Conserved cysteines in the sialyltransferase sialylmotifs form an essential disulfide bond

The sialyltransferase gene family is comprised of 16 cloned enzymes. All me mbers contain two conserved protein domains, termed the S- and L-sialylmoti fs, that participate in substrate binding. Of only six invariant amino acid s, two are cysteines, with one found in each sialylmotif. Although the reco mbinant soluble form of ST6Gal I has six cysteines, quantitative analysis i ndicated the presence of only one disulfide linkage, and thiol reducing age nts dithiothreitol and beta -mercaptoethanol inactivated the enzyme. Analys is of site-directed mutants showed that alanine or serine mutants of invari ant Cys(181) or Cys(332) exhibit no detectable activity, either by direct a ssay or by staining of the transfected cells with Sambucus nigra agglutinin , which recognizes the product NeuAc alpha2,6Gal beta1,4GlcNAc on glycoprot eins. In contrast, alanine mutations of charged residues adjacent to either cysteine showed little or no effect on enzyme activity. Immunofluorescence microscopy showed that although the wild type sialyltransferase is properl y localized in the Gels apparatus, the inactive cysteine mutants are retain ed in the endoplasmic reticulum. The results suggest that the invariant cys teine residues in the L and S-sialylmotifs participate in the formation of an intradisulfide linkage that is essential for proper conformation and act ivity of ST6Gal I.