Link Module Research Articles

Using Molecular Dynamics Simulations To Provide New Insights into Protein Structure on the Nanosecond Timescale: Comparison with Experimental Data and Biological Inferences for the Hyaluronan-Binding Link Module of TSG-6.

Link module domains play an essential role in extracellular matrix assembly and remodeling by binding to the flexible glycosaminoglycan hyaluronan. A high-resolution NMR-structure of the Link module from the protein product of tumor necrosis factor-stimulated gene-6 (Link_TSG6) has been determined, but a fuller appreciation of protein dynamics may be necessary to understand its hyaluronan-binding. Therefore, we have performed a 0.25 μs MD simulation, starting from the lowest-energy NMR-derived solution structure of Link_TSG6, with explicit water and ions, using the CHARMM22 protein force field. The simulation was as good a fit to the NMR data as the ensemble from simulated annealing, except in the β5-β6 loop. Furthermore, analysis revealed that secondary structure elements extended further than previously reported and underwent fast picosecond time scale dynamics, whereas nanosecond dynamics was found in certain loops. In particular, surface side chains proposed to interact with glycosaminoglycans were predicted to be highly mobile and be directed away from the protein surface. Furthermore, the hyaluronan-binding β4-β5 loop remained in a closed conformation, favoring an allosteric interaction mechanism. This enhanced view of the Link module provides general insight into protein dynamics and may be helpful for understanding the dynamic molecular basis of tissue assembly, remodeling, and disease processes.

Ligand-induced Structural Changes of the CD44 Hyaluronan-binding Domain Revealed by NMR

CD44, a major cell surface receptor for hyaluronan (HA), contains a functional domain responsible for HA binding at its N terminus (residues 21-178). Accumulating evidence indicates that proteolytic cleavage of CD44 in its extracellular region (residues 21-268) leads to enhanced tumor cell migration and invasion. Hence, understanding the mechanisms underlying the CD44 proteolytic cleavage is important for understanding the mechanism of CD44-mediated tumor progression. Here we present the NMR structure of the HA-binding domain of CD44 in its HA-bound state. The structure is composed of the Link module (residues 32-124) and an extended lobe (residues 21-31 and 125-152). Interestingly, a comparison of its unbound and HA-bound structures revealed that rearrangement of the beta-strands in the extended lobe (residues 143-148) and disorder of the structure in the following C-terminal region (residues 153-169) occurred upon HA binding, which is consistent with the results of trypsin proteolysis studies of the CD44 HA-binding domain. The order-to-disorder transition of the C-terminal region by HA binding may be involved in the CD44-mediated cell migration.

Versican-thrombospondin-1 binding in vitro and colocalization in microfibrils induced by inflammation on vascular smooth muscle cells

We identified a specific interaction between two secreted proteins, thrombospondin-1 and versican, that is induced during a toll-like receptor-3-dependent inflammatory response in vascular smooth muscle cells. Thrombospondin-1 binding to versican is modulated by divalent cations. This interaction is mediated by interaction of the G1 domain of versican with the N-module of thrombospondin-1 but only weakly with the corresponding N-terminal region of thrombospondin-2. The G1 domain of versican contains two Link modules, which are known to mediate TNFalpha-stimulated gene-6 protein binding to thrombospondin-1, and the related G1 domain of aggrecan is also recognized by thrombospondin-1. Therefore, thrombospondin-1 interacts with three members of the Link-containing hyaladherin family. On the surface of poly-I:C-stimulated vascular smooth muscle cells, versican organizes into fibrillar structures that contain elastin but are largely distinct from those formed by hyaluronan. Endogenous and exogenously added thrombospondin-1 incorporates into these structures. Binding of exogenous thrombospondin-1 to these structures, to purified versican and to its G1 domain is potently inhibited by heparin. At higher concentrations, exogenous thrombospondin-1 delays the poly-I:C induced formation of structures containing versican and elastin, suggesting that thrombospondin-1 negatively modulates this component of a vascular smooth muscle inflammatory response.

Tumour necrosis factor α-stimulated gene-6 inhibits osteoblastic differentiation of human mesenchymal stem cells induced by osteogenic differentiation medium and BMP-2

To better understand the molecular pathogenesis of OPLL (ossification of the posterior longitudinal ligament) of the spine, an ectopic bone formation disease, we performed cDNA microarray analysis on cultured ligament cells from OPLL patients. We found that TSG-6 (tumour necrosis factor alpha-stimulated gene-6) is down-regulated during osteoblastic differentiation. Adenovirus vector-mediated overexpression of TSG-6 inhibited osteoblastic differentiation of human mesenchymal stem cells induced by BMP (bone morphogenetic protein)-2 or OS (osteogenic differentiation medium). TSG-6 suppressed phosphorylation and nuclear accumulation of Smad 1/5 induced by BMP-2, probably by inhibiting binding of the ligand to the receptor, since interaction between TSG-6 and BMP-2 was observed in vitro. TSG-6 has two functional domains, a Link domain (a hyaluronan binding domain) and a CUB domain implicated in protein interaction. The inhibitory effect on osteoblastic differentiation was completely lost with exogenously added Link domain-truncated TSG-6, while partial inhibition was retained by the CUB domain-truncated protein. In addition, the inhibitory action of TSG-6 and the in vitro interaction of TSG-6 with BMP-2 were abolished by the addition of hyaluronan. Thus, TSG-6, identified as a down-regulated gene during osteoblastic differentiation, suppresses osteoblastic differentiation induced by both BMP-2 and OS and is a plausible target for therapeutic intervention in OPLL.

SHAP Potentiates the CD44-mediated Leukocyte Adhesion to the Hyaluronan Substratum

CD44-hyaluronan (HA) interaction is involved in diverse physiological and pathological processes. Regulation of interacting avidity is well studied on CD44 but rarely on HA. We discovered a unique covalent modification of HA with a protein, SHAP, that corresponds to the heavy chains of inter-alpha-trypsin inhibitor family molecules circulating in blood. Formation of the SHAP.HA complex is often associated with inflammation, a well known process involving the CD44-HA interaction. We therefore examined the effect of SHAP on the CD44-HA interaction-mediated lymphocyte adhesion. Under both static and flowing conditions, Hut78 cells (CD44-positive) and CD44-transfected Jurkat cells (originally CD44-negative) adhered preferentially to the immobilized SHAP.HA complex than to HA. The enhanced adhesion is exclusively mediated by the CD44-HA interaction, because it was inhibited by HA, but not IalphaI, and was completely abolished by pretreating the cells with anti-CD44 antibodies. SHAP appears to potentiate the interaction by increasing the avidity of HA to CD44 and altering their distribution on cell surfaces. Large amounts of the SHAP.HA complex accumulate in the hyperplastic synovium of rheumatoid arthritis patients. Leukocytes infiltrated to the synovium were strongly positive for HA, SHAP, and CD44 on their surfaces, suggesting a role for the adhesion-enhancing effect of SHAP in pathogenesis.

TSG-6: a pluripotent inflammatory mediator?

TSG-6 is a multifunctional protein that is up-regulated in many pathological and physiological contexts, where it plays important roles in inflammation and tissue remodelling. For example, it is a potent inhibitor of neutrophil migration and can modulate the protease network through inhibition of plasmin. TSG-6 binds a wide range of GAGs (glycosaminoglycans) [i.e. HA (hyaluronan), chondroitin 4-sulphate, dermatan sulphate, heparin and heparan sulphate] as well as a variety of protein ligands, where these interactions can influence the activities of TSG-6. For example, through its association with HA, TSG-6 can mediate HA cross-linking via several different mechanisms, some of which promote leucocyte adhesion. Binding to heparin, however, enhances the ability of TSG-6 to potentiate the anti-plasmin activity of inter-alpha-inhibitor, which binds non-covalently to TSG-6 via its bikunin chain. Furthermore, although HA and heparin interact with distinct sites on the Link module, the binding of heparin can inhibit subsequent interaction with HA. In addition, the interactions of TSG-6 with HA, heparin and at least some of its protein ligands are sensitive to pH. Therefore it seems that in different tissue micro-environments (characterized, for example, by pH and GAG content), TSG-6 could be partitioned into functional pools with distinct activities.

Overexpression of Hyaluronan Synthase 2 Alters Hyaluronan Distribution and Function in Proximal Tubular Epithelial Cells

The functional consequences of increased renal cortical hyaluronan that is associated with both acute injury and progressive scarring are unclear. The aim of this study was to characterize hyaluronan synthase-2 (HAS2)-driven HA synthesis and determine its effect on renal proximal tubular epithelial cell (PTC) function, because this is known to be the inducible form of HA synthase in this cell type. Overexpression of HAS2 mRNA increased HA generation, which in the supernatant predominantly was HA of large molecular weight, whereas there was an increase in low molecular weight HA in cell-associated fractions. This was associated with increased expression of hyaluronidases, inhibition of HA cable formation concurrent with reduction in HA-dependent monocyte binding, and increased pericellular HA matrix. Overexpression of HAS2 led to enhanced cell migration. HA can be modified by the covalent attachment of heavy chains that are derived from the serum protein inter-alpha-inhibitor (IalphaI), a process that is known to be catalyzed by TNF-alpha-stimulated gene 6 (TSG-6; an inflammation-associated protein). Enhanced migration was abrogated by blocking antibodies to either IalphaI or TSG-6. Addition of recombinant full-length TSG-6 (TSG-6Q) or TSG-6Q_Y94F, a mutant variant with impaired HA binding, increased cell migration. Both of these proteins were able to mediate the covalent transfer of heavy chains, from IalphaI and pre-alpha-inhibitor, onto HA. Addition of the isolated TSG-6-Link module (Link_TSG-6), which binds HA but is unable to form covalent complexes with IalphaI/pre-alpha-inhibitor, had no effect on migration, suggesting that TSG-6-mediated formation of heavy chain-HA complexes is critical in the formation of a pericellular HA matrix.

The DNA‐remodelling activity of DnaD is the sum of oligomerization and DNA‐binding activities on separate domains

The Bacillus subtilis DnaD protein is an essential protein that has been implicated in the primosomal step of DNA replication, and recently in global DNA remodelling. Here we show that DnaD consists of two domains with distinct activities; an N-terminal domain (Nd) with oligomerization activity, and a C-terminal domain (Cd) with DNA-binding activity and a second DNA-induced oligomerization activity. Although Cd can bind to DNA and form large nucleoprotein complexes, it does not exhibit global DNA-remodelling activity. The presence of separate Nd does not restore this activity. Our data suggest that the global DNA-remodelling activity of DnaD is the sum of three separate oligomerization and DNA-binding activities residing on two distinct but linked domains.

Structure of the inflammatory homing receptor CD44 complexed with its pericellular matrix ligand hyaluronan.

The hyaluronan receptor CD44 mediates cell adhesion/migration through tissue matrix during embryonic morphogenesis, tissue repair and leukocyte homing. However, despite their fundamental importance, the precise molecular details of CD44:hyaluronan interactions remain unknown. Here we have determined the crystal structure of the hyaluronan-binding domain complexed with a hyaluronan octasaccharide. This identifies the binding surface as a shallow groove in the main link module lined by a cluster of predominantly hydrophobic residues (tyrosine, alanine and isoleucine) and two key arginines, that together co-ordinate four sugars (GlcUA5-GlcNAc8) of the bound octasaccharide via hydrogen bonds rather than aromatic sugar stacking or ionic interactions. Moreover, we show that the complex experiences a conformational shift within the binding groove that brings an essential arginine residue (Arg45) into contact with ligand. A number of other residues within the link extension previously implicated in ligand-binding, make no apparent contacts with ligand. These data define a molecular interaction of fundamental importance to cell migration as well as providing the basis necessary for rational design of small molecule inhibitors for the treatment of inflammatory disease.

Modular design of soft-switching circuits for two-level and three-level inverters

This paper presents a negative-bus auxiliary resonant circuit (NBARC) and novel mirror symmetrical pair of resonant link modules for soft commutation of two-level and three-level inverters. The NBARC topology reduces the power device counts and requires low device power ratings. While the basic NBARC resonant circuit is conceived and analyzed for zero-voltage switching (ZVS) of two-level inverters, the topology and modular design approach are extended to form a pinched-link resonant stage of ZVS for three-level inverters using mirror-symmetrical pair of resonant modules. The circuit is also designed as an optional module that can be attached to a standard inverter bridge and converted into a soft-switched inverter. Experimental results verify our circuit analysis and implementation.

The N-terminal Module of Thrombospondin-1 Interacts with the Link Domain of TSG-6 and Enhances Its Covalent Association with the Heavy Chains of Inter-α-trypsin Inhibitor

We recently found that leukocytes from thrombospondin-1 (TSP1)-deficient mice exhibit significant reductions in cell surface CD44 relative to those from wild type mice. Because TSG-6 modulates CD44-mediated cellular interactions with hyaluronan, we examined the possibility that TSP1 interacts with TSG-6. We showed that recombinant full-length human TSG-6 (TSG-6Q) and the Link module of TSG-6 (Link_TSG6) bind 125I-TSP1 with comparable affinities. Trimeric recombinant constructs containing the N-modules of TSP1 or TSP2 inhibit binding of TSP1 to TSG-6Q and Link_TSG6, but other recombinant regions of TSP1 do not. Therefore, the N-modules of both TSP1 and TSP2 specifically recognize the Link module of TSG-6. Heparin, which binds to these domains of both proteins, strongly inhibits binding of TSP1 to Link_TSG6 and TSG-6Q, but hyaluronan does not. Inhibition by heparin results from its binding to TSP1, because heparin also inhibits TSP1 binding to Link_TSG6 mutants deficient in heparin binding. Removal of bound Ca2+ from TSP1 reduces its binding to full-length TSG-6. Binding of TSP1 to Link_TSG6, however, is enhanced by chelating divalent cations. In contrast, divalent cations do not influence binding of the N-terminal region of TSP1 to TSG-6Q. This implies that divalent cation dependence is due to conformational effects of calcium-binding to the C-terminal domains of TSP1. TSP1 enhances covalent modification of the inter-alpha-trypsin inhibitor by TSG-6 and transfer of its heavy chains to hyaluronan, suggesting a physiological function of TSP1 binding to TSG-6 in regulation of hyaluronan metabolism at sites of inflammation.

Characterization of the Interaction between Tumor Necrosis Factor-stimulated Gene-6 and Heparin: IMPLICATIONS FOR THE INHIBITION OF PLASMIN IN EXTRACELLULAR MATRIX MICROENVIRONMENTS

TSG-6, the secreted product of tumor necrosis factor-stimulated gene-6, is not constitutively expressed but is up-regulated in various cell-types during inflammatory and inflammation-like processes. The mature protein is comprised largely of contiguous Link and CUB modules, the former binding several matrix components such as hyaluronan (HA) and aggrecan. Here we show that this domain can also associate with the glycosaminoglycan heparin/heparan sulfate. Docking predictions and site-directed mutagenesis demonstrate that this occurs at a site distinct from the HA binding surface and is likely to involve extensive electrostatic contacts. Despite these glycosaminoglycans binding to non-overlapping sites on the Link module, the interaction of heparin can inhibit subsequent binding to HA, and it is possible that this occurs via an allosteric mechanism. We also show that heparin can modify another property of the Link module, i.e. its potentiation of the anti-plasmin activity of inter-alpha-inhibitor (IalphaI). Experiments using the purified components of IalphaI indicate that TSG-6 only binds to the bikunin chain and that this is at a site on the Link module that overlaps the HA binding surface. The association of heparin with the Link module significantly increases the anti-plasmin activity of the TSG-6.IalphaI complex. Changes in plasmin activity have been observed previously at sites of TSG-6 expression, and the results presented here suggest that TSG-6 is likely to contribute to matrix remodeling, at least in part, through down-regulation of the protease network, especially in locations containing heparin/heparan sulfate proteoglycans. The differential effects of HA and heparin on TSG-6 function provide a mechanism for its regulation and functional partitioning in particular tissue microenvironments.

Towards a Structure for a TSG-6·Hyaluronan Complex by Modeling and NMR Spectroscopy: INSIGHTS INTO OTHER MEMBERS OF THE LINK MODULE SUPERFAMILY

The Link module from human TSG-6, a hyaladherin with roles in ovulation and inflammation, has a hyaluronan (HA)-binding groove containing two adjacent tyrosine residues that are likely to form CH-pi stacking interactions with sequential rings in the sugar. We have used this observation to construct a model of a protein.HA complex, which was then tested against existing experimental information and by acquisition of new NMR data sets of [(13)C, (15)N]HA (8-mer) complexed with unlabeled protein. A major finding of this analysis was that acetamido side chains of two GlcNAc rings fit into hydrophobic pockets on either side of the adjacent tyrosines, providing a selectivity mechanism of HA over other polysaccharides. Furthermore, two basic residues have a separation that matches that of glucuronic acids in the sugar, consistent with the formation of salt bridges; NMR experiments at a range of pH values identified protein groups that titrate due to their proximity to a free carboxylate in HA. Sequence alignment and construction of homology models for all human Link modules in their HA-bound states revealed that many of these features are conserved across the superfamily, thus allowing the prediction of functionally important residues. In the case of cartilage link protein, its two Link modules were docked together (using bound HA as a guide), identifying hydrophobic residues likely to form an intra-Link module interface as well as amino acids that could be involved in supporting intermolecular interactions between link proteins and chondroitin sulfate proteoglycans. Here, we propose a mechanism for ternary complex formation that generates higher order helical structures, as may exist in cartilage aggregates.

TSG-6 Protein Binding to Glycosaminoglycans: FORMATION OF STABLE COMPLEXES WITH HYALURONAN AND BINDING TO CHONDROITIN SULFATES

TSG-6 protein, up-regulated in inflammatory lesions and in the ovary during ovulation, shows anti-inflammatory activity and plays an essential role in female fertility. Studies in murine models of acute inflammation and experimental arthritis demonstrated that TSG-6 has a strong anti-inflammatory and chondroprotective effect. TSG-6 protein is composed of the N-terminal link module that binds hyaluronan and a C-terminal CUB domain, present in a variety of proteins. Interactions between the isolated link module and hyaluronan have been studied extensively, but little is known about the binding of full-length TSG-6 protein to hyaluronan and other glycosaminoglycans. We show that TSG-6 protein and hyaluronan, in a temperature-dependent fashion, form a stable complex that is resistant to dissociating agents. The formation of such stable complexes may underlie the activities of TSG-6 protein in inflammation and fertility, e.g. the TSG-6-dependent cross-linking of hyaluronan in the cumulus cell-oocyte complex during ovulation. Because adhesion to hyaluronan is involved in cell trafficking in inflammatory processes, we also studied the effect of TSG-6 on cell adhesion. TSG-6 binding to immobilized hyaluronan did not interfere with subsequent adhesion of lymphoid cells. In addition to immobilized hyaluronan, full-length TSG-6 also binds free hyaluronan and all chondroitin sulfate isoforms under physiological conditions. These interactions may contribute to the localization of TSG-6 in cartilage and to its chondroprotective and anti-inflammatory effects in models of arthritis.

Personal sound link module

A personal sound link module (60) is inserted into a tunnel (40) made through the soft tissue connecting the retro-auricular space (50) with the ear canal (30). The module contains an acoustic transducer (65), located at the distal part (68) of the module, close to or inside the ear canal, an antenna (64) that receives and also potentially sends signals to a remote source, signal processing circuitry (67), telemetry circuitry (69), a power source (66) that powers the module, and possibly a microphone (63). Signals transmitted from a remote source are received through the antenna and telemetry circuitry, processed, and presented to the acoustic transducer, where they are converted to sound waves broadcast into the user's ear canal. The remote source may be a radio station, radio receiver, CD player, DVD player, tape player, audio system, telephone, TV receiver or station, or other source of audio signals intended to be heard privately by the user.

Rapid Plasma Membrane-Endosomal Trafficking of the Lymph Node Sinus and High Endothelial Venule Scavenger Receptor/Homing Receptor Stabilin-1 (Feel-1/Clever-1)

The sinusoidal endothelia of liver, spleen, and lymph node are major sites for uptake and recycling of waste macromolecules through promiscuous binding to a disparate family of scavenger receptors. Among the most complex is stabilin-1, a large multidomain protein containing tandem fasciclin domains, epidermal growth factor-like repeats, and a C-type lectin-like hyaluronan-binding Link module, which functions as an endocytic receptor for acetylated low density lipoprotein and advanced glycation end products. Intriguingly, stabilin-1 has also been reported to mediate both homing of leukocytes across lymph node high endothelial venules and adhesion of metastatic tumor cells to peritumoral lymphatic vessels. Currently, however, it is not clear how stabilin-1 mediates these distinct functions. To address the issue, we have investigated the tissue and subcellular localization of stabilin-1 in detail and assessed the functional status of its Link module. We show that stabilin-1 is almost entirely intracellular in lymph node high endothelial venules, lymphatic sinus endothelium, and cultured endothelial cells but that a finite population, detectable only by fluorescent antibody or fluorescein-labeled (Fl)-acetylated low density lipoprotein uptake, cycles rapidly between the plasma membrane and EEA-1+ve (early endosome antigen 1) early endosomes. In addition, we show using full-length stabilin-1 cDNA and a stabilin-1/CD44 chimera in HeLa cells that intracellular targeting is influenced by the transmembrane domain/cytoplasmic tail, which contains a putative dileucine (DXXLL) Golgi to endosomal sorting signal. Finally, we provide evidence that the stabilin-1 Link domain binds neither hyaluronan nor other glycosaminoglycans. These properties support a role for stabilin-1 as a rapidly recycling scavenger receptor and argue against a role in cell adhesion or lymphocyte homing.

Inhibitory Effects of TSG‐6 Link Module on Leukocyte–Endothelial Cell Interactions In Vitro and In Vivo

The authors investigated whether the anti-inflammatory protein tumor necrosis factor (TNF)-stimulated gene-6 (TSG-6) and its Link module (Link_TSG6) could affect the complex multistep process of leukocyte/endothelial cell (EC) interaction. Mouse mesenteries were inflamed with interleukin (IL)-1beta and the extent of leukocyte rolling, adhesion, and emigration was determined after 2 h. Link_TSG6 and a single-point mutant (termed K13E) were given intraperitoneally together with the cytokine. Human neutrophil chemotaxis and transmigration were determined in vitro in response to IL-8 and/or TNF-alpha. TSG-6, Link_TSG6, and K13E were added to the leukocytes or the EC monolayers. Co-injection of Link_TSG6 with IL-1beta selectively inhibited cell flux, adhesion, and emigration as analyzed in mesenteric postcapillary venules. The fewer cells that rolled in the animals treated with Link_TSG6 displayed a velocity similar to that measured in vehicle-treated mice. In vitro, Link_TSG6 did not affect neutrophil chemotaxis or EC activation but did inhibit neutrophil transmigration across EC monolayers. The latter effect was shared by full-length TSG-6 and observed equally in response to IL-8 or TNF-alpha. These data restrict the site of action for at least some of the anti-inflammatory effects ascribed to TSG-6/Link_TSG6 to the microenvironment of the extravasating leukocyte.

Structural and functional studies on hyaluronan‐protein aggregates produced in vitro

Introduction The proteoglycan aggrecan forms link protein‐stabilized complexes with hyaluronan (HA) that provide cartilage with its load bearing properties. Aggrecan binding to both HA and cartilage link protein are mediated through its N‐terminal G1‐domain, which is composed of an immunoglobulin fold and two contiguous Link modules. Similar aggregates (potentially containing members of the new link protein family) in which the proteoglycan versican substitutes for aggrecan are likely to contribute to the structural integrity of many other tissues.Materials and methods Human cartilage link protein (LP1) and the G1‐domains of human aggrecan and versican were cloned and expressed in Drosophila S2 cells. The recombinant proteins were purified from media using a combination of ion‐exchange chromatography and reverse‐phase HPLC. A microtitre plate assay was used to determine the minimum length of HA necessary for high affinity binding; the interaction of biotinylatd‐LP1 to immobilized G1‐domains was also determined. The ability of the recombinant proteins to form complexes in solution was assessed with HA oligosaccharides of defined length (i.e. HA10‐HA40) using a combination of gel filtration and protein cross‐linking experiments. Cross‐linking products were identified by in‐gel digestion with trypsin followed by MALDI‐TOF mass spectrometry.Results SDS‐PAGE showed the three recombinant proteins to be highly pure and that they all contained N‐linked glycosylation; amino acid sequencing revealed that aggrecan has undergone some differential processing at its N‐terminus. LP1, G1‐aggrecan and G1‐versican were all functionally active showing HA‐binding properties that are similar to those described for the native proteins; e.g. HA10 was the minimum length that could compete effectively for polymeric HA binding. Gel filtration and protein cross‐linking studies reveal that LP1 and G1‐aggrecan interact in both the absence and presence of HA. Interestingly, LP1 and G1‐versican did not interact in the absence of HA yet could be cross‐linked in the presence of HA24 but not HA20 or shorter oligosaccharides. However, when G1‐versican was immobilized on a microtitre plate, it was found to interact with biotinylated‐LP1, which is consistent with recent reports in the literature. Treatment of G1‐aggrecan or G1‐versican with PNGase F (which removes most of the N‐linked glycans from these proteins) did not affect the formation of aggregates in the presence of HA, indicating that glycosylation is not required for structural integrity or functional activity. Surprisingly, for G1‐aggrecan HA of approximately 40 sugar units appears to be the minimum size that can fully accommodate two protein molecules, whereas in the case of versican this occurs with HA between HA20 and HA24.Conclusion Aggrecan and versican appear to have distinct properties both with regard to their interaction with LP1 and HA, consistent with the hypothesis that different hyaladherins bind and capture distinct conformations of HA (Day and Sheehan 2001). Work is now in progress to produce aggrecan/LP1 and versican/LP1 ternary complexes for crystal studies.

Hyaluronan: a simple polysaccharide with structural plasticity and functional diversity

Hyaluronan (HA) is a high‐molecular weight glycosaminoglycan that is involved in an extracellular matrix (ECM) organization and cell adhesion, essential to a wide range of normal physiological processes (e.g. development, immunology and reproduction). Its diverse biological activities may seem surprising for a simple linear polysaccharide composed solely of a repeating disaccharide of glucuronic acid and N‐acetylglucosamine. However, HA is likely to be able to take up many different conformational states, which are transient and rapidly interchanging in solution (Day & Sheehan 2001). It has been suggested that the interaction of HA with different HA‐binding proteins (hyaladherins) may stabilize particular conformations of the polysaccharide leading to HA‐protein complexes with distinct architectures and unique biological properties (Day & Sheehan 2001). Consistent with this hypothesis, recent studies on TSG‐6 (Blundell et al. 2003) and CD44 (Teriete et al. 2004) and molecular modelling of other members of the Link module superfamily reveal significant structural diversity in the HA‐binding domains in these related hyaladherins. Furthermore, there is evidence emerging that the complexes formed between polymeric HA and proteins can exhibit a wide range of higher order structures depending on the hyaladherin(s) involved. In some cases, these complexes may play mainly a structural role in ECM (e.g. the link protein/aggrecan/HA aggregates that provide load bearing function to cartilage), while in other cases they act as cell binding/activation sites (e.g. HA cables formed during inflammation). There is evidence that the function of HA can be modulated by its interaction with proteins. For example, preincubation of HA with TSG‐6 enhances/induces the binding of HA to cell surface CD44 on constitutive/inducible lymphocyte backgrounds (Lesley et al.); these activities are probably caused by the formation of cross‐linked HA fibrils that lead to receptor clustering. HA can also become modified by the covalent attachment of heavy chains (HCs), derived from the interα‐inhibitor, where TSG‐6 plays an essential role in this process (Mukhopadhyay et al.); this reaction is critical for the formation of a HA‐rich ECM required for successful ovulation and fertilization. HC‐HA complexes are also formed during inflammation but their function is not yet clear.

XML에 기반을 둔 C 원시 코드의 주석 관리 시스템

C 언어가 주로 사용되는 미션 크리티컬 응용 소프트웨어 영역에는 문서화, 간결성, 정확성을 갖는 원시 코드가 요구된다. 본 연구는 XML 기반의 C 원시 코드의 주석관리 체계를 제안한다. 이 주석 관리체계는 주석 사용자 모듈, 검토자 모듈, 주석 추출 모듈, 주석 추적 연계 모듈, 주석 태그 정의 모듈, 저장관리 모들을 포함하는 6개 모듈로 구성된다. 본 연구에서 정의된 XML 주석 태그는 IEEE 표준 1028과 IEEE 표준 1012를 적용하는 개발공정 활동의 범주를 포함한다. C 원시 코드에 주석을 삽입하고 추출하기 위하여 XML 스키마가 사용되며. 주석 추출 결과의 시각적 표시 처리를 위하여 XSL-FO가 사용된다. Well documented, simplified and clarified source code is required for the mission critical application software area in which C programing language is generally used. We suggest an XML_based comment management system for C source code. The comment management system is composed of 6 modules including comment user module, reviewer module, comment extraction module, comment traceability link module, comment tag definition module and storage management module. The XML comment tags defined in this paper cover categories of the development process activities applying the IEEE standard 1028 and IEEE standard 1012. The XML Schema Is used to insert comments into C source code and to extract XML tags from C source code and the XSL-FO is used fur the visual display professing o( comment extraction results.