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Christopher L de Graffenried

Publications and source records attributed to Christopher L de Graffenried.

7 recordsLinked to original sources
PubMed2006

Ordered assembly of the duplicating Golgi in Trypanosoma brucei.

The new Golgi in the protozoan parasite Trypanosoma brucei grows near to the old and adjacent to the growing new endoplasmic reticulum exit site. Growth is now shown to be at least a two-stage process, in which a representative matrix marker (GRASP) and enzyme (GntB) are delivered to the site of assembly, followed approximately 10 min later by a COPI component (epsilon-COP) and a trans-Golgi network (TGN) marker (GRIP70). A secretory cargo marker (signal sequence-YFP) appeared early near the new endoplasmic reticulum exit site but did not enter the Golgi until the second stage. Together these data suggest that structural and enzymatic components of the new Golgi stack are laid down first, followed by those needed to move and sort the cargo passing through it.

Animals↗
PubMed2006

Regulating cell surface glycosylation with a small-molecule switch.

Correct localization of Golgi-resident enzymes is essential for the formation of specific glycan epitopes. In this chapter, we describe a method to control the localization, and thus the activity, of an individual glycosyltransferase by administration of a small molecule. Our method takes advantage of the modularity of most Golgi-resident enzymes, which are composed of localization and catalytic domains. These domains can be physically separated and fused to the small molecule binding proteins FRB and FKBP, which dimerize in the presence of rapamycin. In this way, rapamycin serves as a "switch" for enzyme activity.

Catalytic Domain↗
PubMed2004

A small-molecule switch for Golgi sulfotransferases.

The study of glycan function is a major frontier in biology that could benefit from small molecules capable of perturbing carbohydrate structures on cells. The widespread role of sulfotransferases in modulating glycan function makes them prime targets for small-molecule modulators. Here, we report a system for conditional activation of Golgi-resident sulfotransferases using a chemical inducer of dimerization. Our approach capitalizes on two features shared by these enzymes: their requirement of Golgi localization for activity on cellular substrates and the modularity of their catalytic and localization domains. Fusion of these domains to the proteins FRB and FKBP enabled their induced assembly by the natural product rapamycin. We applied this strategy to the GlcNAc-6-sulfotransferases GlcNAc6ST-1 and GlcNAc6ST-2, which collaborate in the sulfation of L-selectin ligands. Both the activity and specificity of the inducible enzymes were indistinguishable from their WT counterparts. We further generated rapamycin-inducible chimeric enzymes comprising the localization domain of a sulfotransferase and the catalytic domain of a glycosyltransferase, demonstrating the generality of the system among other Golgi enzymes. The approach provides a means for studying sulfate-dependent processes in cellular systems and, potentially, in vivo.

Animals↗
PubMed2004

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.

Amino Acid Sequence↗
PubMed2004

The roles of enzyme localisation and complex formation in glycan assembly within the Golgi apparatus.

Cell surface glycans govern numerous cell-cell interactions are therefore key determinants of multicellular biology. They originate from biosynthetic pathways comprising an assembly line of glycosyltransferases within the Golgi compartment. Although the mechanisms of Golgi enzyme localisation are still under debate, the distribution of these enzymes among the Golgi cisternae can dictate the overall structures produced by the cell. Fine-tuning of glycan biosynthetic pathways is further accomplished by specific associations among glycosyltransferases. Together, localisation and association govern the assembly of complex glycans and thereby regulate interactions at the cell surface.

Animals↗
PubMed2003

Golgi localization of carbohydrate sulfotransferases is a determinant of L-selectin ligand biosynthesis.

Sulfation of endothelial glycoproteins by the sulfotransferase GlcNAc6ST-2 is a regulatory modification that promotes binding of the leukocyte adhesion molecule L-selectin. GlcNAc6ST-2 is a member of a family of related enzymes that act on similar carbohydrate substrates in vitro but discrete glycoproteins in vivo. We demonstrate that GlcNAc6ST-1, -2, and -3 have distinct Golgi distributions, with GlcNAc6ST-1 confined to the trans-Golgi network, GlcNAc6ST-3 confined to the early secretory pathway, and GlcNAc6ST-2 distributed throughout the Golgi. Their localization was correlated with preferred activity on either N-linked or O-linked glycoproteins. A chimera comprising the localization domain of GlcNAc6ST-1 fused to the catalytic domain of GlcNAc6ST-2 was confined to the trans-Golgi network and adopted the substrate preference of GlcNAc6ST-1. We propose a model in which Golgi enzyme localization and competition orchestrate the biosynthesis of L-selectin ligands.

Amino Acid Sequence↗