Heparan sulfate biosynthesis enzymes EXT1 and EXT2 affect NDST1 expression and heparan sulfate sulfation - PubMed (original) (raw)

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

Jenny Presto et al. Proc Natl Acad Sci U S A. 2008.

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 N-deacetylase/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.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

Fig. 1.

Protein expression and enzyme activity of NDST1. (A and B) NDST1 protein expression was analyzed by SDS/PAGE (10% gels) and Western blotting after solubilization of the cells in 1% Triton X-100-containing buffer. The separated proteins (20 μg) were blotted to a nitrocellulose membrane, and NDST1 was detected with anti-NDST1 peptide antibody 1A. (C and D) _N_-deacetylase (C) and _N_-sulfotransferase (D) activity of the cell lines examined in A were measured as described in Materials and Methods. Each bar in C and D represents the mean of two samples.

Fig. 2.

Fig. 2.

Disaccharide analysis of HS from the different cell lines. HS disaccharide composition was determined by RPIP HPLC. (A) The relative amount of the different disaccharides shown is the mean value of two determinations of single cell lines. Nac, HexA-GlcNAc; NS, HexA-GlcNS; 6S, HexA-GlcNAc(6OS); 2S, HexA(2OS)-GlcNAc; NS6S, HexA-GlcNS(6OS); NS2S, HexA(2OS)-GlcNS; NS6S2S, HexA (2OS)-GlcNS(6OS). (B) Percentage of disaccharides with different modifications calculated from the data in A.

Fig. 3.

Fig. 3.

Coprecipitation of NDST1 and EXT2. (A) Lysates from NDST1/EXT2-overexpressing cells were incubated with preimmune serum or EXT2 antibodies (N15) with and without prior preincubation with EXT2 blocking peptide. NDST1 was detected after separation of immune complexes by SDS/PAGE (10% gels) and immunoblotting using α-trunc-NDST1 antibodies. (B) Lysates from NDST1/EXT2- or EXT2-overexpressing cells were incubated with α-trunc-NDST1 antibodies and preimmune serum, respectively. EXT2 was detected after separation of immune complexes by SDS/PAGE (10% gels) and immunoblotting using EXT2 antibodies (N15).

Fig. 4.

Fig. 4.

Glycosylation of NDST1 protein. (A) Cell lysates were analyzed by 10% SDS/PAGE and Western blotting as described in the legend to Fig. 1, apart from a longer separation time. (B) Lysates from NDST1/EXT2-overexpressing cells were incubated with the indicated amounts of PNGaseF or endo H followed by SDS/PAGE (7.5% gel) and Western blotting.

Fig. 5.

Fig. 5.

GAGosome model of EXT–NDST1 interactions. NDST1, N1; EXT1, E1; EXT2, E2.

Fig. 6.

Fig. 6.

Validation of the GAGosome model. (A) _N_-deacetylase activity (open bars, as measured by an ELISA method) and _N_-sulfotransferase activity (filled bars) in heart tissue from two control and two EXT2 transgenic mice. (B) _N_-deacetylase activity in heart and kidney tissue from one control and one transgenic mouse, measured in duplicate. (C) _N_-deacetylase activity in fibroblasts from EXT1Gt/Gt and control mice. Each sample was measured in triplicate.

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