Complementation of an Arabidopsis thaliana mutant thatlacks complex asparagine-linked glycans with the human cDNA encodingN-acetylglucosaminyltransferase I.

Abstract

N-Acetylglucosaminyltransferase I (EC 2.4.1.101) initiates the conversion ofhigh-mannose asparaginelinked glycans to complex asparagne-linked glycans in plant as well as in animal cells. This Golgi enzyme is missing in the cgI mutant ofArabidopsis thalrna, and the mutant cells are unable to synthesize complex glycans. Transformation ofcells from the mutant plants with the cDNA encoding human N-acetylglucosaminyltransferase I restores the wild-type phenotype of the plant cells. Fractionation of the subcellular organelles on isopycnic sucrose gradients shows that the human enzyme in the complemented cells bands at the same density, 1.14 g/cm3, typical of Golgi cisternae, as the enzyme in the wild-type plant cells. These results demonstrate that complementation results from the presence of the human enzyme in the plant Golgi apparatus, where it is functionally integrated into the biosynthetic machinery of the plant cell. In addition, given the evolutionary distance between plants and mammals and the great diversity of glycoproteins that are modified in each, there is probably no specific recognition between this Golgi enzyme and the polypeptide domains of the proteins it modifies.

Protein glycosylation in development and disease

“…That glycosylation occurs in the ER-Golgi complex, an organelle dedicated to the production of cell surface and secreted materials, is consistent with this notion. However, plant and animal kingdoms have diverged in their exploitation of GlcNAc-TI-dependent structures, as suggested by the observation that GlcNAc-TI is dispensable for plant growth (9) but required for embryogenesis in mice. (10,11) Glycans are also a common means of adhesion and immune recognition between multicellular organisms and parasitic or symbiotic organisms.…”

Isolation and Characterization of Plant N-Acetyl Glucosaminyltransferase I (GntI) cDNA Sequences. Functional Analyses in the Arabidopsis cgl Mutant and in Antisense Plants

“…Complementation of the complex glycan defect in the Arabidopsis cgl mutant by a cDNA coding for human GnTI has been reported previously (Gomez and Chrispeels, 1994). To check for functionality of the isolated cDNA sequences prior to generating transgenic plants, cauliflower mosaic virus (CaMV)-35S promoter-driven potato and tobacco GntIexpression cassettes were constructed (Fig.…”

Plantation Forestry under Global Warming: Hybrid Poplars with Improved Thermotolerance Provide New Insights on the in Vivo Function of Small Heat Shock Protein Chaperones

“…To overexpress chestnut (Castanea sativa) CsHSP17.5 in poplar, a plant expression cassette was constructed as described by Gomez and Chrispeels (1994). The coding sequence for CsHSP17.5 was obtained via PCR as a 670-pb EcoRI (blunted)-SalI fragment from the corresponding complementary DNA (Soto et al, 1999; EMBL accession no.…”