Inhibition of angiogenesis and angiogenesis-dependent tumor growth by the cryptic kringle fragments of human apolipoprotein(a)

Apolipoprotein(a) (apo(a)) contains tandemly repeated kringle domains that are closely related to plasminogen kringle 4, followed by a single kringle 5-like domain and an inactive protease-like domain. Recently, the anti-angiogenic activities of apo(a) have been demonstrated both in vitro and in vivo. However, its effects on tumor angiogenesis and the underlying mechanisms involved have not been fully elucidated. To evaluate the anti-angiogenic and anti-tumor activities of the apo(a) kringle domains and to elucidate their mechanism of action, we expressed the last three kringle domains of apo(a), KIV-9, KIV-10, and KV, in Escherichia coli. The resultant recombinant protein, termed rhLK68, exhibited a dose-dependent inhibition of basic fibroblast growth factor-stimulated human umbilical vein endothelial cell proliferation and migration in vitro and inhibited the neovascularization in chick chorioallantoic membranes in vivo. The ability of rhLK68 to abrogate the activation of extracellular signal-regulated kinases appears to be responsible for rhLK68-mediated anti-angiogenesis. Furthermore, systemic administration of rhLK68 suppressed human lung (A549) and colon (HCT-15) tumor growth in nude mice. Immunohistochemical examination and in situ hybridization analysis of the tumors showed a significant decrease in the number of blood vessels and the reduced expression of vascular endothelial growth factor, basic fibroblast growth factor, and angiogenin, indicating that suppression of angiogenesis may have played a significant role in the inhibition of tumor growth. Collectively, these results suggest that a truncated apo(a), rhLK68, is a potent anti-angiogenic and anti-tumor molecule.     

Recombinant kringle IV-10 modules of human apolipoprotein(a): structure, ligand binding modes, and biological relevance

The kringle modules of apolipoprotein(a) [apo(a)] of lipoprotein(a) [Lp(a)] are highly homologous with kringle 4 of plasminogen (75-94%) and like the latter are autonomous structural and functional units. Apo(a) contains 14-37 kringle 4 (KIV) repeats distributed into 10 classes (1-10). Lp(a) binds lysine-Sepharose via a lysine binding site (LBS) located in KIV-10 (88% homology with plasminogen K4). However, the W72R substitution that occurs in rhesus monkeys and occasionally in humans leads to impaired lysine binding capacity of KIV-10 and Lp(a). The foregoing has been investigated by determining the structures of KIV-10/M66 (M66 variant) in its unliganded and ligand [epsilon-aminocaproic acid (EACA)] bound modes and the structure of recombinant KIV-10/M66R72 (the W72R mutant). In addition, the EACA liganded structure of a sequence polymorph (M66T in about 42-50% of the human population) was reexamined (KIV-10/T66/EACA). The KIV-10/M66, KIV-10/M66/EACA, and KIV-10/T66/EACA molecular structures are highly isostructural, indicating that the LBS of the kringles is preformed anticipating ligand binding. A displacement of three water molecules from the EACA binding groove and a movement of R35 bringing the guanidinium group close to the carboxylate of EACA to assist R71 in stabilizing the anionic group of the ligand are the only changes accompanying ligand binding. Both EACA structures were in the embedded binding mode utilizing all three binding centers (anionic, hydrophobic, cationic) like plasminogen kringles 1 and 4. The KIV-10/T66/EACA structure determined in this work differs from one previously reported [Mikol, V., Lo Grasso, P. V. and, Boettcher, B. R. (1996) J. Mol. Biol. 256, 751-761], which crystallized in a different crystal system and displayed an unbound binding mode, where only the amino group of EACA interacted with the anionic center of the LBS. The remainder of the ligand extended into solvent perpendicular to the kringle surface, leaving the hydrophobic pocket and the cationic center of the LBS unoccupied. The structure of recombinant KIV-10/M66R72 shows that R72 extends along the ligand binding groove parallel to the expected position of EACA toward the anionic center (D55/D57) and makes a salt bridge with D57. Thus, the R72 side chain mimics ligand binding, and loss of binding ability is the result of steric blockage of the LBS by R72 physically occupying part of the site. The rhesus monkey lysine binding impairment is compared with that of chimpanzee where KIV-10 has been shown to have a D57N mutation instead.     

Baboon lipoprotein(a) binds very weakly to lysine-agarose and fibrin despite the presence of a strong lysine-binding site in apolipoprotein(a) kringle IV type 10

Human apolipoprotein(a) kringle IV type 10 [apo(a) KIV(10)] contains a strong lysine-binding site (LBS) that mediates the interaction of Lp(a) with biological substrates such as fibrin. Mutations in the KIV(10) LBS have been reported in both the rhesus monkey and chimpanzee, and have been proposed to explain the lack of ability of the corresponding Lp(a) species to bind to lysine and fibrin. To further the comparative analyses with other primate species, we sequenced a segment of baboon liver apo(a) cDNA spanning KIV(9) through the protease domain. Like rhesus monkey apo(a), baboon apo(a) lacks a kringle V (KV)-like domain. Interestingly, we found that the baboon apo(a) KIV(10) sequence contains all of the canonical LBS residues. We sequenced the apo(a) KIV(10) sequence from an additional 10 unrelated baboons; 17 of 20 alleles encoded Trp at position 70, whereas only two alleles encoded Arg at this position and thus a defective LBS. Despite the apparent presence of a functional KIV(10) LBS in most of the baboons, none of the Lp(a) in the plasma of the corresponding baboons bound specifically to lysine-Sepharose (agarose) even upon partial purification. Moreover, baboon Lp(a) bound very poorly to plasmin-modified fibrinogen. Expression of baboon and human KIV(10) in bacteria allowed us to verify that these domains bind comparably to lysine and lysine analogues. We conclude that presentation of KIV(10) in the context of apo(a) lacking KV may interfere with the ability of KIV(10) to bind to substrates such as fibrin; this paradigm may also be present in other non-human primates.     

A domain of the Leptospira LigB contributes to high affinity binding of fibronectin

Adhesion of pathogenic Leptospira spp. to mammalian cells is mediated by their adhesins interacting with host cell receptors. In a previous study, we have identified two potential fibronectin (Fn) binding sites in central variable region (LigBCen) and C-terminal variable region (LigBCtv) of LigB, an adhesin of pathogenic Leptospira spp. In this study, we have further localized the Fn-binding site on LigBCen and found a domain of LigB (LigBCen2) (amino acids 1014-1165) strongly bound to Fn. LigBCen2 bound to a 70kDa domain of Fn including N-terminal domain (NTD) and gelatin binding domain (GBD), but with a higher binding affinity to NTD (K(d)=272nM) than to GBD (K(d)=1200nM). Except Fn, LigBCen2 also bound laminin and fibrinogen. LigBCen2 could bind MDCK cells, and blocked the binding of Leptospira on MDCK cells by 45%. These results suggest that LigBCen2 contributed to high affinity binding on NTD or GBD of Fn, laminin, and fibrinogen and mediated Leptospira binding on host cells.     

Actions of the functional upstream domain of protein F1 of Streptococcus pyogenes on the conformation of fibronectin

Fibronectin (Fn), discovered by Harvard's Plasma Protein Program as plasma "cold-insoluble globulin" in the 1940s, has attracted much interest over the past three decades. One of the most interesting features of Fn is its ability to change shape in response to various environmental conditions and interactions with other substances found in the extra-cellular space. Here we examine the potential of the functional upstream domain (FUD) of Streptococcus pyogenes protein F1 to bring about changes in structure of Fn. In particular, we investigate the accessibility of Fn's 10th type III module that contains the integrin binding RGD motif. By use of monoclonal antibodies in a competitive ELISA assay, we found that FUD interacts with the amino-terminal type I modules of Fn to unveil the cell-binding region of Fn. This conformational change was achieved at sub-equimolar ratios of FUD/Fn monomer. We discuss the functional relevance of the interaction for both Fn and S. pyogenes and correlate the results with a conformational model of Fn that arose out of a collaboration between our laboratory and that of John Ferry.     

Characterization of the interaction of recombinant apolipoprotein(a) with modified fibrinogen surfaces and fibrin clots.

Elevated levels of lipoprotein(a) [Lp(a)] in plasma are a significant risk factor for the development of atherosclerotic disease, a property which may arise from the ability of this lipoprotein to inhibit fibrinolysis. In the present study we have quantitated the binding of recombinant forms of apolipoprotein(a) [17K and 12K r-apo(a); containing 8 and 3 copies, respectively, of the major repeat kringle sequence (kringle IV type 2)] to modified fibrinogen surfaces. Iodinated 17K and 12K r-apo(a) bound to immobilized thrombin-modified fibrinogen (i.e., fibrin) surfaces with similar affinities (Kd approximately 1.2-1.6 microM). The total concentration of binding sites (Bmax) present on the fibrin surface was approximately 4-fold greater for the 12K than for the 17K (Bmax values of 0.81 +/- 0.09 nM, and 0.20 +/- 0.01 nM respectively), suggesting that the total binding capacity on fibrin surfaces is reduced for larger apolipoprotein(a) (apo(a)) species. Interestingly, binding of apo(a) to intact fibrin was not detected as assessed by measurement of intrinsic fluorescence of free apo(a) present in the supernatants of sedimented fibrin clots. In other experiments, the total concentration apo(a) binding sites available on plasmin-modified fibrinogen surfaces was shown to be 13.5-fold higher than the number of sites available on unmodified fibrin surfaces (Bmax values of 2.7 +/- 0.3 nM and 0.20 +/- 0.01 nM respectively) while the affinity of apo(a) for these surfaces was similar. The increase in Bmax was correlated with plasmin-mediated exposure of C-terminal lysines since treatment of plasmin-modified fibrinogen surfaces with carboxypeptidase B produced a significant decrease in total binding signal as detected by ELISA (enzyme linked immunosorbent assay). Taken together, these data suggest that apo(a) binds to fibrin with poor affinity (low microM) and that the total concentration of apo(a) binding sites available on modified-fibrinogen surfaces is affected by both apo(a) isoform size and by the increased availability of C-terminal lysines on plasmin-degraded fibrinogen surfaces. However, the low affinity of apo(a) for fibrin indicates that Lp(a) may inhibit fibrinolysis through a mechanism distinct from binding to fibrin, such as binding to plasminogen.     

Binding of transforming growth factor-beta 1 to immobilized human alpha 2-macroglobulin.

Native alpha 2-macroglobulin (alpha 2M) and alpha 2M-methylamine were immobilized in 96-well microtiter plates. 125I-labeled transforming growth factor-beta 1 (TGF-beta 1) bound to both alpha 2M variants; however, greater binding was observed with alpha 2M-methylamine. Binding of 125I-TGF-beta 1 (0.2 nM) to immobilized alpha 2M-methylamine was inhibited by nonradiolabeled TGF-beta 1 (up to 74% with 0.4 microM TGF-beta 1). Approximately 10% of the TGF-beta 1-alpha 2M-methylamine complex was covalent. Treatment of alpha 2M-methylamine with iodoacetamide prior to immobilization completely eliminated covalent TGF-beta 1 binding; the total amount of 125I-TGF-beta 1-alpha 2M-methylamine complex detected was unchanged. The binding of 125I-TGF-beta 1 to immobilized alpha 2M-methylamine was not significantly inhibited by increasing the ionic strength to 1.0 M. Binding and complex dissociation were also unaffected by changes in pH within the range 6.9-8.9. Acidic pH dramatically decreased binding and promoted complex dissociation; no binding of 125I-TGF-beta 1 to immobilized alpha 2M-methylamine was detected at pH 3.5. The interaction of TGF-beta 1 with immobilized alpha 2M-methylamine was not significantly changed by 1.0 mM EDTA or 1.0 mM CaCl2. ZnCl2 (1.0 mM) completely eliminated binding. This result was not due to TGF-beta 1 precipitation or aggregation. Inhibition of 125I-TGF-beta 1 binding to alpha 2M-methylamine was 50% complete (IC50) with 30 microM ZnCl2. Native alpha 2M, thrombospondin, and alpha 2M-methylamine (in solution) decreased binding of 125I-TGF-beta 1 to immobilized alpha 2M-methylamine. The IC50 values for these three proteins were 520, 160, and 79 nM, respectively. The TGF-beta 1-binding activity of native alpha 2M may have reflected, at least in part, trace-contamination with alpha 2M-proteinase complex.     

The Streptococcal Binding Site in the Gelatin-binding Domain of Fibronectin Is Consistent with a Non-linear Arrangement of Modules

Fibronectin-binding proteins (FnBPs) of Staphylococcus aureus and Streptococcus pyogenes mediate invasion of human endothelial and epithelial cells in a process likely to aid the persistence and/or dissemination of infection. In addition to binding sites for the N-terminal domain (NTD) of fibronectin (Fn), a number of streptococcal FnBPs also contain an upstream region (UR) that is closely associated with an NTD-binding region; UR binds to the adjacent gelatin-binding domain (GBD) of Fn. Previously, UR was shown to be required for efficient streptococcal invasion of epithelial cells. Here we show, using a Streptococcus zooepidemicus FnBP, that the UR-binding site in GBD resides largely in the ⁸F1⁹F1 module pair. We also show that UR inhibits binding of a peptide from the α1 chain of type I collagen to ⁸F1⁹F1 and that UR binding to ⁸F1 is likely to occur through anti-parallel β-zipper formation. Thus, we propose that streptococcal proteins that contain adjacent NTD- and GBD-binding sites form a highly unusual extended tandem β-zipper that spans the two domains and mediates high affinity binding to Fn through a large intermolecular interface. The proximity of the UR- and NTD-binding sequences in streptococcal FnBPs is consistent with a non-linear arrangement of modules in the tertiary structure of the GBD of Fn.     

A quantitative assay for the non-covalent association between apolipoprotein[a] and apolipoprotein B: an alternative measure of Lp[a] assembly

Increasing evidence suggests that the assembly of lipoprotein[a] (Lp[a]) proceeds in two steps. In the first step, non-covalent interactions between apolipoprotein[a] (apo[a]) and apolipoprotein B (apoB) of low density lipoprotein (LDL) form a dissociable apo[a]:LDL complex. In the second step, a covalent disulfide linkage forms the stable Lp[a] particle. Several methods are currently used to study the assembly of Lp[a], however, these methods are laborious, time-consuming, and not suitable for a high throughput screening. We report here the development of a rapid and simple assay based on the binding of labeled LDL to a Lp[a]/apo[a] substrate which is immobilized on the surface of a microtiter plate. Quantification of bound LDL provides a measure of the extent of complex formation. Labeled LDL bound to both Lp[a] and apo[a] substrates with similar affinity. Plasma lipoproteins containing apoB as well as free apo[a] were capable of competing with LDL binding. The binding of LDL to Lp[a]/apo[a] was inhibited by L-proline and lysine analogs, which are known to inhibit the non-covalent association between apo[a] and apoB. Using this method we have found that nicotinic acid and captopril are able to inhibit the association of apo[a] with apoB. This method is compatible with automation and can be applied to a high throughput screening of inhibitors of Lp[a] formation.     

Interaction of heparin with fibronectin and isolated fibronectin domains.

Fluorescence polarization, gel exclusion chromatography and affinity chromatography were used to characterize the interaction of heparins of different size with human plasma fibronectin (Fn) and several of its isolated domains. The fluid-phase interaction of Fn with heparin was dominated by the 30 kDa and 40 kDa Hep-2 domains located near the C-terminal ends of the A and B chains respectively. The 30 kDa Hep-2A domain from the heavy chain was indistinguishable from the 40 kDa Hep-2B domain in this respect; the presence of an additional type III homology unit in the latter had no effect on the binding. Evidence was provided that each Hep-2 domain has two binding sites for heparin. The N-terminal Hep-1 domain reacted weakly in fluid phase even though it binds strongly to immobilized heparin. Fn and Hep-2 fragments were rather undiscriminating in their reaction with fluoresceinamine-labelled heparins of different sizes. However, oligosaccharides smaller than the tetradecasaccharide (14-mer) bound Fn with a 5-10-fold lower affinity. These results suggest that the Hep-2 domains of Fn are able to recognize a broad spectrum of oligosaccharides that presumably vary significantly with respect to the amount and spatial distribution of charge.     

Specific interactions between F1 adhesin of Streptococcus pyogenes and N-terminal modules of fibronectin

Protein F1 is a surface protein of Streptococcus pyogenes that mediates high affinity binding to fibronectin (Fn) and facilitates S. pyogenes adherence and penetration into cells. The smallest portion of F1 known to retain the full binding potential of the intact protein is a stretch of 49 amino acids known as the functional upstream domain (FUD). Synthetic and recombinant versions of FUD were labeled with fluorescein isothiocyanate and used in fluorescence anisotropy experiments. These probes bound to Fn or the 70-kDa fragment of Fn with dissociation constants of 8-30 nm. Removal of the N-terminal seven residues of FUD did not cause a change in binding affinity. Further N- or C-terminal truncations resulted in complete loss of binding activity. Analysis of recombinant versions of the 70-kDa fragment that lacked one or several type I modules indicates that residues 1-7 of the 49-mer bind to type I modules I1 and I2 of the 27-kDa subfragment and the C-terminal residues bind to modules I4 and I5. Fluorescein isothiocyanate-labeled 49-mer also bound with lower affinity to large Fn fragments that lack the five type I modules of the 27-kDa fragment but contain the other seven type 1 modules of Fn. These results indicate that, although FUD has a general affinity for type I modules, high affinity binding of FUD to Fn is mediated by specific interactions with N-terminal type I modules.     

Multiple binding sites in fibronectin and the staphylococcal fibronectin receptor.

The binding of fibronectin to Staphylococci exhibits the properties of a ligand-receptor interaction and has been proposed to mediate bacterial adherence to host tissues. To localize staphylococcal-binding sites in fibronectin, the protein was subjected to limited proteolysis and, of the generated fragments, Staphylococci appeared to preferentially bind to the N-terminal fragment. Different fibronectin fragments were isolated and tested for their ability to inhibit 125I-fibronectin binding to Staphylococci. The results indicate that only the N-terminal region effectively competed for fibronectin binding. However, when isolated fragments were adsorbed to microtiter wells, we found that two distinct domains, corresponding to the N-terminal fragment and to the heparin-binding peptide mapping close to the C-terminal end of fibronectin, promoted the attachment of both Staphylococcus aureus Newman and coagulase-negative strain of Staphylococcus capitis 651. These same domains were recognized by purified 125I-labeled staphylococcal receptor, either when immobilized on microtiter wells or probed after adsorption onto nitrocellulose membrane. The heparin-binding domain is comprised of type-III-homology repeats 14, 15 and 16. To determine which repeats participate in this interaction, we isolated and tested repeats type III14 and type III16. We found that the major staphylococcal binding site is located in repeat type III14. The staphylococcal receptor bound the N-terminal domain of fibronectin with a KD of 1.8 nM, whereas the dissociation constant of the receptor molecule for the internal heparin-binding domain was 10 nM. Since the fusion protein ZZ-FR, which contains the active sequences of fibronectin receptor (D1-D3) bound only to the N-terminus, it is reasonable to assume that the bacterial receptor may have additional binding sites outside the D domains, capable of interacting with the internal heparin-binding domain of fibronectin.     

Biochemical studies on the interaction of fibronectin with Ig

We have previously biochemically characterized three separate sites on the fibronectin (Fn) molecule that interact with IgG. These studies have been extended to examine the interaction of Fn with other classes and subclasses of Ig. By ELISA, a preferential quantitative binding order of Fn to the major Ig classes and subclasses was obtained as follows: IgG greater than IgM greater than IgA, IgG1 greater than IgG3 = IgG4 greater than IgG2, and IgA1 = IgA2. Using fragments of Fn obtained by subtilisin digestion followed by IgM and IgA affinity chromatography, immunoblot analysis using monospecific antisera to separate regions of the Fn molecule, and amino acid sequence analysis, these studies indicate that polyclonal IgA and IgM interact with Fn in the same three regions and under the same ionic conditions as previously described for IgG. Site 1 is a 22-kDa fragment that commences at residue 1 of the Fn molecule. Sites 2 (16 kDa) and 3 (26-29 kDa) begin at residues 588 and 1597, respectively. Under physiological conditions a monoclonal antibody that recognizes site 1 completely inhibited the interaction of intact Fn with IgG, IgM, and IgA. Therefore, this is the only physiologically active site in the intact molecule. Aggregated but not monomeric IgG competitively inhibited the binding of Fn to IgG-coated microtiter ELISA plates; thus, this interaction can take place in a fluid-phase system. These results indicate that Fn can potentially interact with immune complexes and aggregates of all Ig in the circulation and thus may play a significant role in both their clearance and deposition in Fn-containing tissues, such as occurs in immune-complex-related disorders.     

Protein F2, a novel fibronectin-binding protein from Streptococcus pyogenes, possesses two binding domains

Binding of the group A streptococcus (GAS) to respiratory epithelium is mediated by the fibronectin (Fn)-binding adhesin, protein F1. Previous studies have suggested that certain GAS strains express Fn-binding proteins that are different from protein F1. In this study, we have cloned, sequenced, and characterized a gene ( prtF2 ) from GAS strain 100076 encoding a novel Fn-binding protein, termed protein F2. Insertional inactivation of prtF2 in strain 100076 abolishes its high-affinity Fn binding. prtF2 -related genes exist in most GAS strains that lack prtF1 (encoding protein F1) but bind Fn with high affinity. These observations suggest that protein F2 is a major Fn-binding protein in GAS. Protein F2 is highly homologous to Fn-binding proteins from Streptococcus dysgalactiae and Strep-tococcus equisimilis , particularly in its carboxy-terminal portion. Two domains are responsible for Fn binding by protein F2. One domain (FBRD) consists of three consecutive repeats, whereas the other domain (UFBD) resides on a non-repeated stretch of approximately 100 amino acids and is located 100 amino acids amino-terminal of FBRD. Each of these domains is capable of binding Fn when expressed as a separate protein. In strain 100076, protein F2 activity is regulated in response to alterations in the concentration of atmospheric oxygen.     

Selection of phage-displayed fab antibodies on the active conformation of ras yields a high affinity conformation-specific antibody preventing the binding of c-Raf kinase to Ras

The Ras proteins cycle in the cell between an inactive state and an active state. In the active state, Ras signals via the switch I region to effectors like c-Raf kinase, leading to cell growth. Since Ras mutations in cancer are often associated with the presence of permanently active Ras, molecules that prevent downstream signaling may be of interest. Here, we show that by selection on the active conformation of Ras, using a recently described large phage antibody repertoire [de Haard et al. (1999) J. Biol. Chem. 274, 18218-18230], a Fab antibody (Fab H2) was identified that exclusively binds to active Ras, and not to inactive Ras. Using surface plasmon resonance (SPR) analysis, the interaction was demonstrated to be of high affinity (7.2 nM). In addition, the interaction with Ras is specific, since binding to the homologous Rap1A protein in BIAcore analysis is at least three orders of magnitude lower, and undetectable in an enzyme-linked immunosorbent assay. The antibody fragment prevents the binding of active Ras to the immobilized Ras-binding domain of c-Raf kinase (Raf-RBD) at an IC(50) value of 135 nM. This value compares well to the K(D) of active Ras-binding to immobilized Raf-RBD using SPR, suggesting identical binding sites. Like the IgG Y13-259, which does not demonstrate preferential binding to either inactive or active Ras, Fab H2 inhibits intrinsic GTPase activity of Ras in vitro. Mapping studies using SPR analysis demonstrate that the binding sites for the antibodies are non-identical. This antibody could be used for dissecting functional differences between Ras effectors. Due to its specificity for active Ras, Fab H2 may well be more selective than previously used anti-Ras antibodies, and thus could be used for gene therapy of cancer with intracellular antibodies.     

A two-domain mechanism for group A streptococcal adherence through protein F to the extracellular matrix.

Streptococcus pyogenes binds to the extracellular matrix (ECM) and a variety of host cells and tissues, causing diverse human diseases. Protein F, a S.pyogenes adhesin that binds fibronectin (Fn), contains two binding domains. A repeated domain (RD2) and an additional domain (UR), located immediately N-terminal to RD2. Both domains are required for maximal Fn binding. In this study, we characterize RD2 and UR precisely and compare their functions and binding sites in Fn. The minimal functional unit of RD2 is of 44 amino acids, with contributions from two adjacent RD2 repeats flanked by a novel 'MGGQSES' motif. RD2 binds to the N-terminal fibrin binding domain of Fn. UR contains 49 amino acids, of which six are from the first repeat of RD2. It binds to Fn with higher affinity than RD2, and recognizes a larger fragment that contains fibrin and collagen binding domains. Expression of UR and RD2 independently on the surface-exposed region of unrelated streptococcal protein demonstrates that both mediate adherence of the bacteria to the ECM. We describe here a mechanism of adherence of a pathogen that involves two pairs of sites located on a single adhesin molecule and directed at the same host receptor.     

Manganese binds to Clostridium difficile Fbp68 and is essential for fibronectin binding

Clostridium difficile is an etiological agent of pseudomembranous colitis and antibiotic-associated diarrhea. Adhesion is the crucial first step in bacterial infection. Thus, in addition to toxins, the importance of colonization factors in C. difficile-associated disease is recognized. In this study, we identified Fbp68, one of the colonization factors that bind to fibronectin (Fn), as a manganese-binding protein (K(D) = 52.70 ± 1.97 nM). Furthermore, the conformation of Fbp68 changed dramatically upon manganese binding. Manganese binding can also stabilize the structure of Fbp68 as evidenced by the increased T(m) measured by thermodenatured circular dichroism and differential scanning calorimetry (CD, T(m) = 58-65 °C; differential scanning calorimetry, T(m) = 59-66 °C). In addition, enhanced tolerance to protease K also suggests greatly improved stability of Fbp68 through manganese binding. Fn binding activity was found to be dependent on manganese due to the lack of binding by manganese-free Fbp68 to Fn. The C-terminal 194 amino acid residues of Fbp68 (Fbp68C) were discovered to bind to the N-terminal domain of Fn (Fbp68C-NTD, K(D) = 233 ± 10 nM, obtained from isothermal titration calorimetry). Moreover, adhesion of C. difficile to Caco-2 cells can be partially blocked if cells are pretreated with Fbp68C, and the binding of Fbp68C on Fn siRNA-transfected cells was significantly reduced. These results raise the possibility that Fbp68 plays a key role in C. difficile adherence on host cells to initiate infection.     

Direct interaction of the extracellular matrix protein DANCE with apolipoprotein(a) mediated by the kringle IV-type 2 domain

Lipoprotein(a) [Lp(a)] consists of LDL and apolipoprotein(a), and has been shown to be a major, independent, risk factor for arteriosclerosis and thrombosis in humans. To further elucidate the (patho)physiological function(s) of Lp(a)/apo(a), we searched for new protein ligands, using the yeast two-hybrid system and employing the highly repetitive kringle IV type 2 (KIV-2) sequence from apo(a) as bait. The extracellular matrix protein DANCE [developmental arteries and neural crest epidermal growth factor (EGF)-like] recently implicated in atherogenesis was identified as an interactor. A direct physical interaction between DANCE and apo(a) was confirmed by co-purification of both recombinant proteins derived from culture supernatants of transiently transfected COS-1 cells. Furthermore, binding of human plasma-derived Lp(a) to recombinant DANCE was also observed. Finally, when monoclonal anti-apo(a) and polyclonal anti-DANCE antibodies were applied to tissue slices of atherosclerotic carotid artery, the two proteins were found to be co-localized in endothelial and smooth muscle cells, suggesting that they occur together in the arterial wall. However, as yet, the in vivo relevance and the possible functional role of this newly identified DANCE:Lp(a)/apo(a) interaction remains speculative.     

A novel microtiter plate based method for identification of B‐cell epitopes

A new type of microtiter plate capable of binding biomolecules covalently in a one step procedure was used to map linear B‐cell epitopes in two different proteins using a peptide‐based solid phase immunoassay. The method was compared with a conventional immobilization method using passive adsorption to microtiter plates. An array of 15‐mer peptides, overlapping by five amino acids, representing the entire sequences of ubiquitin and murine tumor necrosis factor‐α, respectively, was synthesized. The peptides were immobilized covalently using the new, specialized microtiter plates or non‐covalently using conventional ELISA microtiter plates of the high binder type. Subsequently, specific antisera to ubiquitin or murine tumor necrosis factor‐α were added to identify potential linear B‐cell epitopes. All peptides, which were recognized on the conventional microtiter plates, were also recognized on the plates with the covalently bound peptides. In addition, the covalent immobilization method revealed epitopes that were not identified using the method for non‐covalent binding although the peptides were in fact present on the non‐covalent binding surface. The interaction with the hydrophobic surface of the conventional microtiter plate apparently interfered negatively with antibody recognition. The covalently binding microtiter plates described here could be useful for identification of new B‐cell epitopes in protein antigens. Copyright © 1999 European Peptide Society and John Wiley & Sons, Ltd.     

Identifying unfolding intermediates of FN-III(10) by steered molecular dynamics

Experimental studies have indicated that FN-III modules undergo reversible unfolding as a mechanism of elasticity. The unfolding of FN-III modules, including the cell-binding FN-III(10) module, has further been suggested to be functionally relevant by exposing buried cryptic sites or modulating cell binding. While steered molecular dynamics (SMD) simulations have provided one tool to investigate this process, computational requirements so far have limited detailed analysis to the early stages of unfolding. Here, we use an extended periodic box to probe the unfolding of FN-III(10) for extensions longer than 60A. Up to three plateaus, corresponding to three metastable intermediates, were observed in the extension-time profile from SMD stretching of FN-III(10). The first and second plateaus correspond to a twisted and an aligned state prior to unraveling FN-III(10) beta-strands. The third plateau, at an extension of approximately 100A, follows unraveling of FN-III(10) A and B-strands and precedes breaking of inter-strand hydrogen bonds between F and G-strands. The simulations revealed three forced unfolding pathways of FN-III(10), one of which is preferentially selected under physiological conditions. Implications for fibronectin fibrillogenesis are discussed.