Location and Mechanism of α2,6-Sialyltransferase Dimer Formation

Abstract

A significant proportion of the ␣2,6-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 disulfidebonded 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.The sialyltransferases are a large family of glycosyltransferases that function in the Golgi apparatus to transfer NeuAc from the sugar nucleotide donor, CMP-NeuAc, to terminal positions of N-linked and O-linked oligosaccharides of glycoproteins and the oligosaccharides of glycolipids. The sialylated oligosaccharides that are products of these enzymes' action have a variety of important roles in the normal cell and during development and disease (reviewed in Ref. 1). For example, sialylated oligosaccharides function as selectin ligands during inflammation and the homing of lymphocytes; act as receptors for viruses, toxins, and parasites; function in B cell maturation and activation; maintain glycoproteins in the circulation; and play roles in negatively modulating cell adhesion during development and oncogenesis (1).

The Minimal Structural Domains Required for Neural Cell Adhesion Molecule Polysialylation by PST/ST8Sia IV and STX/ST8Sia II

“…Autopolysialylation of the polysialyltransferases may be somewhat different mechanistically and yet still rely on protein-protein interactions in the form of enzyme dimerization or oligomerization. In support of this idea, many glycosyltransferases form dimers (73)(74)(75)(76), and another ␣2,8-sialyltransferase, ST8Sia III, is also capable of autopolysialylation even though it does not polysialylate NCAM or any other glycoprotein substrate (39). We predict that these three ␣2,8-sialyltransferases form dimers or higher order oligomers, and this type of protein-protein interaction allows them to autopolysialylate their own oligosaccharides.…”

Biosynthesis of the Linkage Region of Glycosaminoglycans

“…Interestingly, ␤3GalT1, ␤3GalT5, and the homologous Drosophila protein Brainiac also contain Cys residues in their putative transmembrane domains, but the function of these residues is unknown. One possibility is that they may affect the oligomerization of the protein as recently reported for ␣2,6-sialyltransferase I (36). The transmembrane domain is followed by a segment rich in Ser, Pro, Ala, and Gly, which has been described as the "SPLAG" domain in other transferases (37), and presumably represents a stem region that determines the distance between the catalytic domain and the membrane.…”

Importance of Cys, Gln, and Tyr from the Transmembrane Domain of Human α3/4 Fucosyltransferase III for Its Localization and Sorting in the Golgi of Baby Hamster Kidney Cells

“…Other GTs from the trans- Golgi ␤4-Galactosyltransferase has been shown to form homodimers mediated by Cys-29 and/or His-32 from the TM, and dimerization was associated with Golgi localization (28). On the other hand, ␣2,6-sialyltransferase has been shown to dimerize through Cys-24 from its TM, dimerization not being required for Golgi localization or enzyme activity, but possibly necessary to induce higher multiplicity oligomerization (9). For FT3, it was found that mutation of Cys-29 led to increased levels of expression on the PM (FT3np2).…”

“…The luminal domain of GTs has also been shown to be important for the formation of protein-protein complexes. It has been observed that cis/medial GTs form large insoluble oligomers, contrary to late Golgi GTs that usually exist as dimers (7)(8)(9). Furthermore, changing the targeting of cis/medial Golgi mannosidase II to the endoplasmic reticulum (ER) leads to accumulation of N-acetylglucosaminyltransferase I in the ER (7,10) in a kin-recognition mechanism.…”