Effect of Bisecting GlcNAc and Core Fucosylation on Conformational Properties of Biantennary Complex-Type N-Glycans in Solution

2012 ◽  
Vol 116 (29) ◽  
pp. 8504-8512 ◽  
Author(s):  
Wataru Nishima ◽  
Naoyuki Miyashita ◽  
Yoshiki Yamaguchi ◽  
Yuji Sugita ◽  
Suyong Re
Keyword(s):  
Complex Type ◽  
Conformational Properties ◽  
Core Fucosylation ◽  
Bisecting Glcnac

scholarly journals Enzymatic Synthesis of a Fluorogenic Reporter Substrate and a High-Throughput Assay for Fucosyltransferase VIII Provide a Toolkit to Probe and Inhibit Core Fucosylation

2019 ◽  
Author(s):  
Maxim Soroko ◽  
David Kwan
Keyword(s):  
Enzyme Activity ◽  
Enzymatic Synthesis ◽  
Enzyme Assay ◽  
Enzyme Reaction ◽  
Complex Type ◽  
The Core ◽  
Fluorogenic Probe ◽  
Tissue Lysate ◽  
High Throughput Assay ◽  
Core Fucosylation

We report a straight-forward enzymatic synthesis of the 4-methylumbelliferyl glycoside of a complex-type oligosaccharide substrate for core-fucosylation. We demonstrate the use of this synthetic glycoconjugate in a newly developed enzyme assay to probe the activity and inhibition of fucosyltransferase VIII, which catalyzes the core fucosylation of <i>N</i>-glycans on eukaryotic glycoproteins. In this fucosyltransferase assay, we use the fluorogenic probe and a specific glycosidase in a sequential coupled enzyme reaction to distinguish an unmodified 4-methylumbelliferyl oligosaccharide probe from a fucosylated probe. Our findings show that this strategy is very sensitive and very specific in its detection of enzyme activity and can even be used for analyzing impure tissue lysate samples.

scholarly journals Standardization of PGC-LC-MS-based glycomics for sample specific glycotyping

2019 ◽  
Author(s):  
Christopher Ashwood ◽  
Brian Pratt ◽  
Brendan MacLean ◽  
Rebekah L. Gundry ◽  
Nicolle H. Packer
Keyword(s):  
Predictive Models ◽  
Tissue Sample ◽  
Internal Standard ◽  
Search Space ◽  
Normalization Method ◽  
Porous Graphitized Carbon ◽  
Structural Modifications ◽  
Graphitized Carbon ◽  
Core Fucosylation ◽  
Bisecting Glcnac

<p></p><p>Porous graphitized carbon (PGC) based chromatography achieves high-resolution separation of glycan structures released from glycoproteins. This approach is especially valuable when resolving structurally similar isomers and for discovery of novel and/or sample-specific glycan structures. However, the implementation of PGC-based separations in glycomics studies has been limited because system-independent retention values have not been established to normalize technical variation. To address this limitation, this study combined the use of hydrolyzed dextran as an internal standard and Skyline software for post-acquisition normalization to reduce retention time and peak area technical variation in PGC-based glycan analyses. This approach allowed assignment of system-independent retention values that are applicable to typical PGC-based glycan separations and supported the construction of a library containing >300 PGC-separated glycan structures with normalized glucose unit (GU) retention values. To enable the automation of this normalization method, a spectral MS/MS library was developed of the dextran ladder, achieving confident discrimination against isomeric glycans. The utility of this approach is demonstrated in two ways. First, to inform the search space for bioinformatically predicted but unobserved glycan structures, predictive models for two structural modifications, core-fucosylation and bisecting GlcNAc, were developed based on the GU library. Second, the applicability of this method for the analysis of complex biological samples is evidenced by the ability to discriminate between cell culture and tissue sample types by the normalized intensity of <i>N-</i>glycan structures alone. Overall, the methods and data described here are expected to support the future development of more automated approaches to glycan identification and quantitation.</p><br><p></p>

scholarly journals Standardization of PGC-LC-MS-based glycomics for sample specific glycotyping

2019 ◽  
Author(s):  
Christopher Ashwood ◽  
Brian Pratt ◽  
Brendan MacLean ◽  
Rebekah L. Gundry ◽  
Nicolle H. Packer
Keyword(s):  
Predictive Models ◽  
Tissue Sample ◽  
Internal Standard ◽  
Search Space ◽  
Normalization Method ◽  
Porous Graphitized Carbon ◽  
Structural Modifications ◽  
Graphitized Carbon ◽  
Core Fucosylation ◽  
Bisecting Glcnac

<p></p><p>Porous graphitized carbon (PGC) based chromatography achieves high-resolution separation of glycan structures released from glycoproteins. This approach is especially valuable when resolving structurally similar isomers and for discovery of novel and/or sample-specific glycan structures. However, the implementation of PGC-based separations in glycomics studies has been limited because system-independent retention values have not been established to normalize technical variation. To address this limitation, this study combined the use of hydrolyzed dextran as an internal standard and Skyline software for post-acquisition normalization to reduce retention time and peak area technical variation in PGC-based glycan analyses. This approach allowed assignment of system-independent retention values that are applicable to typical PGC-based glycan separations and supported the construction of a library containing >300 PGC-separated glycan structures with normalized glucose unit (GU) retention values. To enable the automation of this normalization method, a spectral MS/MS library was developed of the dextran ladder, achieving confident discrimination against isomeric glycans. The utility of this approach is demonstrated in two ways. First, to inform the search space for bioinformatically predicted but unobserved glycan structures, predictive models for two structural modifications, core-fucosylation and bisecting GlcNAc, were developed based on the GU library. Second, the applicability of this method for the analysis of complex biological samples is evidenced by the ability to discriminate between cell culture and tissue sample types by the normalized intensity of <i>N-</i>glycan structures alone. Overall, the methods and data described here are expected to support the future development of more automated approaches to glycan identification and quantitation.</p><br><p></p>

scholarly journals High-mannose type N-glycans with core fucosylation and complex-type N-glycans with terminal neuraminic acid residues are unique to porcine islets

PLoS ONE ◽  
2020 ◽  
Vol 15 (11) ◽  
pp. e0241249
Author(s):  
Yoshihide Nanno ◽  
Asif Shajahan ◽  
Roberto N. Sonon ◽  
Parastoo Azadi ◽  
Bernhard J. Hering ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Neuraminic Acid ◽  
Ionization Mass ◽  
Human Pancreas ◽  
Complex Type ◽  
Antibody Mediated Rejection ◽  
Porcine Islets ◽  
Porcine Islet ◽  
High Mannose ◽  
Core Fucosylation

Objectives Islet transplantation is an emerging treatment option for type 1 diabetes but its application is limited by the shortage of human pancreas donors. Characterization of the N- and O-glycan surface antigens that vary between human and genetically engineered porcine islet donors could shed light on targets of antibody mediated rejection. Methods N- and O-glycans were isolated from human and adult porcine islets and analyzed using matrix-assisted laser-desorption time-of-flight mass spectrometry (MALDI-TOF-MS) and electrospray ionization mass spectrometry (ESI-MS/MS). Results A total of 57 porcine and 34 human N-glycans and 21 porcine and 14 human O-glycans were detected from cultured islets. Twenty-eight of which were detected only from porcine islets, which include novel xenoantigens such as high-mannose type N-glycans with core fucosylation and complex-type N-glycans with terminal neuraminic acid residues. Porcine islets have terminal N-glycolylneuraminic acid (NeuGc) residue in bi-antennary N-glycans and sialyl-Tn O-glycans. No galactose-α-1,3-galactose (α-Gal) or Sda epitope were detected on any of the islets. Conclusions These results provide important insights into the potential antigenic differences of N- and O-glycan profiles between human and porcine islets. Glycan differences may identify novel gene targets for genetic engineering to generate superior porcine islet donors.

scholarly journals Enzymatic Synthesis of a Fluorogenic Reporter Substrate and a High-Throughput Assay for Fucosyltransferase VIII Provide a Toolkit to Probe and Inhibit Core Fucosylation

2019 ◽  
Author(s):  
Maxim Soroko ◽  
David Kwan
Keyword(s):  
Enzyme Activity ◽  
Enzymatic Synthesis ◽  
Enzyme Assay ◽  
Enzyme Reaction ◽  
Complex Type ◽  
The Core ◽  
Fluorogenic Probe ◽  
Tissue Lysate ◽  
High Throughput Assay ◽  
Core Fucosylation

We report a straight-forward enzymatic synthesis of the 4-methylumbelliferyl glycoside of a complex-type oligosaccharide substrate for core-fucosylation. We demonstrate the use of this synthetic glycoconjugate in a newly developed enzyme assay to probe the activity and inhibition of fucosyltransferase VIII, which catalyzes the core fucosylation of <i>N</i>-glycans on eukaryotic glycoproteins. In this fucosyltransferase assay, we use the fluorogenic probe and a specific glycosidase in a sequential coupled enzyme reaction to distinguish an unmodified 4-methylumbelliferyl oligosaccharide probe from a fucosylated probe. Our findings show that this strategy is very sensitive and very specific in its detection of enzyme activity and can even be used for analyzing impure tissue lysate samples.

scholarly journals Appropriate aglycone modification significantly expands the glycan substrate acceptability of α1,6-fucosyltransferase (FUT8)

2021 ◽  
Author(s):  
Roushu Zhang ◽  
Qiang Yang ◽  
Bhargavi M Boruah ◽  
Guanghui Zong ◽  
Chao Li ◽  
...  
Keyword(s):  
Substrate Specificity ◽  
Turnover Rate ◽  
Human Neutrophils ◽  
Complex Type ◽  
The Core ◽  
Km Value ◽  
High Mannose ◽  
Overall Efficiency ◽  
Core Fucosylation ◽  
Lysosomal Proteins

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 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.

The conformational properties of calix[4]arenes 1h NMR and molecular mechanics calculations

1989 ◽  
Vol 86 ◽  
pp. 945-954 ◽  
Author(s):  
F. Bayard ◽  
D. Decoret ◽  
D. Pattou ◽  
J. Royer ◽  
A. Satrallah ◽  
...  
Keyword(s):  
Molecular Mechanics ◽  
1H Nmr ◽  
Molecular Mechanics Calculations ◽  
Conformational Properties
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