Contribution of EXT1, EXT2, and EXTL3 to Heparan Sulfate Chain Elongation

Abstract

The exostosin (EXT) family of genes encodes glycosyltransferases involved in heparan sulfate biosynthesis. Five human members of this family have been cloned to date: EXT1, EXT2, EXTL1, EXTL2, and EXTL3. EXT1 and EXT2 are believed to form a Golgi-located hetero-oligomeric complex that catalyzes the chain elongation step in heparan sulfate biosynthesis, whereas the EXTL proteins exhibit overlapping glycosyltransferase activities in vitro, so that it is not apparent what reactions they catalyze in vivo. We used gene-silencing strategies to investigate the roles of EXT1, EXT2, and EXTL3 in heparan sulfate chain elongation. Small interfering RNAs (siRNAs) directed against the human EXT1, EXT2, or EXTL3 mRNAs were introduced into human embryonic kidney 293 cells. Compared with cells transfected with control siRNA, those transfected with EXT1 or EXT2 siRNA synthesized shorter heparan sulfate chains, and those transfected with EXTL3 siRNA synthesized longer chains. We also generated human cell lines overexpressing the EXT proteins. Overexpression of EXT1 resulted in increased HS chain length, which was even more pronounced in cells coexpressing EXT2, whereas overexpression of EXT2 alone had no detectable effect on heparan sulfate chain elongation. Mutations in either EXT1 or EXT2 are associated with hereditary multiple exostoses, a human disorder characterized by the formation of cartilage-capped bony outgrowths at the epiphyseal growth plates. To further investigate the role of EXT2, we generated human cell lines overexpressing mutant EXT2. One of the mutations, EXT2-Y419X, resulted in a truncated protein. Interestingly, the capacity of wild type EXT2 to enhance HS chain length together with EXT1 was not shared by the EXT2-Y419X mutant.

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

“…Also, in HEK293 cells transfected with EXT1 but not with NDST1, a reduction in the endogenous NDST activity could be recorded, whereas no or little effect on NDST activity was seen in EXT2-transfected cells (data not shown). As recently discussed (26), EXT2 appears to be present in a molar excess over EXT1 and the lack of effect of EXT2 overexpression on endogenous NDST1 may simply be caused by the limited availability of NDST1 in HEK 293 cells.…”

“…Two clones expressing EXT1 or EXT2 together with NDST1 and mock-transfected NDST1-expressing cells, and one clone coexpressing EXT1, EXT2, and NDST1 were selected and maintained. HEK 293 cells overexpressing EXT2 have been described (26).…”

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

“…Since overexpression of Ttv-myc with da-Gal4 in an otherwise wild-type animal also reduced synapse size, we conclude that high levels of Ttv protein are detrimental in some way. 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).…”

Pancreatic Reg I Binds MKP-1 and Regulates Cyclin D in Pancreatic-Derived Cells

“…EXT genes are mutated in bone disease, and the function of the EXT and EXTL proteins are in assembly of peptide-glycans, specifically heparan sulfate (8)(9). Specifically, they catalyze the polymerization of glycosaminoglycans into heparan sulfate; EXTL3 actually initializes the event (10). However, EXT proteins have not been linked to cell growth, or cell cycle pathways, so precisely how reg I exerts its mitogenic effect is still unknown.…”