Location and mechanism of alpha 2,6-sialyltransferase dimer formation. Role of cysteine residues in enzyme dimerization, localization, activity, and processing.
A significant proportion of the alpha2,6-sialyltransferase of protein Asn-linked glycosylation (ST6Gal I) forms disulfide-bonded dimers that exhibit decreased activity, but retain the ability to bind asialoglycoprotein substrates. Here, we have investigated the subcellular location and mechanism of ST6Gal I dimer formation, as well as the role of Cys residues in the enzyme's trafficking, localization, and catalytic activity. Pulse-chase analysis demonstrated that the ST6Gal I disulfide-bonded dimer forms in the endoplasmic reticulum. Mutagenesis experiments showed that Cys-24 in the transmembrane region is required for dimerization, while catalytic domain Cys residues are required for trafficking and catalytic activity. Replacement of Cys-181 and Cys-332 generated proteins that are largely retained in the endoplasmic reticulum and minimally active or inactive, respectively. Replacement of Cys-350 or Cys-361 inactivated the enzyme without compromising its localization or processing, suggesting that these amino acids are part of the enzyme's active site. Replacement of Cys-139 or Cys-403 generated proteins that are catalytically active and appear to be more stably localized in the Golgi, since they exhibited decreased cleavage and secretion. The Cys-139 mutant also exhibited increased dimer formation suggesting that ST6Gal I dimers may be critical in the oligomerization process involved in stable ST6Gal I Golgi localization.
Qian
R
R
Department of Biochemistry and Molecular Biology, University of Illinois College of Medicine, Chicago, Illinois 60612, USA.
Chen
C
C
Colley
K J
KJ
D019943:Amino Acid Substitution; D000818:Animals; D001216:Asparagine; D016466:CHO Cells; D019556:COS Cells; D002384:Catalysis; D002522:Chlorocebus aethiops; D002846:Chromatography, Affinity; D006224:Cricetinae; D003553:Cystine; D003569:Cytidine Monophosphate N-Acetylneuraminic Acid / Q000737:chemistry / Q000378:metabolism; D019281:Dimerization; D004220:Disulfides / Q000032:analysis; D004721:Endoplasmic Reticulum / Q000201:enzymology; D006056:Golgi Apparatus / Q000201:enzymology; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D021381:Protein Transport; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D012799:Sialyltransferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D014162:Transfection; D000094802:beta-D-Galactoside alpha 2-6-Sialyltransferase
D004220:Disulfides; D011994:Recombinant Proteins; D003569:Cytidine Monophosphate N-Acetylneuraminic Acid; D003553:Cystine; D001216:Asparagine; D012799:Sialyltransferases; D000094802:beta-D-Galactoside alpha 2-6-Sialyltransferase