Altered Heparan Sulfate Structure in Mice with Deleted NDST3 Gene Function

Abstract

We report the generation and analysis of mutant mice bearing a targeted disruption of the heparan sulfate (HS)-modifying enzyme GlcNAc N-deacetylase/N-sulfotransferase 3 (NDST3). NDST3؊/؊ mice develop normally, are fertile, and show only subtle hematological and behavioral abnormalities in agreement with only moderate HS undersulfation. Compound mutant mice made deficient in NDST2;NDST3 activities also develop normally, showing that both isoforms are not essential for development. In contrast, NDST1؊/؊ ;NDST3 ؊/؊ compound mutant embryos display developmental defects caused by severe HS undersulfation, demonstrating NDST3 contribution to HS synthesis in the absence of NDST1. Moreover, analysis of HS composition in dissected NDST3 mutant adult brain revealed regional changes in HS sulfation, indicating restricted NDST3 activity on nascent HS in defined wild-type tissues. Taken together, we show that NDST3 function is not essential for development or adult homeostasis despite contributing to HS synthesis in a region-specific manner and that the loss of NDST3 function is compensated for by the other NDST isoforms to a varying degree. Heparan sulfate (HS)2 is produced by most mammalian cells as part of membrane and extracellular matrix proteoglycans (1). The chain grows by the copolymerization of GlcA␤1,4 and GlcNAc␣1,4 residues and undergoes modification by one or more of the four NDST isozymes, which remove acetyl groups from subsets of GlcNAc residues and add sulfate to the free amino groups. In vertebrates, ndst1 and ndst2 mRNA are expressed in all embryonic and adult tissues examined, whereas ndst3 and ndst4 transcripts are predominantly expressed during embryonic development and in the adult brain (2). Most subsequent modifications of the HS chain by O-sulfotransferases and a GlcA C5-epimerase depend on the presence of GlcNS residues, making the NDSTs largely responsible for the generation of sulfated ligand binding sites in HS (3-5). In vitro, NDST3 differs biochemically from the other NDST isoforms by possessing a high deacetylase activity but very low sulfotransferase activity (2).Many growth factors and morphogens bind to HS. In some cases, HS-proteoglycans are thought to act as co-receptors for these ligands. Studies in Drosophila melanogaster demonstrated that HS is crucial for embryonic development (6) and that the fly NDST ortholog, Sulfateless, affects signaling mediated by Wingless (Wg), Hedgehog (HH), and fibroblast growth factor (FGF) (7-9). The ability of HS to regulate the activity of morphogens and growth factors is currently best understood for the FGFs. HS was found to be a necessary component of FGF-FGF receptor binding and assembly (10), and global changes in HS expression regulate FGF and FGF receptor assembly during mouse development (11). Due to the multiple developmental processes regulated by the 23 FGFs, including those of the lung, limbs, heart, skeleton, and brain (reviewed in Ref. 12), perturbed HS synthesis results in the generation of FGF-related phenotypes (13,14). The crucial rol...

Synthesis of Heparan Sulfate with Cyclophilin B-binding Properties Is Determined by Cell Type-specific Expression of Sulfotransferases

“…These data are in correspondence with previous studies reporting a higher level of NDST2 relative to NDST1 in mast cells (16), lymphoid tissues, e.g. thymus and spleen, and whole blood leukocytes (14,47), thus indicating that high expression of NDST2 may be a common feature of cells from the hematopoietic line- ages. Although epithelial cells produce high level of HS by comparison with T lymphocytes and monocytes/macrophages, they are devoid of any high affinity binding sites for CyPB.…”

Oversulfated Chondroitin Sulfate Plays Critical Roles in the Neuronal Migration in the Cerebral Cortex

“…This is quite important for the functional analysis of glycosaminoglycan sulfotransferases, because glycosaminoglycans are sulfated by many sulfotransferases that may have overlapping functions. In fact, many of the mutant animals with deleted heparan sulfate sulfotransferase genes displayed unexpectedly mild phenotypes, which might be explained by the compensatory increase in sulfation at other positions in heparan sulfate (37)(38)(39). In this study, we found that knockdown of UST specifically down-regulated the expression of 2-O-sulfated B and D units in the COS-7 cells without a change in the amount of E unit (Fig.…”

Mast Cell Differentiation and Activation Is Closely Linked to Expression of Genes Coding for the Serglycin Proteoglycan Core Protein and a Distinct Set of Chondroitin Sulfate and Heparin Sulfotransferases

“…Hence, NDST activity is a key to the sulfation of heparin, and we therefore chose to study the expression of NDSTs in relation to CTMC-like BMMC maturation. Although four different isoforms of NDSTs (NDST-1-4) have been identified, NDST-1 and NDST-2 appear to be predominant in adult tissue (29). NDST-1 expression was detected already in the early stage (day 0) bone marrow cells, but the level of expression did not increase throughout the process of MC maturation (Fig.…”