Genetic Analysis of the Heparan Modification Network in Caenorhabditis elegans

Department of Genetics, Albert Einstein College of Medicine, Bronx, New York 10461, USA.
Journal of Biological Chemistry (Impact Factor: 4.57). 03/2011; 286(19):16824-31. DOI: 10.1074/jbc.M111.227926
Source: PubMed


Heparan sulfates (HS) are highly modified sugar polymers in multicellular organisms that function in cell adhesion and cellular responses to protein signaling. Functionally distinct, cell type-dependent HS modification patterns arise as the result of a conserved network of enzymes that catalyze deacetylations, sulfations, and epimerizations in specific positions of the sugar residues. To understand the genetic interactions of the enzymes during the HS modification process, we have measured the composition of HS purified from mutant strains of Caenorhabditis elegans. From these measurements we have developed a genetic network model of HS modification. We find the interactions to be highly recursive positive feed-forward and negative feedback loops. Our genetic analyses show that the HS C-5 epimerase hse-5, the HS 2-O-sulfotransferase hst-2, or the HS 6-O-sulfotransferase hst-6 inhibit N-sulfation. In contrast, hse-5 stimulates both 2-O- and 6-O-sulfation and, hst-2 and hst-6 inhibit 6-O- and 2-O-sulfation, respectively. The effects of hst-2 and hst-6 on N-sulfation, 6-O-sulfation, and 2-O-sulfation appear largely dependent on hse-5 function. This core of regulatory interactions is further modulated by 6-O-endosulfatase activity (sul-1). 47% of all 6-O-sulfates get removed from HS and this editing process is dependent on hst-2, thereby providing additional negative feedback between 2-O- and 6-O-sulfation. These findings suggest that the modification patterns are highly sensitive to the relative composition of the HS modification enzymes. Our comprehensive genetic analysis forms the basis of understanding the HS modification network in metazoans.

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Available from: Hannes E Bülow,
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    • "Simultaneous genetic removal of both hst-2 and hst-6 results in almost complete loss of HSN migration suggesting that heparan O-sulfation is needed for HSN migration. Biochemical HS disaccharide analysis has shown increased N-sulfation in hst-2 hst-6 double mutants [35], however, as suggested by this study, glucosamine N-sulfation is not able to functionally compensate for the lack of O-sulfation. Genetic removal of hst-6 and hst-3.1 in hse-5 epimerase mutant background significantly enhances defects in HSN migration whereas removal of hst-2 in hse-5 background does not. "
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    ABSTRACT: Heparan sulfate proteoglycans (HSPGs) play critical roles in the development and adult physiology of all metazoan organisms. Most of the known molecular interactions of HSPGs are attributed to the structurally highly complex heparan sulfate (HS) glycans. However, whether a specific HSPG (such as syndecan) contains HS modifications that differ from another HSPG (such as glypican) has remained largely unresolved. Here, a neural model in C. elegans is used to demonstrate for the first time the relationship between specific HSPGs and HS modifications in a defined biological process in vivo. HSPGs are critical for the migration of hermaphrodite specific neurons (HSNs) as genetic elimination of multiple HSPGs leads to 80% defect of HSN migration. The effects of genetic elimination of HSPGs are additive, suggesting that multiple HSPGs, present in the migrating neuron and in the matrix, act in parallel to support neuron migration. Genetic analyses suggest that syndecan/sdn-1 and HS 6-O-sulfotransferase, hst-6, function in a linear signaling pathway and glypican/lon-2 and HS 2-O-sulfotransferase, hst-2, function together in a pathway that is parallel to sdn-1 and hst-6. These results suggest core protein specific HS modifications that are critical for HSN migration. In C. elegans, the core protein specificity of distinct HS modifications may be in part regulated at the level of tissue specific expression of genes encoding for HSPGs and HS modifying enzymes. Genetic analysis reveals that there is a delicate balance of HS modifications and eliminating one HS modifying enzyme in a compromised genetic background leads to significant changes in the overall phenotype. These findings are of importance with the view of HS as a critical regulator of cell signaling in normal development and disease.
    PLoS ONE 07/2014; 9(7):e102919. DOI:10.1371/journal.pone.0102919 · 3.23 Impact Factor
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    • "sulfotransferases, an epimerase, and a sulfatase (Bernfield et al., 1999; Lee and Chien, 2004) (Figure 5A). The specific modifications made by each enzyme mediate specific roles in various aspects of C. elegans development and physiology , including axon guidance (Attreed et al., 2012; Bü low and Hobert, 2004; Townley and Bü low, 2011). In order to determine whether individual heparan sulfate sugar modifications are important for syndecan's function in axon regeneration, we examined loss-of-function mutants that lack individual modifying enzymes. "
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    ABSTRACT: Growth cones facilitate the repair of nervous system damage by providing the driving force for axon regeneration. Using single-neuron laser axotomy and in vivo time-lapse imaging, we show that syndecan, a heparan sulfate (HS) proteoglycan, is required for growth cone function during axon regeneration in C. elegans. In the absence of syndecan, regenerating growth cones form but are unstable and collapse, decreasing the effective growth rate and impeding regrowth to target cells. We provide evidence that syndecan has two distinct functions during axon regeneration: (1) a canonical function in axon guidance that requires expression outside the nervous system and depends on HS chains and (2) an intrinsic function in growth cone stabilization that is mediated by the syndecan core protein, independently of HS. Thus, syndecan is a regulator of a critical choke point in nervous system repair.
    Cell Reports 07/2014; 8(1). DOI:10.1016/j.celrep.2014.06.008 · 8.36 Impact Factor
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    • "Many pieces of this puzzle and the dynamic interplay still remain to be elucidated. The picture is complex, as the interactions are built up by both negative and positive feedback loops which can be depicted in a network (122), where each member reciprocally affects multiple actions of the other members of the network. Thus changes in the expression of one gene will affect the whole HS biosynthetic machinery of the cell. "
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    ABSTRACT: Proteoglycans (PGs) and in particular the syndecans are involved in the differentiation process across the epithelial-mesenchymal axis, principally through their ability to bind growth factors and modulate their downstream signaling. Malignant tumors have individual proteoglycan profiles, which are closely associated with their differentiation and biological behavior, mesenchymal tumors showing a different profile from that of epithelial tumors. Syndecan-1 is the main syndecan of epithelial malignancies, whereas in sarcomas its expression level is generally low, in accordance with their mesenchymal phenotype and highly malignant behavior. This proteoglycan is often overexpressed in adenocarcinoma cells, whereas mesothelioma and fibrosarcoma cells express syndecan-2 and syndecan-4 more abundantly. Increased expression of syndecan-1 in mesenchymal tumors changes the tumor cell morphology to an epithelioid direction whereas downregulation results in a change in shape from polygonal to spindle-like morphology. Although syndecan-1 plays major roles on the cell-surface, there are also intracellular functions, which are not very well studied. On the functional level, syndecan-1 affects mesenchymal tumor cell proliferation, adhesion, migration and motility, and the effect varies with the different domains of the core protein. Syndecan-1 may exert stimulatory or inhibitory effects, depending on the concentration of various mitogens, enzymes, and signaling molecules, the ratio between the shed and membrane-associated syndecan-1 and histological grade of the tumour. Growth factor signaling seems to be delicately controlled by regulatory loops involving the syndecan expression levels and their sulfation patterns. Overexpression of syndecan-1 modulates the biosynthesis and sulfation of heparan sulfate and it also affects the expression of other PGs. On transcriptomic level, syndecan-1 modulation results in profound effects on genes involved in regulation of cell growth.
    Frontiers in Oncology 12/2013; 3:310. DOI:10.3389/fonc.2013.00310
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