Two N-terminally truncated variants of human β-galactoside α2,6 sialyltransferase I with distinct properties for in vitro protein glycosylation.
Sialic acid groups of protein N-glycans are important determinants of biological activity. Exposed at the end of the glycan chain, they are potential targets for glycan remodeling. Sialyltransferases (STs; EC 2.4.99) are the enzymes that catalyze the sialic acid transfer from a CMP-activated donor on to a carbohydrate acceptor in vivo. Recombinant expression of the full-length human β-galactoside α2,6 sialyltransferase I (ST6Gal-I) was hampered and therefore variants with truncated N-termini were investigated. We report on the distinct properties of two N-terminally truncated versions of ST6Gal-I, namely Δ89ST6Gal-I and Δ108ST6Gal-I, which were successfully expressed in human embryonic kidney cells. The different properties of these enzymes result most probably from the loss of interactions from helix α1 in the Δ108ST6Gal-I variant, which plays a role in acceptor substrate binding. The Kmfor N-acetyl-d-lactosamine was 10-fold increased for Δ108ST6Gal-I (84 mM) as compared to Δ89ST6Gal-I (8.3 mM). The two enzyme variants constitute a suitable tool box for the terminal modification of N-glycans. While the enzyme Δ89ST6Gal-I exhibited both ST (di-sialylation) and sialidase activity on a monoclonal antibody, the enzyme Δ108ST6Gal-I showed only ST activity with specificity for mono-sialylation.
Austrian Centre of Industrial Biotechnology, Petersgasse 14, 8010 Graz, Austria.
Christiane
C
Luley-Goedl
Austrian Centre of Industrial Biotechnology, Petersgasse 14, 8010 Graz, Austria.
Katharina
K
Schmoelzer
Pharma Technical Development Analytics Biologics.
Marco
M
Thomann
Pharma Technical Development Analytics Biologics.
Sebastian
S
Malik
Pharma Technical Development Fermentation, Roche Diagnostics GmbH, Nonnenwald 2, 82377 Penzberg, Germany.
Michael
M
Greif
Austrian Centre of Industrial Biotechnology, Petersgasse 14, 8010 Graz, Austria.
Doris
D
Ribitsch
Pharma Technical Development Fermentation, Roche Diagnostics GmbH, Nonnenwald 2, 82377 Penzberg, Germany.
Christine
C
Jung
Labor Dr. Merk & Kollegen GmbH, Beim Braunland 1, 88416 Ochsenhausen, Germany.
Harald
H
Sobek
Costum Biotech, Roche Diagnostics GmbH, Nonnenwald 2, 82377 Penzberg, Germany.
Alfred
A
Engel
Costum Biotech, Roche Diagnostics GmbH, Nonnenwald 2, 82377 Penzberg, Germany.
Rainer
R
Mueller
Institute of Molecular Biotechnology, Graz University of Technology, Petersgasse 14, 8010 Graz, Austria.
Helmut
H
Schwab
Austrian Centre of Industrial Biotechnology, Petersgasse 14, 8010 Graz, Austria bernd.nidetzky@tugraz.at.|Institute of Biotechnology and Biochemical Engineering, Graz University of Technology, Petersgasse 12/I, 8010 Graz, Austria.
Bernd
B
Nidetzky
D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000378:metabolism; D003001:Cloning, Molecular; D014644:Genetic Variation / Q000235:genetics; D006031:Glycosylation; D057809:HEK293 Cells; D006801:Humans; D008958:Models, Molecular; D011134:Polysaccharides / Q000737:chemistry / Q000378:metabolism; D012799:Sialyltransferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000094802:beta-D-Galactoside alpha 2-6-Sialyltransferase
D000911:Antibodies, Monoclonal; D011134:Polysaccharides; D012799:Sialyltransferases; D000094802:beta-D-Galactoside alpha 2-6-Sialyltransferase
N-terminal truncation; antibody; glycoengineering; human β-galactoside α2,6-sialyltransferase; in vitro protein glycosylation