Purification and characterization of two recombinant human glucuronyltransferases involved in the biosynthesis of HNK-1 carbohydrate in Escherichia coli.
Two glucuronyltransferases (GlcAT-P and GlcAT-S) are involved in the biosynthesis of HNK-1 carbohydrate, which is spatially and temporally regulated in the nervous system. To clarify the enzymatic properties of the respective glucuronyltransferases, we established an expression system for producing large amounts of soluble forms of flag-tagged human GlcAT-P and GlcAT-S in Escherichia coli. Approximately 15 and 6 mg of enzymatically active flag-GlcAT-P and flag-GlcAT-S were purified from E. coli cells in 5 liters of culture medium, respectively. These recombinant enzymes transferred GlcA to a glycoprotein acceptor, asialo-orosomucoid (ASOR), as well as a glycolipid acceptor, paragloboside. The specific activity of the recombinant GlcAT-P (1100 nmol/min/mg) toward a glycoprotein acceptor, ASOR, was comparable to that of the enzyme (4300 nmol/min/mg) purified from rat brain. Phosphatidylinositol (PI) is specifically required for expression of the activity of the recombinant enzymes toward a glycolipid acceptor, paragloboside. The recombinant GlcAT-P was highly specific for the terminal type II structure, Galbeta1-4GlcNAc, while the recombinant GlcAT-S recognized not only the type II structure, Galbeta1-4GlcNAc, but also the type I structure, Galbeta1-3GlcNAc. These acceptor specificities were similar to those of the native enzymes.
Kakuda
Shinako
S
Department of Biological Chemistry, Kyoto University, Kyoto 606-8501, Japan.
Oka
Shogo
S
Kawasaki
Toshisuke
T
D000818:Animals; D001212:Asialoglycoproteins / Q000378:metabolism; D018959:CD57 Antigens / Q000096:biosynthesis*; D004926:Escherichia coli / Q000378:metabolism*; D020723:Glucuronic Acid / Q000378:metabolism; D014453:Glucuronosyltransferase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D006017:Glycolipids / Q000378:metabolism; D006801:Humans; D007141:Immunoglobulin Fc Fragments / Q000235:genetics / Q000378:metabolism; D019006:Neural Cell Adhesion Molecules / Q000235:genetics / Q000378:metabolism; D009961:Orosomucoid / Q000031:analogs & derivatives* / Q000378:metabolism; D010743:Phospholipids / Q000378:metabolism; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D013379:Substrate Specificity; D020349:Surface Plasmon Resonance
D001212:Asialoglycoproteins; D018959:CD57 Antigens; D006017:Glycolipids; D007141:Immunoglobulin Fc Fragments; D019006:Neural Cell Adhesion Molecules; D009961:Orosomucoid; D010743:Phospholipids; D011993:Recombinant Fusion Proteins; C017030:asialoorosomucoid; D020723:Glucuronic Acid; C475472:B3GAT2 protein, human; C111364:galactosylgalactoylxylosylprotein 3-beta-glucuronosyltransferase; D014453:Glucuronosyltransferase