Molecular Mechanism of Substrate Specificity for Heparan Sulfate 2-O-Sulfotransferase

Heparan sulfate (HS) is an abundant polysaccharide in the animal kingdom with essential physiological functions. HS is composed of sulfated saccharides that are biosynthesized through a complex pathway involving multiple enzymes. In vivo regulation of this process remains unclear. HS 2-O-sulfotransferase (2OST) is a key enzyme in this pathway. Here, we report the crystal structure of the ternary complex of 2OST, 3′-phosphoadenosine 5′-phosphate, and a heptasaccharide substrate. Utilizing site-directed mutagenesis and specific oligosaccharide substrate sequences, we probed the molecular basis of specificity and 2OST position in the ordered HS biosynthesis pathway. These studies revealed that Arg-80, Lys-350, and Arg-190 of 2OST interact with the N-sulfo groups near the modification site, consistent with the dependence of 2OST on N-sulfation. In contrast, 6-O-sulfo groups on HS are likely excluded by steric and electrostatic repulsion within the active site supporting the hypothesis that 2-O-sulfation occurs prior to 6-O-sulfation. Our results provide the structural evidence for understanding the sequence of enzymatic events in this pathway.     

N-Sulfation of Heparan Sulfate Regulates Early Branching Events in the Developing Mammary Gland

Branching morphogenesis, a fundamental process in the development of epithelial organs (e.g. breast, kidney, lung, salivary gland, prostate, pancreas), is in part dependent on sulfation of heparan sulfate proteoglycans. Proper sulfation is mediated by biosynthetic enzymes, including exostosin-2 (Ext2), N-deacetylase/N-sulfotransferases and heparan sulfate O-sulfotransferases. Recent conditional knockouts indicate that whereas primary branching is dependent on heparan sulfate, other stages are dependent upon selective addition of N-sulfate and/or 2-O sulfation (Crawford, B .E., Garner, O. B., Bishop, J. R., Zhang, D. Y., Bush, K. T., Nigam, S. K., and Esko, J. D. (2010) PLoS One 5, e10691; Garner, O .B., Bush, K. T., Nigam, S .K., Yamaguchi, Y., Xu, D., Esko, J. D., and Nigam, S. K. (2011) Dev. Biol. 355, 394–403). Here, we analyzed the effect of deleting both Ndst2 and Ndst1. Whereas deletion of Ndst1 has no major effect on primary or secondary branching, deletion of Ndst2 appears to result in a mild increase in branching. When both genes were deleted, ductal growth was variably diminished (likely due to variable Cre-recombinase activity), but an overabundance of branched structures was evident irrespective of the extent of gland growth or postnatal age. “Hyperbranching” is an unusual phenotype. The effects on N-sulfation and growth factor binding were confirmed biochemically. The results indicate that N-sulfation or a factor requiring N-sulfation regulates primary and secondary branching events in the developing mammary gland. Together with previous work, the data indicate that different stages of ductal branching and lobuloalveolar formation are regulated by distinct sets of heparan sulfate biosynthetic enzymes in an appropriate growth factor context.     

Insights into the N-Sulfation Mechanism: Molecular Dynamics Simulations of the N-Sulfotransferase Domain of Ndst1 and Mutants

Sulfation patterns along glycosaminoglycan (GAG) chains dictate their functional role. The N-deacetylase N-sulfotransferase family (NDST) catalyzes the initial downstream modification of heparan sulfate and heparin chains by removing acetyl groups from subsets of N-acetylglucosamine units and, subsequently, sulfating the residual free amino groups. These enzymes transfer the sulfuryl group from 3′-phosphoadenosine-5′-phosphosulfate (PAPS), yielding sulfated sugar chains and 3′-phosphoadenosine-5′-phosphate (PAP). For the N-sulfotransferase domain of NDST1, Lys833 has been implicated to play a role in holding the substrate glycan moiety close to the PAPS cofactor. Additionally, Lys833 together with His716 interact with the sulfonate group, stabilizing the transition state. Such a role seems to be shared by Lys614 through donation of a proton to the bridging oxygen of the cofactor, thereby acting as a catalytic acid. However, the relevance of these boundary residues at the hydrophobic cleft is still unclear. Moreover, whether Lys833, His716 and Lys614 play a role in both glycan recognition and glycan sulfation remains elusive. In this study we evaluate the contribution of NDST mutants (Lys833, His716 and Lys614) to dynamical effects during sulfate transfer using comprehensive combined docking and essential dynamics. In addition, the binding location of the glycan moiety, PAPS and PAP within the active site of NDST1 throughout the sulfate transfer were determined by intermediate state analysis. Furthermore, NDST1 mutants unveiled Lys833 as vital for both the glycan binding and subsequent N-sulfotransferase activity of NDST1.     

Ovarian Cancer Cell Heparan Sulfate 6-O-Sulfotransferases Regulate an Angiogenic Program Induced by Heparin-binding Epidermal Growth Factor (EGF)-like Growth Factor/EGF Receptor Signaling

Heparan sulfate (HS) is a component of cell surface and extracellular matrix proteoglycans that regulates numerous signaling pathways by binding and activating multiple growth factors and chemokines. The amount and pattern of HS sulfation are key determinants for the assembly of the trimolecular, HS-growth factor-receptor, signaling complex. Here we demonstrate that HS 6-O-sulfotransferases 1 and 2 (HS6ST-1 and HS6ST-2), which perform sulfation at 6-O position in glucosamine in HS, impact ovarian cancer angiogenesis through the HS-dependent HB-EGF/EGFR axis that subsequently modulates the expression of multiple angiogenic cytokines. Down-regulation of HS6ST-1 or HS6ST-2 in human ovarian cancer cell lines results in 30–50% reduction in glucosamine 6-O-sulfate levels in HS, impairing HB-EGF-dependent EGFR signaling and diminishing FGF2, IL-6, and IL-8 mRNA and protein levels in cancer cells. These cancer cell-related changes reduce endothelial cell signaling and tubule formation in vitro. In vivo, the development of subcutaneous tumor nodules with reduced 6-O-sulfation is significantly delayed at the initial stages of tumor establishment with further reduction in angiogenesis occurring throughout tumor growth. Our results show that in addition to the critical role that 6-O-sulfate moieties play in angiogenic cytokine activation, HS 6-O-sulfation level, determined by the expression of HS6ST isoforms in ovarian cancer cells, is a major regulator of angiogenic program in ovarian cancer cells impacting HB-EGF signaling and subsequent expression of angiogenic cytokines by cancer cells.     

Oligoribonuclease is a common downstream target of lithium-induced pAp accumulation in Escherichia coli and human cells

We identified Oligoribonuclease (Orn), an essential Escherichia coli protein and the only exonuclease degrading small ribonucleotides (5mer to 2mer) and its human homologue, small fragment nuclease (Sfn), in a screen for proteins that are potentially regulated by 3′-phosphoadenosine 5′-phosphate (pAp). We show that both enzymes are sensitive to micromolar amounts of pAp in vitro. We also demonstrate that Orn can degrade short DNA oligos in addition to its activity on RNA oligos, similar to what was documented for Sfn. pAp was shown to accumulate as a result of inhibition of the pAp-degrading enzyme by lithium, widely used to treat bipolar disorder, thus its regulatory targets are of significant medical interest. CysQ, the E.coli pAp-phosphatase is strongly inhibited by lithium and calcium in vitro and is a main target of lithium toxicity in vivo. Our findings point to remarkable conservation of the connection between sulfur- and RNA metabolism between E.coli and humans.     

The specificity of the malarial VAR2CSA protein for chondroitin sulfate depends on 4-O-sulfation and ligand accessibility

Placental malaria infection is mediated by the binding of the malarial VAR2CSA protein to the placental glycosaminoglycan, chondroitin sulfate. Recombinant subfragments of VAR2CSA (rVAR2) have also been shown to bind specifically and with high affinity to cancer cells and tissues, suggesting the presence of a shared type of oncofetal chondroitin sulfate (ofCS) in the placenta and in tumors. However, the exact structure of ofCS and what determines the selective tropism of VAR2CSA remains poorly understood. In this study, ofCS was purified by affinity chromatography using rVAR2 and subjected to detailed structural analysis. We found high levels of N-acetylgalactosamine 4-O-sulfation (∼80–85%) in placenta- and tumor-derived ofCS. This level of 4-O-sulfation was also found in other tissues that do not support parasite sequestration, suggesting that VAR2CSA tropism is not exclusively determined by placenta- and tumor-specific sulfation. Here, we show that both placenta and tumors contain significantly more chondroitin sulfate moieties of higher molecular weight than other tissues. In line with this, CHPF and CHPF2, which encode proteins required for chondroitin polymerization, are significantly upregulated in most cancer types. CRISPR/Cas9 targeting of CHPF and CHPF2 in tumor cells reduced the average molecular weight of cell-surface chondroitin sulfate and resulted in a marked reduction of rVAR2 binding. Finally, utilizing a cell-based glycocalyx model, we showed that rVAR2 binding correlates with the length of the chondroitin sulfate chains in the cellular glycocalyx. These data demonstrate that the total amount and cellular accessibility of chondroitin sulfate chains impact rVAR2 binding and thus malaria infection.     

The 3′-Phosphoadenosine 5′-Phosphosulfate Transporters,PAPST1 and 2, Contribute to the Maintenance and Differentiation of Mouse Embryonic Stem Cells

Recently, we have identified two 3′-phosphoadenosine 5′-phosphosulfate (PAPS) transporters (PAPST1 and PAPST2), which contribute to PAPS transport into the Golgi, in both human and Drosophila. Mutation and RNA interference (RNAi) of the Drosophila PAPST have shown the importance of PAPST-dependent sulfation of carbohydrates and proteins during development. However, the functional roles of PAPST in mammals are largely unknown. Here, we investigated whether PAPST-dependent sulfation is involved in regulating signaling pathways required for the maintenance of mouse embryonic stem cells (mESCs), differentiation into the three germ layers, and neurogenesis. By using a yeast expression system, mouse PAPST1 and PAPST2 proteins were shown to have PAPS transport activity with an apparent K_m value of 1.54 µM or 1.49 µM, respectively. RNAi-mediated knockdown of each PAPST induced the reduction of chondroitin sulfate (CS) chain sulfation as well as heparan sulfate (HS) chain sulfation, and inhibited mESC self-renewal due to defects in several signaling pathways. However, we suggest that these effects were due to reduced HS, not CS, chain sulfation, because knockdown of mouse N-deacetylase/N-sulfotransferase, which catalyzes the first step of HS sulfation, in mESCs gave similar results to those observed in PAPST-knockdown mESCs, but depletion of CS chains did not. On the other hand, during embryoid body formation, PAPST-knockdown mESCs exhibited abnormal differentiation, in particular neurogenesis was promoted, presumably due to the observed defects in BMP, FGF and Wnt signaling. The latter were reduced as a result of the reduction in both HS and CS chain sulfation. We propose that PAPST-dependent sulfation of HS or CS chains, which is regulated developmentally, regulates the extrinsic signaling required for the maintenance and normal differentiation of mESCs.     

Analysis of Drosophila Glucuronyl C5-Epimerase: IMPLICATIONS FOR DEVELOPMENTAL ROLES OF HEPARAN SULFATE SULFATION COMPENSATION AND 2-O-SULFATED GLUCURONIC ACID*

During the biosynthesis of heparan sulfate (HS), glucuronyl C5-epimerase (Hsepi) catalyzes C5-epimerization of glucuronic acid (GlcA), converting it to iduronic acid (IdoA). Because HS 2-O-sulfotransferase (Hs2st) shows a strong substrate preference for IdoA over GlcA, C5-epimerization is required for normal HS sulfation. However, the physiological significance of C5-epimerization remains elusive. To understand the role of Hsepi in development, we isolated Drosophila Hsepi mutants. Homozygous mutants are viable and fertile with only minor morphological defects, including the formation of an ectopic crossvein in the wing, but they have a short lifespan. We propose that two mechanisms contribute to the mild phenotypes of Hsepi mutants: HS sulfation compensation and possible developmental roles of 2-O-sulfated GlcA (GlcA2S). HS disaccharide analysis showed that loss of Hsepi resulted in a significant impairment of 2-O-sulfation and induced compensatory increases in N- and 6-O-sulfation. Simultaneous block of Hsepi and HS 6-O-sulfotransferase (Hs6st) activity disrupted tracheoblast formation, a well established FGF-dependent process. This result suggests that the increase in 6-O-sulfation in Hsepi mutants is critical for the rescue of FGF signaling. We also found that the ectopic crossvein phenotype can be induced by expression of a mutant form of Hs2st with a strong substrate preference for GlcA-containing units, suggesting that this phenotype is associated with abnormal GlcA 2-O-sulfation. Finally, we show that Hsepi formed a complex with Hs2st and Hs6st in S2 cells, raising the possibility that this complex formation contributes to the close functional relationships between these enzymes.     

A 3D-structural model of unsulfated chondroitin from high-field NMR: 4-sulfation has little effect on backbone conformation

The glycosaminoglycan chondroitin sulfate is essential in human health and disease but exactly how sulfation dictates its 3D-strucutre at the atomic level is unclear. To address this, we have purified homogenous oligosaccharides of unsulfated chondroitin (with and without ¹⁵N-enrichment) and analysed them by high-field NMR to make a comparison published chondroitin sulfate and hyaluronan 3D-structures. The result is the first full assignment of the tetrasaccharide and an experimental 3D-model of the hexasaccharide (PDB code 2KQO). In common with hyaluronan, we confirm that the amide proton is not involved in strong, persistent inter-residue hydrogen bonds. However, in contrast to hyaluronan, a hydrogen bond is not inferred between the hexosamine OH-4 and the glucuronic acid O5 atoms across the β(1→3) glycosidic linkage. The unsulfated chondroitin bond geometry differs slightly from hyaluronan by rotation about the β(1→3) ψ dihedral (as previously predicted by simulation), while the β(1→4) linkage is unaffected. Furthermore, comparison shows that this glycosidic linkage geometry is similar in chondroitin-4-sulfate. We therefore hypothesise that both hexosamine OH-4 and OH-6 atoms are solvent exposed in chondroitin, explaining why it is amenable to sulfation and hyaluronan is not, and also that 4-sulfation has little effect on backbone conformation. Our conclusions exemplify the value of the 3D-model presented here and progress our understanding of glycosaminoglycan molecular properties.     

Blood cells transcriptomics as source of potential biomarkers of articular health improvement: effects of oral intake of a rooster combs extract rich in hyaluronic acid

The aim of the study was to explore peripheral blood gene expression as a source of biomarkers of joint health improvement related to glycosaminoglycan (GAG) intake in humans. Healthy individuals with joint discomfort were enrolled in a randomized, double-blind, placebo-controlled intervention study in humans. Subjects ate control yoghurt or yoghurt supplemented with a recently authorized novel food in Europe containing hyaluronic acid (65 %) from rooster comb (Mobilee™ as commercial name) for 90 days. Effects on functional quality-of-life parameters related to joint health were assessed. Whole-genome microarray analysis of peripheral blood samples from a subset of 20 subjects (10 placebo and 10 supplemented) collected pre- and post-intervention was performed. Mobilee™ supplementation reduced articular pain intensity and synovial effusion and improved knee muscular strength indicators as compared to placebo. About 157 coding genes were differentially expressed in blood cells between supplemented and placebo groups post-intervention, but not pre-intervention (p < 0.05; fold change ≥1.2). Among them, a reduced gene expression of glucuronidase-beta (GUSB), matrix metallopeptidase 23B (MMP23B), xylosyltransferase II (XYLT2), and heparan sulfate 6-O-sulfotransferase 1 (HS6ST1) was found in the supplemented group. Correlation analysis indicated a direct relationship between blood cell gene expression of MMP23B, involved in the breakdown of the extracellular matrix, and pain intensity, and an inverse relationship between blood cell gene expression of HS6ST1, responsible for 6-O-sulfation of heparan sulfate, and indicators of knee muscular strength. Expression levels of specific genes in blood cells, in particular genes related to GAG metabolism and extracellular matrix dynamics, are potential biomarkers of beneficial effects on articular health.

Electronic supplementary material

The online version of this article (doi:10.1007/s12263-014-0417-3) contains supplementary material, which is available to authorized users.     

Microbial Transformations of Natural Antitumor Agents: Products of Rotenone and Dihydrorotenone Transformation by Cunninghamella blakesleeana

Various species of Absidia, Aspergillus, Cunninghamella, Trichothecium, Penicillium, and Phanerochaete were found to transform rotenone to one or more metabolites. Two biotransformation products were isolated from a preparative-scale incubation of rotenone with Cunninghamella blakesleeana and identified as 1′,2′-dihydro-1′,2′-dihydroxyrotenone and 3′-hydroxyrotenone (amorphigenin). The catalytic reduction of the isopropylene side chain of rotenone resulted in the formation of 1′,2′-dihydrorotenone. The latter was transformed by C. blakesleeana to 2′-hydroxy-1′,2′-dihydrorotenone.     

Arylsulfatase B regulates interaction of chondroitin-4-sulfate and kininogen in renal epithelial cells

The enzyme arylsulfatase B (N-acetylgalactosamine 4-sulfatase; ASB; ARSB), which removes 4-sulfate groups from the nonreducing end of chondroitin-4-sulfate (C4S;CSA) and dermatan sulfate, has cellular effects, beyond those associated with the lysosomal storage disease mucopolysaccharidosis VI. Previously, reduced ASB activity was reported in cystic fibrosis patients and in malignant human mammary epithelial cell lines in tissue culture compared to normal cells. ASB silencing and overexpression were associated with alterations in syndecan-1 and decorin expression in MCF-7 cells and in IL-8 secretion in human bronchial epithelial cells. In this report, we present the role of ASB in the regulation of the kininogen–bradykinin axis owing to its effect on chondroitin-4-sulfation and the interaction of C4S with kininogen. Silencing or overexpression of ASB in normal rat kidney epithelial cells in tissue culture modified the content of total sulfated glycosaminoglycans (sGAGs), C4S, kininogen, and bradykinin in spent media and cell lysates. Treatment of the cultured cells with chondroitinase ABC also increased the secretion of bradykinin into the spent media and reduced the C4S-associated kininogen. When ASB was overexpressed, the cellular kininogen that associated with C4S declined, suggesting a vital role for chondroitin-4-sulfation in regulating the kininogen–C4S interaction. These findings suggest that ASB, owing to its effect on chondroitin-4-sulfation, may impact on the kininogen–bradykinin axis and, thereby, may influence blood pressure.Because ASB activity is influenced by several ions, including chloride and phosphate, ASB activity may provide a link between salt responsiveness and the bradykinin-associated mechanism of blood pressure regulation.     

Changes in versican and chondroitin sulfate proteoglycans during structural development of the lung

We have examined whether changes in versican levels, or in the sulfation pattern of its chondroitin sulfate (CS) side chains, are associated with the reduction in perialveolar tissue volumes that characterize lung maturation in late-gestation fetal sheep. Lung tissue was collected from fetuses [90-142 days gestational age (GA)] and lambs (2 wk after term birth). The level and distribution of versican and CS glycosaminoglycans (GAG) were determined using immunohistochemistry, whereas fluorophore-assisted carbohydrate electrophoresis was used to determine changes in CS sulfation patterns. Versican was the predominant CS-containing proteoglycan in the lung and decreased from 19.9 +/- 2.7 arbitrary units at 90 days GA to 6.0 +/- 0.5 arbitrary units at 142 days GA, in close association (P < 0.05) with the reduction in tissue volumes (from 66.0 +/- 4.6 to 25.3 +/- 1.5% at 142 days); similar reductions occurred for both chondroitin-6-sulfate and chondroitin-4-sulfate CS side chains. Hyaluronic acid levels decreased from 3,168 +/- 641 pmol/microg GAG at 90 days GA to 126 +/- 9 pmol/microg GAG at 142 days GA, and the predominant sulfated disaccharide changed from Delta-di-6S at 90 days GA to Delta-di-4S at term. These data indicate that structural development of the lung is closely associated with marked changes in versican levels and the microstructure of CS side chains in perisaccular/alveolar lung tissue.     

Investigation of catalysis by bacterial RNase P via LNA and other modifications at the scissile phosphodiester

We analyzed cleavage of precursor tRNAs with an LNA, 2′-OCH₃, 2′-H or 2′-F modification at the canonical (c₀) site by bacterial RNase P. We infer that the major function of the 2′-substituent at nt −1 during substrate ground state binding is to accept an H-bond. Cleavage of the LNA substrate at the c₀ site by Escherichia coli RNase P RNA demonstrated that the transition state for cleavage can in principle be achieved with a locked C3′ -endo ribose and without the H-bond donor function of the 2′-substituent. LNA and 2′-OCH₃ suppressed processing at the major aberrant m₋₁ site; instead, the m₊₁ (nt +1/+2) site was utilized. For the LNA variant, parallel pathways leading to cleavage at the c₀ and m₊₁ sites had different pH profiles, with a higher Mg²⁺ requirement for c₀ versus m₊₁ cleavage. The strong catalytic defect for LNA and 2′-OCH₃ supports a model where the extra methylene (LNA) or methyl group (2′-OCH₃) causes a steric interference with a nearby bound catalytic Mg²⁺ during its recoordination on the way to the transition state for cleavage. The presence of the protein cofactor suppressed the ground state binding defects, but not the catalytic defects.     

Partial Purification and Characterization of a 3'- Phosphoadenosine 5' -Phosphosulfate: Desulfoglucosinolate Sulfotransferase from Cress (Lepidium sativum)

A 3′ -phosphoadenosine 5′ -phosphosulfate (PAPS):desulfoglucosinolate sulfotransferase (EC 2.8.2-) was extensively purified from light-grown cress (Lepidium sativum L.) seedlings by gel filtration and concanavalin A-Sepharose 4B, Matrex Gel Green A, and Mono Q fast protein liquid chromatography. The purified enzyme, which required bovine serum albumin for stabilization, had a native molecular weight of 31,000 ± 5,000 and an apparent isoelectric point of 5.2. Using PAPS (K_m 60 micromolar) as sulfur donor, it catalyzed the sulfation of desulfobenzylglucosinolate (K_m 82 micromolar), desulfo-p-hydroxybenzylglucosinolate (K_m 670 micromolar), and desulfoallylglucosinolate (K_m 6.5 millimolar) at an optimal pH of 9.0. All other potential substrates tested, including flavonoids, flavonoid glycosides, cinnamic acids, and phenylacetaldoxime, were not sulfated. Sulfotransferase activity was stimulated by MgCl₂, MnCl₂ and reducing agents and inhibited by ZnCl₂, PbNO₃ NiCl₂ and the reaction product PAP. The thiol reagents N-ethylmaleimide, p-chloromercuriphenylsulfonic acid, and 5,5′ -dithio-bis-(2-nitrobenzoic acid) were also potent inhibitors, but the enzyme was protected from covalent modification by β-mercaptoethanol. The kinetics of desulfobenzylglucosinolate sulfation were consistent with a rapid equilibrium ordered mechanism with desulfobenzylglucosinolate binding first and PAPS second.     

A Computational Approach for Identifying the Chemical Factors Involved in the Glycosaminoglycans-Mediated Acceleration of Amyloid Fibril Formation

Background

Amyloid fibril formation is the hallmark of many human diseases, including Alzheimer''s disease, type II diabetes and amyloidosis. Amyloid fibrils deposit in the extracellular space and generally co-localize with the glycosaminoglycans (GAGs) of the basement membrane. GAGs have been shown to accelerate the formation of amyloid fibrils in vitro for a number of protein systems. The high number of data accumulated so far has created the grounds for the construction of a database on the effects of a number of GAGs on different proteins.

Methodology/Principal Findings

In this study, we have constructed such a database and have used a computational approach that uses a combination of single parameter and multivariate analyses to identify the main chemical factors that determine the GAG-induced acceleration of amyloid formation. We show that the GAG accelerating effect is mainly governed by three parameters that account for three-fourths of the observed experimental variability: the GAG sulfation state, the solute molarity, and the ratio of protein and GAG molar concentrations. We then combined these three parameters into a single equation that predicts, with reasonable accuracy, the acceleration provided by a given GAG in a given condition.

Conclusions/Significance

In addition to shedding light on the chemical determinants of the protein∶GAG interaction and to providing a novel mathematical predictive tool, our findings highlight the possibility that GAGs may not have such an accelerating effect on protein aggregation under the conditions existing in the basement membrane, given the values of salt molarity and protein∶GAG molar ratio existing under such conditions.     

Heparan Sulfate 2-O-Sulfotransferase Is Required for Triglyceride-rich Lipoprotein Clearance

Hepatic clearance of triglyceride-rich lipoproteins depends on heparan sulfate and low density lipoprotein receptors expressed on the basal membrane of hepatocytes. Binding and uptake of the lipoproteins by way of heparan sulfate depends on the degree of sulfation of the chains based on accumulation of plasma triglycerides and delayed clearance of triglyceride-rich lipoproteins in mice bearing a hepatocyte-specific alteration of N-acetylglucosamine (GlcNAc) N-deacetylase-N-sulfotransferase 1 (Ndst1) (MacArthur, J. M., Bishop, J. R., Stanford, K. I., Wang, L., Bensadoun, A., Witztum, J. L., and Esko, J. D. (2007) J. Clin. Invest. 117, 153–164). Inactivation of Ndst1 led to decreased overall sulfation of heparan sulfate due to coupling of uronyl 2-O-sulfation and glucosaminyl 6-O-sulfation to initial N-deacetylation and N-sulfation of GlcNAc residues. To determine whether lipoprotein clearance depends on 2-O-and 6-O-sulfation, we evaluated plasma triglyceride levels in mice containing loxP-flanked conditional alleles of uronyl 2-O-sulfotransferase (Hs2st^f/f) and glucosaminyl 6-O-sulfotransferase-1 (Hs6st1^f/f) and the bacterial Cre recombinase expressed in hepatocytes from the rat albumin (Alb) promoter. We show that Hs2st^f/fAlbCre⁺ mice accumulated plasma triglycerides and exhibited delayed clearance of intestinally derived chylomicrons and injected human very low density lipoproteins to the same extent as observed in Ndst1^f/fAlbCre⁺ mice. In contrast, Hs6st1^f/fAlbCre⁺ mice did not exhibit any changes in plasma triglycerides. Chemically modified heparins lacking N-sulfate and 2-O-sulfate groups did not block very low density lipoprotein binding and uptake in isolated hepatocytes, whereas heparin lacking 6-O-sulfate groups was as active as unaltered heparin. Our findings show that plasma lipoprotein clearance depends on specific subclasses of sulfate groups and not on overall charge of the chains.     

Characterization of the 2',3' cyclic phosphodiesterase activities of Clostridium thermocellum polynucleotide kinase-phosphatase and bacteriophage lambda phosphatase

Clostridium thermocellum polynucleotide kinase-phosphatase (CthPnkp) catalyzes 5′ and 3′ end-healing reactions that prepare broken RNA termini for sealing by RNA ligase. The central phosphatase domain of CthPnkp belongs to the dinuclear metallophosphoesterase superfamily exemplified by bacteriophage λ phosphatase (λ-Pase). CthPnkp is a Ni²⁺/Mn²⁺-dependent phosphodiesterase-monoesterase, active on nucleotide and non-nucleotide substrates, that can be transformed toward narrower metal and substrate specificities via mutations of the active site. Here we characterize the Mn²⁺-dependent 2′,3′ cyclic nucleotide phosphodiesterase activity of CthPnkp, the reaction most relevant to RNA repair pathways. We find that CthPnkp prefers a 2′,3′ cyclic phosphate to a 3′,5′ cyclic phosphate. A single H189D mutation imposes strict specificity for a 2′,3′ cyclic phosphate, which is cleaved to form a single 2′-NMP product. Analysis of the cyclic phosphodiesterase activities of mutated CthPnkp enzymes illuminates the active site and the structural features that affect substrate affinity and k_cat. We also characterize a previously unrecognized phosphodiesterase activity of λ-Pase, which catalyzes hydrolysis of bis-p-nitrophenyl phosphate. λ-Pase also has cyclic phosphodiesterase activity with nucleoside 2′,3′ cyclic phosphates, which it hydrolyzes to yield a mixture of 2′-NMP and 3′-NMP products. We discuss our results in light of available structural and functional data for other phosphodiesterase members of the binuclear metallophosphoesterase family and draw inferences about how differences in active site composition influence catalytic repertoire.