Dictyostelium cytosolic fucosyltransferase synthesizes H type 1 trisaccharide in vitro

Abstract

A fucosyltransferase activity has been detected using lacto-N-biose I as acceptor in the lower eukaryote Dictyostelium 2 1. Materials discoideum. This transferase requires divalent cations and is Lacto-N-biose I, LacNAc, GlcNAc, GDP-~-L-fucose, L-Fuc, inhibited by N-ethylmaleimide and detergent treatment. Appar- calculated Km values for GDP-Fuc and lacto-N-biose I are OMe, antipain shaker in axenic medium [6]. At a culture density of 5-8 × 106 cells/ ml, cells were collected by pelleting at 1300 rpm for 4 min, at 4°C. Cell pellet was resuspended with 0.25 M sucrose-TKM buffer (50 mM Tris, 1. Introduction 25 mM KCI, 5 mM MgC12, pH 8.0) by gently pipetting, and spun as above; washing procedure was repeated three times. It was then resuspended to a cell density of 0.5x109 cells/ml in 0.25 M sucroseGlycosyltransferases have been mostly reported as mem-TKM buffer containing protease inhibitors to a final concentration of brane-bound enzymes involved in the biosynthesis of the sac-20 ~tg/ml each antipain, chymostatin, leupeptin, and pepstatin, 0.2 charide chains of glycoproteins and glycolipids [1]. Glycosyl-TIU aprotinin, 37 p.g/ml TLCK, and 1 mM PMSF. The cell suspension (4-6 ml in typical experiments) was lysed by forced passage transferases found in biological fluids reach the extracellular through a polycarbonate filter with a 3 ~tm nominal pore diameter compartment from the Golgi lumen through the secretory (Nucleopore, 25 mm filter diameter), and the lysate obtained was pathway, and proteolytic cleavage eventually makes them referred to as the homogenate. It was spun at 2000×g for 6 min, at rid of the membrane anchor. On the other hand, few cytosolic 4°C, obtaining a supernatant referred to as the postnuclear fraction. glycosyltransferases have recently been demonstrated [2]. Very This fraction was centrifuged at 100000×g for 1 h at 4°C, obtaining a pellet and a supernatant. The latter was referred to as the cytosolic recently, a fucosyltransferase has been purified from the cytofraction; the pellet was resuspended to a protein concentration of 10 sol of the slime mold Dictyostelium discoideum [3]. The en-30 mg/ml with 0.25 M sucrose-TKM buffer containing the same prozyme was originally discovered by its ability to fucosylate tease inhibitors as in the filtration buffer, and referred to as the memthe FP21 glycoprotein [4], that was proved to be mostly cytobrane fraction. Protein content was determined by the procedure of Lowry et al. [7]. COS-7 cells were cultured in Dulbecco's modified solic [5]. The enzyme was also found to use lipid-bound acEagle medium supplemented with 10% fetal bovine serum, 100 U/ml ceptors in vitro, provided they contain a terminal Gal~l-penicillin, 100 p.g/ml streptomycin, and 2 mM L-GIu. 3GIcNAc disaccharide sequence [3,4]. To date, it is not known whether the enzyme utilizes simple sugar acceptors, as do Enzyme assaysmammalian fucosyltransferases, and there is no direct eviDcd,2FucT was determined in a reaction mixture containing, in a final volume of 20 ~1, 0.1 M Tris-HC1 ...

β‐1,3‐galactosyltransferase and α‐1,2‐fucosyltransferase involved in the biosynthesis of type‐1‐chain carbohydrate antigens in human colon adenocarcinoma cell lines

“…Fuc-TIII and HA1,2FT were also assayed upon transfection and expression of the pcDNAI-A1,3/1,4FT and pcDNAI-HA1,2FT plasmids, respectively, in COS-7 cells, using the cell homogenate as the enzyme source, as reported [30].…”

“…Quenched reaction mixtures with 3 H-labeled nucleotide sugars from homogenous experiments were thawed, pooled, chromatographed on Dowex columns (saccharide acceptors) or Sep-Pak C-18 cartridges (phenyl β-galactoside acceptor) and lyophilized. Gal-3 H-labeled products obtained with saccharide acceptors were further purified bv Biogel P2 gel filtration as described [30].…”

“…In addition, Biogel P2 fractions containing the product were pooled, lyophilized, and the material resuspended with 10 µl of water and spotted onto a 5 cm line of an HPTLC plate. After developing, the area corresponding to the disaccharide product was scraped off and the silica gel eluted with water [30].…”

“…Almond meal A-fucosidase digestion: 50 mM sodium acetate, pH 5.0, and 0.6 mU/ml enzyme ; incubation was for 70 h at 37°C. Preparation of radiolabeled reference H type 1 and Le a trisaccharides, and HPTLC analysis were performed as reported [30]. Permethylation was done in a 1-ml screw-cap reaction vial (Pierce) using 200 000 cpm of radioactive samples or 500 nmol unlabeled standard saccharides, each dissolved in 50 µl anhydrous dimethylsulfoxide, as previously reported [30].…”

A Non-Golgi α1,2-Fucosyltransferase That Modifies Skp1 in the Cytoplasm of Dictyostelium

“…A Skp1 ␣1,2-FTase activity (EC 2.4.1.69) has been assayed based on the transfer of [ 3 H]Fuc from GDP-[ 3 H]Fuc to pNO 2 -phenyl-Gal␤1,3GlcNAc (pNP-LNB) or to Gal␤1,3GlcNAc-Skp1 isolated from a GDP-Fuc synthesis mutant (9,10). Highly purified preparations of the enzyme are specific for the O-2 position of Gal that is in ␤1,3 linkage to an underlying HexNAc, which may be either GlcNAc or GalNAc, ␣-or ␤-linked to an aglycone.…”