A Pipeline towards the Biochemical Characterization of the <i>Arabidopsis</i> GT14 Family
Glycosyltransferases (GTs) catalyze the synthesis of glycosidic linkages and are essential in the biosynthesis of glycans, glycoconjugates (glycolipids and glycoproteins), and glycosides. Plant genomes generally encode many more GTs than animal genomes due to the synthesis of a cell wall and a wide...
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| Format: | Article |
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MDPI AG
2021-01-01
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| Series: | International Journal of Molecular Sciences |
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| Online Access: | https://www.mdpi.com/1422-0067/22/3/1360 |
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| author | Lingling Xuan Jie Zhang Weitai Lu Pawel Gluza Berit Ebert Toshihisa Kotake Mengzhu Lu Yuan Zhang Mads H. Clausen Kim L. Johnson Monika S. Doblin Joshua L. Heazlewood Antony Bacic Lili Song Wei Zeng |
| author_facet | Lingling Xuan Jie Zhang Weitai Lu Pawel Gluza Berit Ebert Toshihisa Kotake Mengzhu Lu Yuan Zhang Mads H. Clausen Kim L. Johnson Monika S. Doblin Joshua L. Heazlewood Antony Bacic Lili Song Wei Zeng |
| author_sort | Lingling Xuan |
| collection | DOAJ |
| description | Glycosyltransferases (GTs) catalyze the synthesis of glycosidic linkages and are essential in the biosynthesis of glycans, glycoconjugates (glycolipids and glycoproteins), and glycosides. Plant genomes generally encode many more GTs than animal genomes due to the synthesis of a cell wall and a wide variety of glycosylated secondary metabolites. The <i>Arabidopsis thaliana</i> genome is predicted to encode over 573 GTs that are currently classified into 42 diverse families. The biochemical functions of most of these GTs are still unknown. In this study, we updated the JBEI <i>Arabidopsis</i> GT clone collection by cloning an additional 105 GT cDNAs, 508 in total (89%), into Gateway-compatible vectors for downstream characterization. We further established a functional analysis pipeline using transient expression in tobacco (<i>Nicotiana benthamiana</i>) followed by enzymatic assays, fractionation of enzymatic products by reversed-phase HPLC (RP-HPLC) and characterization by mass spectrometry (MS). Using the GT14 family as an exemplar, we outline a strategy for identifying effective substrates of GT enzymes. By addition of UDP-GlcA as donor and the synthetic acceptors galactose-nitrobenzodiazole (Gal-NBD), β-1,6-galactotetraose (β-1,6-Gal<sub>4</sub>) and β-1,3-galactopentose (β-1,3-Gal<sub>5</sub>) to microsomes expressing individual GT14 enzymes, we verified the β-glucuronosyltransferase (GlcAT) activity of three members of this family (AtGlcAT14A, B, and E). In addition, a new family member (AT4G27480, 248) was shown to possess significantly higher activity than other GT14 enzymes. Our data indicate a likely role in arabinogalactan-protein (AGP) biosynthesis for these GT14 members. Together, the updated <i>Arabidopsis</i> GT clone collection and the biochemical analysis pipeline present an efficient means to identify and characterize novel GT catalytic activities. |
| first_indexed | 2024-03-09T03:18:24Z |
| format | Article |
| id | doaj.art-ea818f14580a446d86bb745c810de5e0 |
| institution | Directory Open Access Journal |
| issn | 1661-6596 1422-0067 |
| language | English |
| last_indexed | 2024-03-09T03:18:24Z |
| publishDate | 2021-01-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | International Journal of Molecular Sciences |
| spelling | doaj.art-ea818f14580a446d86bb745c810de5e02023-12-03T15:15:22ZengMDPI AGInternational Journal of Molecular Sciences1661-65961422-00672021-01-01223136010.3390/ijms22031360A Pipeline towards the Biochemical Characterization of the <i>Arabidopsis</i> GT14 FamilyLingling Xuan0Jie Zhang1Weitai Lu2Pawel Gluza3Berit Ebert4Toshihisa Kotake5Mengzhu Lu6Yuan Zhang7Mads H. Clausen8Kim L. Johnson9Monika S. Doblin10Joshua L. Heazlewood11Antony Bacic12Lili Song13Wei Zeng14Sino-Australia Plant Cell Wall Research Centre, The State Key Laboratory of Subtropical Silviculture, College of Forestry and Biotechnology, Zhejiang A & F University, Lin’an 311300, ChinaSino-Australia Plant Cell Wall Research Centre, The State Key Laboratory of Subtropical Silviculture, College of Forestry and Biotechnology, Zhejiang A & F University, Lin’an 311300, ChinaSino-Australia Plant Cell Wall Research Centre, The State Key Laboratory of Subtropical Silviculture, College of Forestry and Biotechnology, Zhejiang A & F University, Lin’an 311300, ChinaSchool of BioSciences, The University of Melbourne, Parkville, VIC 3010, AustraliaSchool of BioSciences, The University of Melbourne, Parkville, VIC 3010, AustraliaDivision of Life Science, Saitama University, Saitama 338-8642, JapanSino-Australia Plant Cell Wall Research Centre, The State Key Laboratory of Subtropical Silviculture, College of Forestry and Biotechnology, Zhejiang A & F University, Lin’an 311300, ChinaSino-Australia Plant Cell Wall Research Centre, The State Key Laboratory of Subtropical Silviculture, College of Forestry and Biotechnology, Zhejiang A & F University, Lin’an 311300, ChinaCenter for Nanomedicine and Theranostics, Department of Chemistry, Technical University of Denmark, DK-2800 Kgs. Lyngby, DenmarkSino-Australia Plant Cell Wall Research Centre, The State Key Laboratory of Subtropical Silviculture, College of Forestry and Biotechnology, Zhejiang A & F University, Lin’an 311300, ChinaSino-Australia Plant Cell Wall Research Centre, The State Key Laboratory of Subtropical Silviculture, College of Forestry and Biotechnology, Zhejiang A & F University, Lin’an 311300, ChinaSchool of BioSciences, The University of Melbourne, Parkville, VIC 3010, AustraliaSino-Australia Plant Cell Wall Research Centre, The State Key Laboratory of Subtropical Silviculture, College of Forestry and Biotechnology, Zhejiang A & F University, Lin’an 311300, ChinaSino-Australia Plant Cell Wall Research Centre, The State Key Laboratory of Subtropical Silviculture, College of Forestry and Biotechnology, Zhejiang A & F University, Lin’an 311300, ChinaSino-Australia Plant Cell Wall Research Centre, The State Key Laboratory of Subtropical Silviculture, College of Forestry and Biotechnology, Zhejiang A & F University, Lin’an 311300, ChinaGlycosyltransferases (GTs) catalyze the synthesis of glycosidic linkages and are essential in the biosynthesis of glycans, glycoconjugates (glycolipids and glycoproteins), and glycosides. Plant genomes generally encode many more GTs than animal genomes due to the synthesis of a cell wall and a wide variety of glycosylated secondary metabolites. The <i>Arabidopsis thaliana</i> genome is predicted to encode over 573 GTs that are currently classified into 42 diverse families. The biochemical functions of most of these GTs are still unknown. In this study, we updated the JBEI <i>Arabidopsis</i> GT clone collection by cloning an additional 105 GT cDNAs, 508 in total (89%), into Gateway-compatible vectors for downstream characterization. We further established a functional analysis pipeline using transient expression in tobacco (<i>Nicotiana benthamiana</i>) followed by enzymatic assays, fractionation of enzymatic products by reversed-phase HPLC (RP-HPLC) and characterization by mass spectrometry (MS). Using the GT14 family as an exemplar, we outline a strategy for identifying effective substrates of GT enzymes. By addition of UDP-GlcA as donor and the synthetic acceptors galactose-nitrobenzodiazole (Gal-NBD), β-1,6-galactotetraose (β-1,6-Gal<sub>4</sub>) and β-1,3-galactopentose (β-1,3-Gal<sub>5</sub>) to microsomes expressing individual GT14 enzymes, we verified the β-glucuronosyltransferase (GlcAT) activity of three members of this family (AtGlcAT14A, B, and E). In addition, a new family member (AT4G27480, 248) was shown to possess significantly higher activity than other GT14 enzymes. Our data indicate a likely role in arabinogalactan-protein (AGP) biosynthesis for these GT14 members. Together, the updated <i>Arabidopsis</i> GT clone collection and the biochemical analysis pipeline present an efficient means to identify and characterize novel GT catalytic activities.https://www.mdpi.com/1422-0067/22/3/1360glycosyltransferase<i>Arabidopsis</i>plant cell wallglycosylationAGPCAZy |
| spellingShingle | Lingling Xuan Jie Zhang Weitai Lu Pawel Gluza Berit Ebert Toshihisa Kotake Mengzhu Lu Yuan Zhang Mads H. Clausen Kim L. Johnson Monika S. Doblin Joshua L. Heazlewood Antony Bacic Lili Song Wei Zeng A Pipeline towards the Biochemical Characterization of the <i>Arabidopsis</i> GT14 Family International Journal of Molecular Sciences glycosyltransferase <i>Arabidopsis</i> plant cell wall glycosylation AGP CAZy |
| title | A Pipeline towards the Biochemical Characterization of the <i>Arabidopsis</i> GT14 Family |
| title_full | A Pipeline towards the Biochemical Characterization of the <i>Arabidopsis</i> GT14 Family |
| title_fullStr | A Pipeline towards the Biochemical Characterization of the <i>Arabidopsis</i> GT14 Family |
| title_full_unstemmed | A Pipeline towards the Biochemical Characterization of the <i>Arabidopsis</i> GT14 Family |
| title_short | A Pipeline towards the Biochemical Characterization of the <i>Arabidopsis</i> GT14 Family |
| title_sort | pipeline towards the biochemical characterization of the i arabidopsis i gt14 family |
| topic | glycosyltransferase <i>Arabidopsis</i> plant cell wall glycosylation AGP CAZy |
| url | https://www.mdpi.com/1422-0067/22/3/1360 |
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