Domain-specific Modification of Heparan Sulfate by Qsulf1 Modulates the Binding of the Bone Morphogenetic Protein Antagonist Noggin

Abstract

We have reported previously that Noggin is a heparinbinding protein and associates with the cell surface through heparan sulfate proteoglycans, where it remains functional for the binding of bone morphogenetic proteins (BMPs). Here we report that the binding of Noggin to the cell surface is highly selective for heparan sulfate and that specific structural features are required for the interaction. Noggin binds most efficiently to heparin sequences composed of 10 or more monosaccharides; N-, 6-O-, and 2-O-sulfates contribute to this interaction. In addition, we have shown that the developmentally regulated endosulfatase Qsulf1 selectively removes sulfate groups from the 6-O position of sugars within the most highly sulfated S domains of heparan sulfate, whereas 6-O-sulfates in the NA/NS domains are not substrates for the enzyme. The activity of Qsulf1 in cells in culture results in the release of Noggin from the cell surface and a restoration of BMP responsiveness to the cells. This shows that Noggin binds to the S domains of heparan sulfate and provides evidence that, in addition to modulating Wnt signaling in vivo by the release of heparan sulfate bound Wnt, Qsulf1 also modulates BMP signaling by the release of surface-bound Noggin.Heparan sulfate proteoglycans are found ubiquitously both on the surface of cells as well as within the extracellular matrix, where they bind and modify the functions of a diverse array of ligands (1). Loss of function mutations in enzymes of the heparan sulfate biosynthetic pathway have confirmed that this polysaccharide has essential roles during development. Foremost among these is the regulation of cellular responsiveness to a number of growth factors and morphogens that control patterning events (2). Mutations in members of the glypican family of cell-surface heparan sulfate proteoglycans, in both vertebrates and invertebrates, have been specifically associated with loss of bone morphogenetic protein (BMP) 1 activities (3,4). Although the precise molecular mechanisms by which these specific heparan sulfate proteoglycans regulate cellular responsiveness to BMPs in vivo is not known, recent in vitro data does support the conclusion that heparan sulfate can directly augment signaling of BMPs through their receptors (5, 6).Previously we have shown that the BMP antagonist Noggin remains associated with the surface of cells by binding to heparan sulfate, where it remains functional for the inhibition of BMPs (7). We have proposed that the interaction with heparan sulfate in vivo is likely to regulate the range of influence of Noggin by restricting its diffusion, thus providing another mechanism by which cellular responsiveness to BMPs can be regulated by heparan sulfate in vivo (7). This hypothesis supposes that the interaction between Noggin and heparan sulfate is sequence specific and implies that there should be a developmentally regulated mechanism in vivo for the specification of heparan sulfate structures that modulate Noggin binding to the cell surface.Heparan sulfate sequences...

Functional Consequences of the Subdomain Organization of the Sulfs

“…We and others showed that both Sulfs are neutral pH endosulfatases, which remove glucosamine-6-O-sulfate from internal glucosamine residues of highly sulfated subregions within heparin/HSPGs (7,9,10). The ability of these enzymes to modulate the heparin/HSPG interactions of a number of growth factors, morphogens, and chemokines has been confirmed in direct binding assays (9,(11)(12)(13).…”

Organ-specific Sulfation Patterns of Heparan Sulfate Generated by Extracellular Sulfatases Sulf1 and Sulf2 in Mice

“…Total RNAs were extracted using Sepasol I (Nacalai Tesque, Kyoto, Japan) and purified using an RNeasy kit (Qiagen, Hilden, Germany). Total RNAs (5 g) were subjected to reverse-transcription using oligo(dT) [12][13][14][15][16][17][18] and Superscript II (Invitrogen). Quantitative PCR was carried out using a LightCycler and LightCycler FastStart DNA Master SYBR Green I reagent (Roche Diagnostics).…”

“…Therefore, Sulf1 and Sulf2 are thought to be key regulators of cell proliferation, differentiation, and migration and are also implicated in cancer progression and metastasis (22,23). Biochemical studies have demonstrated that Sulf1 and Sulf2 are most active in the UA2S-GlcNS6S trisulfated disaccharide unit, which is present in the NS domain (9,11,13,16,24,25). In addition, weaker 6-O-desulfation activity is also detectable in the UA-GlcNS6S-disulfated disaccharide unit (11,24,25).…”

Dynamic expression patterns of 6‐O endosulfatases during zebrafish development suggest a subfunctionalisation event for sulf2

“…Similarly, VEGF binding and activity requires moderately 6-O sulfated HS (Ashikari-Hada et al, 2005;Robinson et al, 2006) and treatment of heparin with human SULF2 abolishes its binding to VEGF (Uchimura et al, 2006). Although the exact nature of the structural HS requirement has not been decoded for BMP4 activity, it has been suggested that strict levels of 6-O sulfation are critical for BMP regulation since in-vitro studies show that exposure to Quail Sulf1 leads to the dissociation of BMP from its inhibitor noggin, allowing BMP to freely associate with its receptor and promote signalling (Viviano et al, 2004).…”