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

Abstract

We report on the isolation and characterization of full-length cDNA sequences coding for N-acetylglucosaminyltransferase I (GnTI) from potato (Solanum tuberosum L.), tobacco (Nicotiana tabacum L.), and Arabidopsis. The deduced polypeptide sequences show highest homology among the solanaceous species (93% identity between potato and tobacco compared with about 75% with Arabidopsis) but share only weak homology with human GnTI (35% identity). In contrast to the corresponding enzymes from animals, all plant GnTI sequences identified are characterized by a much shorter hydrophobic membrane anchor and contain one putative N-glycosylation site that is conserved in potato and tobacco, but differs in Arabidopsis. Southern-blot analyses revealed that GntI behaves as a single-copy gene. Northern-blot analyses showed that GntI-mRNA expression is largely constitutive. Arabidopsis cgl mutants deficient in GnTI activity also possess GntI mRNA, indicating that they result from point mutations. GntI-expression constructs were tested for the ability to relieve the GnTI block in protoplasts of the Arabidopsis cgl mutant and used to obtain transgenic potato and tobacco plants that display a substantial reduction of complex glycan patterns. The latter observation indicates that production of heterologous glycoproteins with little or no antigenic glycans can be achieved in whole plants, and not in just Arabidopsis, using antisense technology.

Dolichol Biosynthesis and Its Effects on the Unfolded Protein Response and Abiotic Stress Resistance in Arabidopsis

“…Genes encoding some of the enzymes functioning at various steps of the protein glycosylation pathway have been characterized. These include, for example, GNTI (for N-ACETYL GLUCOSAMINYLTRANSFER-ASE I, encoded by COMPLEX GLYCAN1 [CGL1]) (von Schaewen et al, 1993;Wenderoth and von Schaewen, 2000;Strasser et al, 2005), GMII (an a-mannosidase II, encoded by HYBRID GLY-COSYLATION1 [HGL1]) (Strasser et al, 2006), STT3a (Koiwa et al, 2003), DGL1 (for DEFECTIVE GLYCOSYLATION1), an Arabidopsis homolog of an oligosaccharyltransferase complex subunit (Lerouxel et al, 2005), CYT1 (for CYTOKINESIS-DEFECTIVE1, mannose-1-phosphate guanylyltransferase) (Lukowitz et al, 2001), GCS1/KNF (glucosidase I), and RSW3 (glucosidase II; glucosidases I and II trim the terminal glucose of N-glycans). Mutations in these genes have some overlapping, yet differential, effects on plant growth, development, and stress responses.…”

RISAP Is a TGN-Associated RAC5 Effector Regulating Membrane Traffic during Polar Cell Growth in Tobacco

“…A cDNA encoding a Golgi-targeted YFP fusion protein (St-GnT1 1-70 :YFP) was obtained by fusing a nucleotide sequence coding for the membrane anchor of a potato (Solanum tuberosum) N-acetylglucosaminyltransferase (Wenderoth and von Schaewen, 2000; amino acids 1 to 70) to the 59 end of the YFP cDNA.…”

“…The ER as a whole is rapidly moving with the cytoplasmic streaming (LovyWheeler et al, 2007). Golgi stacks visualized by YFP attached to the membrane anchor of a potato (Solanum tuberosum) N-acetylglucosaminyltransferase (St-GNT1 1-70 :YFP; Wenderoth and von Schaewen, 2000) are also highly motile. Interestingly, they are evenly distributed throughout most of the cytoplasm but are excluded not only from the CZ but also from a small subapical region of granular cytoplasm into which the ER extends ( Figure 10A, yellow underlining).…”

Reduced Immunogenicity of Arabidopsis hgl1 Mutant N-Glycans Caused by Altered Accessibility of Xylose and core Fucose Epitopes

“…Equal amounts were separated by 10 -12% SDS-PAGE (reducing conditions) and blotted to nitrocellulose prior to reversible Ponceau S staining (0.3% (w/v) in 3% TCA). After blocking with 2% (w/v) nonfat dry milk in Trisbuffered saline containing Tween 20 (TBST; 20 mM Tris, pH 7.4, 150 mM NaCl, 0.1% (v/v) Tween 20), the blots were incubated with crude polyclonal rabbit antisera raised either against PHA-L (␣-PHA-L) (26,34) or against HRP (␣-HRP, purchased from Sigma) (30), diluted 1:10,000 in TBST or 1:20,000 in 2ϫ TBST, respectively, including 2% (w/v) nonfat dry milk as described previously (8) or with affinity-selected fractions thereof. For IgE detection, blot membranes were blocked with 2% nonfat dry milk in TBST for 1 h at room temperature and incubated either with diluted patient sera (1:10 in TBST, 2% (w/v) nonfat dry milk) for 2 h or with undiluted patient sera supplemented with 2% (w/v) nonfat dry milk overnight at room temperature.…”