Hyaluronan Binding Domain Research Articles

Mapping the Hyaluronan-binding Site on the Link Module from Human Tumor Necrosis Factor-stimulated Gene-6 by Site-directed Mutagenesis

Link modules are hyaluronan-binding domains found in extracellular proteins involved in matrix assembly, development, and immune cell migration. Previously we have expressed the Link module from the inflammation-associated protein tumor necrosis factor-stimulated gene-6 (TSG-6) and determined its tertiary structure in solution. Here we generated 21 Link module mutants, and these were analyzed by nuclear magnetic resonance spectroscopy and a hyaluronan-binding assay. The individual mutation of five amino acids, which form a cluster on one face of the Link module, caused large reductions in functional activity but did not affect the Link module fold. This ligand-binding site in TSG-6 is similar to that determined previously for the hyaluronan receptor, CD44, suggesting that the location of the interaction surfaces may also be conserved in other Link module-containing proteins. Analysis of the sequences of TSG-6 and CD44 indicates that the molecular details of their association with hyaluronan are likely to be significantly different. This comparison identifies key sequence positions that may be important in mediating hyaluronan binding, across the Link module superfamily. The use of multiple sequence alignment and molecular modeling allowed the prediction of functional residues in link protein, and this approach can be extended to all members of the superfamily.

Xenopus brevican is expressed in the notochord and the brain during early embryogenesis.

A complete cDNA encoding the Xenopus laevis homologue of the aggrecan/versican family member, brevican (Xbcan) was cloned from an embryonic stage 42 cDNA library. In the deduced amino acid sequence, 1152 in length, similarity to the hyaluronan-binding (link) domains of brevicans from other species were present in the N-terminal region as well as EGF-, lectin- and complement regulatory protein-like domains in the C-terminal part, the latter three being characteristic for brevican found within the extracellular matrix (J. Biol. Chem. 269 (1994) 10119). Indeed, Xbcan was secreted into the extracellular space as a soluble protein when expressed in oocytes. No cDNAs encoding a GPI-anchored bcan variant could be isolated from that cDNA library. During embryonic development, the expression of this gene was first observed in the notochord of neurula stage embryos. In addition to this, in tailbuds, Xbcan was also found to be expressed within the fifth and sixth rhombomere of the hindbrain. In tadpole stage embryos, expression was furthermore observed in periventricular regions of the developing brain and the rostral part of the spinal cord.

Proteoglycans analyzed by composite gel electrophoresis and immunoblotting.

Proteoglycans are the protein products of diverse genes posttranslationally modified with highly negatively charged side chains, commonly known as glycosami-noglycans. The latter consist of repeating disaccharides capable of forming polymers of varying size, which, depending on the specific disaccharide composition, are known as the chondroitin sulfates, the iduronate-containing dermatan sulfates, keratan sul-fate, and heparan sulfate. Some of the more complex proteoglycans, such as aggrecan, are decorated by additional nonglycosaminoglycan oligosaccharides (). Aggrecan contains additionally a hyaluronan-binding domain that in the extracellular matrix facilitates formation of large noncovalently-bound complexes containing one hyaluronan molecule and numerous aggrecans (). Analysis of proteoglycans by standard SDS-PAGE is complicated by the presence of the negatively charged side chains, which prevent the linerarization of molecules usually achieved by treatment with a mercaptoethanol and SDS. Consequently, in SDS-PAGE most proteoglycans with multiple glycosaminoglycan side chains, if they enter the gel at all, migrate as broad bands and appear as smears due to size heterogeneity and large electroendosmotic effects particularly when the acrylamide composition of the PAGE gel is held to a minimum.

Hyaluronate receptors mediating glioma cell migration and proliferation.

The extracellular matrix (ECM) of the central nervous system (CNS) is enriched in hyaluronate (HA). Ubiquitous receptors for HA are CD44 and the Receptor for HA-Mediated Motility known as RHAMM. In the present study, we have investigated the potential role of CD44 and RHAMM in the migration and proliferation of human astrocytoma cells. HA-receptor expression in brain tumor cell lines and surgical specimens was determined by immunocytochemistry and western blot analyses. The ability of RHAMM to bind ligand was determined through cetylpyridinium chloride (CPC) precipitations of brain tumor lysates in HA-binding assays. The effects of HA, CD44 blocking antibodies, and RHAMM soluble peptide on astrocytoma cell growth and migration was determined using MTT and migration assays. Our results show that the expression of the HA-receptors, CD44, and RHAMM, is virtually ubiquitous amongst glioma cell lines, and glioma tumor specimens. There was a gradient of expression amongst gliomas with high grade gliomas expressing more RHAMM and CD44 than did lower grade lesions or did normal human astrocytes or non-neoplastic specimens of human brain. Specific RHAMM variants of 85- and 58-kDa size were shown to bind avidly to HA following CPC precipitations. RHAMM soluble peptide inhibited glioma cell line proliferation in a dose-dependent fashion. Finally, while anti-CD44 antibodies did not inhibit the migration of human glioma cells, soluble peptides directed at the HA-binding domain of RHAMM inhibited glioma migration both on and off an HA-based ECM. These data support the notion that HA-receptors contribute to brain tumor adhesion, proliferation, and migration, biological features which must be better understood before more effective treatment strategies for these tumors can be found.

Coordinate regulation of cadherin and integrin function by the chondroitin sulfate proteoglycan neurocan.

N-cadherin and beta1-integrins play decisive roles in morphogenesis and neurite extension and are often present on the same cell. Therefore, the function of these two types of adhesion systems must be coordinated in time and space to achieve the appropriate cell and tissue organization. We now show that interaction of the chondroitin sulfate proteoglycan neurocan with its GalNAcPTase receptor coordinately inhibits both N-cadherin- and beta1-integrin-mediated adhesion and neurite outgrowth. Furthermore, the inhibitory activity is localized to an NH(2)-terminal fragment of neurocan containing an Ig loop and an HA-binding domain. The effect of neurocan on beta1-integrin function is dependent on a signal originating from the cadherin cytoplasmic domain, possibly mediated by the nonreceptor protein tyrosine kinase Fer, indicating that cadherin and integrin engage in direct cross-talk. In the developing chick, neural retina neurocan is present in the inner plexiform layer from day 7 on, and the GalNAcPTase receptor becomes restricted to the inner nuclear layer and the ganglion cell layer (as well as the fiber layer), the two forming a sandwich. These data suggest that the coordinate inhibition of cadherin and integrin function on interaction of neurocan with its receptor may prevent cell and neurite migration across boundaries.

Neurocan Is a Heparin Binding Proteoglycan

Neurocan and brevican are related chondroitin sulfate proteoglycans which are mainly expressed in the central nervous system. Neurocan and the secreted brevican variant are composed of globular N-terminal hyaluronan binding domains, central O-linked oligosaccharide attachment regions, and globular C-terminal domains. Interaction studies of mouse brain proteoglycans revealed that neurocan, but not brevican, was retained on a heparin affinity matrix. Also a recombinantly produced C-terminal fragment of neurocan, expressed by HEK 293 cells, was retained by the heparin affinity matrix. The substitution of this fragment with a chondroitin sulfate chain did not inhibit binding to the heparin affinity matrix at physiological NaCl concentrations, but decreased the NaCl concentration necessary for elution. Two potential consequences of the heparin binding ability of neurocan are an enforcement of the interaction with other heparin binding molecules and a directed secretion by polarized cells.

Lecticans: organizers of the brain extracellular matrix.

Lecticans are a family of chondroitin sulfate proteoglycans, encompassing aggrecan, versican, neurocan and brevican. These proteoglycans are characterized by the presence of a hyaluronan-binding domain and a C-type lectin domain in their core proteins. Through these domains, lecticans interact with carbohydrate and protein ligands in the extracellular matrix and act as linkers of these extracellular matrix molecules. In adult brain, lecticans are thought to interact with hyaluronan and tenascin-R to form a ternary complex. We propose that the hyaluronan-lectican-tenascin-R complex constitutes the core assembly of the adult brain extracellular matrix, which is found mainly in pericellular spaces of neurons as 'perineuronal nets'.

Requirements for Signal Delivery Through CD44: Analysis Using CD44-Fas Chimeric Proteins

CD44 is a transmembrane glycoprotein involved in various cell adhesion events, including lymphocyte migration, early hemopoiesis, and tumor metastasis. To examine the requirements of CD44 for signal delivery through the extracellular domain, we constructed a chimeric CD44 protein fused to the intracellular domain of Fas on its C-terminus. In cells expressing the CD44-Fas fusion protein, apoptosis could be induced by treatment with certain anti-CD44 mAbs alone, especially those recognizing the epitope group d, which has been previously shown to play a role in ligand binding, indicating that ligation of a specific region of the CD44 extracellular domain results in signal delivery. Of note was that appropriate ligation of the epitope h also resulted in the generation of apoptotic signal, although this region was not thought to be involved in ligand binding. In contrast, the so-called blocking anti-CD44 mAbs (epitope group f) that can abrogate the binding of hyaluronate (HA) failed to induce apoptosis even after further cross-linking with the secondary Ab, indicating that a mere mAb-induced oligomerization of the chimeric proteins is insufficient for signal generation. However, these blocking mAbs were instead capable of inhibiting apoptosis induced by nonblocking mAb (epitope group h). Furthermore, a chimeric protein bearing a mutation in the HA binding domain and hence lacking the ability to recognize HA was incapable of mediating the mAb-induced apoptosis, suggesting that the functional integrity of the HA binding domain is crucial to the signal generation in CD44.

Versican/PG-M isoforms in vascular smooth muscle cells.

The expression of increased amounts of proteoglycans in the extracellular matrix may play a role in vascular stenosis and lipid retention. The large chondroitin sulfate proteoglycan versican is synthesized by vascular smooth muscle cells (SMCs), accumulates during human atherosclerosis and restenosis, and has been shown to bind LDLs. We recently demonstrated that adult rat aortic SMCs express several versican mRNAs. Four versican splice variants, V0, V1, V2, and V3, have recently been described, which differ dramatically in length. These variants differ in the extent of modification by glycosaminoglycan chains, and V3 may lack glycosaminoglycan chains. In this study, we characterized versican RNAs from rat SMCs by cloning, sequencing, and hybridization with domain-specific probes. DNA sequence was obtained for the V3 isoform, and for a truncated V0 isoform. By hybridization of polyadenylated RNA with domain-specific probes, we determined that the V0, V1, and V3 isoforms are present in vascular SMCs. We confirmed the presence of the V3 isoform in polyadenylated RNA and in RT-PCR products by hybridization with an oligonucleotide that spans the splice junction between the hyaluronan-binding domain and the epidermal growth factor-like domain. In addition, a novel splice variant was cloned by PCR amplification from both rat and human SMC RNA. This appears to be an incompletely spliced variant, retaining the final intron. PCR analysis shows that this intron can be retained in both V1 and V3 isoforms. The predicted translation product of this variant would have a different carboxy-terminus than previously described versican isoforms.

Modulation of hyaluronan receptor (CD44) function in vivo in a murine model of rheumatoid arthritis.

To determine how in vivo modulation of CD44 function by antibodies influences disease severity in a murine model of rheumatoid arthritis. Mice with proteoglycan (PG)-induced arthritis were subjected to systemic treatment with 3 different monoclonal antibodies against CD44. Joint swelling and serum levels of hyaluronan (HA) and soluble CD44 (sCD44) were monitored. Inflammatory leukocyte infiltration in the joints, cell surface CD44 expression, and leukocyte adhesion to HA were compared. The effects of anti-CD44 treatment on the immune status of arthritic animals were also determined. Antibody IRAWB14, which enhances HA binding, aggravated the inflammatory symptoms, while KM201, which blocks ligand binding, reduced the severity of arthritis. The most effective suppression of inflammation was noted upon treatment with antibody IM7, whose epitope lies outside the HA binding domain of CD44. Serum levels of sCD44 increased, and HA levels decreased, in response to IM7. KM201 and IM7 treatment reduced, but IRAWB14 treatment enhanced, the adhesion of leukocytes to HA. However, these antibodies had little effect on PG-specific immune responses. Each antibody acted in vivo by virtue of its combined effects on CD44-HA binding and CD44 shedding. The dramatic reduction in arthritis severity effected by IM7 treatment was associated with extensive shedding of cell surface CD44 molecules. Loss of CD44 appears to be a major factor in preventing CD44- and HA-dependent cell-matrix interactions at the inflammatory site. Our study indicates a critical role for CD44 in the pathology of joint inflammation and reveals a unique mechanism of receptor down-regulation, which can be used therapeutically.

LYVE-1, a new homologue of the CD44 glycoprotein, is a lymph-specific receptor for hyaluronan.

The extracellular matrix glycosaminoglycan hyaluronan (HA) is an abundant component of skin and mesenchymal tissues where it facilitates cell migration during wound healing, inflammation, and embryonic morphogenesis. Both during normal tissue homeostasis and particularly after tissue injury, HA is mobilized from these sites through lymphatic vessels to the lymph nodes where it is degraded before entering the circulation for rapid uptake by the liver. Currently, however, the identities of HA binding molecules which control this pathway are unknown. Here we describe the first such molecule, LYVE-1, which we have identified as a major receptor for HA on the lymph vessel wall. The deduced amino acid sequence of LYVE-1 predicts a 322-residue type I integral membrane polypeptide 41% similar to the CD44 HA receptor with a 212-residue extracellular domain containing a single Link module the prototypic HA binding domain of the Link protein superfamily. Like CD44, the LYVE-1 molecule binds both soluble and immobilized HA. However, unlike CD44, the LYVE-1 molecule colocalizes with HA on the luminal face of the lymph vessel wall and is completely absent from blood vessels. Hence, LYVE-1 is the first lymph-specific HA receptor to be characterized and is a uniquely powerful marker for lymph vessels themselves.

Characterization of a functional hyaluronan-binding domain from the human CD44 molecule expressed in Escherichia coli.

The CD44 molecule is a widely distributed cell surface receptor for the extracellular matrix glycosaminoglycan hyaluronan. The ligand-binding site which is located in the membrane distal portion of the molecule encompasses a region of approximately 100 amino acids termed the Link domain, a structural unit that is conserved among members of the Hyaladherin superfamily which includes cartilage link protein, aggrecan, and tumor necrosis factor-stimulated gene-6 (TSG-6). In contrast to these other Hyaladherins, however, the ligand-binding domain of CD44 appears to extend beyond the Link domain to involve additional basic residues located toward the membrane proximal region. Furthermore, recent molecular modeling studies indicate that within the CD44 Link domain itself, the spatial arrangement of critical residues involved in HA binding is likely to differ significantly from the prototypic TSG-6 Link module. In order to obtain material to solve the CD44 solution structure we have developed an optimized method for the expression and purification of functionally active CD44 ectodomains encompassing both the Link module and the additional downstream HA-binding residues in Escherichia coli. Here we describe the details of the method which involves solubilization of recombinant CD44 from inclusion bodies in 8 M urea, followed by refolding and purification of intact monomers using size-exclusion and reverse-phase chromatography. We show the method yields CD44 molecules that (1) retain reactivity with a panel of conformation-sensitive antibodies, (2) possess similar hyaluronan-binding characteristics to authentically folded CD44 molecules expressed in eukaryotic cells, and (3) display one-dimensional NMR spectra that indicate the presence of a single conformational species. This method should enable sufficient amounts of functional CD44 Link module to be produced for comprehensive structural analyses by multidimensional NMR spectroscopy.

SPACR, a Novel Interphotoreceptor Matrix Glycoprotein in Human Retina That Interacts with Hyaluronan

SPACR (sialoprotein associated with cones and rods), is the major 147-150-kDa glycoprotein present in the insoluble interphotoreceptor matrix of the human retina. Immunocytochemistry localizes SPACR to the matrix surrounding rods and cones (Acharya, S., Rayborn, M. E., and Hollyfield, J. G. (1998) Glycobiology 8, 997-1006). From affinity-purified SPACR, we obtained seven peptide sequences showing 100% identity to the deduced sequence of IMPG1, a purported chondroitin 6-sulfate proteoglycan core protein, which binds peanut agglutinin and is localized to the interphotoreceptor matrix. We show here that SPACR is the most prominent 147-150-kDa band present in the interphotoreceptor matrix and is the gene product of IMPG1. SPACR is not a chondroitin sulfate proteoglycan, since it is not a product of chondroitinase ABC digestion and does not react to a specific antibody for chondroitin 6-sulfate proteoglycan. Moreover, the deduced amino acid sequence reveals no established glycosaminoglycan attachment site. One hyaluronan binding motif is present in the predicted sequence of SPACR. We present evidence that SPACR has a functional hyaluronan binding domain, suggesting that interactions between SPACR and hyaluronan may serve to form the basic macromolecular scaffold, which comprises the insoluble interphotoreceptor matrix.

Epitope Specificity of CD44 for Monoclonal Antibody–Dependent Facilitation of Marrow Engraftment in a Canine Model

AbstractPrimary graft rejection after marrow transplantation occurs more frequently in patients receiving HLA-haploidentical compared with HLA-identical sibling transplants. Both human and experimental animal data suggest that the cells responsible for this phenomenon are either host natural killer (NK) cells, T cells, or both. To investigate the mechanisms of graft rejection, we have developed a canine model of marrow transplantation, which uses DLA-nonidentical unrelated donors in the absence of postgrafting immunosuppression. In this model most animals rejected their marrow grafts after a preparative regimen of 9.2 Gy total body irradiation (TBI). However, engraftment of DLA-nonidentical marrow can be facilitated when the recipients are pretreated with monoclonal antibody (MoAb) S5, which recognizes CD44. In this report, we extended these observations by first cloning the canine CD44 and, next, mapping the epitope recognized by S5, which was located in a region conserved among human and canine CD44 and was distinct from the hyaluronan binding domain. However, in vitro binding of S5 caused a conformational change in CD44, which allowed increased hyaluronan binding. Then, we reexamined the in vivo model of marrow transplantation and compared results with MoAb S5 to those with two other anti-CD44 MoAbs, IM7 and S3. Only MoAb S5 significantly increased the engraftment rate of DLA-nonidentical unrelated marrow, whereas the two other anti-CD44 MoAbs were ineffective. The enhanced in vivo effect was not related to differences in the MoAbs' avidities, since both S5 and IM7 had equivalent binding to CD44, but most likely related to the specific epitope that S5 recognizes. Thus, this study shows that the effect of the anti-CD44 MoAb S5 in facilitating engraftment is epitope specific and if one is to use an anti-CD44 to facilitate engraftment of marrow in humans, one cannot assume that any anti-CD44 would work.

Epitope Specificity of CD44 for Monoclonal Antibody–Dependent Facilitation of Marrow Engraftment in a Canine Model

Primary graft rejection after marrow transplantation occurs more frequently in patients receiving HLA-haploidentical compared with HLA-identical sibling transplants. Both human and experimental animal data suggest that the cells responsible for this phenomenon are either host natural killer (NK) cells, T cells, or both. To investigate the mechanisms of graft rejection, we have developed a canine model of marrow transplantation, which uses DLA-nonidentical unrelated donors in the absence of postgrafting immunosuppression. In this model most animals rejected their marrow grafts after a preparative regimen of 9.2 Gy total body irradiation (TBI). However, engraftment of DLA-nonidentical marrow can be facilitated when the recipients are pretreated with monoclonal antibody (MoAb) S5, which recognizes CD44. In this report, we extended these observations by first cloning the canine CD44 and, next, mapping the epitope recognized by S5, which was located in a region conserved among human and canine CD44 and was distinct from the hyaluronan binding domain. However, in vitro binding of S5 caused a conformational change in CD44, which allowed increased hyaluronan binding. Then, we reexamined the in vivo model of marrow transplantation and compared results with MoAb S5 to those with two other anti-CD44 MoAbs, IM7 and S3. Only MoAb S5 significantly increased the engraftment rate of DLA-nonidentical unrelated marrow, whereas the two other anti-CD44 MoAbs were ineffective. The enhanced in vivo effect was not related to differences in the MoAbs' avidities, since both S5 and IM7 had equivalent binding to CD44, but most likely related to the specific epitope that S5 recognizes. Thus, this study shows that the effect of the anti-CD44 MoAb S5 in facilitating engraftment is epitope specific and if one is to use an anti-CD44 to facilitate engraftment of marrow in humans, one cannot assume that any anti-CD44 would work.

The hyaluronan receptor for RHAMM in noradrenergic fibers contributes to axon growth capacity of locus coeruleus neurons in an intraocular transplant model

The hyaluronan receptor for hyaluronic acid-mediated motility (RHAMM) plays a role in cell migration and motility in many systems. Recent observations on the involvement of RHAMM in neurite motility in vitro suggest that it might also be important in axon outgrowth in situ. This was addressed directly by investigating both RHAMM expression in the rat CNS and the ability of anti-RHAMM reagents to interfere with tissue growth and axon outgrowth in intraocular brainstem transplants. By western blotting, anti-RHAMM antibody detected a RHAMM isoform of 75 000 mol. wt in both whole brain homogenate and synaptosome preparations, and a 65 000 mol. wt isoform in synaptosomes. Immunofluorescence of adult brain sections revealed RHAMM-like immunoreactivity in varicose fibers that were also positive for the noradrenergic marker dopamine- β-hydroxylase. Not all noradrenergic fibers contained RHAMM, nor was RHAMM detected in other monoaminergic fiber types. Lesions of noradrenergic fiber systems with β-halobenzylamine- N-(2-chloroethyl)- N-ethyl-2-bromobenzylamine (DSP-4) eliminated RHAMM-positive fibers, but noradrenergic axons that sprouted extensively after this treatment were strongly RHAMM-positive. To assess RHAMM's role in fiber outgrowth, fetal brainstem tissue containing noradrenergic neurons was grafted into the anterior chamber of the eye. Treatment of grafts with anti-RHAMM antibody caused significant inhibition of tissue growth and axon outgrowth, as did a peptide corresponding to a hyaluronan binding domain of RHAMM. These agents had no such effects on transplants containing serotonergic and dopaminergic neurons. These results suggest that RHAMM, an extracellular matrix receptor previously shown to contribute to migratory and contact behavior of cells, may also be important in the growth and/or regenerative capacity of central noradrenergic fibers originating from the locus coeruleus.

Expression of a Cleaved Brain-Specific Extracellular Matrix Protein Mediates Glioma Cell InvasionIn Vivo

Malignant gliomas (primary brain tumors) aggressively invade the surrounding normal brain. This invasive ability is not demonstrated by brain metastases of nonglial cancers. The brain-specific, brain-enriched hyaluronan binding (BEHAB)/brevican gene, which encodes an extracellular hyaluronan-binding protein, is consistently expressed by human glioma and is not expressed by tumors of nonglial origin (Jaworski et al., 1996). BEHAB/brevican can be cleaved into an N-terminal fragment that contains a hyaluronan-binding domain (HABD) and a C-terminal fragment (Yamada et al., 1995). Here, using antisera to peptides in the predicted N-terminal and C-terminal proteolytic fragments, we demonstrate that the BEHAB/brevican protein is cleaved in invasive human and rodent gliomas. A role for this protein in glioma cell invasion was tested by transfecting a noninvasive cell line with the BEHAB/brevican gene. The noninvasive 9L glioma cell was transfected with either full-length BEHAB/brevican or the HABD and tested for invasion in in vitro and in vivo invasion assays. Although both constructs increased invasion in vitro, only the HABD increased invasion by tumors growing in vivo. Experimental intracranial tumors from full-length transfectants showed no increase in invasion over control tumors, whereas tumors from HABD transfectants showed a marked potentiation of tumor invasion, producing new tumor foci at sites distant from the main tumor mass. This work demonstrates a role for a brain-specific extracellular matrix protein in glioma invasion, opening new therapeutic avenues for a uniformly fatal disease.

Identification, partial characterization, and distribution of versican and link protein in bovine dental pulp.

The dynamics of changes in the cellularity and extracellular matrix composition of dental pulp varies considerably during tooth development and maturation. In this paper, we studied matrix proteoglycans where we hypothesized that they played important roles in structural, spatial, and transport aspects of pulpal development and maintenance. The pulpal tissue was collected from partially erupted bovine incisors, pulverized, and then extracted with 6 M guanidine-HCl. The extract was subjected to anion column chromatography (DEAE-8HR), and the fractions collected were screened by dot-blot immunoassay by means of monoclonal antibodies generated against 4- and 6-sulfated chondroitin sulfate isomers, and keratan sulfate, 2-B-6, 3-B-3, and 5-D-4, respectively. The chondroitin-6-sulfate was the major glycosaminoglycan species and occurred as a large-molecular-weight proteoglycan (> 500 kDa). After further purification, it was subjected to agarose/acrylamide composite gel electrophoresis, and it migrated as a single band stained with Stains-All. The band was immunopositive against antibody 3-B-3 by Western blot analysis. The partial amino acid sequence analyses of the core protein clearly indicated this molecule to be versican. The presence of link protein was also confirmed by Western blot analysis with an anti-link protein monoclonal antibody, 8-A-4. Furthermore, immunohistochemical study indicated that the distributions of versican and link protein coincide in the dental pulp and are enriched in the peripheral area of the tissue just beneath the odontoblast layer. Since the dental pulp contains hyaluronan, versican may bind to hyaluronan via its hyaluronan-binding domain, where this association is stabilized by link protein. This complex, then, could form large hydrated proteoglycan aggregates that fill the extracellular space, support odontoblasts, and/or facilitate the transport function of metabolites and nutrients within the tissue.

Identification of Hyaluronan-binding Domains of Aggrecan

Aggrecan, a large cartilage proteoglycan, interacts with hyaluronan (HA), to form aggregates which function to resist compression in joints. The N-terminal region of aggrecan contains two structurally related globular domains, G1 and G2 separated by IGD domain. The G1 domain consists of three subdomains, A, B, and B', structural features characteristic to many other HA-binding proteoglycans. Here, we studied the interaction of aggrecan domains with HA using recombinant proteins expressed in 293 cells, an embryonal kidney cell line. Deglycosylation of the recombinant aggrecan fragment reduced the HA binding activity. We found that both the B and B' subdomains were required for HA binding and that a single module of A, B, or B' was unable to bind HA. The A subdomain increased the HA binding activity of the B-B' region. The G2 domain had no HA binding activity confirming previous reports. Studies of HA-binding properties using a BIAcoreTM biosensor system revealed that the KD of recombinant aggrecan fragment (AgW) consisting of G1, IGD, and G2 was 0.226 microM, whereas the KD of another HA-binding protein, native bovine link protein, is 0.089 microM. In contrast, AgMut11 which lacked subdomain A showed little HA binding activity. AgMut12 consisting of only B-B' had a 3.4-fold lower affinity and AgMut13 containing A-B-B' was 1.5-fold lower than AgW. These results suggest that carbohydrates are essential for high level aggrecan binding to HA and that the A subdomain of aggrecan functions in a cooperative manner with subdomains B and B'.

Overlapping sites on the Link module of human TSG-6 mediate binding to hyaluronan and chondroitin-4-sulphate

Link modules are hyaluronan-binding domains that are involved in the formation and stability of extracellular matrix and cell migration. We have examined the glycosaminoglycan specificity of the Link module from the arthritis-associated protein, human TSG-6, by microtitre plate-based assays employing biotinylated-hyaluronan or mono-biotinylated Link module. This domain was found to interact specifically with chondroitin-4-sulphate (C4S), with similar affinity to hyaluronan, but not with chondroitin-6-sulphate or heparin. Competition experiments indicate that C4S and hyaluronan have overlapping binding surfaces on the TSG-6 Link module. Disease-associated changes in C4S expression may influence the localisation and biological role of TSG-6.