N-glycosylation of tomato nuclease TBN1 produced in N. benthamiana and its effect on the enzyme activity

Kód výsledku v IS VaVaI

<a href="https://www.isvavai.cz/riv?ss=detail&h=RIV%2F86652036%3A_____%2F18%3A00497108" target="_blank" >RIV/86652036:_____/18:00497108 - isvavai.cz</a>

Nalezeny alternativní kódy

RIV/60077344:_____/18:00497108 RIV/60461373:22330/18:43915976

Výsledek na webu

<a href="http://dx.doi.org/10.1016/j.plantsci.2018.08.011" target="_blank" >http://dx.doi.org/10.1016/j.plantsci.2018.08.011</a>

DOI - Digital Object Identifier

<a href="http://dx.doi.org/10.1016/j.plantsci.2018.08.011" target="_blank" >10.1016/j.plantsci.2018.08.011</a>

Jazyk výsledku

angličtina

Název v původním jazyce

N-glycosylation of tomato nuclease TBN1 produced in N. benthamiana and its effect on the enzyme activity

Popis výsledku v původním jazyce

A unique analysis of an enzyme activity versus structure modification of the tomato nuclease R-TBN1 is presented. R-TBN1, the non-specific nuclease belonging to the S1-P1 nuclease family, was recombinantly produced in N. benthamiana. The native structure is posttranslationally modified by N-glycosylation at three sites. In this work, it was found that this nuclease is modified by high-mannose type N-glycosylation with a certain degree of macro-and microheterogeneity. To monitor the role of N-glycosylation in its activity, hypo-and hyperglycosylated nuclease mutants, R-TBN1 digested by alpha-mannosidase, and R-TBN1 deglycosylated by PNGase F were prepared. Deglycosylated R-TBN1 and mutant N94D/N112D were virtually inactive. Compared to R-TBN1 wt, both N94D and N112D mutants showed about 60% and 10% of the activity, respectively, while the N186D, D36S, and D36S/E104 N mutants were equally or even more active than R-TBN1 wt. The partial demannosylation of R-TBN1 did not affect the nuclease aktivity, moreover, a little shift in substrate specificity was observed. The results show two facts: 1) which sites must be occupied by a glycan for the proper folding and stability and 2) how N. benthamiana glycosylates the foreign nuclease. At the same time, the modifications can be interesting in designing the nuclease activity or specificity through its glycosylation.

Název v anglickém jazyce

N-glycosylation of tomato nuclease TBN1 produced in N. benthamiana and its effect on the enzyme activity

Popis výsledku anglicky

A unique analysis of an enzyme activity versus structure modification of the tomato nuclease R-TBN1 is presented. R-TBN1, the non-specific nuclease belonging to the S1-P1 nuclease family, was recombinantly produced in N. benthamiana. The native structure is posttranslationally modified by N-glycosylation at three sites. In this work, it was found that this nuclease is modified by high-mannose type N-glycosylation with a certain degree of macro-and microheterogeneity. To monitor the role of N-glycosylation in its activity, hypo-and hyperglycosylated nuclease mutants, R-TBN1 digested by alpha-mannosidase, and R-TBN1 deglycosylated by PNGase F were prepared. Deglycosylated R-TBN1 and mutant N94D/N112D were virtually inactive. Compared to R-TBN1 wt, both N94D and N112D mutants showed about 60% and 10% of the activity, respectively, while the N186D, D36S, and D36S/E104 N mutants were equally or even more active than R-TBN1 wt. The partial demannosylation of R-TBN1 did not affect the nuclease aktivity, moreover, a little shift in substrate specificity was observed. The results show two facts: 1) which sites must be occupied by a glycan for the proper folding and stability and 2) how N. benthamiana glycosylates the foreign nuclease. At the same time, the modifications can be interesting in designing the nuclease activity or specificity through its glycosylation.

Druh

J_imp - Článek v periodiku v databázi Web of Science

CEP obor

—

OECD FORD obor

10608 - Biochemistry and molecular biology

Projekt

Výsledek vznikl pri realizaci vícero projektů. Více informací v záložce Projekty.

Návaznosti

P - Projekt vyzkumu a vyvoje financovany z verejnych zdroju (s odkazem do CEP)

Rok uplatnění

2018

Kód důvěrnosti údajů

S - Úplné a pravdivé údaje o projektu nepodléhají ochraně podle zvláštních právních předpisů

Název periodika

Plant Science

ISSN

0168-9452

e-ISSN

—

Svazek periodika

276

Číslo periodika v rámci svazku

NOV 2018

Stát vydavatele periodika

IE - Irsko

Počet stran výsledku

10

Strana od-do

152-161

Kód UT WoS článku

000449310500015

EID výsledku v databázi Scopus

2-s2.0-85052477021