Molecular Basis of N-acetylglucosaminyltransferase I Deficiency in Arabidopsis Thaliana Plants Lacking Complex N-glycans

Overview

Journal Biochem J

Specialty Biochemistry

Date 2004 Nov 13

PMID 15537386

Citations 59

Authors

Richard Strasser

Johannes Stadlmann

Barbara Svoboda

Friedrich Altmann

Josef Glossl

Lukas Mach

Affiliations

Soon will be listed here.

Abstract

GnTI (N-acetylglucosaminyltransferase I) is a Golgi-resident enzyme essential for the processing of high-mannose to hybrid and complex N-glycans. The Arabidopsis thaliana cgl mutant lacks GnTI activity and as a consequence accumulates oligomannosidic structures. Molecular cloning of cgl GnTI cDNA revealed a point mutation, which causes a critical amino acid substitution (Asp144-->Asn), thereby creating an additional N-glycosylation site. Heterologous expression of cgl GnTI in insect cells confirmed its lack of activity and the use of the N-glycosylation site. Remarkably, introduction of the Asp144-->Asn mutation into rabbit GnTI, which does not result in the formation of a new N-glycosylation site, led to a protein with strongly reduced, but still detectable enzymic activity. Expression of Asn144 rabbit GnTI in cgl plants could partially restore complex N-glycan formation. These results indicate that the complete deficiency of GnTI activity in cgl plants is mainly due to the additional N-glycan, which appears to interfere with the proper folding of the enzyme.

Citing Articles

Engineering for production of active cannabinoid synthases via secretory pathway optimization.

Bolanos-Martinez O, Urbanetz A, Maresch D, Strasser R, Vimolmangkang S Biotechnol Rep (Amst). 2024; 45:e00865.

PMID: 39691101 PMC: 11647631. DOI: 10.1016/j.btre.2024.e00865.

Elucidation of the late steps in the glycan-dependent ERAD of soluble misfolded glycoproteins.

Schoberer J, Vavra U, Shin Y, Grunwald-Gruber C, Strasser R Plant J. 2024; 121(1):e17185.

PMID: 39642157 PMC: 11712024. DOI: 10.1111/tpj.17185.

Hexosamine biosynthesis and related pathways, protein N-glycosylation and O-GlcNAcylation: their interconnection and role in plants.

Chen Y, Cheng W Front Plant Sci. 2024; 15:1349064.

PMID: 38510444 PMC: 10951099. DOI: 10.3389/fpls.2024.1349064.

The tobacco GNTI stem region harbors a strong motif for homomeric protein complex formation.

Schoberer J, Izadi S, Kierein C, Vavra U, Konig-Beihammer J, Ruocco V Front Plant Sci. 2023; 14:1320051.

PMID: 38089803 PMC: 10715278. DOI: 10.3389/fpls.2023.1320051.

Impact of mutations on the plant-based production of recombinant SARS-CoV-2 RBDs.

Ruocco V, Vavra U, Konig-Beihammer J, Bolanos Martinez O, Kallolimath S, Maresch D Front Plant Sci. 2023; 14:1275228.

PMID: 37868317 PMC: 10588190. DOI: 10.3389/fpls.2023.1275228.

References

Boyes D, Zayed A, Ascenzi R, McCaskill A, Hoffman N, Davis K . Growth stage-based phenotypic analysis of Arabidopsis: a model for high throughput functional genomics in plants. Plant Cell. 2001; 13(7):1499-510. PMC: 139543. DOI: 10.1105/tpc.010011. View

Hamilton C . A binary-BAC system for plant transformation with high-molecular-weight DNA. Gene. 1997; 200(1-2):107-16. DOI: 10.1016/s0378-1119(97)00388-0. View

Bakker H, Lommen A, Jordi W, Stiekema W, Bosch D . An Arabidopsis thaliana cDNA complements the N-acetylglucosaminyltransferase I deficiency of CHO Lec1 cells. Biochem Biophys Res Commun. 1999; 261(3):829-32. DOI: 10.1006/bbrc.1999.1117. View

Strasser R, Altmann F, Mach L, Glossl J, Steinkellner H . Generation of Arabidopsis thaliana plants with complex N-glycans lacking beta1,2-linked xylose and core alpha1,3-linked fucose. FEBS Lett. 2004; 561(1-3):132-6. DOI: 10.1016/S0014-5793(04)00150-4. View

Chen W, Unligil U, Rini J, Stanley P . Independent Lec1A CHO glycosylation mutants arise from point mutations in N-acetylglucosaminyltransferase I that reduce affinity for both substrates. Molecular consequences based on the crystal structure of GlcNAc-TI. Biochemistry. 2001; 40(30):8765-72. DOI: 10.1021/bi015538b. View

Ioffe E, Stanley P . Mice lacking N-acetylglucosaminyltransferase I activity die at mid-gestation, revealing an essential role for complex or hybrid N-linked carbohydrates. Proc Natl Acad Sci U S A. 1994; 91(2):728-32. PMC: 43022. DOI: 10.1073/pnas.91.2.728. View

von Schaewen A, Sturm A, ONeill J, Chrispeels M . Isolation of a mutant Arabidopsis plant that lacks N-acetyl glucosaminyl transferase I and is unable to synthesize Golgi-modified complex N-linked glycans. Plant Physiol. 1993; 102(4):1109-18. PMC: 158895. DOI: 10.1104/pp.102.4.1109. View

Clough S, Bent A . Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J. 1999; 16(6):735-43. DOI: 10.1046/j.1365-313x.1998.00343.x. View

Zhu S, Hanneman A, Reinhold V, Spence A, Schachter H . Caenorhabditis elegans triple null mutant lacking UDP-N-acetyl-D-glucosamine:alpha-3-D-mannoside beta1,2-N-acetylglucosaminyltransferase I. Biochem J. 2004; 382(Pt 3):995-1001. PMC: 1133976. DOI: 10.1042/BJ20040793. View

10.

Mucha J, Svoboda B, Frohwein U, Strasser R, Mischinger M, Schwihla H . Tissues of the clawed frog Xenopus laevis contain two closely related forms of UDP-GlcNAc:alpha3-D-mannoside beta-1,2-N-acetylglucosaminyltransferase I. Glycobiology. 2001; 11(9):769-78. DOI: 10.1093/glycob/11.9.769. View

11.

Mitterbauer R, Poppenberger B, Raditschnig A, Lucyshyn D, Lemmens M, Glossl J . Toxin-dependent utilization of engineered ribosomal protein L3 limits trichothecene resistance in transgenic plants. Plant Biotechnol J. 2006; 2(4):329-40. DOI: 10.1111/j.1467-7652.2004.00075.x. View

12.

Stanley P, Chaney W . Control of carbohydrate processing: the lec1A CHO mutation results in partial loss of N-acetylglucosaminyltransferase I activity. Mol Cell Biol. 1985; 5(6):1204-11. PMC: 366847. DOI: 10.1128/mcb.5.6.1204-1211.1985. View

13.

Strasser R, Altmann F, Glossl J, Steinkellner H . Unaltered complex N-glycan profiles in Nicotiana benthamiana despite drastic reduction of beta1,2- N -acetylglucosaminyltransferase I activity. Glycoconj J. 2004; 21(5):275-82. DOI: 10.1023/B:GLYC.0000045099.29038.04. View

14.

Wilson I, Zeleny R, Kolarich D, Staudacher E, Stroop C, Kamerling J . Analysis of Asn-linked glycans from vegetable foodstuffs: widespread occurrence of Lewis a, core alpha1,3-linked fucose and xylose substitutions. Glycobiology. 2001; 11(4):261-74. DOI: 10.1093/glycob/11.4.261. View

15.

Lerouge P, Rayon C, Gomord V, Faye L . N-glycoprotein biosynthesis in plants: recent developments and future trends. Plant Mol Biol. 1998; 38(1-2):31-48. View

16.

Metzler M, Gertz A, Sarkar M, SCHACHTER H, Schrader J, Marth J . Complex asparagine-linked oligosaccharides are required for morphogenic events during post-implantation development. EMBO J. 1994; 13(9):2056-65. PMC: 395055. DOI: 10.1002/j.1460-2075.1994.tb06480.x. View

17.

Gomez L, Chrispeels M . Complementation of an Arabidopsis thaliana mutant that lacks complex asparagine-linked glycans with the human cDNA encoding N-acetylglucosaminyltransferase I. Proc Natl Acad Sci U S A. 1994; 91(5):1829-33. PMC: 43257. DOI: 10.1073/pnas.91.5.1829. View

18.

KORNFELD R, Kornfeld S . Assembly of asparagine-linked oligosaccharides. Annu Rev Biochem. 1985; 54:631-64. DOI: 10.1146/annurev.bi.54.070185.003215. View

19.

Horton R, Cai Z, Ho S, Pease L . Gene splicing by overlap extension: tailor-made genes using the polymerase chain reaction. Biotechniques. 1990; 8(5):528-35. View

20.

Nishikawa Y, Pegg W, Paulsen H, SCHACHTER H . Control of glycoprotein synthesis. Purification and characterization of rabbit liver UDP-N-acetylglucosamine:alpha-3-D-mannoside beta-1,2-N-acetylglucosaminyltransferase I. J Biol Chem. 1988; 263(17):8270-81. View