Pigment epithelium-derived factor binds to hyaluronan. Mapping of a hyaluronan binding site

Pigment epithelium-derived factor (PEDF) is a multifunctional serpin with antitumorigenic, antimetastatic, and differentiating activities. PEDF is found within tissues rich in the glycosaminoglycan hyaluronan (HA), and its amino acid sequence contains putative HA-binding motifs. We show that PEDF coprecipitation with glycosaminoglycans in media conditioned by human retinoblastoma Y-79 cells decreased after pretreatments with hyaluronidase, implying an association between HA and PEDF. Direct binding of human recombinant PEDF to highly purified HA was demonstrated by coprecipitation in the presence of cetylpyridinium chloride. Binding of PEDF to HA was concentration-dependent and saturable. The PEDF-HA interactions were sensitive to increasing NaCl concentrations, indicating an ionic nature of these interactions and having affinity higher than PEDF-heparin. Competition assays showed that PEDF can bind heparin and HA simultaneously. PEDF chemically modified with fluorescein retained the capacity for interacting with HA but lacked heparin affinity, suggesting one or more distinct HA-binding regions on PEDF. The HA-binding region was examined by site-directed mutagenesis. Single-point and cumulative alterations at basic residues within the putative HA-binding motif K189A/K191A/R194A/K197A drastically reduced the HA-binding activity without affecting heparin- or collagen I binding of PEDF. Cumulative alterations at sites critical for heparin binding (K146A/K147A/R149A) decreased HA affinity but not collagen I binding. Thus these clusters of basic residues (BXBXXBXXB and BX3AB2XB motifs) in PEDF are functional regions for binding HA. In the spatial PEDF structure they are located in distinct areas away from the collagen-binding site. The HA-binding activity of PEDF may contribute to deposition in the extracellular matrix and to its reported antitumor/antimetastatic effects.     

Competitive binding of heparin with hyaluronan to a specific motif in SPACR

The critical hyaluronan binding motif (HABM) in sialoprotein associated with cones and rods (SPACR) has already been determined. As sialoproteoglycan associated with cones and rods, another interphotoreceptor matrix molecule, binds to chondroitin sulfate and heparin with or without the employment of HABMs, respectively, we evaluated and compared the binding of these glycosaminoglycans to SPACR. A western blotting study in combination with inhibition assays showed that heparin bound specifically to SPACR. A series of GST fusion proteins covering the whole SPACR molecule narrowed down the region responsible for the binding. Finally, a site-directed mutagenesis assay demonstrated that the critical HABM also acts as a specific binding site for heparin. These results were supported with mutual inhibitions by hyaluronan and heparin in analyses using GST fusion proteins and native SPACR derived from retina. Thus, these glycosaminoglycans bind to SPACR in a different manner than to sialoproteoglycan associated with cones and rods. The competitive binding between hyaluronan and heparin to SPACR, mediated through the identical HABM, may dominate the functions of SPACR, in turn involving physiological and pathological processes involved in retinal development, aging and other related disorders.     

Hyaluronan-binding motif identified by panning a random peptide display library

The glycosaminoglycan hyaluronan (HA) is involved in a variety of functions such as cell migration, adhesion, activation of intracellular signaling, metastasis, inflammation and wound repair. These functions of HA are mediated via HA-binding proteins (HABPs). To derive details of the HA-binding site in HABPs, here, we panned a random peptide display library expressed on the E. coli flagellin protein using HA-coated plates. Using this random peptide display library, 40 positive clones were obtained and the nucleotide sequences were determined. As a result, an Arg-Arg sequence, in addition to the known B-X7-B motif, was found to bind to HA. A binding experiment using the IAsys resonant mirror biosensor verified that a peptide containing an Arg-Arg sequence binds to HA.     

Identification of a common hyaluronan binding motif in the hyaluronan binding proteins RHAMM, CD44 and link protein.

We have previously identified two hyaluronan (HA) binding domains in the HA receptor, RHAMM, that occur near the carboxyl-terminus of this protein. We show here that these two HA binding domains are the only HA binding regions in RHAMM, and that they contribute approximately equally to the HA binding ability of this receptor. Mutation of domain II using recombinant polypeptides of RHAMM demonstrates that K423 and R431, spaced seven amino acids apart, are critical for HA binding activity. Domain I contains two sets of two basic amino acids, each spaced seven residues apart, and mutation of these basic amino acids reduced their binding to HA--Sepharose. These results predict that two basic amino acids flanking a seven amino acid stretch [hereafter called B(X7)B] are minimally required for HA binding activity. To assess whether this motif predicts HA binding in the intact RHAMM protein, we mutated all basic amino acids in domains I and II that form part of these motifs using site-directed mutagenesis and prepared fusion protein from the mutated cDNA. The altered RHAMM protein did not bind HA, confirming that the basic amino acids and their spacing are critical for binding. A specific requirement for arginine or lysine residues was identified since mutation of K430, R431 and K432 to histidine residues abolished binding. Clustering of basic amino acids either within or at either end of the motif enhanced HA binding activity while the occurrence of acidic residues between the basic amino acids reduced binding. The B(X7)B motif, in which B is either R or K and X7 contains no acidic residues and at least one basic amino acid, was found in all HA binding proteins molecularly characterized to date. Recombinant techniques were used to generate chimeric proteins containing either the B(X7)B motifs present in CD44 or link protein, with the amino-terminus of RHAMM (amino acids 1-238) that does not bind HA. All chimeric proteins containing the motif bound HA in transblot analyses. Site-directed mutations of these motifs in CD44 sequences abolished HA binding. Collectively, these results predict that the motif of B(X7)B as a minimal binding requirement for HA in RHAMM, CD44 and link protein, and occurs in all HA binding proteins described to date.     

Studies on the interaction between hyaluronan and a rat colon cancer cell line

Binding studies with 125I-Tyr labelled hyaluronan (HA) on a cultured rat colon cancer cell line were performed to characterize the association of HA to tumour cells in vitro. Results show a specific and saturable binding (Kd= 1.36nM) which indicates the presence of an HA binding receptor on the tumour cells. There is a specific constant increase of cell-associated HA over time, which indicates that HA is specifically taken up by the cells through endocytosis. The binding of 125I-Tyr labelled HA was more effectively inhibited by unlabelled HA of high MW in relation to low MW species of the polysaccharide indicating that the receptor binds HA of high MW with greater affinity than low MW species. In competition experiments, the HA-binding could not be inhibited by other polysaccharides such as chondroitin sulphate and heparin. Nor could ligands for scavenger receptors and antibodies directed towards ICAM-1, CD 44 and RHAMM (Receptor for HA Mediated Motility) significantly inhibit the association of HA to tumour cells.     

Unmasking a hyaluronan-binding site of the BX(7)B type in the H3 heavy chain of the inter-alpha-inhibitor family.

The inter-alpha-inhibitor (I alpha I) family gathers together several plasma protease inhibitors such as I alpha I and pre-alpha-inhibitor (P alpha I) that are variously assembled from a set of polypeptide chain precursors designated H1P to H3P. In addition to their protease inhibitory activity, a major physiological function of I alpha I family members is hyaluronan (HA) binding and HA-dependent stabilization of the extracellular matrix surrounding various cell types. Also, binding of HA to these molecules has been shown to be an important event in tumor cell proliferation and rheumatoid arthritis. However, how HA and I alpha I family members first recognize each other has so far remained elusive. The so-called BX7B domain found in some HA-binding proteins is an HA-binding site in which B represents a basic amino-acid residue and X represents any nonacidic residue. This domain has now been identified in the N-terminal end of H3P that is a precursor of P alpha I. A series of wild-type or mutant recombinant H3P chains produced with a mouse cDNA expressed in Escherichia coli allowed us to demonstrate that this domain binds HA in a noncovalent fashion. Furthermore, unmasking this HA-binding activity required most of H3P to be trimmed off at its C-terminal end. The latter observation was confirmed with a natural, mature H3 chain purified from human plasma. Indeed, a thermolysin-generated, N-terminal fragment of this H3 chain strongly bound HA whereas the intact H3 chain did not. Therefore, in vivo, the HA-binding activity of the mature H3 chain within P alpha I may vary with the folding and/or fragmentation of this protein.     

Plasticity of the TSG-6 HA-binding loop and mobility in the TSG-6-HA complex revealed by NMR and X-ray crystallography

Tumour necrosis factor-stimulated gene-6 (TSG-6) is a glycosaminoglycan-binding protein expressed during inflammatory and inflammation-like processes. Previously NMR structures were calculated for the Link module of TSG-6 (Link_TSG6) in its free state and when bound to an octasaccharide of hyaluronan (HA(8)). Heparin was found to compete for HA binding even though it interacts at a site that is distinct from the HA-binding surface. Here we present crystallography data on the free protein, and (15)N NMR relaxation data for the uncomplexed and HA(8)-bound forms of Link_TSG6. Although the Link module is comparatively rigid overall, the free protein shows a high degree of mobility in the beta4/beta5 loop and at the Cys47-Cys68 disulfide bond, both of which are regions involved in HA binding. When bound to HA(8), this dynamic behaviour is dampened, but not eliminated, suggesting a degree of dynamic matching between the protein and sugar that may decrease the entropic penalty of complex formation. A further highly dynamic residue is Lys54, which is distant from the HA-binding site, but was previously shown to be involved in heparin binding. When HA is bound, Lys54 becomes less mobile, providing evidence for an allosteric effect linking the HA and heparin-binding sites. A mechanism is suggested involving the beta2-strand and alpha2-helix. The crystal structure of free Link_TSG6 contains five molecules in the asymmetric unit that are highly similar to the NMR structure and support the dynamic behaviour seen near the HA-binding site: they show little or no electron density for the beta4/beta5 loop and display multiple conformations for the Cys47-Cys68 disulfide bond. The crystal structures were used in docking calculations with heparin. An extended interface between a Link_TSG6 dimer and heparin 11-mer was identified that is in excellent agreement with previous mutagenesis and calorimetric data, providing the basis for further investigation of this interaction.     

Binding of Lactose and Galactose to Native and Iodinated Ricin D

The binding of lactose and galactose to native and iodinated ricin D was investigated by equilibrium dialysis and ultraviolet difference spectroscopy. The results provided direct evidence that native ricin D has two independent saccharide binding sites with different affinities, of which the high-affinity (HA-) binding site is able to bind with both lactose and galactose while the low-affinity (LA-) binding site binds only with lactose. In contrast, the iodinated ricin D possesses only one binding site both for lactose and galactose with high affinity.

By UV-difference spectroscopic analysis we found that there is one tyrosyl residue at or near the HA-binding site in ricin D which may be involvled in binding with saccharide. This tyrosyl residue was not iodinated in the presence of lactose but was iodinated in the absence of lactose and was perturbed by an addition of lactose even after iodination.

From these results, it was inferred that the binding site abolished by the iodination is the LA-binding site and this may be due to the conformational alteration of the LA-binding site caused by the iodination of the tyrosyl residue(s) present near the LA-binding site.     

Evidence for activation of Amh gene expression by steroidogenic factor 1

The anti-Müllerian hormone gene (Amh) is responsible for regression in males of the Müllerian ducts. The molecular mechanism of regulation of chicken Amh expression is poorly understood. To investigate the regulation of chicken Amh expression, we have cloned Amh cDNAs from quail and duck as well as the promoter regions of the gene from chicken, quail, and duck. The expression patterns of Amh during embryonic development in these three species were found to be similar, suggesting that the regulatory mechanisms of Amh expression are conserved. The sequence of the proximal promoter of Amh contains a putative binding site for steroidogenic factor 1 (SF1), the protein product of which can up-regulate Amh in mammals. We showed here that SF1 is able to activate the chicken Amh promoter and binds to its putative SF1 binding site. These results suggest that SF1 plays a role in regulation of Amh expression in avian species.     

Multiple sequence-specific DNA binding activities are eluted from chicken nuclei at low ionic strengths.

DNA sequence-specific binding proteins eluted from chicken erythrocyte and thymus nuclei, and fractionated as described by Emerson and Felsenfeld (19), have been investigated by filter binding and footprint analyses. The erythrocyte nuclear protein fraction specifically binds to at least two sites within the 5' flanking chromatin hypersensitive site of the chicken beta A-globin gene, and to a site 5' to the human beta-globin gene. The major chicken beta A globin gene binding site [G)18CGGGTGG) and the human beta-globin gene binding site [TA)6(T)8C(T)4) occur at or near sequences which are hypersensitive to S1 nuclease cleavage in supercoiled plasmids. Downstream, the second chicken beta A-globin gene binding site includes the beta-globin gene CACCC consensus sequence. Filter binding studies also show other sequence specific binding activities to human N-ras and human (but not chicken) c-myc gene sequences.     

Expression and purification of RHC–EGFP fusion protein and its application in hyaluronic acid assay

Hyaluronan is a widely distributed glycosaminoglycan which has multiple functions. Hyaluronic acid (HA) accumulation has been reported in many human diseases. Understanding the role of hyaluronan and its binding proteins in the pathobiology of disease will facilitate the development of novel therapeutics for many critical diseases. Current techniques described for the analysis of HA are mainly for HA quantification in solutions, not for the direct detection of HA in tissues or on cell surfaces. In our study, a fusion protein, named C-terminal domain of RHAMM–enhanced green fluorescence protein (RHC–EGFP), combined the HA-binding domain, C-terminal of receptor for hyaluronan-mediated motility, with EGFP, a widely used enhanced green fluorescence protein, was expressed and purified from Escherichia coli with high purity. Based on the sensitivity and convenience of fluorescence detection, methods for direct assay of HA in solutions, on cell surface or in tissues were established using RHC–EGFP. The binding specificity was also confirmed by competitive binding experiment and hyaluronidase degradation experiment. Our results provide an alternative choice for the specific and convenient assay of HA in various samples, and maybe helpful for further understanding of the fundamental and comprehensive functions of HA.     

Specific interaction between the initiator protein (Rep) and origin of plasmid ColE2-P9

The replication initiator protein (Rep) of plasmid ColE2-P9 (ColE2) is multifunctional. We are interested in how Rep binds to the origin (Ori) to perform various functions. We used the wild type and variants of Rep to study the Rep-Ori interaction by both in vitro and in vivo approaches, including biochemical analyses of protein-DNA interactions and an in vivo replication assay. We identified three regions (I, II, and III) of Rep, located in the C-terminal half, and three corresponding binding sites (I, II, and III) in Ori which are important for Rep-Ori interaction. We showed that region I, containing a putative helix-turn-helix motif, is necessary and sufficient for specific Ori recognition, interacting with site I of the origin DNA from the major groove. Region II interacts with site II of the origin DNA, from the adjacent minor groove in the left half of Ori, and region III interacts with site III, next to the template sequence for primer synthesis, which is one and one-half turn apart from site I on the opposite surface of the origin DNA. A putative linker region located between the two DNA binding domains (regions II and III) was identified, which might provide Rep an extended conformation suitable for binding to the two separate sites in Ori. Based on the results presented in this paper, we propose a model for Rep-Ori interaction in which Rep binds to Ori as a monomer.     

Divergent RNA binding specificity of yeast Puf2p

PUF proteins bind mRNAs and regulate their translation, stability, and localization. Each PUF protein binds a selective group of mRNAs, enabling their coordinate control. We focus here on the specificity of Puf2p and Puf1p of Saccharomyces cerevisiae, which copurify with overlapping groups of mRNAs. We applied an RNA-adapted version of the DRIM algorithm to identify putative binding sequences for both proteins. We first identified a novel motif in the 3' UTRs of mRNAs previously shown to associate with Puf2p. This motif consisted of two UAAU tetranucleotides separated by a 3-nt linker sequence, which we refer to as the dual UAAU motif. The dual UAAU motif was necessary for binding to Puf2p, as judged by gel shift, yeast three-hybrid, and coimmunoprecipitation from yeast lysates. The UAAU tetranucleotides are required for optimal binding, while the identity and length of the linker sequences are less critical. Puf1p also binds the dual UAAU sequence, consistent with the prior observation that it associates with similar populations of mRNAs. In contrast, three other canonical yeast PUF proteins fail to bind the Puf2p recognition site. The dual UAAU motif is distinct from previously known PUF protein binding sites, which invariably possess a UGU trinucleotide. This study expands the repertoire of cis elements bound by PUF proteins and suggests new modes by which PUF proteins recognize their mRNA targets.     

Characterization of SPACR, a sialoprotein associated with cones and rods present in the interphotoreceptor matrix of the human retina: immunological and lectin binding analysis

Rod and cone photoreceptors project from the outer retinal surface into a carbohydrate-rich interphotoreceptor matrix (IPM). Unique IPM glycoconjugates are distributed around rods and cones. Wheat germ agglutinin (WGA) strongly decorates the rod matrix domains and weakly decorates the cone matrix domains. This study characterizes the major WGA-binding glycoprotein in the human IPM, which we refer to as SPACR (sialoprotein associated with cones and rods). SPACR, which has a molecular weight of 147 kDa, was isolated and purified from the IPM by lectin affinity chromatography. A polyclonal antibody to SPACR was prepared that colocalizes in tissue preparations with WGA-binding domains in the IPM. Sequential digestion of SPACR with N- and O- glycosidases results in a systematic increase in electrophorectic mobility, indicating the presence of both N- and O-linked glycoconjugates. Complete deglycosylation results in a reduction in the relative molecular mass of SPACR by about 30%. Analysis of lectin binding allowed us to identify some of the structural characteristics of SPACR glycoconjugates. Treatment with neuraminidase exposes Galbeta1- 3GalNAc disaccharide as indicated by positive peanut agglutinin (PNA) staining, accompanied by the loss of WGA staining. Maackia amurensis agglutinins (MAA-1 and MAA-2), specific for sialic acid in alpha2-3 linkage to Gal, bind SPACR, while Sambucus nigra agglutinin (SNA), specific for alpha2-6 linked sialic acid, does not, indicating that the dominant glycoconjugate determinant on SPACR is the O-linked carbohydrate, NeuAcalpha2-3Galbeta1-3GalNAc. The abundance of sialic acid in SPACR suggests that this glycoprotein may contribute substantially to the polyanionic nature of the IPM. The carbohydrate chains present on SPACR could also provide sites for extensive crosslinking and participate in the formation of the ordered IPM lattice that surrounds the elongate photoreceptors projecting from the outer retinal surface.      

Identification and characterization of a novel peritrophic matrix protein, Ae-Aper50, and the microvillar membrane protein, AEG12, from the mosquito, Aedes aegypti

Immuno-screening of an adult Aedes aegypti midgut cDNA expression library with anti-peritrophic matrix antibodies identified cDNAs encoding a novel peritrophic matrix protein, termed Ae. aegypti Adult Peritrophin 50 (Ae-Aper50), and the epithelial cell-surface membrane protein, AEG12. Both genes are expressed exclusively in the midguts of adult female mosquitoes and their expression is strongly induced by blood feeding. Ae-Aper50 has a predicted secretory signal peptide and five chitin-binding domains with intervening mucin-like domains. Localization of Ae-Aper50 to the peritrophic matrix was demonstrated by immuno-electron microscopy. Recombinant Ae-Aper50 expressed in baculovirus-infected insect cells binds chitin in vitro. Site-directed mutagenesis was used to study the role that cysteine residues from a single chitin-binding domain play in the binding to a chitin substrate. Most of the cysteine residues proved to be critical for binding. AEG12 has a putative secretory signal peptide at the amino-terminus and a putative glycosyl-phosphatidylinositol (GPI) anchor signal at its carboxyl-terminus and the protein was localized by immuno-electron microscopy to the midgut epithelial cell microvilli.     

The inflammation-associated protein TSG-6 cross-links hyaluronan via hyaluronan-induced TSG-6 oligomers

Tumor necrosis factor-stimulated gene-6 (TSG-6) is a hyaluronan (HA)-binding protein that plays important roles in inflammation and ovulation. TSG-6-mediated cross-linking of HA has been proposed as a functional mechanism (e.g. for regulating leukocyte adhesion), but direct evidence for cross-linking is lacking, and we know very little about its impact on HA ultrastructure. Here we used films of polymeric and oligomeric HA chains, end-grafted to a solid support, and a combination of surface-sensitive biophysical techniques to quantify the binding of TSG-6 into HA films and to correlate binding to morphological changes. We find that full-length TSG-6 binds with pronounced positive cooperativity and demonstrate that it can cross-link HA at physiologically relevant concentrations. Our data indicate that cooperative binding of full-length TSG-6 arises from HA-induced protein oligomerization and that the TSG-6 oligomers act as cross-linkers. In contrast, the HA-binding domain of TSG-6 (the Link module) alone binds without positive cooperativity and weaker than the full-length protein. Both the Link module and full-length TSG-6 condensed and rigidified HA films, and the degree of condensation scaled with the affinity between the TSG-6 constructs and HA. We propose that condensation is the result of protein-mediated HA cross-linking. Our findings firmly establish that TSG-6 is a potent HA cross-linking agent and might hence have important implications for the mechanistic understanding of the biological function of TSG-6 (e.g. in inflammation).     

BEHAB, a new member of the proteoglycan tandem repeat family of hyaluronan-binding proteins that is restricted to the brain [published erratum appears in J Cell Biol 1997 Apr 21;137(2):521]

Hyaluronan (HA) is a ubiquitous component of the extracellular matrix of all tissues. In the mammalian central nervous system (CNS) HA is present throughout development and into adulthood. While the functions of HA are likely to be mediated by HA-binding proteins, no cell or tissue specific HA-binding proteins have been reported. In an effort to characterize the composition of the extracellular matrix of the CNS, we sought to identify neural HA-binding proteins. We report here the isolation and characterization of a cDNA with a high degree of sequence homology to members of the proteoglycan tandem repeat (PTR) family of HA-binding proteins. Unlike other HA-binding proteins, the expression of this cDNA is restricted to the CNS. We propose the name BEHAB, Brain Enriched HyAluronan Binding protein, for this gene. The expression of BEHAB mRNA is developmentally regulated; expression is first detected in the late embryonic period and peaks during the first two postnatal weeks. In the embryo, BEHAB is expressed at highest levels in mitotically active cells. The sequence of BEHAB has long stretches of identity between rat and cat, suggesting that the encoded protein is functionally important. The size and sequence of BEHAB are consistent with the possibility that it could serve a function like link protein, stabilizing interactions between HA and brain proteoglycans. These observations suggest that existence of other tissue specific HA-binding proteins.     

Unbinding of hyaluronan accelerates the enzymatic activity of bee hyaluronidase

Hyaluronan (HA), a polymeric glycosaminoglycan ubiquitously present in higher animals, is hydrolyzed by hyaluronidases (HAases). Here, we used bee HAase as a model enzyme to study the HA-HAase interaction. Located in close proximity to the active center, a bulky surface loop, which appears to obstruct one end of the substrate binding groove, was found to be functionally involved in HA turnover. To better understand kinetic changes in substrate interaction, binding of high molecular weight HA to catalytically inactive HAase was monitored by means of quartz crystal microbalance technology. Replacement of the delimiting loop by a tetrapeptide interconnection increased the affinity for HA up to 100-fold, with a K(D) below 1 nm being the highest affinity among HA-binding proteins surveyed so far. The experimental data of HA-HAase interaction were further validated showing best fit to the theoretically proposed sequential two-site model. Besides the one, which had been shown previously in course of x-ray structure determination, a previously unrecognized binding site works in conjunction with an unbinding loop that facilitates liberation of hydrolyzed HA.     

Crystallographic location of two Zn(2+)-binding sites in the avian cytochrome bc(1) complex

The chicken mitochondrial ubiquinol cytochrome c oxidoreductase (bc(1) complex) is inhibited by Zn(2+) ions, but with higher K(i) ( approximately 3 microM) than the corresponding bovine enzyme. When equilibrated with mother liquor containing 200 microM ZnCl(2) for 7 days, the crystalline chicken bc(1) complex specifically binds Zn(2+) at 4 sites representing two sites on each monomer in the dimer. These two sites are close to the stigmatellin-binding site, taken to be center Q(o) of the Q-cycle mechanism, and are candidates for the inhibitory site. One binding site is actually in the hydrophobic channel between the Q(o) site and the bulk lipid phase, and may interfere with quinone binding. The other is in a hydrophilic area between cytochromes b and c(1), and might interfere with the egress of protons from the Q(o) site to the intermembrane aqueous medium. No zinc was bound near the putative proteolytic active site of subunits 1 and 2 (homologous to mitochondrial processing peptidase) under these conditions.