Refolding of human beta-1-2 GlcNAc transferase (GnT1) and the role of its unpaired Cys 121.
Human beta1-2N-acetylglucosaminyltransferase (hGnT1) lacking the first 103 amino acids was expressed as a maltose binding protein (MBP) fusion protein in inclusion bodies (IBs) in Escherichia coli and refolded using an oxido-shuffling method. GnT1 mutants were prepared by replacing a predicted unpaired cysteine (C121) with alanine (C121A), serine (C121S), threonine (C121T) or aspartic acid (C121D). A double mutant R120A/C121H, was generated to mimic Gly14, the Caenorhabditis elegans GnT1 counterpart to hGNT1. Each mutant hGnT1 was constructed as an MBP fusion protein and resultant IBs were isolated and refolded. Wild type hGnT1 and mutants C121A, C121S and R120A/C121H transferred UDP-GlcNAc to the glycoprotein acceptor Man(5)-RNAse B, whereas mutants C121T and C121D were inactive. These findings indicated that cysteine 121 has a structural role in maintaining active site geometry of hGnT1, rather than a catalytic role, and illustrates for the first time the potential utility of E. coli as an expression system for hGnT1.
Saribas
A Sami
AS
Neose Technologies, Inc., 102 Rock Road, Horsham, PA 19044, USA.
Johnson
Karl
K
Liu
Li
L
Bezila
Dan
D
Hakes
David
D
D000818:Animals; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism; D002384:Catalysis; D020134:Catalytic Domain; D003001:Cloning, Molecular; D003545:Cysteine / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D058619:Maltose-Binding Proteins; D008958:Models, Molecular; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism; D009154:Mutation; D017351:N-Acetylglucosaminyltransferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011134:Polysaccharides / Q000032:analysis; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012260:Ribonucleases / Q000737:chemistry / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D002352:Carrier Proteins; D058619:Maltose-Binding Proteins; D050505:Mutant Proteins; D011134:Polysaccharides; D011993:Recombinant Fusion Proteins; D017351:N-Acetylglucosaminyltransferases; C063057:UDP-N-acetylglucosamine-peptide beta-N-acetylglucosaminyltransferase; D012260:Ribonucleases; C029600:ribonuclease B; D003545:Cysteine