Anthony J. Day

Hyaluronan-binding proteins : tying up the giant

The ubiquitous glycosaminoglycan (GAG) hyaluronan has diverse biological roles in vertebrates. These include acting as a vital structural component of connective tissues, the formation of loose hydrated matrices that allow cells to divide and migrate (e.g. during development), immune cell adhesion and activation, and a role in intracellular signaling (1–3). This wide range of activities may seem surprising for an unbranched polysaccharide comprised entirely of a repeating disaccharide, D-glucuronic acid( 133)N-acetyl-D-glucosamine( 134), which (unlike other GAGs) is neither attached to a protein core nor Oor N-sulfated. Such diversity results in fact from the large number of hyaluronan-binding proteins (often termed hyaladherins) that exhibit significant differences in their tissue expression, cellular localization, specificity, affinity, and regulation. Therefore, characterization of the molecular basis of hyaluronan recognition by proteins and how this is modulated in vivo is an important key to understanding the biology of this GAG. In this article, we review the structural organization of vertebrate hyaladherins and how this may contribute to their different biological activities.

Hyaluronan and homeostasis: a balancing act.

Published, JBC Papers in Press, November 20, 2001, DOI 10.1074/jbc.R100037200 Markku I. Tammi‡, Anthony J. Day§, and Eva A. Turley¶ From the ‡Department of Anatomy, University of Kuopio, FIN-70211, Kuopio, Finland, §Medical Research Council Immunochemistry Unit, Department of Biochemistry, University of Oxford, Oxford OX1 3QU, United Kingdom, and ¶London Regional Cancer Center, London, Ontario N6A 4L6, Canada

PTX3 plays a key role in the organization of the cumulus oophorus extracellular matrix and in in vivo fertilization

PTX3 is a prototypic long pentraxin that plays a non-redundant role in innate immunity against selected pathogens and in female fertility. Here, we report that the infertility of Ptx3–/– mice is associated with severe abnormalities of the cumulus oophorus and failure of in vivo, but not in vitro, oocyte fertilization. PTX3 is produced by mouse cumulus cells during cumulus expansion and localizes in the matrix. PTX3 is expressed in the human cumulus oophorus as well. Cumuli from Ptx3–/– mice synthesize normal amounts of hyaluronan (HA), but are unable to organize it in a stable matrix. Exogenous PTX3 restores a normal cumulus phenotype. Incorporation in the matrix of inter-α-trypsin inhibitor is normal in Ptx3–/– cumuli. PTX3 does not interact directly with HA, but it binds the cumulus matrix hyaladherin tumor necrosis factor α-induced protein 6 (TNFAIP6, also known as TSG6) and thereby may form multimolecular complexes that can cross-link HA chains. Thus, PTX3 is a structural constituent of the cumulus oophorus extracellular matrix essential for female fertility.

Impaired cumulus mucification and female sterility in tumor necrosis factor-induced protein-6 deficient mice

Mucification of the cumulus layer around the oocyte is an obligatory process for female fertility. Tumor necrosis factor-induced protein-6 (TNFIP6 or TSG6) has been shown to be specifically expressed during this process. We have generated TNFIP6-deficient mice and tested the ability of their cumulus cells to undergo mucification. Cumulus cell-oocyte complexes fail to expand in TNFIP6-deficient female mice because of the inability of the cumulus cells to assemble their hyaluronan-rich extracellular matrix. The impaired cumulus matrix formation is due to the lack of covalent complexes between hyaluronan and the heavy chains of the inter-α-trypsin inhibitor family. As a consequence, TNFIP6-deficient females are sterile. Cultured TNFIP6-deficient cumulus cell-oocyte complexes also fail to expand when stimulated with dibutyryl cyclic AMP or epidermal growth factor. Recombinant TNFIP6 is able to catalyze the covalent transfer of heavy chains to hyaluronan in a cell-free system, restore the expansion of Tnfip6-null cumulus cell-oocyte complexes in vitro, and rescue the fertility in Tnfip6-null females. These results provide clear evidence that TNFIP6 is a key catalyst in the formation of the cumulus extracellular matrix and indispensable for female fertility.

Solution Structure of the Link Module: A Hyaluronan-Binding Domain Involved in Extracellular Matrix Stability and Cell Migration

Link modules are hyaluronan-binding domains found in proteins involved in the assembly of extracellular matrix, cell adhesion, and migration. The solution structure of the Link module from human TSG-6 was determined and found to consist of two alpha helices and two antiparallel beta sheets arranged around a large hydrophobic core. This defines the consensus fold for the Link module superfamily, which includes CD44, cartilage link protein, and aggrecan. The TSG-6 Link module was shown to interact with hyaluronan, and a putative binding surface was identified on the structure. A structural database search revealed close similarity between the Link module and the C-type lectin domain, with the predicted hyaluronan-binding site at an analogous position to the carbohydrate-binding pocket in E-selectin.

Structure-function relationships of the complement components

The primary amino acid sequences of the 20 complement components and control proteins, found in plasma, and of many of the cell-surface molecules associated with the control of the complement system are known from recent cDNA cloning studies. This has indicated that most of these proteins contain a number of well-defined domains of 40-80 amino acids: between two and 30 domains of one type are found in some of the proteins, while others show a mosaic structure composed of more than three different types of domain. The same types of domain are found in a growing number of non-complement proteins such as blood clotting factors and certain cell adhesion molecules. The main purpose of a recent meeting was to assess and correlate the data emerging from structure comparison and prediction techniques and from functional and physicochemical studies of isolated domains and whole proteins.

TSG-6: a multifunctional protein associated with inflammation.

TSG-6 expression is upregulated in many cell types in response to a variety of proinflammatory mediators and growth factors. This protein is detected in several inflammatory disease states (e.g. rheumatoid arthritis) and in the context of inflammation-like processes, such as ovulation, and is often associated with extracellular matrix remodelling. TSG-6 has anti-inflammatory and chondroprotective effects in various models of inflammation and arthritis, which suggest that it is a component of a negative feedback loop capable of downregulating the inflammatory response. Growing evidence also indicates that TSG-6 acts as a crucial factor in ovulation by influencing the expansion of the hyaluronan-rich cumulus extracellular matrix in the preovulatory follicle. TSG-6 is a member of the Link module superfamily and binds to hyaluronan (a vital component of extracellular matrix), as well as other glycosaminoglycans, via its Link module. In addition, TSG-6 forms both covalent and non-covalent complexes with inter-α-inhibitor (a serine protease inhibitor present at high levels in serum) and potentiates its anti-plasmin activity.

Three-dimensional structure of a complement control protein module in solution.

The complement control protein (CCP) modules (also known as short consensus repeats) are defined by a consensus sequence within a stretch of about 60 amino acid residues. These modules have been identified more than 140 times in over 20 proteins, including 12 proteins of the complement system. The solution structure of the 16th CCP module from human complement factor H has been determined by a combination of 2-dimensional nuclear magnetic resonance spectroscopy and restrained simulated annealing. In all, 548 structurally important nuclear Overhauser enhancement cross-peaks were quantified as distance restraints and, together with 41 experimentally measured angle restraints, were incorporated into a simulated annealing protocol to determine a family of closely related structures that satisfied the experimental observations. The CCP structure is shown to be based on a beta-sandwich arrangement; one face made up of three beta-strands hydrogen-bonded to form a triple-stranded region at its centre and the other face formed from two separate beta-strands. Both faces of the molecule contribute highly conserved hydrophobic side-chains to a compact core. The regions between the beta-strands are composed of both well-defined turns and less well-defined loops. Analysis of CCP sequence alignments, in light of the determined structure, reveals a high degree of conservation amongst residues of obvious structural importance, while almost all insertions, deletions or replacements observed in the known sequences are found in the less well-defined loop regions. On the basis of these observations it is postulated that models of other CCP modules that are based on the structure presented here will be accurate. Certain families of CCP modules differ from the consensus in that they contain extra cysteine residues. As a test of structural consensus, the extra disulphide bridges are shown to be easily accommodated within the determined CCP model.

Structures of the Cd44-Hyaluronan Complex Provide Insight Into a Fundamental Carbohydrate-Protein Interaction.

Regulation of transient interactions between cells and the ubiquitous matrix glycosaminoglycan hyaluronan is crucial to such fundamental processes as embryonic development and leukocyte homing. Cd44, the primary cell surface receptor for hyaluronan, binds ligand via a lectin-like fold termed the Link module, but only after appropriate functional activation. The molecular details of the Cd44-hyaluronan interaction and hence the structural basis for this activation are unknown. Here we present the first crystal structure of Cd44 complexed with hyaluronan. This reveals that the interaction with hyaluronan is dominated by shape and hydrogen-bonding complementarity and identifies two conformational forms of the receptor that differ in orientation of a crucial hyaluronan-binding residue (Arg45, equivalent to Arg41 in human CD44). Measurements by NMR indicate that the conformational transition can be induced by hyaluronan binding, providing further insight into possible mechanisms for regulation of Cd44.

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.