Heparan Sulfate Synthesized by Mouse Embryonic Stem Cells Deficient in NDST1 and NDST2 Is 6-O-Sulfated but Contains No N-Sulfate Groups

Abstract

Heparan sulfate structure differs significantly between various cell types and during different developmental stages. The diversity is created during biosynthesis by sulfotransferases, which add sulfate groups to the growing chain, and a C5-epimerase, which converts selected glucuronic acid residues to iduronic acid. All these modifications are believed to depend on initial glucosamine N-sulfation carried out by the enzyme glucosaminyl N-deacetylase/N-sulfotransferase (NDST). Here we report that heparan sulfate synthesized by mouse embryonic stem cells deficient in NDST1 and NDST2 completely lacks N-sulfation but still contains 6-O-sulfate groups, demonstrating that 6-O-sulfation can occur without prior N-sulfation. Reverse transcriptase-PCR analysis indicates that all three identified 6-O-sulfotransferases are expressed by the cells, 6-O-sulfotransferase-1 being the dominating form. The 6-O-sulfated polysaccharide lacking N-sulfate groups also contains N-unsubstituted glucosamine units, raising questions about how these units are generated.

Specific Glycosaminoglycans Modulate Neural Specification of Mouse Embryonic Stem Cells

“…One of the hallmarks of early differentiation is the response to autocrine signals, many of which are dependent on HS, for example, the FGF-mediated system described in this study. Indeed, our previous study demonstrated that wild-type ES cells make a low sulfated species of HS [25] in comparison with many other embryonic tissues [39]. This is in good agreement with the ''ground state'' hypothesis in which naïve pluripotency is maintained by protection from Erk signaling [33].…”

Lowered Expression of Heparan Sulfate/Heparin Biosynthesis Enzyme N-Deacetylase/N-Sulfotransferase 1 Results in Increased Sulfation of Mast Cell Heparin

“…After incubation, cells were pelleted by centrifugation for 10 min at 300 ϫ g, washed with cold PBS, incubated in 2 ml of solubilization buffer (50 mM Tris-HCl, pH 7.5, 1% Triton X-100, 0.10 M NaCl), and centrifuged at 800 ϫ g for 15 min. The supernatant containing radiolabeled macromolecules was recovered, and 35 S-labeled glycosaminoglycans were isolated from the solubilized cell lysate on a 0.3-ml column of DEAE-Sephacel (Amersham Biosciences), equilibrated with 50 mM Tris-HCl, pH 7.4, 0.1% Triton X-100, 0.10 M NaCl. After washing the column with equilibration buffer followed by a second washing step with 50 mM acetate buffer, pH 4.0, containing 0.1% Triton X-100 and 0.10 M NaCl, the 35 S-labeled glycosaminoglycans were eluted with 50 mM acetate buffer, pH 4.0, containing 0.1% Triton X-100 and 2 M NaCl.…”

“…Cells were put on cytospin glasses and stained with May-Grünwald/Giemsa for morphological examination of MC differentiation. In addition, DNA was purified and analyzed by PCR, as described previously (35), to define the genotype of the cultures.…”

“…To be able to determine the cause of the early embryonic lethality, we recently established embryonic stem (ES) cell lines deficient for both NDST1 and NDST2 and showed that the HS produced lacks N-sulfation but contains low levels of 6-O-sulfate groups (35). In the present paper, we have studied the role of NDST1 and NDST2 in MC development in vitro.…”

“…Crosses between NDST2 Ϫ/Ϫ mice and NDST1 ϩ/Ϫ and analyses of their offspring demonstrated that lack of both isoforms results in early embryonic lethality (35). To be able to determine the cause of the early embryonic lethality, we recently established embryonic stem (ES) cell lines deficient for both NDST1 and NDST2 and showed that the HS produced lacks N-sulfation but contains low levels of 6-O-sulfate groups (35).…”