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J. Biol. Chem., Vol. 276, Issue 18, 15200-15207, May 4, 2001

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Conserved Cysteines in the Sialyltransferase Sialylmotifs Form an Essential Disulfide Bond^*

Arun K. Datta, Roger Chammas^¶, and James C. Paulson^§

From the  Department of Molecular Biology and Molecular and Experimental Medicine, Scripps Research Institute, San Diego, California 92037 and the ^¶ Department of Molecular Genetics, University of California, San Diego, California 92121

The sialyltransferase gene family is comprised of 16 cloned enzymes. All members contain two conserved protein domains, termed the S- and L-sialylmotifs, that participate in substrate binding. Of only six invariant amino acids, two are cysteines, with one found in each sialylmotif. Although the recombinant soluble form of ST6Gal I has six cysteines, quantitative analysis indicated the presence of only one disulfide linkage, and thiol reducing agents dithiothreitol and -mercaptoethanol inactivated the enzyme. Analysis of site-directed mutants showed that alanine or serine mutants of invariant Cys¹⁸¹ or Cys³³² exhibit no detectable activity, either by direct assay or by staining of the transfected cells with Sambucus nigra agglutinin, which recognizes the product NeuAc2,6Gal1,4GlcNAc on glycoproteins. 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 properly localized in the Golgi apparatus, the inactive cysteine mutants are retained in the endoplasmic reticulum. The results suggest that the invariant cysteine residues in the L- and S-sialylmotifs participate in the formation of an intradisulfide linkage that is essential for proper conformation and activity of ST6Gal I.

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