Glucose Trimming ofN-Glycan in Endoplasmic Reticulum Is Indispensable for the Growth ofRaphanus sativusSeedling (kaiware radish)

Abstract

Recently I found that glycosidase inhibitors such as castanospermine, deoxynojirimycin, swainsonine, 2-acetamindo 2,3-dideoxynojirimycin, and deoxymannojirimycin change the N-glycan structure of root glycoproteins, and that the glucosidase inhibitors castanospermine and deoxynojirimycin suppress the growth of Raphanus sativus seedlings (Mega, T., J. Biochem., 2004). The present study undertook to see whether the growth suppression is due to the inhibition of glucose trimming in endoplasmic reticulum (ER). The study, using three glucosidase inhibitors, castanospermine, Nmethyl deoxynojirimycin, and deoxynojirimycin, upon the growth of R. sativus foliage leaf, made clear that glucose trimming is indispensable for plant growth, because the inhibition of glucose trimming correlated with leaf growth. On the other hand, processing inhibition in the Golgi apparatus by other glycosidase inhibitors had little effect on plant growth, although Nglycan processing was disrupted depending on inhibitor specificity. These results suggest that N-glycan processing after glucosidase processing is dispensable for plant growth and cell differentiation.Key words: castanospermine; deoxynojirimycin; N-glycan; glycosidase inhibitors; Raphanus sativusThe functions of plant N-glycan remain unclarified, 1) although plant glycoproteins, glycosidases, and lectins have been well studied. To identify the functions of enzymes, two approaches are usually taken: the use of mutants lacking the target enzyme activity, and the use of enzyme inhibitors. The method using enzyme inhibitors is easy to apply and enables easy control of the degree of an effect by regulating the amount administered. But we must consider the effects of inhibitors on as yet unidentified enzymes or interactions in living organisms.It has been reported that the glucosidase I mutant of Arabidopsis cannot form seeds, 2) and produces a diminished amount of cellulose.3) The Arabidopsis glucosidase II mutant can grow with a radial swollen root and is heat labile.4) In a previous study, R. sativus seed was germinated in the presence of glycosidase inhibitors, and the effect of these inhibitors on the Nglycan structure in the root was examined. Structural analysis showed that glycosidase inhibitors inhibited N-glycan processing in both the ER and the Golgi apparatus, as shown in Scheme 1. 5) That is, glycosidase inhibitors inhibit the processing glycosidases according to inhibitor specificity. For example, dMNJ (deoxymannojirimycin) let almost all the N-glycans in a plant retain oligomannose-type structures, but, germination of seeds and growth were normal. Glucosidase inhibitors such as CST and DNJ inhibit glucosidase I and/or II of plants, and also plant growth. Usually in cultured cells these inhibitors influence the N-glycan structure but not growth.These glucosidase inhibitors are also known to inhibit sucrase and -glucosidase.6,7) It is reasonable to speculate that they inhibit one or more glucose related enzymes which lead to the growth inhibition, because (a) sucrose has an ...

Plant-TypeN-Glycans Containing Fucose and Xylose in Bryophyta (Mosses) and Tracheophyta (Ferns)

“…Oligosaccharides released in hydrazine were N-acetylated, pyridylaminated, and analyzed by HPLC after purification by phenol-chloroform extraction and Dowex 50 NH 4þ filtration with H 2 O according to the conventional method used in Hase's laboratory, [17][18][19] and as also described in my previous papers. 10,14) SF-HPLC was performed with an Asahipak NH2P-50 column (2:0 Â 150 mm) at a flow rate of 75 ml/min. RP-HPLC was performed with a Cosmosil 5C18-P column (1:5 Â 250 mm) at a flow rate of 150 ml/ min.…”

“…10,14) These results indicate that the final N-glycan structure is not directly associated with plant growth or differentiation. However, M3FX-type N-glycans are supposed to have important unknown functions, because almost all the plants have retained M3FX-type glycans (M3FX, Gn2M3FX, and Le a 2M3FX) as the main N-glycans during evolution.…”

“…13) In my study using glycosidase inhibitors on germination, the seedling and leaf growth of R. sativus showed that ER-glucosidase inhibition inhibited the growth of the plant, although the inhibition of N-glycan processing in Golgi by deoxymannojirimycin, swainsonine, and 1,2-dideoxy-2-acetamido-nojirimysin had no eminent effect except for a change in the N-glycan structure. 10,14) These results indicate that the final N-glycan structure is not directly associated with plant growth or differentiation. However, M3FX-type N-glycans are supposed to have important unknown functions, because almost all the plants have retained M3FX-type glycans (M3FX, Gn2M3FX, and Le a 2M3FX) as the main N-glycans during evolution.…”

“…The M3FX, Gn2M3FX, and Le a 2M3FX amounts were calculated from the area of the peak on RP-HPLC or SF-HPLC. Total oligosaccharide amounts were estimated from the total area of the all peaks that appeared between glucose units 5 and 13 in the SF-HPLC profile, because most of the peaks that appeared in these areas were found to be derived from N-glycans in the previous analyzes of R. sativus (10,14). The yield of the analysis varied greatly (roughly 20-60%), although the relative area of each peak was constant.…”

“…1 is assumed to be almost universal. 5,6) N-Glycan processing byglucosidases I and II in ER is important in plants, [7][8][9][10] whereas N-glycan processing in the Golgi apparatus has little effect on plant growth, as determined in the varous studies: Arabidopsis mutants that lack N-acetylglucosaminyltransferase I 11) or both fucosyltrasferase and xylosyltransferase activities 12) grow normally and produce normal seeds. Fucosyltransferase and xylosyltransferase knockout plants of Bryopsida (Physcomitrella patens) grow normally.…”