Heparan Sulfate 2-O-sulfotransferase Research Articles

New tools for carbohydrate sulfation analysis: heparan sulfate 2-O-sulfotransferase (HS2ST) is a target for small-molecule protein kinase inhibitors.

Sulfation of carbohydrate residues occurs on a variety of glycans destined for secretion, and this modification is essential for efficient matrix-based signal transduction. Heparan sulfate (HS) glycosaminoglycans control physiological functions ranging from blood coagulation to cell proliferation. HS biosynthesis involves membrane-bound Golgi sulfotransferases, including HS 2-O-sulfotransferase (HS2ST), which transfers sulfate from the cofactor PAPS (3′-phosphoadenosine 5′-phosphosulfate) to the 2-O position of α-l-iduronate in the maturing polysaccharide chain. The current lack of simple non-radioactive enzyme assays that can be used to quantify the levels of carbohydrate sulfation hampers kinetic analysis of this process and the discovery of HS2ST inhibitors. In the present paper, we describe a new procedure for thermal shift analysis of purified HS2ST. Using this approach, we quantify HS2ST-catalysed oligosaccharide sulfation using a novel synthetic fluorescent substrate and screen the Published Kinase Inhibitor Set, to evaluate compounds that inhibit catalysis. We report the susceptibility of HS2ST to a variety of cell-permeable compounds in vitro, including polyanionic polar molecules, the protein kinase inhibitor rottlerin and oxindole-based RAF kinase inhibitors. In a related study, published back-to-back with the present study, we demonstrated that tyrosyl protein sulfotranferases are also inhibited by a variety of protein kinase inhibitors. We propose that appropriately validated small-molecule compounds could become new tools for rapid inhibition of glycan (and protein) sulfation in cells, and that protein kinase inhibitors might be repurposed or redesigned for the specific inhibition of HS2ST.

Mice Deficient in Heparan Sulfate 6-O-Sulfotransferase-1 Exhibit Defective Heparan Sulfate Biosynthesis, Abnormal Placentation, and Late Embryonic Lethality

Heparan sulfate (HS) plays critical roles in a variety of developmental, physiological, and pathogenic processes due to its ability to interact in a structure-dependent manner with numerous growth factors that participate in cellular signaling. The divergent structures of HS glycosaminoglycans are the result of the coordinate actions of several N- and O-sulfotransferases, C5-epimerase, and 6-O-endosulfatases. We have shown that 6-O-sulfation of the glucosamine residues in HS are catalyzed by the sulfotransferases HS6ST-1, -2, and -3. To determine the biological and physiological importance of HS6ST-1, we now describe the creation of transgenic mice that lack this sulfotransferase. Most of our HS6ST-1-null mice died between embryonic day 15.5 and the perinatal stage, and those mice that survived were considerably smaller than their wild-type littermates. Some of these HS6ST-1-null mice exhibited development abnormalities, and histochemical and molecular analyses of these mice revealed an approximately 50% reduction in the number of fetal microvessels in the labyrinthine zone of the placenta relative to that in the wild-type mice. Because we observed a modest reduction in VEGF-A mRNA and protein in the tissues of HS6ST-1-null mice, an HS-dependent defect in cytokine signaling probably contributes to increased embryonic lethality and decreased growth. Biochemical studies of the HS chains isolated from various organs of our HS6ST-1-null mice revealed a marked reduction of GlcNAc(6SO(4)) and HexA-GlcNSO(3)(6SO(4)) levels and a reduced ability to bind Wnt2. Thus, despite the presence of three closely related 6-O-sulfotransferase genes in the mouse genome, HS6ST-1 is the primary one used in HS biosynthesis in most tissues.

Mutational Study of Heparan Sulfate 2-O-Sulfotransferase and Chondroitin Sulfate 2-O-Sulfotransferase

Heparan sulfate (HS) and chondroitin sulfate (CS) are highly sulfated polysaccharides with a wide range of biological functions. Heparan sulfate 2-O-sulfotransferase (HS-2OST) transfers the sulfo group from 3'-phosphoadenosine 5'-phosphosulfate (PAPS) to the 2-OH position of the hexauronic acid that is adjacent to N-sulfated glucosamine, whereas chondroitin sulfate 2-O-sulfotransferase (CS-2OST) transfers the sulfo group to the hexauronic acid that is adjacent to N-acetylated galactosamine. Here we report a systematic mutagenesis study of HS-2OST and CS-2OST based on their structural homology to estrogen sulfotransferase and HS 3-O-sulfotransferase isoform 3 (3-OST3), for which crystal structures exist. We have identified six residues possibly involved in binding to PAPS. HS-2OST carrying mutations of these residues lacks sulfotransferase activity and the ability to bind 3'-phosphoadenosine 5'-phosphate, a PAPS analogue, as determined by isothermal titration calorimetry. Similar residues involved in binding to PAPS were also identified in CS-2OST. Additional residues that participate in carbohydrate substrate binding were also identified in both enzymes. Mutations at these residues led to the loss of sulfotransferase activity but maintained the ability to bind to phosphoadenosine 5'-phosphate. The catalytic function of HS-2OST appears to involve two histidine residues (His140 and His142), whereas only one histidine (His168) of CS 2-OST is likely to be critical. This unique feature of HS 2-OST catalytic residues directed us to characterize the Drosophila heparan sulfate 2-O-sulfotransferase. The results from this study provide insight into the differences and similarities various residues play in the biological roles of the HS-2OST and CS-2OST enzymes.

Distinctive Expression Patterns of Heparan Sulfate O-Sulfotransferases and Regional Differences in Heparan Sulfate Structure in Chick Limb Buds

The skeletal tissue development and patterning in chick limb buds are known to be under the spacio-temporal control of various heparin-binding cell growth factors such as fibroblast growth factors and bone morphogenetic proteins. Different structural regions on heparan sulfate (HS) chains of proteoglycans could be implicated in regional differences in the binding capacities of these cell growth factors, by which they could selectively interact with targeted cells and regulate their signaling in those processes. In this study we first demonstrated by cDNA cloning that one heparan sulfate 2-O-sulfotransferase (HS2ST) and two isoforms of heparan sulfate 6-O-sulfotransferase (HS6ST-1 and -2) occurred in chick embryos and had different substrate specificities each other. We next showed by whole mount in situ hybridization that the HS6ST-1 and HS6ST-2 transcripts were preferentially localized to the anterior proximal region and at the posterior proximal region of the limb bud, respectively, whereas the HS2ST transcript was distributed rather uniformly throughout the bud. Analyses of the structures of HS from different regions of the wing buds have shown variation in that 6-O-sulfated residues are more abundant in the proximal than distal region, whereas iduronosyl 6-O-sulfated residues are abundant in the anterior proximal region and glucuronosyl 6-O-sulfated residues in the posterior proximal region. These results suggest that HS with different sulfation patterns created with multiple sulfotransferase activities provides an appropriate extracellular environment for morphogenetic signal transduction.

From Morphogen Gradients to Axon Guidance

Heparan sulfate proteoglycans (HSPs) interact with many cell-adhesion proteins and signaling proteins (see the Perspective by Blair ). Two groups investigated HSP interactions with key signaling proteins. Paine-Saunders et al. determined that the bone morphogenic protein (BMP) antagonist Noggin interacted with HSPs and was sequestered at the cell surface when expressed in Chinese hamster ovary cell lines that synthesized HSPs. Surface localization of Noggin appeared to be dependent on heparin sulfate, because Noggin was released from the cell surface by the addition of excess soluble heparin or when expressed in cells with mutations in enzymes essential for heparin sulfate biosynthesis. The heparin-bound Noggin retained biological activity as a BMP antagonist, and the interaction with HSPs may be one mechanism by which gradients of BMP activity are established in vivo. A second group, Ihrie et al. , analyzed how sulfation of specific sites on heparin sulfate contributes to the control of axon guidance in the Xenopus optic pathway. Axon targeting was disrupted when exogenous heparin sulfates were applied or when sulfation of the polysaccharides in the 2- O and 6- O positions was inhibited pharmacologically. Analysis of the expression of the enzymes required for sulfation at the 2- O [heparan sulfate 2- O sulfotransferase (HS2ST)] and 6- O (HS6ST) positions showed broad distribution of the HS2ST modification, but a highly restricted occurrence of HS6ST. Thus, the structure of the HSPs also plays a key role in controlling the interaction between these proteins and those that are important for mediating axon guidance. S. S. Blair, Wnts, signaling and sulfates. Science's STKE (2001), http://stke.sciencemag.org/cgi/content/full/OC_sigtrans;2001/101/pe32 [Abstract] [Full Text] S. Paine-Saunders, B. L. Viviano, A. N. Economides, S. Saunders, Heparan sulfate proteoglycans retain Noggin at the cell surface. J. Biol. Chem. 277 , 2089-2096 (2002). [Abstract] [Full Text] A. Ihrie, E. A. Yates, J. E. Turnbull, C. E. Holt, Specific heparan sulfate structures involved in retinal axon targeting. Development 129 , 61-70 (2002). [Online Journal]