Preparation of gellan sulfate as an artificial ligand for removal of extra domain A containing fibronectin

The extra domain A containing fibronectin (EDA(+)FN) concentration in plasma of rheumatoid arthritis (RA) is abnormally higher than the normal level. We synthesized various gellan-sulfate (GS) candidates as artificial ligands for removing EDA(+)FN from plasma. The interaction between these artificial ligands and EDA(+)FN was evaluated using affinity constants (KA), which were determined by surface plasmon resonance measurement. The KA (3.6×10⁸ per M) of GS-25 [degree of substitution for sulfonation (DS)=25%] with EDA(+)FN was higher than those of other molecules: GS-16 (DS=16%) at 8.3×10⁷ per M, and GS-35 (DS=35%) at 1.7×10⁸ per M. Furthermore, GSs displayed selectivity of EDA(+)FN for binding with plasma FN (KA_EDA(+)FN/KA_{plasma FN}>2). The removal ratio in plasma was measured by using GS-immobilized gel. Removals of 66, 11, 7.7, 6.2, 6.9, and 12% for EDA(+)FN, plasma FN, fibrinogen, albumin, immunoglobulin G (IgG) and antithrombin III from the patient-model plasma were, respectively, achieved with GS-25-immobilized gel. These results suggest that GS may be used as a selective artificial ligand for EDA(+)FN removal from plasma in RA treatment.     

Hydrodynamics of cryogelation

Cryogel, prevalent in the plasma of rheumatoid arthritis patients, is a plasma fibronectin (pFN)-extra domain A containing FN [EDA(+)FN]-fibrinogen (Fbg) aggregate formed by the addition of heparin (Hep) at low temperature. Although EDA(+)FN is not usually present in normal plasma, its prevalence in rheumatic patients induces cryogelation. In this study, we determined the hydrodynamic radius (R_h) ratio (R_h/R_h30) of the cryogel component by dynamic light scattering in vitro. R_h/R_h30 was normalized to R_h at 30 °C (R_h30) at several temperatures. The R_h/R_h30 of Fbg was found to increase only by self-aggregation, whereas the R_h/R_h30 of FNs does not increase in response to temperature changes. The R_h/R_h30 of the Fbg/FN aggregate is increased by the addition of Hep, and the R_h/R_h30 (12.5) of the Hep-induced EDA(+)FN/Fbg aggregate is greater than that (2.5) of the pFN/Fbg aggregate. These results suggest that cryogelation requires Fbg self-aggregation and the interaction between EDA(+)FN and Hep.     

Specific interactions between cryogel components: role of extra domain A containing fibronectin in cryogelation

Cryogel is a physical gel formed by heterophilic aggregation of extra domain A containing fibronectin [EDA(+)FN], plasma fibronectin (pFN), fibrinogen (Fbg) and heparin (Hep), which are found in high concentrations in the blood of patients suffering from rheumatoid arthritis. In this study, we clarify the specific interactions between cryogel components in terms of the affinity constant (K(A)), obtained by surface plasmon resonance (SPR). It is found that Fbg self-interactions occur at lower temperatures, and that K(A) of Fbg-Hep changes with temperature. Specifically, K(A) (2.0 x 10(8) [M(-1)]) of Fbg-Hep at 5 degrees C increases significantly from that (1.0x10(7) [M(-1)]) at 40 degrees C. K(A) of EDA(+)FN-Hep increases with temperature, by approximately 100-fold between 40 degrees C (K(A)=10(12) [M(-1)]) and 20 degrees C (K(A)=10(10) [M(-1)]). Although K(A) of the FN fragments of Hep-binding domain containing an EDA region [EDA(+)HBD(+)] and Hep increases with temperatures above 30 degrees C, K(A)s of HBD(+)-Hep and EDA(+)-Hep are not temperature-dependent. Therefore, EDA(+)HBD(+), formed as a special structure for high Hep affinity, exhibits temperature-dependent interaction with Hep. These results suggest that the main role of EDA(+)FN in cryogelation is to support the interaction with Hep.     

A combinatorial approach for directing the amount of fibronectin fibrils assembled by cells that uses surfaces derivatized with mixtures of fibronectin and cell binding domains

Fibrillar fibronectin (FN) has the crucial role of attracting and attaching cells as well as molecules that mediate tissue repair during wound healing. A previous study demonstrated higher extracellular staining of FN fibrils in cells cultured on surfaces tethered with an equimolar mixture of a FN binding domain and FN's cell binding domain, III1-2 and III9-10 respectively, than on surfaces with III9-10 alone. The effect of varying surface amounts of III1-2 and III9-10 on the quantity of FN fibrils formed by NIH-3T3 fibroblasts was examined. GST tagged III1-2 and III9-10 were conjugated to polyurethane surfaces and ELISAs were used to identify the experimental design space or the range of concentrations of GST-III1-2 and GST-III9-10 that demarcated the limits of protein loading on the surface. When GST-III1-2 was fixed and GST-III9-10 varied within the design space, the amount of FN fibrils measured by immunoblotting detergent insoluble cell lysates was dependent on the ratio of III9-10 to III1-2. When the total protein concentration was fixed and the mixture composition of GST-III1-2 and GST-III9-10 varied such that it optimally covered the design space, a parabolic relationship between FN fibril amount and the ratio of III9-10 to III1-2 was obtained. This relationship had a maximum value when the surface was bonded to equal amounts of III1-2 and III9-10 (P<0.05). Thus the ratio of III9-10 to III1-2 can be utilized to direct the quantity of FN fibrils formed on surfaces.     

Extra domain A and type III connecting segment of fibronectin in assembly and cleavage

To determine the role of the extra domain A (EDA) and type III connecting segment (IIICS) of fibronectin in fiber assembly, topographical distribution and proteolytic cleavage, eight full-length human fibronectin cDNA variants (aa0, aa64, aa89, and aa120 variations in the IIICS with or without the EDA) tagged with the V5 epitope were cloned from human endothelial cells and were expressed in CHO-K1 cells. All eight variants were assembled on cell surfaces. However, only the EDA(+) variants, regardless of the type of the IIICS domain, formed extensive fibrous networks. In contrast, the EDA(-)/aa64 and EDA(-)/aa89 variants were present predominantly as a soluble form. Western analysis of both soluble and cell-associated fibronectin/V5 variants showed that aa64, aa89, and aa120 variants with or without the EDA domain produced the major 50- to 62-kDa C-terminal fragments, whereas the aa0 variants did not, suggesting that the IIICS domain provides proteolytic cleavage sites.     

P1 variant antithrombins Glasgow (393 Arg to His) and Pescara (393 Arg to Pro) have increased heparin affinity and are resistant to catalytic cleavage by elastase Implications for the heparin activation mechanism

The heparin affinity of normal and two P1 variants of antithrombin-III (AT) was studied by gradient elution with NaCl in Tris buffer on heparin-Sepharose. At pH 7.4 normal AT eluted art [Na⁺] 0.78 mol/l and the variants both showed increased affinity with AT Pescara eluting at [Na⁺] 0.86 mol/l and AT Glasgow at [Na⁺] 0.92 mol/l. We have earlier proposed a model for heparin activation in which the native state of AT maintains a salt bridge involving the P1 Arg-393 residue. Binding of heparin induces a higher heparin affinity conformation in which the salt bridge is disrupted to reveal the reactive centre for inhibition of thrombin. The Glasgow and Pescara variants, lacking a reactive centre P1 basic residue, would be unable to form this salt bridge, and we suggested that the high affinity conformation which they adopt as their native state would resemble the heparin induced conformation. To examine this model, we measured the heparin induced fluorescence of two P1 variants and tested the susceptibility of their reactive loops to catalytic cleavage. Both variants had fluorescence spectra indistinguishable from normal AT. In the absence of heparin, neither variant was more susceptible than normal to catalytic cleavage by human neutrophil elastase. These findings suggest that the conformation of these P1 variants is different to that of fully heparinized normal AT.     

A peptide from the first fibronectin domain of NCAM acts as an inverse agonist and stimulates FGF receptor activation, neurite outgrowth and survival

Neural cell adhesion molecule (NCAM) contributes to axon growth and guidance during development and learning and memory in adulthood. Although the Ig domains mediate homophilic binding, outgrowth activity localizes to two membrane proximal fibronectin-like domains. The first of these contains a site identified as a potential FGF receptor (FGFR) activation motif (FRM) important for NCAM stimulation of neurite outgrowth, but its activity has hitherto remained hypothetical. Here, we have tested the effects of a domain-specific antibody and peptides corresponding to the FRM in cellular assays in vitro. The first fibronectin domain antibody inhibited NCAM-stimulated outgrowth, indicating the importance of the domain for NCAM function. Monomeric FRM peptide behaved as an inverse agonist; low concentrations specifically inhibited neurite outgrowth stimulated by NCAM and cellular responses to FGF2, while saturating concentrations stimulated FGFR-dependent neurite outgrowth equivalent to NCAM itself. Dendrimeric FRM peptide was 125-fold more active and stimulated FGFR activation, FGFR-dependent and FGF-mimetic neurite outgrowth and cell survival (but not proliferation). We conclude that the FRM peptide contains NCAM-mimetic bioactivity accounted for by stimulation of FGF signalling pathways at the level of or upstream from FGF receptors, and discuss the possibility that FRM comprises part of an FGFR activation site on NCAM.     

Diversity in peptide recognition by the SH2 domain of SH2B1

SH2B1 is a multidomain protein that serves as a key adaptor to regulate numerous cellular events, such as insulin, leptin, and growth hormone signaling pathways. Many of these protein‐protein interactions are mediated by the SH2 domain of SH2B1, which recognizes ligands containing a phosphorylated tyrosine (pY), including peptides derived from janus kinase 2, insulin receptor, and insulin receptor substrate‐1 and ?2. Specificity for the SH2 domain of SH2B1 is conferred in these ligands either by a hydrophobic or an acidic side chain at the +3 position C‐terminal to the pY. This specificity for chemically disparate species suggests that SH2B1 relies on distinct thermodynamic or structural mechanisms to bind to peptides. Using binding and structural strategies, we have identified unique thermodynamic signatures for each peptide binding mode, and several SH2B1 residues, including K575 and R578, that play distinct roles in peptide binding. The high‐resolution structure of the SH2 domain of SH2B1 further reveals conformationally plastic protein loops that may contribute to the ability of the protein to recognize dissimilar ligands. Together, numerous hydrophobic and electrostatic interactions, in addition to backbone conformational flexibility, permit the recognition of diverse peptides by SH2B1. An understanding of this expanded peptide recognition will allow for the identification of novel physiologically relevant SH2B1/peptide interactions, which can contribute to the design of obesity and diabetes pharmaceuticals to target the ligand‐binding interface of SH2B1 with high specificity.     

The C-terminal ligand-binding domain of Clostridium difficile toxin A (TcdA) abrogates TcdA-specific binding to cells and prevents mouse lethality

We have investigated the ability of a recombinant protein (REP231), derived from Clostridium difficile toxin A C-terminal domain, to protect against toxin A (TcdA) intoxication in vitro and in vivo. REP231 was cloned, expressed and purified by thyroglobulin affinity chromatography, and demonstrated identical binding properties to TcdA. Immunofluorescence experiments and in vitro cytotoxicity assays using mouse teratocarcinoma cells F9 showed that specific binding of TcdA to F9 cells through its C-terminal domain is essential for producing cytotoxic effects. TcdA binding and cytotoxicity was inhibited by REP231 and a monoclonal antibody directed against the C-terminal domain. Toxin B did not bind to F9 cells and was consequently inactive in cytotoxicity assays. Inhibition studies with lectins and a Le^x-specific antibody supported earlier findings that a terminal galactose is part of the bound saccharide but excluded Le^x as a receptor for TcdA. Mice immunised with REP231 were protected against a threefold lethal dose of TcdA. Thus, REP231 appeared to be a suitable candidate to develop an alternative therapeutic agent, which is able to neutralise carbohydrate-mediated TcdA binding and might act as a vaccine.     

Characterization of the high affinity heparin binding site of the Alzheimer's disease βA4 amyloid precursor protein (APP) and its enhancement by zinc(II)

The Alzheimer's disease βA4 amyloid precursor protein (APP) has been shown to be involved in a diverse set of biological protein precursor-like proteins (APLP1 and APLP2) belong to a superfamily of proteins that are probably functionally related. In order to characterize the cell adhesion properties of APP the brain specific isoform APP₆₉₅ was purified and used to assess the binding to herparin, a structural and functional analogue of the glycosaminoglycan heparan sulfate. We show that APP binds in a time dependent and saturable manner to heparin. The salt concentration of 620 mM at which APP elutes from heparin Sepharose is greater than physiological. Tha apparent equilibrium constant for dissociation was determined to be 300 pM for APP binding to heparin Sepharose. A high affinity heparin binding site was identified within a region conversed in rodent and human APP, APLP1 and APLP2. This binding site was located between residues 316-337 of APP₆₉₅ which is within the carbohydrate domain of APP. We also demonstrate an interaction between this heparin binding site and the zinc(II) binding site which is conserved in all members of the APP superfamily. We show by using an automated surface plasmon resonance biosensor (BIAcore, Pharmacia) that the affinity for heparin is increased two- to four-fold in the presence of micromolar zinc(II). The identification of zinc-enhanced binding of APP to heparin sulfate side chains of proteoglycans offers a molecular link between zinc(II), as a putative environmental toxin for Alzheimer's disease, and aggregation of amyloid βA4 protein.     

Structure - function analysis of factor VII activating protease (FSAP): Sequence determinants for heparin binding and cellular functions

Factor VII activating protease (FSAP) is associated with cardiovascular diseases and liver fibrosis. To understand the regulation of its proteolytic activity we have characterized recombinant FSAP-mutants over-expressed in HEK-293 cells. The secreted FSAP-protein concentration correlated inversely with the enzymatic activity of the FSAP-mutants. Over-expression of enzymatically active FSAP decreased cell viability, whereas inactive variants were expressed and secreted in adequate amounts. The naturally occurring G534E-variant exhibited reduced proteolytic activity. The ΔEGF-3 mutant showed diminished binding to and activation by heparin. Hence, regulation of FSAP activity is dependent on its EGF-3 domain and over-expression of active variants induces cell death.     

Base coupling in sequence-specific site recognition by the ETS domain of murine PU.1

The ETS domain of murine PU.1 tolerates a large number of DNA cognates bearing a central consensus 5'-GGAA-3' that is flanked by a diverse combination of bases on both sides. Previous attempts to define the sequence selectivity of this DNA binding domain by combinatorial methods have not successfully predicted observed patterns among in vivo promoter sequences in the genome, and have led to the hypothesis that energetic coupling occurs among the bases in the flanking sequences. To test this hypothesis, we determined, using thermodynamic cycles, the complex stabilities and base coupling energies of the PU.1 ETS domain for a set of 26 cognate variants (based on the lambdaB site of the Ig(lambda)2-4 enhancer, 5'-AATAAAAGGAAGTGAAACCAA-3') in which flanking sequences up to three bases upstream and/or two bases downstream of the core consensus are substituted. We observed that both cooperative and anticooperative coupling occurs commonly among the flanking sequences at all the positions investigated. This phenomenon extends at least three bases in the 5' side and is, at least on our experimental data, due exclusively to pairwise interactions between the flanking bases, and not changes in the local environment of the DNA groove floor. Energetic coupling also occurs between the flanking sides across the core consensus, suggesting long-range conformational effects along the DNA target and/or in the protein. Our data provide an energetic explanation for the pattern of flanking bases observed among in vivo promoter sequences and reconcile the apparent discrepancies raised by the combinatorial experiments. We also discuss the significance of base coupling in light of an indirect readout mechanism in ETS/DNA site recognition.     

A proof‐of‐concept study in engineering synthetic protein for selective recognition of substrate‐free polyubiquitin

Similar to substrate‐conjugated polyubiquitin, unanchored polyubiquitin chains are emerging as important regulators for diverse biological processes. The affinity purification of unanchored polyubiquitin from various organisms has been reported, however, tools able to distinguish unanchored polyubiquitin chains with different isopeptide linkages have not yet been described. Toward the goal of selectively identifying and purifying unanchored polyubiquitin chains linked through different Lysines, Scott et al. developed a novel strategy in their study [Proteomics 2016, 16, 1961–1969]. They designed a linker‐optimized ubiquitin‐binding domain hybrid (t‐UBD) containing two UBDs, a ZnFCUBP domain, and a linkage‐selective UBA domain, to specifically recognize unanchored Lys48‐linked polyubiquitin chains. Subsequently, a series of assays has proved the feasibility of this novel strategy for the purification of endogenous substrate‐free Lys48‐linked polyubiquitin chains from mammalian cell extracts. Their research not only provides a tool for purifying unanchored polyubiquitin with different isopeptide linkages, but also paves the way for generating reagents to study the function of unanchored polyubiquitin chains of different linkages in the future. The design of UBD hybrids for defined unanchored polyubiquitin (Lys48‐polyubiquitin) in this study also set an excellent example for future methodology studies regarding monitoring in vivo dynamic changes in the patterns of ubiquitination.     

Molecular domain organization of BldD, an essential transcriptional regulator for developmental process of Streptomyces coelicolor A3(2)

A homodimeric protein, BldD is a key regulator for developmental process of Streptomyces coelicolor and the bldD mutant exhibits severely pleiotropic defects in the antibiotic production and morphological differentiation of the bacterium. In the present work, we approached domain organization of BldD, to structurally and functionally characterize the protein as a DNA-binding protein. We first observed a proteolytic cleavage of BldD by the cytoplasmic extracts of S. coelicolor, which was highly dependent on the developmental stage of the bacterium. The resulting fragment of BldD was identified by mass spectrometry as the N-terminal domain resistant to the proteolysis. Recombinant proteins corresponding to the intact BldD, the N-terminal domain (residues 1-79) and the rest part (C-terminal domain; residues 80-167) were used for comparative analyses by several spectroscopic, thermodynamic, and biochemical experiments, respectively. The results of circular dichroism and nuclear magnetic resonance spectroscopies certified each of the two determined domains could be regarded as an individual folding unit possessing an independent thermodynamic cooperativity. Structural interaction between the two domains was little observed in the DNA-free and DNA-bound states. Strikingly, it was revealed by gel permeation chromatography, chemical crosslink, gel mobility shift, and NMR-monitored DNA-binding experiments, that only the N-terminal domain is responsible for the dimerization as well as DNA-binding of BldD. Detailed inspection of the present results suggests that BldD function in a unique and complicated mode to totally regulate the diverse developmental stages of S. coelicolor.     

Distinct requirements for heparin and alpha-dystroglycan binding revealed by structure-based mutagenesis of the laminin alpha2 LG4-LG5 domain pair

Laminin-2 (alpha2beta1gamma1) is found in basement membranes surrounding muscle and peripheral nerve cells. Several types of cellular receptors bind to the laminin G-like (LG) domains at the C terminus of the alpha2 chain, the interaction with alpha-dystroglycan (alpha-DG) being particularly important in muscle. We have used site-directed mutagenesis and in vitro binding assays to map the binding sites on the laminin alpha2 chain LG4-LG5 domain pair for alpha-DG, heparin and sulfatides. Calcium-dependent alpha-DG recognition requires the calcium ion in LG4, but not the one in LG5, as well as basic residues in both LG domains. Heparin and sulfatides also bind to basic residues in both LG domains, but there is little overlap in the binding sites for alpha-DG and heparin/sulfatides. The results should prove useful for the molecular dissection of laminin-receptor interactions in vivo.     

Acyl-CoA binding domain containing 3 (ACBD3) recruits the protein phosphatase PPM1L to ER-Golgi membrane contact sites

The metal-dependent protein phosphatase family (PPM) governs a number of signaling pathways. PPM1L, originally identified as a negative regulator of stress-activated protein kinase signaling, was recently shown to be involved in the regulation of ceramide trafficking at ER-Golgi membrane contact sites. Here, we identified acyl-CoA binding domain containing 3 (ACBD3) as an interacting partner of PPM1L. We showed that this association, which recruits PPM1L to ER-Golgi membrane contact sites, is mediated by a GOLD (Golgi dynamics) domain in ACBD3. These results suggested that ACBD3 plays a pivotal role in ceramide transport regulation at the ER-Golgi interface.

Structured summary of protein interactions

ACBD3 and PPM1Lcolocalize by fluorescence microscopy (View interaction)FYCO1physically interacts with PPM1L by pull down (View interaction)SEC14L2physically interacts with PPM1L by pull down (View interaction)ACBD3physically interacts with PPM1L by pull down (View interaction)SEC14L1physically interacts with PPM1L by pull down (View interaction)PPM1Lphysically interacts with ACBD3 by two hybrid (View interaction)     

Inhibition of cell proliferation in Saccharomyces cerevisiae by expression of human NAD+ ADP-ribosyltransferase requires the DNA binding domain ("zinc fingers").

Summary Constitutive expression of human nuclear NAD⁺: protein ADP-ribosyltransferase (polymerizing) [pADPRT; poly(ADP-ribose)polymerase; EC 2.4.2.30] as an active enzyme in Saccharomyces cerevisiae, under the control of the alcohol dehydrogenase promoter, was only possible with simultaneous inhibition of ADP-ribosylation by 3-methoxybenzamide. Induction of fully active pADPRT from the inducible galactose epimerase promoter resulted in inhibition of cell division and morphological changes reminiscent of cell cycle mutants. Expression of a pADPRT cDNA truncated at its 5end had no influence on cell proliferation at all. Obviously the amino-terminal part of the DNA binding domain containing the first zinc finger, which is essential for inducibility of pADPRT activity by DNA breaks, is also required for inhibition of cell growth on expression in yeast. Full-length as well as truncated pADPRT molecules were directed to the cell nucleus where the fully active enzyme produced large amounts of poly(ADP-ribose) by automodification. Since pADPRT turned out to be the only target for ADP-ribosylation in these cells, elevated levels of poly(ADP-ribose) were the most likely cause of inhibition of cell division, presumably resulting from interaction with chromosomal proteins.     

Effect of the C-terminal domain of Vibrio proteolyticus chitinase A on the chitinolytic activity in association with pH changes

Aims: To reveal the cause of the difference in activity of chitinase A from Vibrio proteolyticus and chitinase A from a strain of Vibrio carchariae (a junior synonym of Vibrio harveyi), we investigated the pH‐dependent activity of full‐length V. proteolyticus chitinase A and a truncated recombinant corresponding to the V. harveyi form of chitinase A. Methods and Results: After overexpression in Escherichia coli strain DH5α, the full‐length and truncated recombinant chitinases were purified by ammonium sulphate precipitation and anion exchange column chromatography. Chitinase activity was measured at various pH values using α‐crystal and colloidal chitins as the substrate. The pH‐dependent patterns of the relative specific activities for α‐crystal chitin differed between the full‐length and truncated recombinant chitinases, whereas those for colloidal chitin were similar to each other. Conclusion: The difference in the activity of V. proteolyticus chitinase A and V. harveyi chitinase A might be partly due to a change in the pH dependence of the chitinase activities against α‐crystal chitin, resulting from C‐terminal processing. Significance and Impact of Study: The present results are important findings for not only ecological studies on the genus Vibrio in association with survival strategies, but also phylogenetic studies.