The stem region of the sulfotransferase GlcNAc6ST-1 is a determinant of substrate specificity.
The GlcNAc-6-sulfotransferases are a family of Golgi-resident enzymes that modulate glycan function. Two members of this family, GlcNAc6ST-1 and -2, collaborate in the biosynthesis of ligands for the leukocyte adhesion molecule L-selectin. Although their biochemical properties are similar in vitro, the enzymes have distinct glycoprotein substrate preferences in vivo. The sulfotransferases share similar overall architecture with the exception of an extended stem region in GlcNAc6ST-1 that is absent in GlcNAc6ST-2. In this study we probed the importance of the stem region with respect to substrate preference, localization, and oligomerization. Analysis of truncation mutants demonstrated that perturbation of the stem region of GlcNAc6ST-1 affects the cellular substrate preference of the enzyme without altering its retention within the Golgi. A chimeric enzyme comprising the stem region of GlcNAc6ST-1 inserted between the catalytic and transmembrane domains of GlcNAc6ST-2 had the same substrate preference as native GlcNAc6ST-1. In cells, GlcNAc6ST-1 exists as a dimer; two cysteine residues within the stem and transmembrane domain were found to be critical for dimerization. However, disruption of the dimer by mutagenesis did not affect either localization or substrate preference. Collectively, these results indicate that the stem region of GlcNAc6ST-1 influences substrate specificity, independent of its role in dimerization or Golgi retention.
de Graffenried
Christopher L
CL
Department of Chemistry, University of California, Berkeley, California 94720, USA.
Bertozzi
Carolyn R
CR
D000595:Amino Acid Sequence; D019281:Dimerization; D017353:Gene Deletion; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D017434:Protein Structure, Tertiary; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D015238:Sulfotransferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D000097811:Carbohydrate Sulfotransferases
D015238:Sulfotransferases; D000097811:Carbohydrate Sulfotransferases