scholarly journals Arthrobacter Endo-β-N-Acetylglucosaminidase Shows Transglycosylation Activity on Complex-Type N-Glycan Oxazolines: One-Pot Conversion of Ribonuclease B to Sialylated Ribonuclease C

ChemBioChem ◽  
2010 ◽  
Vol 11 (10) ◽  
pp. 1350-1355 ◽  
Author(s):  
Wei Huang ◽  
Qiang Yang ◽  
Midori Umekawa ◽  
Kenji Yamamoto ◽  
Lai-Xi Wang
Keyword(s):  
Complex Type ◽  
One Pot ◽  
Transglycosylation Activity ◽  
Ribonuclease B

Chemoenzymatic Synthesis of the Immunoglobulin Domain of Tim-3 Carrying a Complex-Type N-Glycan by Using a One-pot Ligation

2013 ◽  
Vol 52 (37) ◽  
pp. 9733-9737 ◽  
Author(s):  
Yuya Asahina ◽  
Shigehiro Kamitori ◽  
Toshifumi Takao ◽  
Nozomu Nishi ◽  
Hironobu Hojo
Keyword(s):  
Chemoenzymatic Synthesis ◽  
Complex Type ◽  
One Pot ◽  
Immunoglobulin Domain

Novel endo-β-N-acetylglucosaminidases from Tannerella species hydrolyze multibranched complex-type N-glycans with different specificities

Glycobiology ◽  
2020 ◽  
Vol 30 (11) ◽  
pp. 923-934
Author(s):  
Shou Takashima ◽  
Masaki Kurogochi ◽  
Kenji Osumi ◽  
Shu-ichi Sugawara ◽  
Mamoru Mizuno ◽  
...  
Keyword(s):  
Sialic Acid ◽  
Recombinant Proteins ◽  
Hydrolysis Reaction ◽  
Glycan Structure ◽  
Complex Type ◽  
Substrate Specificities ◽  
Transglycosylation Activity ◽  
High Mannose ◽  
Hydrolysis Activity

Abstract Endo-β-N-acetylglucosaminidases are enzymes that hydrolyze the N,N′-diacetylchitobiose unit of N-glycans. Many endo-β-N-acetylglucosaminidases also exhibit transglycosylation activity, which corresponds to the reverse of the hydrolysis reaction. Because of these activities, some of these enzymes have recently been used as powerful tools for glycan remodeling of glycoproteins. Although many endo-β-N-acetylglucosaminidases have been identified and characterized to date, there are few enzymes that exhibit hydrolysis activity toward multibranched (tetra-antennary or more) complex-type N-glycans on glycoproteins. Therefore, we searched for novel endo-β-N-acetylglucosaminidases that exhibit hydrolysis activity toward multibranched complex-type N-glycans in this study. From database searches, we selected three candidate enzymes from Tannerella species—Endo-Tsp1006, Endo-Tsp1263 and Endo-Tsp1457—and prepared them as recombinant proteins. We analyzed the hydrolysis activity of these enzymes toward N-glycans on glycoproteins and found that Endo-Tsp1006 and Endo-Tsp1263 exhibited hydrolysis activity toward complex-type N-glycans, including multibranched N-glycans, preferentially, whereas Endo-Tsp1457 exhibited hydrolysis activity toward high-mannose-type N-glycans exclusively. We further analyzed substrate specificities of Endo-Tsp1006 and Endo-Tsp1263 using 18 defined glycopeptides as substrates, each having a different N-glycan structure. We found that Endo-Tsp1006 preferred N-glycans with galactose or α2,6-linked sialic acid residues in their nonreducing ends as substrates, whereas Endo-Tsp1263 preferred N-glycans with N-acetylglucosamine residues in their nonreducing ends as substrates.

scholarly journals Cell-free glycoengineering of the recombinant SARS-CoV-2 spike glycoprotein

2021 ◽  
Author(s):  
Johannes Ruhnau ◽  
Valerian Grote ◽  
Mariana Juarez-Osorio ◽  
Dunja Bruder ◽  
Erdmann Rapp ◽  
...  
Keyword(s):  
Expression System ◽  
Protein Glycosylation ◽  
Spike Glycoprotein ◽  
Complex Type ◽  
One Pot ◽  
Subunit Vaccines ◽  
Vaccine Candidates ◽  
Wide Range ◽  
The Impact

AbstractThe baculovirus-insect cell expression system is readily utilized to produce viral glycoproteins for research as well as for subunit vaccines and vaccine candidates, for instance against SARS-CoV-2 infections. However, the glycoforms of recombinant proteins derived from this expression system are inherently different from mammalian cell-derived glycoforms with mainly complex-type N-glycans attached, and the impact of these differences in protein glycosylation on the immunogenicity is severely underinvestigated. This applies also to the SARS-CoV-2 spike glycoprotein, which is the antigen target of all licensed vaccines and vaccine candidates including virus like particles and subunit vaccines that are variants of the spike protein. Here, we expressed the transmembrane-deleted human β-1,2 N-acetlyglucosamintransferases I and II (MGAT1∆TM and MGAT2∆TM) and the β-1,4-galactosyltransferase (GalT∆TM) in E. coli to in-vitro remodel the N-glycans of a recombinant SARS-CoV-2 spike glycoprotein derived from insect cells. In a cell-free sequential one-pot reaction, fucosylated and afucosylated paucimannose-type N-glycans were converted to complex-type galactosylated N-glycans. In the future, this in-vitro glycoengineering approach can be used to efficiently generate a wide range of N-glycans on antigens considered as vaccine candidates for animal trials and preclinical testing to better characterize the impact of N-glycosylation on immunity and to improve the efficacy of protein subunit vaccines.

scholarly journals Cell-Free Glycoengineering of the Recombinant SARS-CoV-2 Spike Glycoprotein

2021 ◽  
Vol 9 ◽  
Author(s):  
Johannes Ruhnau ◽  
Valerian Grote ◽  
Mariana Juarez-Osorio ◽  
Dunja Bruder ◽  
Reza Mahour ◽  
...  
Keyword(s):  
Expression System ◽  
Protein Glycosylation ◽  
Spike Glycoprotein ◽  
Complex Type ◽  
One Pot ◽  
Subunit Vaccines ◽  
Vaccine Candidates ◽  
Wide Range ◽  
The Impact

The baculovirus-insect cell expression system is readily utilized to produce viral glycoproteins for research as well as for subunit vaccines and vaccine candidates, for instance against SARS-CoV-2 infections. However, the glycoforms of recombinant proteins derived from this expression system are inherently different from mammalian cell-derived glycoforms with mainly complex-type N-glycans attached, and the impact of these differences in protein glycosylation on the immunogenicity is severely under investigated. This applies also to the SARS-CoV-2 spike glycoprotein, which is the antigen target of all licensed vaccines and vaccine candidates including virus like particles and subunit vaccines that are variants of the spike protein. Here, we expressed the transmembrane-deleted human β-1,2 N-acetlyglucosamintransferases I and II (MGAT1ΔTM and MGAT2ΔTM) and the β-1,4-galactosyltransferase (GalTΔTM) in E. coli to in-vitro remodel the N-glycans of a recombinant SARS-CoV-2 spike glycoprotein derived from insect cells. In a cell-free sequential one-pot reaction, fucosylated and afucosylated paucimannose-type N-glycans were converted to complex-type galactosylated N-glycans. In the future, this in-vitro glycoengineering approach can be used to efficiently generate a wide range of N-glycans on antigens considered as vaccine candidates for animal trials and preclinical testing to better characterize the impact of N-glycosylation on immunity and to improve the efficacy of protein subunit vaccines.

Chemoenzymatic Synthesis of the Immunoglobulin Domain of Tim-3 Carrying a Complex-Type N-Glycan by Using a One-pot Ligation

2013 ◽  
Vol 125 (37) ◽  
pp. 9915-9919 ◽  
Author(s):  
Yuya Asahina ◽  
Shigehiro Kamitori ◽  
Toshifumi Takao ◽  
Nozomu Nishi ◽  
Hironobu Hojo
Keyword(s):  
Chemoenzymatic Synthesis ◽  
Complex Type ◽  
One Pot ◽  
Immunoglobulin Domain
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