Enzymatic Synthesis of Glycans and Glycoconjugates

Rexer, Thomas; Laaf, Dominic; Gottschalk, Johannes; Frohnmeyer, Hannes; Rapp, Erdmann; Elling, Lothar

doi:10.1007/10_2020_148

Cited by 14 publications

(21 citation statements)

References 298 publications

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“…Hyaluronic acid (HA) is an unbranched glycosaminoglycan composed of disaccharides units ([-3)GlcNAc(β1-4)GlcA(β1-] n ) with a molecular size up to 10 7 Da. [1] Because of its viscoelastic and nonpathogenic characteristics, the omnipresent glycosaminoglycan is used in many medical and cosmetic applications like the treatment of arthritis or during eye surgery. [2][3][4][5] With an increased variety of applications and demand, [1] the production of defined HA especially for biomedical applications stays in focus.…”

Section: Introductionmentioning

confidence: 99%

“…[1] Because of its viscoelastic and nonpathogenic characteristics, the omnipresent glycosaminoglycan is used in many medical and cosmetic applications like the treatment of arthritis or during eye surgery. [2][3][4][5] With an increased variety of applications and demand, [1] the production of defined HA especially for biomedical applications stays in focus. Common industrial processes like extraction from rooster combs or fermentation with Streptococcus end up into a highly dispersed HA product, which also has the risk of contamination of avian proteins or endotoxins, respectively.…”

Section: Introductionmentioning

confidence: 99%

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Integration of a Nucleoside Triphosphate Regeneration System in the One‐pot Synthesis of UDP‐sugars and Hyaluronic Acid

et al. 2021

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Enzymatic synthesis is a promising approach to produce hyaluronic acid (HA) products with defined molecular weight (MW) and dispersity. We here report on the evaluation of nucleoside triphosphate (NTP) regeneration system for the synthesis of the precursors uridine-5'-diphosphate α-d-N-acetylglucosamine (UDP-GlcNAc) and uridine-5'-diphosphate α-dglucuronic acid (UDP-GlcA) and integration in the one-pot synthesis of HA. By integration of polyphosphate kinase from Ruegeria pomeroyi (RpPPK2-3) into the enzyme cascade to synthesize UDP-GlcA we reached 89 % yield after 1 h with a regeneration cycle number (RCN) of 17. With the UDP-GlcNAc cascade, the ATP concentration was lowered 250-fold and reached 94 % yield after 1 h with an RCN of 234. Integration of the RpPPK2-3 regeneration system in one-pot HA synthesis afforded optimization of MgCl 2 and ATP starting concentrations. With 25 mM MgCl 2 and 0.1 mM ATP, a HA concentration of 0.81 g L À 1 with an average MW of 1.17 MDa and dispersity of about 1.08 and an RCN of 75 was obtained. We conclude that integration of NTP regeneration with RpPPK2-3 expanded the enzymatic possibilities to produce HA with lower range MW.

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Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Integration of a Nucleoside Triphosphate Regeneration System in the One‐pot Synthesis of UDP‐sugars and Hyaluronic Acid

et al. 2021

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“…The enrichment was based on the upregulation of gdp mannose-4,6-dehydratase (AAEL014961), which converts GDPmannose to GDP-4-dehydro-6-deoxy-D-mannose, the first of three steps for the conversion of GDP-mannose to GDP-fucose. This result suggests an induction caused by the presence of Wolbachia in insect cells to generate glycoconjugates, as GDPmannose is a precursor for these molecules (Stick and Williams, 2009;Rexer et al, 2020), that may be used as elements to form new bacterial structures (García-del Portillo, 2020). An important FIGURE 5 | Overview of significantly enriched GO [-log10 (P-value)] terms of ontological interaction network in biological processes of up-regulated (red) and down-regulated (blue) genes in ZIKV infected versus non-infected mosquitoes.…”

Section: Wolbachia Endosymbiosis Interfere In Glycoconjugates Productmentioning

confidence: 98%

Comprehensive Quantitative Proteome Analysis of Aedes aegypti Identifies Proteins and Pathways Involved in Wolbachia pipientis and Zika Virus Interference Phenomenon

et al. 2021

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Zika virus (ZIKV) is a global public health emergency due to its association with microcephaly, Guillain-Barré syndrome, neuropathy, and myelitis in children and adults. A total of 87 countries have had evidence of autochthonous mosquito-borne transmission of ZIKV, distributed across four continents, and no antivirus therapy or vaccines are available. Therefore, several strategies have been developed to target the main mosquito vector, Aedes aegypti, to reduce the burden of different arboviruses. Among such strategies, the use of the maternally-inherited endosymbiont Wolbachia pipientis has been applied successfully to reduce virus susceptibility and decrease transmission. However, the mechanisms by which Wolbachia orchestrate resistance to ZIKV infection remain to be elucidated. In this study, we apply isobaric labeling quantitative mass spectrometry (MS)-based proteomics to quantify proteins and identify pathways altered during ZIKV infection; Wolbachia infection; co-infection with Wolbachia/ZIKV in the A. aegypti heads and salivary glands. We show that Wolbachia regulates proteins involved in reactive oxygen species production, regulates humoral immune response, and antioxidant production. The reduction of ZIKV polyprotein in the presence of Wolbachia in mosquitoes was determined by MS and corroborates the idea that Wolbachia helps to block ZIKV infections in A. aegypti. The present study offers a rich resource of data that may help to elucidate mechanisms by which Wolbachia orchestrate resistance to ZIKV infection in A. aegypti, and represents a step further on the development of new targeted methods to detect and quantify ZIKV and Wolbachia directly in complex tissues.

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“…CC-BY-NC-ND 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted April 30, 2021. ; https://doi.org/10.1101/2021.04.30.442139 doi: bioRxiv preprint Landuyt et al, 2019;Rexer et al, 2020a). In our study, recombinant human β-1,2 Nacetlyglucosamintransferases I and II (MGAT1ΔTM and MGAT2 ΔTM) and the β-1,4galactosyltransferase (GalTΔTM) expressed in E. coli were utilized to convert insect cellderived paucimannose structures of recombinant SARS-CoV-2 spike glycoprotein to typical mammalian, complex-type galactosylated structures in a cell-free one-pot reaction (Fujiyama et al, 2001;Boeggeman et al, 2003;Bendiak, 2014;Ramakrishnan and Qasba, 2014;Stanley, 2014 1).…”

Section: Introductionmentioning

confidence: 99%

“…Over the past years the establishment of protocols for expression of eukaryotic and bacterial glycosyltransferases has facilitated the processing of glycans in cell-free one-pot reactions. As a platform technology, the corresponding in-vitro glycoengineering approaches have the potential to tailor the glycoform of proteins independent of the expression systems used (Van Landuyt et al, 2019;Rexer et al, 2020a). In our study, recombinant human β-1,2 N-acetlyglucosamintransferases I and II (MGAT1∆TM and MGAT2 ∆TM) and the β-1,4-galactosyltransferase (GalT∆TM) expressed in E. coli were utilized to convert insect cell-derived paucimannose structures of recombinant SARS-CoV-2 spike glycoprotein to typical mammalian, complex-type galactosylated structures in a cell-free one-pot reaction (Fujiyama et al, 2001;Boeggeman et al, 2003;Bendiak, 2014;Ramakrishnan and Qasba, 2014;Stanley, 2014).…”

Section: Introductionmentioning

confidence: 99%

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

Ruhnau

Grote

Juarez-Osorio

et al. 2021

Preprint

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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 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.

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Enzymatic Synthesis of Glycans and Glycoconjugates

Cited by 14 publications

References 298 publications

Integration of a Nucleoside Triphosphate Regeneration System in the One‐pot Synthesis of UDP‐sugars and Hyaluronic Acid

Integration of a Nucleoside Triphosphate Regeneration System in the One‐pot Synthesis of UDP‐sugars and Hyaluronic Acid

Comprehensive Quantitative Proteome Analysis of Aedes aegypti Identifies Proteins and Pathways Involved in Wolbachia pipientis and Zika Virus Interference Phenomenon

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

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