The Stem Region of the Sulfotransferase GlcNAc6ST-1 Is a Determinant of Substrate Specificity

Abstract

The GlcNAc-6-sulfotransferases are a family of Golgiresident 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.

Expression of Long-form N-Acetylglucosamine-6-O-Sulfotransferase 1 in Human High Endothelial Venules

“…2 (left panel), immunoblotting with anti-FLAG of samples not treated with PNGase F showed multiple immunoreactive bands migrating at ~60 kDa for the long form and ~55 kDa for the short form. In addition, immunoreactive species migrating at >100 kDa, the molecular weight of enzyme homodimers, were also detected with both forms of the enzyme, as described previously (de Graffenried and Bertozzi 2004). The appearance of multiple bands is consistent with a previous study using HeLa cells transfected with wild-type or mutant forms of GlcNAc6ST-1, which demonstrated that at least three of four potential N-glycosylation sites were glycosylated (Desko et al 2009).…”

Cloning and Recombinant Expression of Active Full-length Xylosyltransferase I (XT-I) and Characterization of Subcellular Localization of XT-I and XT-II

“…The way in which our XT-I and XT-II data can be reconciled with the kin recognition theory should be investigated in further studies. The present investigations reported that for some glycoyltransferases, oligomerization of the stem region was responsible for their Golgi retention (41), whereas others still showed Golgi retention after the disruption of the protein-protein interactions (42,43). We could only hypothesize that the truncated stem region of XT-I leads to decreased formation of aggregates and therefore to impaired Golgi retention.…”

GlcNAc6ST-1-mediated decoration of MAdCAM-1 protein with L-selectin ligand carbohydrates directs disease activity of ulcerative colitis

“…What determines scaffold protein‐dependent preference of GlcNAc6ST‐1 is unknown. Many factors influence the cellular substrate preferences of Golgi enzymes, such as Golgi distribution, association with other proteins, and intrinsic substrate binding activity 36. In the context of distribution, GlcNAc6ST‐1 resides in the trans‐Golgi network, whereas GlcNAc6ST‐2 is more broadly distributed and more strongly represented in the earlier parts of the Golgi 37.…”