data suggest that in undifferentiated HT-29 cells, glycoproteins with high-mannose-type oligosaccharides are delivered to a degradative compartment containing swainsonine-sensitive alpha-mannosidase(s), whereas in differentiated HT-29 cells glycoproteins enter a compartment in which alpha-mannosidase II (Golgi apparatus) is present. Thus, this apparent dual effect of swainsonine on N-glycan trimming may reflect differences in the intracellular traffic of glycoproteins as a function of the state of enterocytic differentiation of HT-29 cells.     

The distribution of glycan structures in individual N-glycosylation sites in animal and plant glycoproteins

Glycopeptides representing each individual N-glycosylation site in six animal and plant glycoproteins (ovoinhibitor and ovotransferrin, orosomucoid, antitrypsin, phaseolin, and phytohemagglutinin) have been isolated and compared by mass spectrometric analysis. Since the isolation step separates each individual peptide regardless of the nature of the glycan attached to it, it is possible to observe the entire spectrum of glycans associated with each site from the mass spectrum of the corresponding glycopeptide. The three glycosylation sites in ovoinhibitor have very similar but not identical glycans; they are significantly different from those observed in the single site of ovotransferrin. The three sites in serum antitrypsin also have quite similar glycans, whereas the five sites in orosomucoid show considerable variation in both the nature and the relative amount of glycans. The two plant glycoproteins each have two sites with very different glycan structures. Except for the first and third glycosylation sites of antitrypsin which were found to have remarkably homogeneous glycans (97 and 90% of a biantennary complex structure), all the individual glycosylation sites contained heterogeneous mixtures of glycan structures. The results support the proposition that each N-linked glycan in a glycoprotein is affected by its unique protein environment to such an extent that each one may be displayed to the processing enzymes as a unique structural entity. On the basis of a limited number of observations of the glycan interfering with chymotryptic but not tryptic cleavage in the proximity of the glycan attachment site, it is proposed that hydrophobic interactions between the protein and the glycan may be involved in the conformational modulation of the glycans.     

Calnexin Improves the Folding Efficiency of Mutant Rhodopsin in the Presence of Pharmacological Chaperone 11-cis-Retinal

The lectin chaperone calnexin (Cnx) is important for quality control of glycoproteins, and the chances of correct folding of a protein increase the longer the protein interacts with Cnx. Mutations in glycoproteins increase their association with Cnx, and these mutant proteins are retained in the endoplasmic reticulum. However, until now, the increased interaction with Cnx was not known to increase the folding of mutant glycoproteins. Because many human diseases result from glycoprotein misfolding, a Cnx-assisted folding of mutant glycoproteins could be beneficial. Mutations of rhodopsin, the glycoprotein pigment of rod photoreceptors, cause misfolding resulting in retinitis pigmentosa. Despite the critical role of Cnx in glycoprotein folding, surprisingly little is known about its interaction with rhodopsin or whether this interaction could be modulated to increase the folding of mutant rhodopsin. Here, we demonstrate that Cnx preferentially associates with misfolded mutant opsins associated with retinitis pigmentosa. Furthermore, the overexpression of Cnx leads to an increased accumulation of misfolded P23H opsin but not the correctly folded protein. Finally, we demonstrate that increased levels of Cnx in the presence of the pharmacological chaperone 11-cis-retinal increase the folding efficiency and result in an increase in correct folding of mutant rhodopsin. These results demonstrate that misfolded rather than correctly folded rhodopsin is a substrate for Cnx and that the interaction between Cnx and mutant, misfolded rhodopsin, can be targeted to increase the yield of folded mutant protein.     

Recovery of intact 2-aminobenzamide-labeled O-glycans released from glycoproteins by hydrazinolysis

The initial step in quantitative analysis of O-linked glycans of glycoproteins is to release them in high yield, nonselectively, unmodified, and with a free reducing terminus. In contrast to other techniques, hydrazinolysis can meet these criteria. However, when analyzing pools of O-linked glycans as described in the accompanying article by L. Royle et al. (2002, Anal. Biochem. 304), some peeling of the glycans was observed. Critical steps in the sample preparation and glycan recovery were therefore evaluated by analyzing and identifying both intact O-glycans and degraded products. Synthetic O-glycopeptides were characterized by mass spectrometry. Released glycans were identical to those on the glycopeptide. O-Linked glycans from a range of glycoproteins of increasing complexity, namely, bovine serum fetuin, glycophorin A, and previously uncharacterized glycopeptides isolated from human salivary mucin Muc5B, were also analyzed. Quantitative analysis of the glycan profile confirmed that there was <2% peeling of O-glycans released by hydrazinolysis conditions of 60 degrees C for 6 h, and recovered using the optimised procedure now described. This demonstrated that O-glycans can be prepared by hydrazinolysis without degradation and, as part of an analytical strategy, makes the analysis of O-glycans attached to low-microgram levels of naturally occurring glycoproteins feasible.     

Isolation of a mutant Arabidopsis plant that lacks N-acetyl glucosaminyl transferase I and is unable to synthesize Golgi-modified complex N-linked glycans.

The complex asparagine-linked glycans of plant glycoproteins, characterized by the presence of beta 1-->2 xylose and alpha 1-->3 fucose residues, are derived from typical mannose9(N-acetylglucosamine)2 (Man9GlcNAc2) N-linked glycans through the activity of a series of glycosidases and glycosyl transferases in the Golgi apparatus. By screening leaf extracts with an antiserum against complex glycans, we isolated a mutant of Arabidopsis thaliana that is blocked in the conversion of high-manne to complex glycans. In callus tissues derived from the mutant plants, all glycans bind to concanavalin A. These glycans can be released by treatment with endoglycosidase H, and the majority has the same size as Man5GlcNAc1 glycans. In the presence of deoxymannojirimycin, an inhibitor of mannosidase I, the mutant cells synthesize Man9GlcNAc2 and Man8GlcNAc2 glycans, suggesting that the biochemical lesion in the mutant is not in the biosynthesis of high-mannose glycans in the endoplasmic reticulum but in their modification in the Golgi. Direct enzyme assays of cell extracts show that the mutant cells lack N-acetyl glucosaminyl transferase I, the first enzyme in the pathway of complex glycan biosynthesis. The mutant plants are able to complete their development normally under several environmental conditions, suggesting that complex glycans are not essential for normal developmental processes. By crossing the complex-glycan-deficient strain of A. thaliana with a transgenic strain that expresses the glycoprotein phytohemagglutinin, we obtained a unique strain that synthesizes phytohemagglutinin with two high-mannose glycans, instead of one high-mannose and one complex glycan.     

High-mannose-type glycan modifications of dihydrofolate reductase using glycan-methotrexate conjugates

Various high-mannose-type glycan modifications of dihydrofolate reductase (DHFR) were achieved by ligand-based approach using glycan-methotrexate (MTX) conjugates as tight binding glycan bearing ligands for DHFR. The resulting glycan-MTX conjugates and the corresponding artificial glycoproteins could be useful as oligosaccharide- and glycoprotein-probes to perform quantitative analysis of glycan recognizing protein such as lectins, glycosyltransferases or glycosidases. Moreover, artificial glycoproteins having two different high-mannose-type glycans were developed for the first time by a combination of two different types of glycan modification strategies.