Purification and enzymatic characterization of tobacco leaf β-N-acetylhexosaminidase

Ryšlavá, Helena; Valenta, Rudolf; Hýsková, Veronika; Křížek, Tomáš; Liberda, Jiřı́; Coufal, Pavel

doi:10.1016/j.biochi.2014.09.006

Cited by 8 publications

(7 citation statements)

References 36 publications

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“…Although β‐N‐acetylhexosaminidase shows relatively high activity in tobacco leaves, its function, and hence its substrates, in plants are not entirely clear (Ryslava et al . 2014). Chitooligosaccharides do not occur in plant metabolism, but chitinases are important for pathogenesis‐related proteins synthesized in response to biotic stress.…”

Section: Discussionmentioning

confidence: 99%

“…The role of a-mannosidase is particularly important for oligosaccharide chain processing in glycoproteins (Strasser 2016); both heat shock and viral infection could also affect this process. Although b-N-acetylhexosaminidase shows relatively high activity in tobacco leaves, its function, and hence its substrates, in plants are not entirely clear (Ryslava et al 2014). Chitooligosaccharides do not occur in plant metabolism, but chitinases are important for pathogenesis-related proteins synthesized in response to biotic stress.…”

Section: Suggested Roles Of Glycosidases During Heat Shock and Viral Stressmentioning

confidence: 99%

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Effects of heat treatment on metabolism of tobacco plants infected with Potato virus Y

et al. 2021

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Many factors affect successful virus propagation and plant defence responses. Heat shock protein (Hsp) expression after heat shock plays an ambiguous role in viral infection. On the one hand, Hsp70 participates in plant defence response; on the other hand, Hsp70 could interact with viral proteins and facilitate virus propagation.• Here, we studied metabolic adaptations of Nicotiana tabacum L. subjected to heat shock (42°C, 2 h) before or after inoculating the plants with Potato virus Y (potyvirus). RT-qPCR and ELISA were used for potyvirus quantification. Hsp70 and Hsp90 isoforms were analysed by Western blotting. Salicylic, quinic and chlorogenic acid content was determined by LC-MS. The activity of Hatch-Slack enzymes (as markers of potyviral infection in tobacco) and glycosidases was assayed.• Application of heat shock before or after inoculation showed accelerated potyviral propagation in comparison with only inoculated plants. Plants exposed to heat shock and concurrently inoculated showed higher potyviral content, higher amount of Hsp70, together with late decline of quinic acid content and low chlorogenic acid content. Spread of potyviral infection correlated with enhanced salicylic acid content and activities of enzymes of the Hatch-Slack cycle, aand b-galactosidase, a-mannosidase, a-glucosidase and b-N-acetylhexosaminidase.• Heat shock proteins accelerate potyviral propagation. The lower weight cytosolic and mitochondrial Hsp70 (~50-75 kDa) persist throughout the viral infection. Also, the plant defense response results in increase of salicylic and chlorogenic acids but decrease of quinic acid content.

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Section: Discussionmentioning

confidence: 99%

Section: Suggested Roles Of Glycosidases During Heat Shock and Viral Stressmentioning

confidence: 99%

Effects of heat treatment on metabolism of tobacco plants infected with Potato virus Y

et al. 2021

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“…The interplay of pathogen/parasite-associated glycans with plant immunity is a fairly unexplored research area, which is in contrast to the strong focus on glycans in research regarding animal parasitic worms (Hokke and Yazdanbakhsh, 2005;Tundup et al, 2012). A role for HEXOs in plant defense responses is further supported by the chitinolytic activity of several plant HEXOs (Gutternigg et al, 2007;Strasser et al, 2007;Ryšlavá et al, 2014;Hyskova, 2015). Where the chitinolytic activity of HEXOs in other organisms could play a role in the turnover of chitin in the cell wall (fungi), exoskeleton (insects), or cuticle (nematodes), plants might employ these enzymes to protect themselves against the constant threat of fungi, insects, and nematodes.…”

Section: Discussionmentioning

confidence: 99%

β-Hexosaminidases Along the Secretory Pathway of Nicotiana benthamiana Have Distinct Specificities Toward Engineered Helminth N-Glycans on Recombinant Glycoproteins

et al. 2021

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Secretions of parasitic worms (helminths) contain a wide collection of immunomodulatory glycoproteins with the potential to treat inflammatory disorders, like autoimmune diseases. Yet, the identification of single molecules that can be developed into novel biopharmaceuticals is hampered by the limited availability of native parasite-derived proteins. Recently, pioneering work has shown that helminth glycoproteins can be produced transiently in Nicotiana benthamiana plants while simultaneously mimicking their native helminth N-glycan composition by co-expression of desired glycosyltransferases. However, efficient “helminthization” of N-glycans in plants by glyco-engineering seems to be hampered by the undesired truncation of complex N-glycans by β-N-acetyl-hexosaminidases, in particular when aiming for the synthesis of N-glycans with antennary GalNAcβ1-4GlcNAc (LacdiNAc or LDN). In this study, we cloned novel β-hexosaminidase open reading frames from N. benthamiana and characterized the biochemical activity of these enzymes. We identified HEXO2 and HEXO3 as enzymes responsible for the cleavage of antennary GalNAc residues of N-glycans on the model helminth glycoprotein kappa-5. Furthermore, we reveal that each member of the HEXO family has a distinct specificity for N-glycan substrates, where HEXO2 has strict β-galactosaminidase activity, whereas HEXO3 cleaves both GlcNAc and GalNAc. The identification of HEXO2 and HEXO3 as major targets for LDN cleavage will enable a targeted genome editing approach to reduce undesired processing of these N-glycans. Effective knockout of these enzymes could allow the production of therapeutically relevant glycoproteins with tailor-made helminth N-glycans in plants.

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“…Some plant β-N-acetylhexosaminidases (such as from Arabidopsis thaliana, wheat, jack bean) prefer longer chitooligosaccharides to shorter ones [16,20]. Also tobacco β-N-acetylhexosaminidase is able to degrade chitooligomers; N, N'-diacetylchitobiose and N, N', N''triacetylchitotriose with the reaction rate reduced only to 60% and 34%, respectively compared to artificial substrate pNP-GlcNAc [21]. The highest reaction rate and the highest affinity to the substrate is usually achieved with pNP-GlcNAc, which is commonly used for easy colorimetric β-N-acetylhexosaminidase activity determination.…”

mentioning

confidence: 99%

“…On the other hand, the testing of reaction rate in the presence of chitooligomers is more complicated, because the products must be chromatographically separated and for the sensitive quantification they are often labeled by fluorescent tag that can also affect properties of the substrate. Another alternative is the separation of products by capillary electrophoresis, which enables quick analysis in very small reaction volumes [21,22].…”

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confidence: 99%

Different Roles of ÃÂ²-N-Acetylhexosaminidase in Metabolism

Hýsková¹

2015

Biochem Anal Biochem

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typically found in plants and invertebrate [16]. These structures are characterized by the presence of fucose and/or xylose, which can be transferred to the oligosaccharide core only after removal of the terminal GlcNAc (catalyzed by β-N-acetylhexosaminidase) from the complex oligosaccharide. It is interesting that this step of N-glycan processing occurs in plants in vacuole rather than Golgi apparatus.

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Purification and enzymatic characterization of tobacco leaf β-N-acetylhexosaminidase

Cited by 8 publications

References 36 publications

Effects of heat treatment on metabolism of tobacco plants infected with Potato virus Y

Effects of heat treatment on metabolism of tobacco plants infected with Potato virus Y

β-Hexosaminidases Along the Secretory Pathway of Nicotiana benthamiana Have Distinct Specificities Toward Engineered Helminth N-Glycans on Recombinant Glycoproteins

Different Roles of ÃÂ²-N-Acetylhexosaminidase in Metabolism

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Purification and enzymatic characterization of tobacco leaf β-N-acetylhexosaminidase

Cited by 8 publications

References 36 publications

Effects of heat treatment on metabolism of tobacco plants infected with Potato virus Y

Effects of heat treatment on metabolism of tobacco plants infected with Potato virus Y

β-Hexosaminidases Along the Secretory Pathway of Nicotiana benthamiana Have Distinct Specificities Toward Engineered Helminth N-Glycans on Recombinant Glycoproteins

Different Roles of ÃÂ²-N-Acetylhexosaminidase in Metabolism

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Different Roles of ÃÂ²-N-Acetylhexosaminidase in Metabolism