Abstract
Heparan sulfate (HS) is a co-receptor for a number of growth factors, morphogens, and adhesion proteins. HS biosynthetic modifications may determine the strength and outcome of HS-ligand interactions. We previously described the phenotype of mice with a gene-trap mutation in Hs2st, encoding the key HS 2-O-sulfotransferase enzyme in HS polymer modification. In contrast to the early developmental failure of embryos lacking HS, the onset of abnormalities in the Hs2st(-/-) mice occurs only after midgestation, the most dramatic being the complete failure of kidney development. Uronate 2-O-sulfates were not detected in the mutant HS, indicating a complete loss of function of Hs2st. However, the domain structure of the mutant HS is conserved, and compensatory increases in N- and 6-O-sulfation maintain the overall charge density. The apparent affinities of the mutant HS for hepatocyte growth factor/scatter factor and fibronectin were unchanged but were reduced for fibroblast growth factor-1 and -2. Surprisingly, the Hs2st(-/-) cells were able to mount an apparently normal signaling response to fibroblast growth factor-1 and -2 as well as to hepatocyte growth factor/scatter factor.
Highlights
Many key events in embryogenesis depend on growth and differentiation factors that specify lineage commitment and control the expansion and morphogenesis of maturing cell populations
No significant size differences were seen between the WT and mutant heparan sulfate (HS) chains on Sepharose CL-6B, in both cases the cellular HS was consistently longer than the mediumderived HS (Kav values of 0.45 and 0.5, respectively, corresponding to ϳ30 and 20 kDa by reference to the calibration of Wasteson [27]) (Fig. 1)
The increase in 6S was confined to GlcNAc to N-sulfoglucosamine (GlcNS) residues that are predominantly located in S domains
Summary
Materials—Glasgow modified Eagle’s medium, Dulbecco’s modified Eagle’s medium and fetal calf serum were from Life Technologies, Inc. Molecular Size and Domain Structure of HS chains—Each glycosaminoglycan pool was digested with chondroitinase ABC, and the products were separated by gel filtration on a Sepharose CL-6B column (66 ϫ 1.5 cm), eluted with 0.2 M NH4HCO3 at 12 ml hϪ1. Disaccharides (Ͼ95% of total HS counts) were recovered from a Bio-Gel P-2 column (150 ϫ 1 cm) eluted at 4 ml hϪ1 in 0.1 M NH4HCO3 with collection of 1-ml fractions. A combination of heparinases I, II, and III (16 milli-international units each) was added to each well for a further hour After this time, (i) growth factor was added at 1 ng/ml, (ii) serum-free medium was exchanged for medium supplemented with 10% fetal calf serum, or (iii) serum-free medium was retained for a further 10 min at 37 °C
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