Identification of the N-glycosylation sites on recombinant bovine CD38 expressed in Pichia pastoris: their impact on enzyme stability and catalytic activity.
Bovine CD38, a type II glycoprotein, contains two potential N-glycosylation sites (Asn-201 and Asn-268) in its extracellular domain. This contrasts with the other mammalian members of the ADP-ribosyl cyclase family, such as human CD38 and BST-1/CD157, in which four such sites are present. Our study was designed to determine the occupancy of these sites in a recombinant form of this ecto-enzyme and to evaluate its impact on the protein stability and catalytic functions. To that end we have successfully expressed the hydrosoluble ecto-domain of bovine CD38 (bCD38; residues 32-278), and corresponding glycosylation mutants, in the methylotrophic yeast Pichia pastoris. The secreted proteins were purified to homogeneity by affinity chromatography on immobilized Cibacron blue. We found by site-directed mutagenesis and mass spectrometry that bCD38 was a monoglycosylated protein at Asn-201. The expression yield of the deglycosylated mutants was not significantly affected, indicating that glycosylation at Asn-201 was not required for a proper processing and secretion of this protein by P. pastoris. Significant alterations in the kinetic parameters of NAD(+) were observed for the deglycosylated mutants. The thermostability of the recombinant enzyme was also influenced by mutation at position 201. Interestingly both parameters were dependent on the nature of the mutant and a stable deglycosylated mutant N201D of bCD38 could be produced that can be further used for structural studies.
Muller-Steffner
Hélène
H
Faculté de Pharmacie, Université de Strasbourg, 67400 Illkirch, France. steffner@bioorga.u-strasbg.fr
Kuhn
Isabelle
I
Argentini
Manuela
M
Schuber
Francis
F
D051997:ADP-ribosyl Cyclase 1 / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics*; D000595:Amino Acid Sequence; D000818:Animals; D002384:Catalysis; D002417:Cattle; D004795:Enzyme Stability; D006031:Glycosylation; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase / Q000378:metabolism; D010843:Pichia / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics
D011994:Recombinant Proteins; D051997:ADP-ribosyl Cyclase 1; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase