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-l inked glycosylation (ST6Gal 1) forms di. sulfide-bonded dimers that exhibit decreased activity, but retain the ability to bind asialoglycoprotein subs trates. Here, we have investigated the subcellular location and mechanism o f ST6Gal I dimer formation, as well as the role of Cys residues in the enzy me's trafficking, localization, and catalytic activity. Pulse-chase analysi s demonstrated that the ST6Gal I disulfide-bonded dimer forms in the endopl asmic reticulum. Mutagenesis experiments showed that Cys-24 in the transmem brane region is required for dimerization, while catalytic domain Cys resid ues are required for trafficking and catalytic activity. Replacement of Cys -181 and Cys-332 generated proteins that are largely retained in the endopl asmic reticulum and minimally active or inactive, respectively. Replacement of Cys-350 or Cys-361 inactivated the enzyme without compromising its loca lization or processing, suggesting that these amino acids are part of the e nzyme's active site. Replacement of Cys-139 or Cys-403 generated proteins t hat are catalytically active and appear to be more stably localized in the Golgi, since they exhibited decreased cleavage and secretion. The Cys-139 m utant also exhibited increased dimer formation suggesting that ST6Gal I dim ers may be critical in the oligomerization process involved in stable ST6Ga l I Golgi localization.