Studies on synthetic pathway of xylose‐containing N‐linked oligosaccharides deduced from substrate specificities of the processing enzymes in sycamore cells (Acer pseudoplatanus L.)

Abstract

We measured the activities of a-I ,3-mannosyl-glycoprotein ~-l,2-N-acetylglucosaminyltransferase, a-1,6-mannosyl-glycoprotein p-1,2-N-acetylglucosaminyltransferase, ~-3,4-mannosyl-glycoprotein p-1,2-xylosyltransferase and glycoprotein 3-a-~-fucosyltransferase in the Golgi fraction of suspension-cultured cells of sycamore (Acer pseudoplatanus L.) using fluorescence-labelled oligosaccharides as acceptor substrates for these transferase reactions. The structures of the pyridylaminated oligosaccharides produced by these reactions were analyzed by two-dimensional sugar mapping using high-performance liquid chromatography. We demonstrated that The biosynthesis of oligosaccharides in animal cells has been well characterized [l]. This pathway starts from the transfer of Glc3Man9GlcNAc, to nascent polypeptide chains in the endoplasmic reticulum. The processing of Glc3Man9GlcNAcz begins with the removal of three glucose and four mannose residues by sequential reactions of a-glucosidases and aCorrespondence to N . Takahashi, Nagoya City University, College of Nursing, Mizuho-ku, Nagoya, Japan 467.Abbreviations. GlcNAc transferase I, a-I ,3-mannosyl-glycoprotcin ~-1,2-N-acetyIglucosaminyltransferase; GlcNAc transferase 11, a-1,6-mannosyl-glycoprotein ~-1,2-N-acetylglucosaminyltransferase; xylosyl transferase, ~-1,4-mannosyl-glycoprotein B-1,2-xylosyltransferase; fucosyl transferase, glycoprotein 3-a-~-fucosyltransferase; Fuc, fucose; UDP-Xyl, uridine(5')diphospho(l)-/?-xylose; GDP-Fuc, guanosine(5')diphospho-a-L-fucose.Enzymes. a-l,3-Mannosyl-glycoprotein ~-1,2-N-acetylglucosami- mannosidases. The product of these reactions, Man5-GlcNAc,, is the precursor of all N-acetyllactosamine-type oligosaccharides [2]. It is the substrate of GlcNAc transferase I which produces the first N-acetyllactosamine-type intermediate, GlcNAcMan,GlcNAcz. Various N-acetyllactosamine-type oligosaccharides found in animal glycoproteins are produced from GlcNAcMan5GlcNAcz after further modification by a-mannosidase 11, GlcNAc transferase, fucosyl transferase, galactosyl transferase and sialyl transferase. The structures of many oligosaccharides in plant glycoproteins have been elucidated during the last decade [3 -141. These works have revealed that xylose-containing oligosaccharides are widely distributed throughout the plant kingdom, although, xylose-containing N-linked oligosaccharides were also found in glycoproteins from gastropods [15, 161 and Chlorophyceae [17]. The biosynthesis of the oligosaccharide chains in plant glycoproteins has been recently investigatedThe biosynthesis of N-linked oligosaccharide in rice xamylase containing GlcNAc,Man3XylGlcNAcz has been par-

In the Biosynthesis of N‐Glycans in Connective Tissue of the Snail Lymnaea Stagnalis of Incorporation GlcNAc by β2GlcNAc‐transferase I is an essential prerequisite for the action of β2GlcNAc‐transferase II and β2Xyl‐transferase

“…The bean cotyledon xylosyltransferase acts on glycopeptide acceptors having a terminal Bl-Zlinked GlcNAc residue on the Manal-3-arm and it was suggested that xylose in vivo is introduced after the action of both DGlcNAc-T I and a-mannosidase I1 since GlcNAcMan,GlcNAc,Asn was a poor xylose acceptor [lo]. For sycamore cell DXyl-T similar results have been reported, and this DXyl-T also required acceptors having the structural element GlcNAcpl-2Manal-3Man/3-R [26]. The substrate specificities of both of these plant p2Xyl-Ts are similar to that of the snail connective tissue DXyl-T.…”

“…This is the first report of an animal xylosyltransferase acting on N-linked oligosaccharide chains. N-glycans containing xylose are common in the plant kingdom and plant xylosyltransferases involved in the biosynthesis of these glycoproteins have been described for Phaseolus vulgaris cotyledons [lo] and sycamore cells of Acer pseudoplatanus [26]. The bean cotyledon xylosyltransferase acts on glycopeptide acceptors having a terminal Bl-Zlinked GlcNAc residue on the Manal-3-arm and it was suggested that xylose in vivo is introduced after the action of both DGlcNAc-T I and a-mannosidase I1 since GlcNAcMan,GlcNAc,Asn was a poor xylose acceptor [lo].…”

Distribution of xylosylation and fucosylation in the plant Golgi apparatus

“…According to the present results that clearly indicate in which Golgi compartment most PI +2 xylose and a1 +3 fucose residues are added to plant complex glycans and considering what has already been published on N-glycosylation in rice (Hayashi et al, 1990), bean (Sturm et a/., 1987) and sycamore (Tezuka et al, 1992), we propose a model for plant N-linked glycan processing in the Golgi apparatus. After a trimming of three glucoses and possibly one mannose residue in the ER, the glycoprotein is transported through the cis and the medial cisternae of the Golgi apparatus where the glycan can progressively be further matured to a G I C N A~~M~~, ( G I C N A C )~ structure.…”

Structural requirements for Arabidopsisβ1,2‐xylosyltransferase activity and targeting to the Golgi

“…Furthermore, AtXylT activity was strongly decreased in the absence of MnCl 2 , or after addition of EDTA in the assay. Therefore, AtXylT has properties similar to most other xylosyltransferases (Johnson and Chrispeels, 1987;Tezuka et al, 1992) or glycosyltransferases characterized to date, except for soybean XylT which does not appear to require divalent cations (Zeng et al, 1997).…”

“…In plants, substrate specificities have been analyzed for most glycosyltransferases responsible for N-glycan maturation (Johnson and Chrispeels, 1987;Tezuka et al, 1992) and a distribution of the most typical plant glycosyltransferases in the Golgi was proposed from an indirect approach based on immunolocalization of their products. For instance, using antibodies against the main glycoepitopes present in plant N-glycans we have shown that b1,2-xylose is added mainly in the medial Golgi cisternae, a1,3-fucosylation occurs mostly in the trans Golgi (Fitchette et al 1994), while the plant Lewis a antennae biosynthesis is a late Golgi event occurring in the trans Golgi and trans Golgi network (TGN) (Fitchette et al, 1999).…”