Heparan sulfate biosynthesis enzymes EXT1 and EXT2 affect NDST1 expression and heparan sulfate sulfation

Abstract

Heparan sulfate (HS) proteoglycans influence embryonic development and adult physiology through interactions with protein ligands. The interactions depend on HS structure, which is determined largely during biosynthesis by Golgi enzymes. How biosynthesis is regulated is more or less unknown. During polymerization of the HS chain, carried out by a complex of the exostosin proteins EXT1 and EXT2, the first modification enzyme, glucosaminyl Ndeacetylase/N-sulfotransferase (NDST), introduces N-sulfate groups into the growing polymer. Unexpectedly, we found that the level of expression of EXT1 and EXT2 affected the amount of NDST1 present in the cell, which, in turn, greatly influenced HS structure. Whereas overexpression of EXT2 in HEK 293 cells enhanced NDST1 expression, increased NDST1 N-glycosylation, and resulted in elevated HS sulfation, overexpression of EXT1 had opposite effects. Accordingly, heart tissue from transgenic mice overexpressing EXT2 showed increased NDST activity. Immunoprecipitaion experiments suggested an interaction between EXT2 and NDST1. We speculate that NDST1 competes with EXT1 for binding to EXT2. Increased NDST activity in fibroblasts with a gene trap mutation in EXT1 supports this notion. These results support a model in which the enzymes of HS biosynthesis form a complex, or a GAGosome.

Specific Glycosaminoglycans Modulate Neural Specification of Mouse Embryonic Stem Cells

“…In related analyses, we have also characterized HS from Ext1 þ/À ES cells (generated alongside Ext1 À/À cells used in this study), which also display shorter chain length and alterations in disaccharide composition (our unpublished data), distinct from the sulfation changes described in this study. 46Cext1RNAi:5 and Ext1 þ/À ES cells are likely to have different levels of Ext1 enzyme activity, and the above findings correlate well with those demonstrating that altered levels of Ext1 enzyme influenced levels of N-deacetylase N-sulfotransferase 1 activity and subsequent HS structure, including increased O-sulfation [36]. We hope that this data will help refine the GAGosome theory, in which the GAG biosynthetic enzymes exist in a physical complex in the Golgi with inter-related activities [36,37].…”

The Dominating Role of N-Deacetylase/N-Sulfotransferase 1 in Forming Domain Structures in Heparan Sulfate

“…One plausible approach to reduce the product heterogeneity is to direct NDST-1 to a specific GlcNAc residue. Presto et al (27) discovered the interaction of one HS polymerase (EXT-2) and NDST-1. Interestingly, the authors discovered that HS isolated HEK 293 cells overexpressing EXT-2 was highly sulfated and more closed to heparin than HS (Heparin is considered structurally more homogeneous than HS).…”

“…Whether different isoforms have distinct capability of generating the N-S domains and N-Ac is currently unknown. In addition, the core protein of proteoglycans as well as potential molecular chaperons or the supra molecular complex involved in HS biosynthetic enzymes (27) …”

Cell Type-Specific Requirements for Heparan Sulfate Biosynthesis at the Drosophila Neuromuscular Junction: Effects on Synapse Function, Membrane Trafficking, and Mitochondrial Localization

“…Ttv is part of a copolymerase with Sotv (Han et al, 2004;Izumikawa et al, 2006); excessive levels of Ttv may enhance HS polymerization (Busse et al, 2007) or interfere with the activity of this protein complex. In addition to potential changes in HS chain length produced by overexpression of Ttv, these polymers may have aberrant levels or patterns of sulfation (Presto et al, 2008).…”