Malcolm Lyon

The Interaction of the Transforming Growth Factor-βs with Heparin/Heparan Sulfate Is Isoform-specific

We have undertaken a comparative study of the interaction of the three mammalian transforming growth factor-βs (TGF-β) with heparin and heparan sulfate. TGF-β1 and -β2, but not -β3, bind to heparin and the highly sulfated liver heparan sulfate. These polysaccharides potentiate the biological activity of TGF-β1 (but not the other isoforms), whereas a low sulfated mucosal heparan sulfate fails to do so. Potentiation is due to antagonism of the binding and inactivation of TGF-β1 by α2-macroglobulin, rather than by modulation of growth factor-receptor interactions. TGF-β2·α2-macroglobulin complexes are more refractory to heparin/heparan sulfate, and those involving TGF-β3 cannot be affected. Comparison of the amino acid sequences of the TGF-β isoforms strongly implicates the basic amino acid residue at position 26 of each monomer as being a vital binding determinant. A model is proposed in which polysaccharide binding occurs at two distinct sites on the TGF-β dimer. Interaction with heparin and liver heparan sulfate may be most effective because of the ability of the dimer to co-operatively engage two specific sulfated binding sequences, separated by a distance of approximately seven disaccharides, within the same chain.

A New Map of Glycosaminoglycan and C3b Binding Sites on Factor H

Human complement factor H, consisting of 20 complement control protein (CCP) modules, is an abundant plasma glycoprotein. It prevents C3b amplification on self surfaces bearing certain polyanionic carbohydrates, while complement activation progresses on most other, mainly foreign, surfaces. Herein, locations of binding sites for polyanions and C3b are reexamined rigorously by overexpressing factor H segments, structural validation, and binding assays. As anticipated, constructs corresponding to CCPs 7–8 and 19–20 bind well in heparin-affinity chromatography. However, CCPs 8–9, previously reported to bind glycosaminoglycans, bind neither to heparin resin nor to heparin fragments in gel-mobility shift assays. Introduction of nonnative residues N-terminal to a construct containing CCPs 8–9, identical to those in proteins used in the previous report, converted this module pair to an artificially heparin-binding one. The module pair CCPs 12–13 does not bind heparin appreciably, notwithstanding previous suggestions to the contrary. We further checked CCPs 10–12, 11–14, 13–15, 10–15, and 8–15 for ability to bind heparin but found very low affinity or none. As expected, constructs corresponding to CCPs 1–4 and 19–20 bind C3b amine coupled to a CM5 chip (Kds of 14 and 3.5 μM, respectively) or a C1 chip (Kds of 10 and 4.5 μM, respectively). Constructs CCPs 7–8 and 6–8 exhibit measurable affinities for C3b according to surface plasmon resonance, although they are weak compared with CCPs 19–20. Contrary to expectations, none of several constructs encompassing modules from CCP 9 to 15 exhibited significant C3b binding in this assay. Thus, we propose a new functional map of factor H.

HSulf-2, an extracellular endoglucosamine-6-sulfatase, selectively mobilizes heparin-bound growth factors and chemokines: effects on VEGF, FGF-1, and SDF-1

BackgroundHeparin/heparan sulfate (HS) proteoglycans are found in the extracellular matrix (ECM) and on the cell surface. A considerable body of evidence has established that heparin and heparan sulfate proteoglycans (HSPGs) interact with numerous protein ligands including fibroblast growth factors, vascular endothelial growth factor (VEGF), cytokines, and chemokines. These interactions are highly dependent upon the pattern of sulfation modifications within the glycosaminoglycan chains. We previously cloned a cDNA encoding a novel human endosulfatase, HSulf-2, which removes 6-O-sulfate groups on glucosamine from subregions of intact heparin. Here, we have employed both recombinant HSulf-2 and the native enzyme from conditioned medium of the MCF-7-breast carcinoma cell line. To determine whether HSulf-2 modulates the interactions between heparin-binding factors and heparin, we developed an ELISA, in which soluble factors were allowed to bind to immobilized heparin.ResultsOur results show that the binding of VEGF, FGF-1, and certain chemokines (SDF-1 and SLC) to immobilized heparin was abolished or greatly diminished by pre-treating the heparin with HSulf-2. Furthermore, HSulf-2 released these soluble proteins from their association with heparin. Native Sulf-2 from MCF-7 cells reproduced all of these activities.ConclusionOur results validate Sulf-2 as a new tool for deciphering the sulfation requirements in the interaction of protein ligands with heparin/HSPGs and expand the range of potential biological activities of this enzyme.

Modified expression of the ADAMTS enzymes and tissue inhibitor of metalloproteinases 3 during human intervertebral disc degeneration

OBJECTIVE Intervertebral disc degeneration is linked to loss of extracellular matrix (ECM), particularly the early loss of aggrecan. A group of metalloproteinases called aggrecanases are important mediators of aggrecan turnover. The present study was undertaken to investigate the expression of the recognized aggrecanases and their inhibitor, tissue inhibitor of metalloproteinases 3 (TIMP-3), in human intervertebral disc tissue. METHODS Twenty-four nondegenerated and 30 degenerated disc samples were analyzed for absolute messenger RNA (mRNA) copy number of ADAMTS 1, 4, 5, 8, 9, and 15 and TIMP-3 by real-time reverse transcription-polymerase chain reaction. Thirty-six formalin-fixed embedded intervertebral disc samples of varying grades of degeneration were used for immunohistochemical analyses. In addition, samples from 8 subjects were analyzed for the presence of matrix metalloproteinase (MMP)- and aggrecanase-generated aggrecan products. RESULTS Messenger RNA for all the aggrecanases other than ADAMTS-8 was identified in intervertebral disc tissue, as was mRNA for TIMP-3. Levels of mRNA expression of ADAMTS 1, 4, 5, and 15 were significantly increased in degenerated tissue compared with nondegenerated tissue. All these aggrecanases and TIMP-3 were also detected immunohistochemically in disc tissue, and numbers of nucleus pulposus cells staining positive for ADAMTS 4, 5, 9, and 15 were significantly increased in degenerated tissue compared with nondegenerated tissue. Aggrecan breakdown products generated by MMP and aggrecanase activities were also detected in intervertebral disc tissue. CONCLUSION The aggrecanases ADAMTS 1, 4, 5, 9, and 15 may contribute to the changes occurring in the ECM during intervertebral disc degeneration. Targeting these enzymes may be a possible future therapeutic strategy for the prevention of intervertebral disc degeneration and its associated morbidity.

The molecular phenotype of heparan sulfate in the Hs2st-/- mutant mouse.

Heparan sulfate (HS) is a co-receptor for a number of growth factors, morphogens, and adhesion proteins. HS biosynthetic modifications may determine the strength and outcome of HS-ligand interactions. We previously described the phenotype of mice with a gene-trap mutation inHs2st, encoding the key HS 2-O-sulfotransferase enzyme in HS polymer modification. In contrast to the early developmental failure of embryos lacking HS, the onset of abnormalities in the Hs2st −/− mice occurs only after midgestation, the most dramatic being the complete failure of kidney development. Uronate 2-O-sulfates were not detected in the mutant HS, indicating a complete loss of function of Hs2st. However, the domain structure of the mutant HS is conserved, and compensatory increases in N- and 6-O-sulfation maintain the overall charge density. The apparent affinities of the mutant HS for hepatocyte growth factor/scatter factor and fibronectin were unchanged but were reduced for fibroblast growth factor-1 and -2. Surprisingly, theHs2st −/− cells were able to mount an apparently normal signaling response to fibroblast growth factor-1 and -2 as well as to hepatocyte growth factor/scatter factor.

Endocan is a novel chondroitin sulfate/dermatan sulfate proteoglycan that promotes hepatocyte growth factor/scatter factor mitogenic activity.

Proteoglycans that modulate the activities of growth factors, chemokines, and coagulation factors regulate in turn the vascular endothelium with respect to processes such as inflammation, hemostasis, and angiogenesis. Endothelial cell-specific molecule-1 is mainly expressed by endothelial cells and regulated by pro-inflammatory cytokines (Lassalle, P., Molet, S., Janin, A., Heyden, J. V., Tavernier, J., Fiers, W., Devos, R., and Tonnel, A. B. (1996) J. Biol. Chem. 271, 20458–20464). We demonstrate that this molecule is secreted as a soluble dermatan sulfate (DS) proteoglycan. This proteoglycan represents the major form either secreted by cell lines or circulating in the human bloodstream. Because this proteoglycan is specifically secreted by endothelial cells, we propose to name it endocan. The glycosaminoglycan component of endocan consists of a single DS chain covalently attached to serine 137. Endocan dose-dependently increased the hepatocyte growth factor/scatter factor (HGF/SF)-mediated proliferation of human embryonic kidney cells, whereas the nonglycanated form of endocan did not. Moreover, DS chains purified from endocan mimicked the endocan-mediated increase of cell proliferation in the presence of HGF/SF. Overall, our results demonstrate that endocan is a novel soluble dermatan sulfate proteoglycan produced by endothelial cells. Endocan regulates HGF/SF-mediated mitogenic activity and may support the function of HGF/SF not only in embryogenesis and tissue repair after injury but also in tumor progression.

Hepatocyte growth factor/scatter factor binds with high affinity to dermatan sulfate

We have demonstrated by affinity chromatography that hepatocyte growth factor/scatter factor (HGF/SF) binds strongly to dermatan sulfate (DS), with a similar ionic strength dependence to that previously seen with heparan sulfate (HS). Analysis of binding kinetics on a biosensor yields an equilibrium dissociation constant,K D , of 19.7 nm. This corresponds to a 10–100-fold weaker interaction than that with HS, primarily due to a faster dissociation rate of the complex. The smallest DS oligosaccharide with significant affinity for HGF/SF by affinity chromatography appears to be an octasaccharide. A sequence comprising unsulfated iduronate residues in combination with 4-O-sulfated N-acetylgalactosamine is sufficient for high affinity binding. The presence of 2-O-sulfation on the iduronate residues does not appear to be inhibitory. These observations concur with our previous suggestions, from analyses of HS binding (Lyon, M., Deakin, J. A., Mizuno, K., Nakamura, T., and Gallagher, J.T. (1994) J. Biol. Chem. 269, 11216–11223), that N-sulfation of hexosamines and 2-O-sulfation of iduronates are not absolute requirements for glycosaminoglycan binding to HGF/SF. This is the first described example of a high affinity interaction between a growth factor and DS, and is likely to have significant implications for the biological activity of this paracrine-acting factor.

Highly Sensitive Sequencing of the Sulfated Domains of Heparan Sulfate

The heparan sulfates (HS) are hypervariable linear polysaccharides that act as membrane co-receptors for growth factors, chemokines, and extracellular matrix proteins. In most instances, the molecular basis of protein recognition by HS is poorly understood. We have sequenced 75% of the sulfated domains (S-domains) of fibroblast HS, including all of the major ones. This analysis revealed tight coupling of N- and 2-O-sulfation and a low frequency but precise positioning of 6-O-sulfates, which are required functional groups for HS-mediated activation of the fibroblast growth factors. S-domain sequencing was conducted using a novel and highly sensitive method based on a new way of reading the sequence from high performance liquid chromatography separation profiles of metabolically labeled HS-saccharides following specific chemical and enzymatic scission. The implications of the patterns seen in the sulfated domains for better understanding of the synthesis and function of HS are discussed.

Heparan Sulfate Undergoes Specific Structural Changes during the Progression from Human Colon Adenoma to Carcinoma in Vitro

We report a detailed analysis of heparan sulfate (HS) structure using a model of human colon carcinogenesis. Metabolically radiolabeled HS was isolated from adenoma and carcinoma cells. The chain length of HS was the same in both cell populations (M r 20,000; 45–50 disaccharides), and the chains contained on average of two sulfated domains (S domains), identified by heparinase I scission. This enzyme produced fragments of approximate size 7 kDa, suggesting that the S domains were evenly spaced in the intact HS chain. The degree of polymer sulfation and the patterns of sulfation were strikingly different between the two HS species. When compared with adenoma HS, the iduronic acid 2-O-sulfate content of the carcinoma-derived material was reduced by 33%, and the overall level of N-sulfation was reduced by 20%. However, the level of 6-O-sulfation was increased by 24%, and this was almost entirely attributable to an enhanced level of N-sulfated glucosamine 6-O-sulfate, a species whose data implied was mainly located in the mixed sequences of alternating N-sulfated and N-acetylated disaccharides. The results indicate that in the transition to malignancy in human colon adenoma cells, the overall molecular organization of HS is preserved, but there are distinct modifications in both the S domains and their flanking mixed domains that may contribute to the aberrant behavior of the cancer cell.

Structure Shows that a Glycosaminoglycan and Protein Recognition Site in Factor H is Perturbed by Age-Related Macular Degeneration-Linked Single Nucleotide Polymorphism.

A common single nucleotide polymorphism in the factor H gene predisposes to age-related macular degeneration. Factor H blocks the alternative pathway of complement on self-surfaces bearing specific polyanions, including the glycosaminoglycan chains of proteoglycans. Factor H also binds C-reactive protein, potentially contributing to noninflammatory apoptotic processes. The at risk sequence contains His (rather than Tyr) at position 402 (384 in the mature protein), in the seventh of the 20 complement control protein (CCP) modules (CCP7) of factor H. We expressed both His402 and Tyr402 variants of CCP7, CCP7,8, and CCP6-8. We determined structures of His402 and Tyr402 CCP7 and showed them to be nearly identical. The side chains of His/Tyr402 have similar, solvent-exposed orientations far from interfaces with CCP6 and -8. Tyr402 CCP7 bound significantly more tightly than His402 CCP7 to a heparin affinity column as well as to defined-length sulfated heparin oligosaccharides employed in gel mobility shift assays. This observation is consistent with the position of the 402 side chain on the edge of one of two glycosaminoglycan-binding surface patches on CCP7 that we inferred on the basis of chemical shift perturbation studies with a sulfated heparin tetrasaccharide. According to surface plasmon resonance measurements, Tyr402 CCP6-8 binds significantly more tightly than His402 CCP6-8 to immobilized C-reactive protein. The data support a causal link between H402Y and age-related macular degeneration in which variation at position 402 modulates the response of factor H to age-related changes in the glycosaminoglycan composition and apoptotic activity of the macula.