ABSTRACT
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.
Subject(s)
Cysteine/chemistry , N-Acetylglucosaminyltransferases/chemistry , Protein Folding , Animals , Carrier Proteins/genetics , Carrier Proteins/metabolism , Catalysis , Catalytic Domain , Cloning, Molecular , Cysteine/genetics , Cysteine/metabolism , Electrophoresis, Polyacrylamide Gel , Escherichia coli/genetics , Humans , Maltose-Binding Proteins , Models, Molecular , Mutant Proteins/chemistry , Mutant Proteins/metabolism , Mutation , N-Acetylglucosaminyltransferases/genetics , N-Acetylglucosaminyltransferases/metabolism , Polysaccharides/analysis , Protein Structure, Tertiary , Rabbits , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Ribonucleases/chemistry , Ribonucleases/metabolism , Spectrometry, Mass, Matrix-Assisted Laser Desorption-IonizationABSTRACT
Heparan sulfate/heparin N-deacetylase/N-sulfotransferase-1 (NDST-1) is a critical enzyme involved in heparan sulfate/heparin biosynthesis. This dual-function enzyme modifies the GlcNAc-GlcA disaccharide repeating sugar backbone to make N-sulfated heparosan. N-sulfation is an absolute requirement for the subsequent epimerization and O-sulfation steps in heparan sulfate/heparin biosynthesis. We have expressed rat liver (r) NDST-1 in Saccharomyces cerevisiae as a soluble protein. The yeast-expressed enzyme has both N-deacetylase and N-sulfotransferase activities. N-acetyl heparosan, isolated from Escherichia coli K5 polysaccharide, de-N-sulfated heparin (DNSH) and completely desulfated N-acetylated heparan sulfate (CDSNAcHS) are all good substrates for the rNDST-1. However, N-desulfated, N-acetylated heparin (NDSNAcH) is a poor substrate. The rNDST-1 was partially purified on heparin Sepharose CL-6B. Purified rNDST-1 requires Mn(2+) for its enzymatic activity, can utilize PAPS regenerated in vitro by the PAPS cycle (PAP plus para-nitrophenylsulfate in the presence of arylsulfotransferase IV), and with the addition of exogenous PAPS is capable of producing 60-65% N-sulfated heparosan from E. coli K5 polysaccharide or Pasteurella multocida polysaccharide.
