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...

Glypicans represent a family of six cell surface heparan sulfate proteoglycans in vertebrates. Although no specific in vivo functions have thus far been described for these proteoglycans, spontaneous mutations in the human and induced deletions in the mouse glypican-3 (Gpc3) gene result in severe malformations and both pre- and postnatal overgrowth, known clinically as the Simpson-Golabi-Behmel syndrome (SGBS). Mice carrying mutant alleles of Gpc3 created by either targeted gene disruption or gene trapping display a wide range of phenotypes associated with SGBS including renal cystic dysplasia, ventral wall defects, and skeletal abnormalities that are consistent with the pattern of Gpc3 expression in the mouse embryo. Previous studies in Drosophila have implicated glypicans in the signaling of decapentaplegic, a BMP homolog. Our experiments with mice show a significant relationship between vertebrate BMP signaling and glypican function; GPC3-deficient animals were mated with mice haploinsufficient for bone morphogenetic protein-4 (Bmp4) and their offspring displayed a high penetrance of postaxial polydactyly and rib malformations not observed in either parent strain. This previously unknown link between glypican-3 and BMP4 function provides evidence of a role for glypicans in vertebrate limb patterning and skeletal development and suggests a mechanism for the skeletal defects seen in SGBS.

Heparan sulfate is ubiquitous at the cell surface, where it is expressed predominantly on proteoglycans of either the transmembrane syndecan family or the glycosylphosphatidylinositol (GPI)-anchored glypican family, and has been proposed to function as a "coreceptor" for a number of "heparin-binding" growth factors. Although little is known about functional differences between individual members of the glypican gene family, mutations in both the Drosophila gene dally and the human gene for glypican-3 strongly suggest that at least some glypicans do function in cellular growth control and morphogenesis. In particular, deletion of the human glypican-3 gene is responsible for Simpson-Golabi-Behmel syndrome, and its associated pre- and postnatal tissue overgrowth, increased risk of embryonal tumors during early childhood, and numerous visceral and skeletal anomalies. We have identified and characterized, by sequencing of EST clones and products of rapid amplification of cDNA ends (RACE), an mRNA that encodes a 572-amino-acid member of the glypican gene family (glypican-5) that is most related (50% amino acid similarity, 39% identity) to glypican-3. Glypican-5 mRNA is detected as a 3.9- and 4.4-kb transcript in adult and neonatal mouse brain total RNA, and in situ hybridization results localize transcript primarily to restricted regions of the developing central nervous system, limb, and kidney in patterns consistent with a role in the control of cell growth or differentiation. Interestingly, glypican-5 localizes to 13q31-32 of the human genome, deletions of which are associated with human 13q- syndrome, a developmental disorder with a pattern of defects that shows significant overlap with the pattern of glypican-5 expression.

In mmals, ceil-surface heparan sulfate is required for the action of basic fibroblast growth factor, fibronectin, antithrombln m, as well as other effectors. The syndecans, a gene family offour transmembrane proteoglycans that participates in these interactions, are the major source of this heparan sulfate. Based on the conserved transmembrane and cytoplasmic domains ofthe ma syndecans, a single syndecan-like gene was detected and localized in the Drosophila genome. As in mammals, Drosophila syndecan is a heparan sulfate proteoglycan expressed at the cell surface that can be shed from cultured cells. The single Drosophila syndecan is expressed in embryonic tissues that correspond with those tissues in mammals that express distinct members of the syndecan family predominantly. Conservation of this class of molecules suggests that Drosophila, like mammals, uses cellsurface heparan sulfate as a receptor or coreceptor for extracellular effector molecules.

The glypicans are a family of glycosylphosphatidylinositol (GPI)-anchored proteoglycans that, by virtue of their cell-surface localization and possession of heparan sulfate chains, may regulate the responses of cells to numerous heparin-binding growth factors, cell adhesion molecules, and extracellular matrix components. Mutations in one glypican cause a syndrome of human birth defects, suggesting important roles for these proteoglycans in development. Glypican-1, the first-discovered member of this family, was originally found in cultured fibroblasts, and later shown to be a major proteoglycan of the mature and developing brain. Here we examine the pattern of glypican-1 mRNA and protein expression more widely in the developing rodent, concentrating on late embryonic and early postnatal stages. High levels of glypican-1 expression were found throughout the brain and skeletal system. In the brain, glypican-1 mRNA was widely, and sometimes only transiently, expressed by zones of neurons and neuroepithelia. Glypican-1 protein localized strongly to axons and, in the adult, to synaptic terminal fields as well. In the developing skeletal system, glypican-1 was found in the periosteum and bony trabeculae in a pattern consistent with expression by osteoblasts, as well as in the bone marrow. Glypican-1 was also observed in skeletal and smooth muscle, epidermis, and in the developing tubules and glomeruli of the kidney. Little or no expression was observed in the developing heart, lung, liver, dermis, or vascular endothelium at the stages examined. The tissue-, cell type-, and in some cases stage-specific expression of glypican-1 revealed in this study are likely to provide insight into the functions of this proteoglycan in development.

Bone morphogenetic proteins (BMPs) are expressed broadly and regulate a diverse array of developmental events in vivo. Essential to many of these functions is the establishment of activity gradients of BMP, which provide positional information that influences cell fates. Secreted polypeptides, such as Noggin, bind BMPs and inhibit their function by preventing interaction with receptors on the cell surface. These BMP antagonists are assumed to be diffusible and therefore potentially important in the establishment of BMP activity gradients in vivo. Nothing is known, however, about the potential interactions between Noggin and components of the cell surface or extracellular matrix that might limit its diffusion. We have found that Noggin binds strongly to heparin in vitro, and to heparan sulfate proteoglycans on the surface of cultured cells. Noggin is detected only on the surface of cells that express heparan sulfate, can be specifically displaced from cells by heparin, and can be directly cross-linked to a cell surface proteoglycan in culture. Heparan sulfate-bound Noggin remains functional and can bind BMP4 at the plasma membrane. A Noggin mutant with a deletion in a putative heparin binding domain has reduced binding to heparin and does not bind to the cell surface but has preserved BMP binding and antagonist functions. Our results imply that interactions between Noggin and heparan sulfate proteoglycans in vivo regulate diffusion and therefore the formation of gradients of BMP activity.

Loss of function mutations in the gene encoding the heparan sulfate proteoglycan Glypican-3 (GPC3) causes an X-linked disorder in humans known as Simpson-Golabi-Behmel Syndrome (SGBS). This disorder includes both pre- and postnatal overgrowth, a predisposition to certain childhood cancers, and a complex assortment of congenital defects including skeletal abnormalities. In this study, we have identified a previously unrecognized delay in endochondral ossification associated with the loss of Gpc3 function. Gpc3 knockout animals show a marked reduction in calcified trabecular bone, and an abnormal persistence of hypertrophic chondrocytes at embryonic day 16.5 (E16.5). These hypertrophic chondrocytes down-regulate Type X collagen mRNA expression and undergo apoptosis, suggesting a normal progression of hypertrophic chondrocyte cell fate. However, replacement of these cells by mineralized bone is delayed in association with a marked delay in the appearance of osteoclasts in the bone in vivo. This delay in vivo correlates with a significant reduction in the capacity to form osteoclasts from bone marrow macrophage precursors in vitro in response to M-CSF and RANKL, and with a reduction in the numbers of bone-marrow-derived cells expressing the markers CD11b and Gr-1. Together, these results indicate selective impairment in the development of the common hematopoietic lineage from which monocyte/macrophages and PMNs are derived. This is the first report of a requirement for heparan sulfate, and specifically Gpc3, in the lineage-specific differentiation of these cell types in vivo.

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