Regulation of heparan sulfate 6-O-sulfation by beta-secretase activity

The enzymes involved in glycosaminoglycan chain biosynthesis are mostly Golgi resident proteins, but some are secreted extracellularly. For example, the activities of heparan sulfate 6-O-sulfotransferase (HS6ST) and heparan sulfate 3-O-sulfotransferase are detected in the serum as well in the medium of cell lines. However, the biological significance of this is largely unknown. Here we have investigated by means of monitoring green fluorescent protein (GFP) fluorescence how C-terminally GFP-tagged HS6STs that are stably expressed in CHO-K1 cell lines are secreted/shed. Brefeldin A and monensin treatments revealed that the N-terminal hydrophobic domain of HS6ST3 is processed in the endoplasmic reticulum or cis/medial Golgi. Treatment of HS6ST3-GFP-expressing cells with various protease inhibitors revealed that the cell-permeable beta-secretase inhibitor N-benzyloxycarbonyl-Val-Leu-leucinal (Z-VLL-CHO) specifically inhibits HS6ST secretion, although this effect was specific for HS6ST3 but not for HS6ST1 and HS6ST2. However, Z-VLL-CHO treatment did not increase the molecular size of the HS6ST3-GFP that accumulated in the cell. Z-VLL-CHO treatment also induced the intracellular accumulation of SP-HS6ST3(-TMD)-GFP, a modified secretory form of HS6ST3 that has the preprotrypsin leader sequence as its N-terminal hydrophobic domain. Diminishment of beta-secretase activity by coexpressing the amyloid precursor protein of a Swedish mutant, a potent beta-secretase substrate, also induced intracellular HS6ST3-GFP accumulation. Moreover, Z-VLL-CHO treatment increased the 6-O-sulfate (6S) levels of HS, especially in the disaccharide unit of hexuronic acid-GlcNS(6S). Thus, the HS6ST3 enzyme in the Golgi apparatus and therefore the 6-O sulfation of heparan sulfates in the cell are at least partly regulated by beta-secretase via an indirect mechanism.     

Interaction of hemojuvelin with neogenin results in iron accumulation in human embryonic kidney 293 cells

Type 2 hereditary hemochromatosis (HH) or juvenile hemochromatosis is an early onset, genetically heterogeneous, autosomal recessive disorder of iron overload. Type 2A HH is caused by mutations in the recently cloned hemojuvelin gene (HJV; also called HFE2) (Papanikolaou, G., Samuels, M. E., Ludwig, E. H., MacDonald, M. L., Franchini, P. L., Dube, M. P., Andres, L., MacFarlane, J., Sakellaropoulos, N., Politou, M., Nemeth, E., Thompson, J., Risler, J. K., Zaborowska, C., Babakaiff, R., Radomski, C. C., Pape, T. D., Davidas, O., Christakis, J., Brissot, P., Lockitch, G., Ganz, T., Hayden, M. R., and Goldberg, Y. P. (2004) Nat. Genet. 36, 77-82), whereas Type 2B HH is caused by mutations in hepcidin. HJV is highly expressed in both skeletal muscle and liver. Mutations in HJV are implicated in the majority of diagnosed juvenile hemochromatosis patients. In this study, we stably transfected HJV cDNA into human embryonic kidney 293 cells and characterized the processing of HJV and its effect on iron homeostasis. Our results indicate that HJV is a glycosylphosphatidylinositol-linked protein and undergoes a partial autocatalytic cleavage during its intracellular processing. HJV co-immunoprecipitated with neogenin, a receptor involved in a variety of cellular signaling processes. It did not interact with the closely related receptor DCC (deleted in Colon Cancer). In addition, the HJV G320V mutant implicated in Type 2A HH did not co-immunoprecipitate with neogenin. Immunoblot analysis of ferritin levels and transferrin-55Fe accumulation studies indicated that the HJV-induced increase in intracellular iron levels in human embryonic kidney 293 cells is dependent on the presence of neogenin in the cells, thus linking these two proteins to intracellular iron homeostasis.     

6-O-sulfation of heparan sulfate differentially regulates various fibroblast growth factor-dependent signalings in culture

Heparan sulfate (HS) interacts with diverse heparin-binding growth factors and thereby regulates their bioactivities. These interactions depend on the structures characterized by the sulfation pattern and isomer of uronic acid residues. One of the biosynthetic modifications of HS, namely 6-O-sulfation, is catalyzed by three isoforms of HS6-O-sulfotransferase. We generated HS6ST-1- and/or HS6ST-2-deficient mice (6ST1-KO, 6ST2-KO, and double knock-out (dKO)) that exhibited different phenotypes. We examined the effects of HS 6-O-sulfation in heparin-binding growth factor signaling using fibroblasts derived from these mutant mice. Mouse embryonic fibroblasts (MEF) prepared from E14.5 dKO mice produced HS with little 6-O-sulfate, whereas 2-O-sulfation in HS from dKO-MEF (dKO-HS) was increased by 1.9-fold. HS6-O-sulfotransferase activity in the dKO-MEF was hardly detected, and HS2-O-sulfotransferase activity was 1.5-fold higher than that in wild type (WT)-MEFs. The response of dKO-MEFs to fibroblast growth factors (FGFs) was distinct from that of WT-MEFs; in dKO-MEFs, FGF-4- and FGF-2-dependent signalings were reduced to approximately 30 and 60% of WT-MEFs, respectively, and FGF-1-dependent signaling was moderately reduced compared with that of WT-MEFs but only at the lower FGF-1 concentrations. Analysis with a surface plasmon resonance biosensor demonstrated that the apparent affinity of dKO-HS for FGF-4 was markedly reduced and was also reduced for FGF-1. In contrast, the affinity of dKO-HS for FGF-2 was 2.5-fold higher than that of HS from WT-MEFs. Thus, 6-O-sulfate in HS may regulate the signalings of some of HB-GFs, including FGFs, by inducing different interactions between ligands and their receptors.     

Structural diversity of N-sulfated heparan sulfate domains: distinct modes of glucuronyl C5 epimerization, iduronic acid 2-O-sulfation, and glucosamine 6-O-sulfation

The N-sulfated regions (NS domains) represent the modified sequences of heparan sulfate chains and mediate interactions of the polysaccharide with proteins. We have investigated the relationship between the type/extent of polymer modification and the length of NS domains in heparan sulfate species from human aorta, bovine kidney, and cultured NMuMG and MDCK cells. C5 epimerization of D-glucuronic acid to L-iduronic acid was found to be extensive and essentially similar in all heparan sulfate species studied, regardless of domain size, whereas the subsequent 2-O-sulfation of the formed iduronic acid residues varies appreciably. In aorta heparan sulfate, up to 90% of the formed iduronate residues were 2-O-sulfated, whereas in kidney heparan sulfate 2-O-sulfation occurred only in 相似文献   

Overexpressing human membrane proteins in stably transfected and clonal human embryonic kidney 293S cells

X-ray crystal structures of human membrane proteins, although potentially of extremely great impact, are highly underrepresented relative to those of prokaryotic membrane proteins. One key reason for this is that human membrane proteins can be difficult to express at a level, and at a quality, suitable for structural studies. This protocol describes the methods that we use to overexpress human membrane proteins from clonal human embryonic kidney 293 (HEK293S) cells lacking N-acetylglucosaminyltransferase I (GnTI(-)), and was recently used in our 2.1-? X-ray crystal structure determination of human RhCG. Upon identification of highly expressing cell lines, suspension cell cultures are scaled up in a facile manner either using spinner flasks or cellbag bioreactors, resulting in a final purified yield of ～0.5 mg of membrane protein per liter of medium. The protocol described here is reliable and cost effective, can be used to express proteins that would otherwise be toxic to mammalian cells and can be completed in 8-10 weeks.     

Isolation and physicochemical properties of tankyrase of human embryonic kidney cells of line 293

We have isolated and purified endogenous cytosolic tankyrase from human embryonic kidney cells of line 293. Our data confirm a model of De Rycker and Price who consider that tankyrase is a master scaffolding protein capable of regulating assembly of large protein complexes. We have also studied kinetic characteristics of tankyrase in the complex, pH dependence of the enzyme activity, and its physicochemical properties.     

Distinct arachidonate-releasing functions of mammalian secreted phospholipase A2s in human embryonic kidney 293 and rat mastocytoma RBL-2H3 cells through heparan sulfate shuttling and external plasma membrane mechanisms

We analyzed the ability of a diverse set of mammalian secreted phospholipase A(2) (sPLA(2)) to release arachidonate for lipid mediator generation in two transfected cell lines. In human embryonic kidney 293 cells, the heparin-binding enzymes sPLA(2)-IIA, -IID, and -V promote stimulus-dependent arachidonic acid release and prostaglandin E(2) production in a manner dependent on the heparan sulfate proteoglycan glypican. In contrast, sPLA(2)-IB, -IIC, and -IIE, which bind weakly or not at all to heparanoids, fail to elicit arachidonate release, and addition of a heparin binding site to sPLA(2)-IIC allows it to release arachidonate. Heparin nonbinding sPLA(2)-X liberates arachidonic acid most likely from the phosphatidylcholine-rich outer plasma membrane in a glypican-independent manner. In rat mastocytoma RBL-2H3 cells that lack glypican, sPLA(2)-V and -X, which are unique among sPLA(2)s in being able to hydrolyze phosphatidylcholine-rich membranes, act most likely on the extracellular face of the plasma membrane to markedly augment IgE-dependent immediate production of leukotriene C(4) and platelet-activating factor. sPLA(2)-IB, -IIA, -IIC, -IID, and -IIE exert minimal effects in RBL-2H3 cells. These results are also supported by studies with sPLA(2) mutants and immunocytostaining and reveal that sPLA(2)-dependent lipid mediator generation occur by distinct (heparanoid-dependent and -independent) mechanisms in HEK293 and RBL-2H3 cells.     

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.     

Undersulfation of heparan sulfate restricts differentiation potential of mouse embryonic stem cells

Heparan sulfate proteoglycans, present on cell surfaces and in the extracellular matrix, interact with growth factors and morphogens to influence growth and differentiation of cells. The sulfation pattern of the heparan sulfate chains formed during biosynthesis in the Golgi compartment will determine the interaction potential of the proteoglycan. The glucosaminyl N-deacetylase/N-sulfotransferase (NDST) enzymes have a key role during biosynthesis, greatly influencing total sulfation of the heparan sulfate chains. The differentiation potential of mouse embryonic stem cells lacking both NDST1 and NDST2 was studied using in vitro differentiation protocols, expression of differentiation markers, and assessment of the ability of the cells to respond to growth factors. The results show that NDST1 and NDST2 are dispensable for mesodermal differentiation into osteoblasts but necessary for induction of adipocytes and neural cells. Gene expression analysis suggested a differentiation block at the primitive ectoderm stage. Also, GATA4, a primitive endoderm marker, was expressed by these cells. The addition of FGF4 or FGF2 together with heparin rescued the differentiation potential to neural progenitors and further to mature neurons and glia. Our results suggest that the embryonic stem cells lacking both NDST1 and NDST2, expressing a very low sulfated heparan sulfate, can take the initial step toward differentiation into all three germ layers. Except for their potential for mesodermal differentiation into osteoblasts, the cells are then arrested in a primitive ectoderm and/or endoderm stage.     

Overexpression of different isoforms of glucosaminyl N-deacetylase/N-sulfotransferase results in distinct heparan sulfate N-sulfation patterns

Functional interactions of heparan sulfate (HS) with selected proteins depend on distinct saccharide sequences which are generated during biosynthesis of the polysaccharide. Glucosaminyl N-deacetylase/N-sulfotransferases (NDSTs) catalyze both the N-deacetylation and N-sulfation reactions that initiate the modification of the (GlcNAc-GlcA)(n) polysaccharide backbone. The N-acetyl/N-sulfate exchange is restricted to certain regions of the polysaccharide chains, and only these can be further modified by glucuronyl C5-epimerization and O-sulfation at various positions. To investigate whether NDST isoforms influenced differently the structure of HS, murine NDST-1 was overexpressed in human kidney 293 cells, and the structure of the HS produced was compared to HS from NDST-2 overexpressing cells [Cheung, W. F., Eriksson, I., Kusche-Gullberg M., Lindahl, U., and Kjellén, L. (1996) Biochemistry 35, 5250-5256]. The level of N-sulfation increased from 40% in control cells to 60% and 80%, respectively, in NDST-1 and NDST-2 transfected cells. Interestingly, the increase in N-sulfation was accompanied by an increased chain length, while no effect on IdoA content or O-sulfation was seen. The most extended N-sulfated domains were found in HS synthesized by NDST-2 transfected cells. Since both the N-deacetylase and the N-sulfotransferase activities were lower in these cells than in the NDST-1 overexpressing cells, we conclude that, in addition to the level of enzyme expression, the NDST isoform also is important in determining the N-sulfation pattern in HS.     

Heparan sulfate in chronic kidney diseases: Exploring the role of 3-O-sulfation

One of the main feature of chronic kidney disease is the development of renal fibrosis. Heparan Sulfate (HS) is involved in disease development by modifying the function of growth factors and cytokines and creating chemokine gradients. In this context, we aimed to understand the function of HS sulfation in renal fibrosis. Using a mouse model of renal fibrosis, we found that total HS 2-O-sulfation was increased in damaged kidneys, whilst, tubular staining of HS 3-O-sulfation was decreased. The expression of HS modifying enzymes significantly correlated with the development of fibrosis with HS3ST1 demonstrating the strongest correlation. The pro-fibrotic factors TGFβ1 and TGFβ2/IL1β significantly downregulated HS3ST1 expression in both renal epithelial cells and renal fibroblasts. To determine the implication of HS3ST1 in growth factor binding and signalling, we generated an in vitro model of renal epithelial cells overexpressing HS3ST1 (HKC8-HS3ST1). Heparin Binding EGF like growth factor (HB-EGF) induced rapid, transient STAT3 phosphorylation in control HKC8 cells. In contrast, a prolonged response was demonstrated in HKC8-HS3ST1 cells. Finally, we showed that both HS 3-O-sulfation and HB-EGF tubular staining were decreased with the development of fibrosis. Taken together, these data suggest that HS 3-O-sulfation is modified in fibrosis and highlight HS3ST1 as an attractive biomarker of fibrosis progression with a potential role in HB-EGF signalling.     

Latent membrane protein 1 is dispensable for Epstein-Barr virus replication in human embryonic kidney 293 cells

Epstein Barr Virus (EBV) replicates in oral epithelial cells and gains entry to B-lymphocytes. In B-lymphocytes, EBV expresses a restricted subset of genes, the Latency III program, which converts B-lymphocytes to proliferating lymphoblasts. Latent Membrane Protein 1 (LMP1) and the other Latency III associated proteins are also expressed during virus replication. LMP1 is essential for virus replication and egress from Akata Burkitt Lymphoma cells, but a role in epithelial cell replication has not been established. Therefore, we have investigated whether LMP1 enhances EBV replication and egress from HEK293 cells, a model epithelial cell line used for EBV recombinant molecular genetics. We compared wild type (wt) and LMP1-deleted (LMP1Δ) EBV bacterial artificial chromosome (BAC) based virus replication and egress from HEK293. Following EBV immediate early Zta protein induction of EBV replication in HEK293 cells, similar levels of EBV proteins were expressed in wt- and LMP1Δ-infected HEK293 cells. LMP1 deletion did not impair EBV replication associated DNA replication, DNA encapsidation, or mature virus release. Indeed, virus from LMP1Δ-infected HEK293 cells was as infectious as EBV from wt EBV infected HEK cells. Trans-complementation with LMP1 reduced Rta expression and subsequent virus production. These data indicate that LMP1 is not required for EBV replication and egress from HEK293 cells.     

Soluble tankyrase located in cytosol of human embryonic kidney cell line 293

We studied the subcellular localization of tankyrase in primary and immortalized human cell cultures. In embryonic kidney cell line 293 the enzyme was excluded from the nuclei and distributed in fractions of soluble cytosolic proteins and low-density microsomes. Newly revealed cytosolic tankyrase in its poly(ADP-ribosyl)ated form was passed through a Sepharose 2B column and eluted as an apparently monomeric protein. The cytosolic localization of the enzyme correlated with its relatively high activity in the 293 cell line in comparison to eight other studied cell types.     

Presence of unsulfated heparan chains on the heparan sulfate proteoglycan of human colon carcinoma cells. Implications for heparan sulfate proteoglycan biosynthesis

We provide direct evidence for the presence of unsulfated, but fully elongated heparan glycosaminoglycans covalently linked to the protein core of a heparan sulfate proteoglycan synthesized by human colon carcinoma cells. Chemical and enzymatic studies revealed that a significant proportion of these chains contained glucuronic acid and N-acetylated glucosamine moieties, consistent with N-acetylheparosan, an established precursor of heparin and heparan sulfate. The presence of unsulfated chains was not dependent upon the exogenous supply of sulfate since their synthesis, structure, or relative amount did not vary with low exogenous sulfate concentrations. Culture in sulfate-free medium also failed to generate undersulfated heparan sulfate-proteoglycan, but revealed an endogenous source of sulfate which was primarily derived from the catabolism of the sulfur-containing amino acids methionine and cysteine. Furthermore, the presence of unsulfated chains was not due to a defect in the sulfation process because pulse-chase experiments showed that they could be converted into the fully sulfated chains. However, their formation was inhibited by limiting the endogenous supply of hexosamine. The results also indicated the coexistence of the unsulfated and sulfated chains on the same protein core and further suggested that the sulfation of heparan sulfate may occur as an all or nothing phenomenon. Taken together, the results support the current biosynthetic model developed for the heparin proteoglycan in which unsulfated glycosaminoglycans are first elongated on the protein core, and subsequently modified and sulfated. These data provide the first evidence for the presence of such an unsulfated precursor in an intact cellular system.     

ARG1基因对人胚肾细胞293砷染毒前后增殖及凋亡的影响

目的:观察ARG1基因对不同浓度的亚砷酸钠(NaAsO2)染毒后的293细胞的增殖及凋亡的影响,并讨论ARG1的抗砷性.方法:将ARG1表达质粒pcDNA3.1-ARG1及空载体对照质粒pcDNA3.1分别经阳离子脂质(Lipofectamine2000)介导转染293细胞后,荧光共聚焦显微镜观察细胞转染率;将实验组pcDNA3.1.ARG1-293细胞、空白对照组pcDNA3.1-293细胞及阴性对照组293细胞用不同浓度的NaAsO2浓度染毒48h后,使用MTT法检测ARG1基因对砷染毒后293细胞的增殖的影响;将实验组、空白对照纽及阴性对照组按不同浓度NaAsO2染毒,加入Annexin V FITC/PI凋亡试剂,采用流式细胞仪检测细胞的凋亡情况.结果:(1)荧光共聚焦显微镜观察转染结果显示转染率可达75-85%;(2)NaAsO2在1-8 μ mol/L时其对实验组细胞增值的抑制率明显低于对照组,而在浓度>8μ mol/L时则没有明显差异;(3)实验组细胞在NaAsO2浓度<8 μ mol/L其凋亡率明显低于对照组.结论:ARG1能够降低NaAsO2对293细胞增值的抑制率,减轻其对293细胞的促凋亡作用,主要是在低浓度时发挥作用.     

Thrombopoietin from human embryonic kidney cells causes increased thrombocytopoiesis in sublethally irradiated mice.

Previous work showed that mice treated with platelet-specific antiserum prior to whole-body irradiation did not suffer the degree or duration of thrombocytopenia as did irradiated control mice. We now report that a partially purified preparation of a thrombocytopoiesis-stimulating factor (TSF or thrombopoietin) mimics the biological effects of platelet-specific antiserum treatment in hematopoietically suppressed mice. Male C3H mice were exposed to 3.0 or 4.5 Gy of 137Cs gamma radiation and injected with a total dose of 4 units (U) of TSF. Human serum albumin (HSA) and rabbit anti-mouse platelet serum-injected mice, along with unirradiated mice, served as controls. Packed cell volumes (PCV), RBC counts, WBC counts, platelet counts, and percentage 35S incorporation into platelets were measured in mice at various days (7-14) following treatment. The results showed that irradiated mice treated with TSF had increased 35S uptake into platelets and higher platelet counts than HSA-treated controls. Also, PCV, RBC counts, and WBC counts of irradiated mice treated with TSF were significantly higher than values for HSA-treated mice. Additional experiments using 40,000 U/mouse of Interleukin-6 (IL-6), 227 U/mouse of granulocyte macrophage-colony stimulating factor (GM-CSF), or a combination of GM-CSF and IL-6 did not show increased platelet counts or 35S incorporation into platelets on Days 10 and 14 when compared to other mice treated with control substances. These results suggest that the radioprotective effects of platelet antibodies reported previously may be due to the release and action of thrombopoietin. These studies also demonstrate that thrombopoietin therapy will modulate the severe thrombocytopenia that occurs in radiation-induced bone marrow suppression.     

Sulf loss influences N-, 2-O-, and 6-O-sulfation of multiple heparan sulfate proteoglycans and modulates fibroblast growth factor signaling

Sulf1 and Sulf2 are two heparan sulfate 6-O-endosulfatases that regulate the activity of multiple growth factors, such as fibroblast growth factor and Wnt, and are essential for mammalian development and survival. In this study, the mammalian Sulfs were functionally characterized using overexpressing cell lines, in vitro enzyme assays, and in vivo Sulf knock-out cell models. Analysis of subcellular Sulf localization revealed significant differences in enzyme secretion and detergent solubility between the human isoforms and their previously characterized quail orthologs. Further, the activity of the Sulfs toward their native heparan sulfate substrates was determined in vitro, demonstrating restricted specificity for S-domain-associated 6S disaccharides and an inability to modify transition zone-associated UA-GlcNAc(6S). Analysis of heparan sulfate composition from different cell surface, shed, glycosylphosphatidylinositol-anchored and extracellular matrix proteoglycan fractions of Sulf knock-out cell lines established differential effects of Sulf1 and/or Sulf2 loss on nonsubstrate N-, 2-O-, and 6-O-sulfate groups. These findings indicate a dynamic influence of Sulf deficiency on the HS biosynthetic machinery. Real time PCR analysis substantiated differential expression of the Hs2st and Hs6st heparan sulfate sulfotransferase enzymes in the Sulf knock-out cell lines. Functionally, the changes in heparan sulfate sulfation resulting from Sulf loss were shown to elicit significant effects on fibroblast growth factor signaling. Taken together, this study implicates that the Sulfs are involved in a potential cellular feed-back mechanism, in which they edit the sulfation of multiple heparan sulfate proteoglycans, thereby regulating cellular signaling and modulating the expression of heparan sulfate biosynthetic enzymes.