2D Selective-TOCSY–DQFCOSY and HSQC–TOCSY NMR experiments for assignment of a homogeneous asparagine-linked triantennary complex type undecasaccharide

Sato, Hajime; Fukae, Kazuhiro; Kajihara, Yasuhiro

doi:10.1016/j.carres.2008.03.008

Cited by 11 publications

(6 citation statements)

References 22 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The NMR signal assignment (Fig. 6A) of glycan 6 was in good agreement with a similar triantennary derivative with two GlcNAc moieties at the reducing terminus (70), and the results further confirmed the homogeneity of the asialylated triantennary N-glycan (6). The twodimensional 1 H/ 13 C HSQC spectrum (Fig.…”

Section: Generation and Characterization Of Glycosynthase Mutants Of supporting

confidence: 71%

“…4. Previously, the components and structures of N-glycans from bovine fetuin have been well characterized by chromatographic separation, HPAEC-PAD analysis, and detailed NMR studies (67)(68)(69)(70). The majority (Ͼ80%) of the N-glycans were triantennary N-glycans with varied degrees of ␣2,6-and/or ␣2,3-sialylations at the terminus.…”

Section: Generation and Characterization Of Glycosynthase Mutants Of mentioning

confidence: 99%

See 1 more Smart Citation

Endo-F3 Glycosynthase Mutants Enable Chemoenzymatic Synthesis of Core-fucosylated Triantennary Complex Type Glycopeptides and Glycoproteins

Giddens

Lomino

Amin

et al. 2016

Journal of Biological Chemistry

115

View full text Add to dashboard Cite

Chemoenzymatic synthesis is emerging as a promising approach to the synthesis of homogeneous glycopeptides and glycoproteins highly demanded for functional glycomics studies, but its generality relies on the availability of a range of enzymes with high catalytic efficiency and well defined substrate specificity. We describe in this paper the discovery of glycosynthase mutants derived from Elizabethkingia meningoseptica endoglycosidase F3 (Endo-F3) of the GH18 family, which are devoid of the inherent hydrolytic activity but are able to take glycan oxazolines for transglycosylation. Notably, the Endo-F3 D165A and D165Q mutants demonstrated high acceptor substrate specificity toward ␣1,6-fucosyl-GlcNAc-Asn or ␣1, 6-fucosyl-GlcNAc-polypeptide in transglycosylation, enabling a highly convergent synthesis of core-fucosylated, complex CD52 glycopeptide antigen. The Endo-F3 mutants were able to use both bi-and triantennary glycan oxazolines as substrates for transglycosylation, in contrast to previously reported endoglycosidases derived from Endo-S, Endo-M, Endo-D, and Endo-A mutants that could not recognize triantennary N-glycans. Using rituximab as a model system, we have further demonstrated that the Endo-F3 mutants are highly efficient for glycosylation remodeling of monoclonal antibodies to produce homogeneous intact antibody glycoforms. Interestingly, the new triantennary glycan glycoform of antibody showed much higher affinity for galectin-3 than that of the commercial antibody. The Endo-F3 mutants represent the first endoglycosidase-based glycosynthases capable of transferring triantennary complex N-glycans, which would be very useful for glycoprotein synthesis and glycosylation remodeling of antibodies.Protein glycosylation is one of the most prevalent posttranslational modifications, found in almost all living organisms ranging from bacteria to eukaryotes (1). Glycosylation can profoundly affect a protein's intrinsic properties, such as folding, stability, intracellular trafficking, and immunogenicity. In addition, the oligosaccharide components of glycoproteins can participate directly in a number of important biological recognition processes, including cell adhesion, signaling, hostpathogen interactions, and immune responses (2-8). It is well documented that subtle changes in glycosylation can lead to a significant impact on the biological functions and, in the case of therapeutic glycoproteins, such as monoclonal antibodies, the in vivo stability and therapeutic efficacy (7, 9 -12). Natural and recombinant glycoproteins are usually produced as mixtures of glycoforms that differ only in the structures of pendant glycans, from which pure glycoforms are extremely difficult to isolate by current chromatographic techniques. As a result, synthetic homogeneous glycopeptides and glycoproteins emerge as indispensable tools for functional studies and for drug/vaccine discoveries. Many elegant chemical and biochemical strategies have been explored for making homogeneous glycoproteins and mimics, including total chemic...

show abstract

Section: Generation and Characterization Of Glycosynthase Mutants Of supporting

confidence: 71%

Section: Generation and Characterization Of Glycosynthase Mutants Of mentioning

confidence: 99%

Endo-F3 Glycosynthase Mutants Enable Chemoenzymatic Synthesis of Core-fucosylated Triantennary Complex Type Glycopeptides and Glycoproteins

Giddens

Lomino

Amin

et al. 2016

Journal of Biological Chemistry

115

View full text Add to dashboard Cite

show abstract

“…The strong shift for H-4 can be taken as confirmation for a bisecting residue on the core β-mannose and so shares a trend observed with data on a bisected mammalian glycan (36), while the data are not compatible with a C-2 modification. The presence of β- d -Gal p 4″ and some α- d -Gal p C linked to mannose as well as some α- l -Fuc p linked to galactose is shown by relevant chemical shifts typical for such residues [4.6-4.3 (34,36), 5.5-5.1 (37), and 5.3-5.2 ppm (38) respectively for the anomeric H-1]; for the latter, it is noteworthy that older NMR data was interpreted as showing α1,2-fucosylation of mannose (39), but our data overall indicate the presence of a bisecting Fucα1,2Gal motif. Indeed, the proton signals of the β- d -Gal p 4″ show the most pronounced shift variations, which may be caused by the fucose or methylfucose bound in substoichiometric amounts to this unit, while the α-galactose is assumed to be linked to position 2 of the α- d -Man p 4 .…”

Section: Resultsmentioning

confidence: 99%

Bisecting Galactose as a Feature of N-Glycans of Wild-type and Mutant Caenorhabditis elegans

Yan

Brecker

Jin

et al. 2015

Molecular & Cellular Proteomics

View full text Add to dashboard Cite

The N-glycosylation of the model nematode Caenorhabiditis elegans has proven to be highly variable and rather complex; it is an example to contradict the existing impression that ‘simple’ organisms possess also a rather simple glycomic capacity. In previous studies in a number of laboratories, N-glycans with up to four fucose residues have been detected. However, although the linkage of three fucose residues to the N,N′-diacetylchitobiosyl core has been proven by structural and enzymatic analyses, the nature of the fourth fucose has remained uncertain. By constructing a triple mutant with deletions in the three genes responsible for core fucosylation (fut-1, fut-6 and fut-8), we have produced a nematode strain lacking products of these enzymes, but still retaining maximally one fucose residue on its N-glycans. Using mass spectrometry and HPLC in conjunction with chemical and enzymatic treatments as well as NMR, we examined a set of α-mannosidase-resistant N-glycans. Within this glycomic sub-pool, we can reveal that the core β-mannose can be trisubstituted and so carries not only the ubiquitous α1,3- and α1,6-mannose residues, but also a ‘bisecting’ β-galactose which is substoichiometrically modified with fucose or methylfucose. In addition, the α1,3-mannose can also be α-galactosylated. Not only do these residues constitute novel modifications of N-glycans, but these features may well be targets of nematoxic proteins produced as defence molecules by various fungal species.

show abstract

“…2c and d). These sequences have already been reported for the analysis of complex oligosaccharides but without multiplexed acquisition [17,18]. Many more interesting applications can be envisaged.…”

Section: Principlementioning

confidence: 97%

An alternative scheme for the multiplexed acquisition of 1D and 2D NMR spectra

Plainchont

Martinez

Tisse

et al. 2010

Journal of Magnetic Resonance

View full text Add to dashboard Cite

2D Selective-TOCSY–DQFCOSY and HSQC–TOCSY NMR experiments for assignment of a homogeneous asparagine-linked triantennary complex type undecasaccharide

Cited by 11 publications

References 22 publications

Endo-F3 Glycosynthase Mutants Enable Chemoenzymatic Synthesis of Core-fucosylated Triantennary Complex Type Glycopeptides and Glycoproteins

Endo-F3 Glycosynthase Mutants Enable Chemoenzymatic Synthesis of Core-fucosylated Triantennary Complex Type Glycopeptides and Glycoproteins

Bisecting Galactose as a Feature of N-Glycans of Wild-type and Mutant Caenorhabditis elegans

An alternative scheme for the multiplexed acquisition of 1D and 2D NMR spectra

Contact Info

Product

Resources

About