Glycosaminoglycans inhibit the adherence and the spreading of osteoclasts and their precursors: role in osteoclastogenesis and bone resorption.

The bone microenvironment (e.g. glycosaminoglycans (GAGs), growth factors) plays a major role in bone resorption, especially in the formation of osteoclasts which differentiate from the hematopoietic lineage in the presence of RANKL. Previous studies revealed that GAGs may influence osteoclastogenesis, but data are very controversial, some studies showing an inhibitory effect of GAGs on osteoclastic differentiation whereas others demonstrated a stimulatory effect. To clarify their activities, we investigated the effect of 5 families of GAGs in three different models of human/mouse osteoclastogenesis. The present data revealed that heparin inhibited osteoclastogenesis in these three models, which was confirmed by a decrease in mRNA expression of osteoclastic markers and by an inhibition of the bone resorption capacity. We also demonstrated in RAW 264.7 cells that other families of GAGs different from heparin inhibited RANKL-induced osteoclastogenesis, and that this inhibition was dependent on the length and the level of sulfation of GAGs. In the present work, heparin did not bind to RANKL and did not modulate RANKL signaling. Heparin acted at 2 distinct steps of osteoclastogenesis from human CD14(+) cells: first, heparin strongly decreased the adherence of osteoclast precursors, and secondly inhibited osteoclasts to spread and to be active. Furthermore, the second action of heparin was reversible as the removal of heparin at the end of the culture time allowed the condensed cells to spread out and showed the formation of morphological active osteoclasts. The present work clearly evidences that GAGs inhibit osteoclastogenesis in vitro and strengthens the therapeutic interest of defined GAGs in osteolytic diseases.

Evidence that heparin saccharides promote FGF2 mitogenesis through two distinct mechanisms.

Heparin-like saccharides play an essential role in binding to both fibroblast growth factors (FGF) and their receptors at the cell surface. In this study we prepared a series of heparin oligosaccharides according to their size and sulfation level. We then investigated their affinity for FGF2 and their ability to support FGF2 mitogenesis of heparan sulfate-deficient cells expressing FGFR1c. Tetra- and hexasaccharides bound FGF2, but failed to dimerize the growth factor. Nevertheless, these saccharides promoted FGF2-mediated cell growth. Furthermore, whereas enzymatic removal of the non-reducing end 2-O-sulfate group had little effect on the 1:1 interaction with FGF2, it eliminated the mitogenic activity of these saccharides. This evidence supports the symmetric two-end model of ternary complex formation. In contrast, even at very low concentrations, octasaccharide and larger heparin fragments conferred a potent mitogenic activity that was independent of terminal 2-O-sulfation. This correlated with the ability to dimerize FGF2 in an apparently cooperative manner. This data suggests that potent mitogenic signaling results from heparin-mediated trans-dimerization of FGF2, consistent with the asymmetric model of ternary complex formation. We propose that, depending on saccharide structure, there are different architectures and modes of ternary complex assembly that differ in stability and/or efficiency of transmembrane signaling.

Towards GAG glycomics: analysis of highly sulfated heparins by MALDI-TOF mass spectrometry.

Glycomics is a developing field that provides structural information on complex populations of glycans isolated from tissues, cells and organs. Strategies employing matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) are central to glycomic analysis. Current MALDI-based glycomic strategies are capable of efficiently analyzing glycoprotein and glycosphingolipid glycomes but little attention has been paid to devising glycomic methodologies suited to the analysis of glycosaminoglycan (GAG) polysaccharides which pose special problems for MALDI analysis because of their high level of sulfation and large size. In this paper, we describe MALDI strategies that have been optimized for the analysis of highly sulfated GAG-derived oligosaccharides. A crystalline matrix norharmane, as well as an ionic liquid 1-methylimidazolium alpha-cyano-4-hydroxycinnamate (ImCHCA), have been used for the analysis of heparin di-, tetra-, hexa- and decasaccharides carrying from 2 to 13 sulfate groups. Information about the maximum number of sulfate groups is obtained using the ionic liquid whereas MALDI-TOF/TOF MS/MS experiments using norharmane allowed the determination of the nature of the glycosidic backbone, and more precise information about the presence and the position in the sequence of N-acetylated residues.

Neuropilin-1 binds vascular endothelial growth factor 165, placenta growth factor-2, and heparin via its b1b2 domain.

Neuroplin-1 (NRP1), a receptor for vascular endothelial growth factor (VEGF) family members, has three distinct extracellular domains, a1a2, b1b2, and c. To determine the VEGF(165) and placenta growth factor 2 (PlGF-2)-binding sites of NRP1, recombinant NRP1 domains were expressed in mammalian cells as Myc-tagged, soluble proteins, and used in co-precipitation experiments with 125I-VEGF165 and 125I-PlGF-2. Anti-Myc antibodies immunoprecipitated 125I-VEGF165 and 125I-PlGF-2 in the presence of the b1b2 but not of the a1a2 and c domains. Neither b1 nor b2 alone was capable of binding 125I-VEGF165. In competition experiments, VEGF165 competed PlGF-2 binding to the NRP1 b1b2 domain, suggesting that the binding sites of VEGF165 and PlGF-2 overlap. The presence of the a1a2 domain greatly enhanced VEGF165, but not PlGF-2 binding to b1b2. Heparin enhanced the binding of both 125I-VEGF165 and 125I-PlGF-2 to the b1b2 domain by 20- and 4-fold, respectively. A heparin chain of at least 20-24 monosaccharides was necessary for binding. In addition, the b1b2 domain of NRP1 could bind heparin directly, requiring heparin oligomers of at least 8 monosaccharide units. It was concluded that an intact b1b2 domain serves as the VEGF165-, PlGF-2-, and heparin-binding sites in NRP1, and that heparin is a critical component for regulating VEGF165 and PlGF-2 interactions with NRP1 by physically interacting with both receptor and ligands.

Domain-specific modification of heparan sulfate by Qsulf1 modulates the binding of the bone morphogenetic protein antagonist Noggin.

We have reported previously that Noggin is a heparin-binding 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.

The heparin binding properties of heparin cofactor II suggest an antithrombin-like activation mechanism.

The serpin heparin cofactor II (HCII) is a glycosaminoglycan-activated inhibitor of thrombin that circulates at a high concentration in the blood. The antithrombotic effect of heparin, however, is due primarily to the specific interaction of a fraction of heparin chains with the related serpin antithrombin (AT). What currently prevents selective therapeutic activation of HCII is the lack of knowledge of the determinants of glycosaminoglycan binding specificity. In this report we investigate the heparin binding properties of HCII and conclude that binding is nonspecific with a minimal heparin length of 13 monosaccharide units required and affinity critically dependent on ionic strength. Rapid kinetics of heparin binding indicate an induced fit mechanism that involves a conformational change in HCII. Thus, HCII binds to heparin in a manner analogous to the interaction of AT with low affinity heparin. A fully allosteric 2000-fold heparin activation of thrombin inhibition by HCII is demonstrated for heparin chains up to 26 monosaccharide units in length. We conclude that the heparin-binding mechanism of HCII is closely analogous to that of AT and that the induced fit mechanism suggests the potential design or discovery of specific HCII agonists.