Purification and characterization of a soluble form of the recombinant human galactose-beta1,3-glucuronosyltransferase I expressed in the yeast Pichia pastoris.
The galactose-beta1,3-glucuronosyltransferase I (GlcAT-I) catalyzes the transfer of glucuronic acid from UDP-alpha-D-glucuronic acid onto the terminal galactose of the trisaccharide glycosaminoglycan-protein linker region of proteoglycans. This enzyme plays a key role in the process of proteoglycan assembly since the completion of the linkage region is essential for the conversion of a core protein into a functional proteoglycan. To investigate the enzymatic properties of human GlcAT-I, we established an expression system for producing a soluble form of enzyme in the methylotrophic yeast Pichia pastoris and developed a three-step purification procedure using a combination of anion exchange, cation exchange and heparin chromatographies. This procedure yielded 1.6 mg homogeneous enzyme from 200 ml yeast cell culture, with a specific activity value of 1.5 micromol/min/mg protein. Analysis of the specificity of GlcAT-I towards Galbeta1-3Gal and Galbeta1-4GlcNAc derivatives known as substrates of the beta1,3-glucuronosyltransferases, showed that the enzyme exhibited a strict selectivity towards Galbeta1-3Gal structures. Thus, the large source of purified active enzyme allowed the determination of the kinetic parameters of GlcAT-I towards the donor substrate UDP-GlcA and the acceptor substrate digalactoside Galbeta1-3Gal.
Lattard
Virginie
V
UMR 7561 CNRS-Université Henri Poincaré Nancy I, Faculté de Médecine, BP 184, 54505 Vandoeuvre-lès-Nancy Cedex, France.
Fondeur-Gelinotte
Magali
M
Gulberti
Sandrine
S
Jacquinet
Jean-Claude
JC
Boudrant
Joseph
J
Netter
Patrick
P
Magdalou
Jacques
J
Ouzzine
Mohamed
M
Fournel-Gigleux
Sylvie
S
D003001:Cloning, Molecular; D005697:Galactosides / Q000737:chemistry / Q000378:metabolism; D014453:Glucuronosyltransferase / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification*; D006801:Humans; D007700:Kinetics; D010843:Pichia / Q000201:enzymology / Q000235:genetics*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification*; D012995:Solubility; D013379:Substrate Specificity; D014535:Uridine Diphosphate Glucuronic Acid / Q000737:chemistry / Q000378:metabolism
D005697:Galactosides; D011994:Recombinant Proteins; D014535:Uridine Diphosphate Glucuronic Acid; C067806:glucuronyltransferase GlcAT-1; D014453:Glucuronosyltransferase