Article |
Address correspondence to Hiroshi Nakato, Dept. of Genetics, Cell Biology, and Development, University of Minnesota, 6-160 Jackson Hall, 321 Church St. SE, Minneapolis, MN 55455. Tel.: (612) 625-1727. Fax: (612) 626-5652. email: nakat003{at}umn.edu
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Abstract |
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Key Words: 3-O sulfation; heparan sulfate proteoglycan; Notch signaling; transgenic RNAi; Drosophila
Abbreviations used in this paper: ACV, anterior cross vein; A/P, anteriorposterior; AT, antithrombin; BDGP, Berkeley Drosophila Genome Project; ct, cut; Dl, Delta; dpp, decapentaplegic; dx, deltex; D/V, dorsalventral; E(spl)C, Enhancer of split Complex; gD, glycoprotein D; HS, heparan sulfate; HS3ST, HS 3-O sulfotransferase; HSPG, HS proteoglycan; HSV, herpes simplex virus; IR, inverted repeat; kuz, kuzbanian; N, Notch; NECD, extracellular domain of N protein; neur, neuralized; NICD, intracellular domain of N protein; RNAi, RNA interference; sca, scabrous; Ser, Serrate; SMC, sensory organ mother cell; ST, sulfotransferase; Su(H), Suppressor of Hairless; UAS, upstream activating sequence; vg, vestigial; wg, wingless.
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Introduction |
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The first and still the most notable example showing the importance of sequence-specific sulfation in HSprotein interactions comes from analyses of 3-O sulfated HS. The 3-O sulfate group is the rarest component in HS, and 3-O sulfation occurs late in the HS biosynthesis pathway (for review see Rosenberg et al., 1997). In humans, there are at least seven HS 3-O sulfotransferases (HS3STs) that produce at least two distinct forms of 3-O sulfated HS (Shworak et al., 1997, 1999; Shukla et al., 1999; Xia et al., 2002). One form of 3-O sulfated HS plays a critical role in the blood coagulation cascade. HS3ST-1 generates HSAT+: HS with a specific 3-O sulfated motif that binds to antithrombin (AT). In therapeutic heparin administration, this interaction leads to inactivation of thrombin, resulting in inhibition of blood coagulation (Rosenberg et al., 1997; Shworak et al., 1997). However, a recent report showed that Hs3st1/ knockout mice do not exhibit an obvious procoagulant phenotype even though HSAT+ levels in these mice are largely reduced (HajMohammadi et al., 2003). Instead, these mice showed postnatal lethality and intrauterine growth retardation, implying other physiological roles for 3-O sulfation. A different form of 3-O sulfated HS is involved in the entry of HSV-1 into host cells and the fusion of infected cells. HS3ST-3 isoforms generate HSgD+: HS with a different 3-O sulfated sequence that mediates the cellular entry of HSV-1. HSgD+ on the surface of animal cells interacts with a viral coat protein, glycoprotein D (gD), to promote fusion of the HSV-1 envelope with the cell membrane of host cells (Shukla et al., 1999). Multinucleated giant cells (polykaryocytes), resulting from virus-induced cell fusion, are a hallmark of HSV-1 infection. It has also been demonstrated that CHO cells expressing HS3ST-3 fuse with CHO cells that express viral glycoproteins gB, gD, gH, and gL, suggesting that 3-O sulfated HS has a crucial role both in the entry and spreading of HSV-1 (Tiwari et al., 2004). However, an endogenous ligand for this HS sequence is not known. Thus, although sequence-specific 3-O sulfation clearly has key roles in defining interactions between HS and specific binding proteins, its in vivo relevance is poorly understood. Interestingly, sequences homologous to mammalian HS3ST genes are found in the genome of Drosophila melanogaster and Caenorhabditis elegans despite the very dissimilar blood coagulation systems that occur in lower animals. The existence of HS3ST homologues in these organisms suggests that 3-O sulfation has a role in evolutionarily conserved processes, such as the developmental pathways mediated by secreted factors or integral membrane proteins.
One of the central and widely deployed developmental pathways in the animal kingdom is Notch (N) signaling (for reviews see Panin and Irvine, 1998; Artavanis-Tsakonas et al., 1999; Mumm and Kopan, 2000; Baron et al., 2002). In Drosophila, the core components of this pathway are the receptor N, and the ligands Delta (Dl) and Serrate (Ser). Upon binding the ligands, N undergoes Presenilin-dependent processing, which results in the release of the intracellular domain of N (NICD) from the membrane. Subsequently, this domain translocates to the nucleus where it forms a complex with Suppressor of Hairless (Su(H)) and Mastermind to activate the expression of target genes such as Enhancer of split Complex (E(spl)C). During maturation of N protein, this molecule undergoes extensive modulation mediated by various molecules such as a glycosyltransferase, Fringe; extracellular modulator, Scabrous (Sca); and metalloproteinase, Kuzbanian (Kuz). Furthermore, proper transport to the cell surface as well as internalization of N protein are required for the normal activity of this molecule. Multiple genes are known to affect N signaling through roles in membrane trafficking. These include Shibire (dynamin), Ca-P60A (Ca2+ ATPase), heatshock cognate 70, Warthog (rab6), Numb, and N-ethylmaleimidesensitive fusion protein (for review see Baron et al., 2002).
To examine the in vivo importance of a specific sulfation of HS for the interaction with binding proteins, we studied the function of 3-O sulfated HS during Drosophila development. We isolated cDNA clones for two Drosophila Hs3sts (Hs3st-A and -B), and characterized their structures, expression, and functions. Transfectional analyses showed that Hs3st-B, like HS3ST-3 isoforms, mediated cellular entry of HSV-1 that is triggered by HSgD+. We found that reduction of Hs3st-B function using the transgenic RNA interference (RNAi) technique caused neurogenic phenotypes that are characteristic of N signaling mutants. Hs3st-B genetically interacts with various N signaling components and regulates expression of N target genes. In addition, levels of N protein were markedly decreased by reduction of Hs3st-B. These findings show that Hs3st-B is a novel regulator of the N signaling pathway and controls pattern formation. We also observed that the Hs3st-B RNAi affects the number and size of endosomal/lysosomal compartments, suggesting the role of 3-O sulfated HS in intracellular trafficking of N protein.
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Results |
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We subcloned Drosophila and representative mammalian HS3ST cDNAs into the pcDNA3.1 expression vector and determined the enzymatic ability to create functional HSgD+ by an in vivo assay in which the sequence specific activity of HS3ST-3 isoforms generates HSgD+ and renders CHO cells susceptible to infection by HSV-1 (Shukla et al., 1999). Thus, CHO cells were transfected with HS3ST expression constructs, and then exposed to a recombinant form of HSV-1, and virus entry was monitored (Fig. 1 C). Negligible viral entry was detected in cells expressing Hs3st-A, the empty vector control, or HS3ST-1, which does not produce HSgD+ (Shukla et al., 1999). In contrast, expression of Hs3st-B conveyed cellular susceptibility to HSV-1 entry, as did the HS3ST-3Apositive control. Moreover, coexpression of HSV-1 gD with Hs3st-B reduced cellular susceptibility (unpublished data) through the ability of cellular gD to interfere with viral entry via HSgD+ (Shukla et al., 1999). Together, these results demonstrate that Hs3st-B, like HS3ST-3A, preferentially generates HSgD+. Thus, the two Drosophila HS3ST enzymes exhibit distinct yet phylogenetically conserved sequence specificities.
mRNA expression of Hs3sts
To determine the temporal and spatial expression pattern of the Hs3st mRNAs, we performed RT-PCR and in situ RNA hybridization. RT-PCR revealed that Hs3st-A mRNA is expressed at low levels in all stages examined (Fig. 2 A). Its transcription levels were especially low in larval and pupal stages. In contrast, Hs3st-B mRNA is abundantly expressed throughout development (Fig. 2 A). We next examined the spatial distribution of Hs3st-B in embryos and larval imaginal tissues by in situ hybridization (Fig. 2 B). In these tissues, Hs3st-B was widely but not evenly expressed. Hs3st-B expression was particularly enriched in the salivary glands in embryos, the morphogenetic furrow of eye discs, and the brain lobes of the larval CNS. In the wing disc, Hs3st-B mRNA was detected at high levels in the wing pouch region (Fig. 2 C). We failed to detect any signals from Hs3st-A mRNA (unpublished data), probably due to the low level of expression.
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In this work, we focused our analysis on the function of Hs3st-B. To test the effectiveness of Hs3st-B transgenic RNAi, we examined the ability of IR-Hs3st-B to reduce endogenous Hs3st-B mRNA expression. To prepare RNA from actin-GAL4/UAS-IR-Hs3st-B animals, larval stages were reared at 18°C, and then shifted to 25°C for 24 h before RNA preparation. RT-PCR analysis showed that expression of UAS-IR-Hs3st-B dramatically reduced the Hs3st-B mRNA level, whereas it had no effect on mRNA levels of Hs3st-A and ribosomal protein 49 (Fig. 3 A), confirming the target specificity of the transgenic RNAi technique.
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Hs3st-B regulates N target genes during wing margin formation
During wing development, the N pathway activates a set of genes controlling wing margin formation, such as cut (ct), wg, and vestigial (vg), in a stripe of cells along the dorsalventral (D/V) boundary (Fig. 4, A, C, and E; Couso et al., 1995; Kim et al., 1995; Rulifson and Blair, 1995; Micchelli et al., 1997). To ask if Hs3st-B regulates N signaling, we examined expression of Ct, Wg, and the vg boundary enhancer, vgBE-lacZ, in the Hs3st-B transgenic RNAi animals. Expression of IR-Hs3st-B under the control of dpp-GAL4 completely eliminated expression of these genes in the central region of the disc (Fig. 4, B, D, and F). We also examined expression of E(spl)m8, the m8 gene from E(spl)C, which is induced along the D/V boundary by N signaling, although E(spl)m8 is not required for wing margin formation (Fig. 4 G; de Celis et al., 1996). We found that the Hs3st-B transgenic RNAi also abolished E(spl)m8-lacZ expression (Fig. 4 H). Because transcription of vg and E(spl)m8 along the D/V boundary is induced by direct interaction between their regulatory sequences and Su(H) DNA binding protein (Lecourtois and Schweisguth, 1995; Kim et al., 1996), our results suggest that Hs3st-B is required for the Su(H)-mediated N pathway. Thus, Hs3st-B regulates N signaling during wing margin formation, which is consistent with its phenotypic effects and genetic interactions.
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Hs3st-B transgenic RNAi affects N protein levels
The activity of N protein is regulated at multiple steps including proteolytic processing, glycosylation, intracellular transport to the cell surface, and degradation (for review see Baron et al., 2002). To gain insight into the mechanism for control of N function by Hs3st-B, we examined N protein distribution in wing discs from Hs3st-B transgenic RNAi animals using antibodies against the NECD or NICD. In the third instar larva of wild type, N is evenly expressed throughout the wing disc with the highest protein levels on the apical surface (Fig. 6, A and C; Fehon et al., 1991). We found that expression of IR-Hs3st-B by dpp-GAL4 significantly reduced signals for both NECD (Fig. 6, B and B') and NICD (Fig. 6, D and D') at the A/P boundary. The reduction of N protein levels was not caused by decreased expression of the N gene because Hs3st-B RNAi did not affect levels and patterns of N-lacZ expression in the wing disc (Fig. 6, F and F'; de Celis et al., 1997).
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Hs3st-B transgenic RNAi affects distribution and morphology of intracellular membrane compartments
Two lines of evidence prompted us to examine the effects of Hs3st-B RNAi on vesicle trafficking. First, several mutations in genes involved in intracellular trafficking or endocytosis compromise N signaling (for review see Baron et al., 2002). Second, a mammalian homologue of Hs3st-B, HS3ST-3, has a critical role in membrane fusion. 3-O sulfated HS generated by HS3ST-3 mediates fusion of the envelope of HSV-1 and the host cell membrane (Shukla et al., 1999). Furthermore, CHO cells expressing HS3ST-3 fuse with other CHO cells that express viral glycoproteins, resulting in the generation of polykaryocytes (Tiwari et al., 2004).
We studied the effects of Hs3st-B RNAi on the morphology of endosomal/lysosomal compartments. Wing discs from Hs3st-B RNAi animals (UAS-IR-Hs3st-B/+; dpp-GAL4,UAS-GFP/+) were cultured in medium containing an endocytosis marker (Texas red dextran) or a lysosomal marker (LysoTracker). We found that IR-Hs3st-B expression strikingly increased the number and size of both dextran and LysoTracker-uptaking vesicular puncta (Fig. 6, IK). Such enlarged endosomal/lysosomal compartments may lead to a higher rate of protein degradation. We tested whether or not impairment of N signaling causes the same changes of the endosomal/lysosomal compartments and found that expression of either a dominant-negative form of N or an N transgenic RNAi construct driven by dpp-GAL4 does not induce such phenomena (unpublished data). Thus, the changes in number and size of the endosomal/lysosomal compartments seem to be directly induced by the Hs3st-B RNAi and not through a reduction of N signaling. In summary, these results suggested one possible mechanism in which reduction of N protein levels in Hs3st-B RNAi is caused by defects in trafficking to the cell surface and/or to the endosomal/lysosomal compartments.
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Discussion |
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Mechanisms by which 3-O sulfated HS regulates Drosophila N signaling
What is the mechanism for control of the N signaling system by HS 3-O sulfation? Although our work implicated Hs3st-B in N signaling, there is no direct evidence reported to date of a requirement of HS in this pathway. However, there are several heparin-binding proteins that have been shown to regulate N signaling. Therefore, Hs3st-B may affect N activity through these molecules. First, Kuz, a Drosophila member of the ADAM family, has a domain that is highly conserved among members of the family. Because one of the mammalian homologues, ADAM12, was shown to interact with HS through this domain (Iba et al., 2000), Kuz could also potentially associate with HSPGs. Kuz cleaves the NECD, and this cleavage is required for activation of the N receptor (Lieber et al., 2002). Therefore, it is possible that 3-O sulfated HS interacts with Kuz and regulates the processing of N. The second candidate whose activity may be regulated by 3-O sulfated HS is Sca. Sca is a secreted fibrinogen-related protein that binds to heparin in vitro (Lee et al., 1996). Previous papers showed that Sca has an ability to directly bind and stabilize N protein on the cell surface (Powell et al., 2001). Finally, it is also possible that 3-O sulfated HS affects the N pathway through the activity of Wg, a known heparin-binding factor. It has been suggested that Wg binds to the specific EGF repeats of the NECD(Wesley, 1999) and that this binding inhibits interactions between N and its ligands, Dl and Ser, during development (Brennan et al., 1999). Therefore, 3-O sulfation of HS might control N signaling by modulating the NWg interaction.
Another model that explains how Hs3st-B regulates N protein levels was suggested by our experiments showing that Hs3st-B RNAi causes aberrant intracellular trafficking events. Previous work has shown that the activity of N signaling is very sensitive to alterations in membrane trafficking (for review see Baron et al., 2002). For example, phosphocholine cytidylyltransferase 1 is involved in the formation of phospholipid membranes and positively regulates N signaling (Weber et al., 2003). Phosphocholine cytidylyltransferase 1 mutant cells show an increase in endocytic activity and have enlarged endosomes, resulting in abnormal subcellular localization of N protein. In addition, mutations in warthog, which encodes a Drosophila homologue of rab6 and regulates trafficking from the Golgi to the trans-Golgi network, result in defective N signaling (Purcell and Artavanis-Tsakonas, 1999). Thus, some genes that are involved in controlling membrane trafficking are known to affect N signal transduction. Together with our observation, these findings suggest that a reduction of N protein by the Hs3st-B RNAi may be caused by abnormal sorting of N protein or enhanced activities of endocytosis/protein degradation. Interestingly, mammalian HS3ST-3 mediates the fusion of the viral envelope with the host cell membranes (Shukla et al., 1999), as well as the fusion between host cell membranes (Tiwari et al., 2004), suggesting a possible role of 3-O sulfated HS in membrane fusion during trafficking events. It is also possible that the Hs3st-B RNAi independently affects N signaling and intracellular trafficking.
Given that the role of HS in N signaling has not been reported, it is possible that the substrate of Hs3st-B is not HS, but other glycoconjugates such as sugar chains of N protein itself. The O-linked sugar chains on the EGF repeats of N protein play a key role in its activity (for review see Haines and Irvine, 2003), and further modification of this sugar chain (i.e., sulfation) may give a potential regulatory mechanism. It has been shown that fringe (N-acetylglucosaminyltransferase), O-fucosyltransferase 1, and fringe connection (UDP-sugar transporter) are involved in glycosylation of N and affect N signal transduction (for review see Haines and Irvine, 2003). Therefore, it is important to know the effects of Hs3st-B RNAi on modification of glycoconjugates other than HS. Also, we cannot rule out the possibility that Hs3st-B RNAi induces a phenomenon that does not reflect functions of endogenous HS chains. For example, inappropriately modified HS may exert an inhibitory effect on N signaling, even though HSPGs do not normally have any role in this pathway. Further studies on the function of other HS biosynthetic enzymes and HSPG core protein genes in N signaling and membrane trafficking will reveal the molecular basis underlying the control of these events by 3-O sulfated HS. In particular, identification of the ligand of 3-O sulfated HS will be important to understand the in vivo functions of this class of enzymes.
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Materials and methods |
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Cloning and enzymatic assay of Hs3sts
Two genomic loci for putative Drosophila Hs3sts (Hs3st-A ;CG33147, and B; CG7890) were identified in the Drosophila genome database using the tBlastn program of the BDGP. An Hs3st-A cDNA clone (GH20068) containing the entire protein coding region was obtained from the BDGP. To isolate an Hs3st-B cDNA clone, 4 x 105 colonies from a Drosophila embryonic cDNA library were screened using Hs3st-B genomic DNA as a probe (Brown and Kafatos, 1988). Hybridization was performed as described by Sambrook et al. (1989). One positive clone was isolated, and the inserted cDNA sequence was determined using a Big DyeTM Thermal Cycle Sequencing Ready Reaction Kit and the ABI PRISM 310 Analyzer.
To characterize the enzymatic sequence specificities of Hs3sts, expression constructs were generated by inserting an EcoRVXhoI fragment of Hs3st-A and a HindIIIEcoRI fragment of Hs3st-B cDNAs into the polylinker of pcDNA3.1 (Life Technologies). pcDNA3.1-based expression plasmids containing mouse HS3ST-1 or human HS3ST-3A were described previously (Shworak et al., 1997; Liu et al., 1999). Determination of CHO cell susceptibility to HSV-1 entry was performed as previously described (Montgomery et al., 1996; Shukla et al., 1999).
RT-PCR analyses
Total RNA was isolated from wild-type and actin-GAL4/UAS-IR-Hs3st-B larvae using the FastRNA Kit (BIO101) and FastPrep FP120 (BIO101). Isolated RNA was reverse-transcribed using SuperScriptTM First-Strand Synthesis System for RT-PCR (Life Technologies). PCR experiments were performed using the synthesized cDNA as a template and the following primers: Hs3st-AFW (5'-GCTGCGATGACCACGACAAC-3') and Hs3st-ARV (5'-TTCACCCGATGCTCAATGCC-3') for Hs3st-A detection; Hs3st-BFW (5'-CTACTTCGTCACCAAGGAGG-3') and Hs3st-BRV (5'-GCATAGACGCCGATCTTCAC-3') for Hs3st-B detection; and rp49FW (5'-ATGACCATCCGCCCAGCATACAGG-3') and rp49RV (5'-TCGTTCTCTTGAGAACGCAGGCGA-3') for ribosomal protein 49 detection.
Construction of Hs3st transgenic RNAi flies
Transgenic RNAi flies of Hs3st-A and -B were obtained using the inducible RNAi method (Kennerdell and Carthew, 2000). A 500-bp-long cDNA fragment from the first methionine was amplified by PCR and inserted as an IR into a modified pBluescript vector, pSC1, which possesses an IR formation site consisting of paired CpoI and SfiI restriction sites. In all cases, IRs were constructed in a head-to-head orientation. IR-containing fragments were cut out with NotI and were subcloned into pUAST, a transformation vector. Transformation of Drosophila embryos was performed using w1118 as a recipient strain.
Scanning EM
Adult flies were sequentially dehydrated in 25, 50, 75, and 100% ethanol, for 2 h each. The 100% dehydration was repeated three times. The preparation was subjected to critical point drying before being mounted on stubs and viewed on an S-4700 field emission scanning electron microscope (HITACHI).
In situ hybridization and immunostaining
In situ RNA hybridization was performed as described previously (Kamimura et al., 2001). Antibody staining was performed as described previously (Fujise et al., 2001) using rabbit antiß-galactosidase (1:500; Cappel), mouse anti-Wg (4D4; 1:20; Developmental Studies Hybridoma Bank [DSHB]), mouse anti-Ct (2B10; 1:20; DSHB), mouse anti-NECD (C458.2H; 1:20; DSHB), mouse anti-NICD (C17.9C6; 1:10; DSHB), and mouse anti-Dl (C594.9B; 1:200). The primary antibodies were detected with Alexa Fluorconjugated secondary antibodies (1:500; Molecular Probes).
Labeling the endosomal and lysosomal compartments
To visualize late endosomes, wing discs from third instar larvae were incubated in 0.5 mM of Texas red dextran (lysine fixable, MW3000; Molecular Probes) for 5 min in M3 medium at 25°C and then washed five times for 2 min with ice-cold M3 medium. Afterwards, these discs were incubated in M3 medium for 60 min at 25°C and fixed in PFA for 20 min at RT before mounting in VECTASHIELD Mounting Medium (Vector Laboratories). To visualize lysosomes, wing discs were incubated in 100 nM LysoTracker red (Molecular Probes) at 25°C for 2 min in PBS. Subsequently, the discs were washed two times with PBS and incubated in PBS at 25°C for 20 min. These discs were mounted in PBS for observation.
Online supplemental material
Fig. S1 shows the genetic interactions between Hs3st-B and various N signaling components during formation of the wing and notum. Online supplemental material is available at http://www.jcb.org/cgi/content/full/jcb.200403077/DC1.
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Acknowledgments |
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This work was supported in part by the National Institutes of Health (grant HD042769 to H. Nakato and grant AI053774 to P.G. Spear) and the Human Frontier Science Program to H. Nakato and N.W. Shworak. K. Kamimura is supported by the Japan Society for the Promotion of Science for Young Scientists.
Submitted: 12 March 2004
Accepted: 12 August 2004
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