Neutron and X-ray scattering studies on the human complement protein properdin provide an analysis of the thrombospondin repeat

Properdin is a regulatory glycoprotein of the alternative pathway of the complement system of immune defense. It is responsible for the stabilization of the C3 convertase complex formed between C3b and the Bb fragment of factor B. Neutron and X-ray solution scattering experiments were performed on the dimeric and trimeric forms of properdin. These have RG values of 9.1 and 10.7 nm, respectively. The scattering curves were compared with Debye sphere modeling simulations for properdin. Good agreements were obtained for models similar to published electron micrographs showing that the properdin trimer has a triangular structure with sides of 26 nm. Such a structure also accounted for sedimentation coefficient data on properdin. Primary structure analyses for mouse and human properdin have shown that this contains six homologous motifs known as the thrombospondin repeat (TSR), which is the second most abundant domain type found in the complement proteins. Sequences for these 12 TSRs were aligned with 19 others found in thrombospondin and the late complement components. Three distinct groups of TSRs were identified, namely, the TSRs found in thrombospondin and properdin, the TSRs mostly found at the N-terminus of the late complement components, and the TSRs found at the C-terminus of the late components. Averaged secondary structure predictions suggested that all three groups contain similar backbone structures with two amphipathic turn regions and one hydrophilic beta-strand region. The mean dimensions of the TSRs of properdin in solution were determined to be approximately 4 nm X 1.7 nm X 1.7 nm, showing that these are elongated in structure.     

Extended and flexible domain solution structure of the extracellular matrix protein anosmin-1 by X-ray scattering, analytical ultracentrifugation and constrained modelling

Kallmann's syndrome corresponds to a loss of sense of smell and hypogonadotrophic hypogonadism. Defects in anosmin-1 result in the X-linked inherited form of Kallmann's syndrome. Anosmin-1 is an extracellular matrix protein comprised of an N-terminal, cysteine-rich (Cys-box) domain and a whey acidic protein-like (WAP) domain, followed by four fibronectin type III (FnIII) domains. The solution structures of recombinant proteins containing the first three domains (PIWF1) and all six domains (PIWF4) were determined by X-ray scattering and analytical ultracentrifugation. Guinier analyses showed that PIWF1 and PIWF4 have different radii of gyration (R(G)) values of 3.1 nm and 6.7 nm, respectively, but similar cross-sectional radii of gyration (R(XS)) values of 1.5 nm and 1.9 nm, respectively. Distance distribution functions showed that the maximum lengths of PIWF1 and PIWF4 were 11 nm and 23 nm, respectively. Analytical ultracentrifugation gave sedimentation coefficients of 2.52 S and 3.55 S for PIWF1 and PIWF4, respectively. The interpretation of the scattering data by constrained modelling requires homology models for all six domains in anosmin-1. While models were already available for the WAP and FnIII domains, searches suggested the Cys-box domain may resemble the cysteine-rich region of the insulin-like growth factor receptor. Automated constrained molecular modelling based on joining the anosmin-1 domains with structurally randomised linkers resulted in 10,000 models for anosmin-1. A trial-and-error search showed that about 0.1-1.4% of these models fitted the X-ray data. The best models showed that the three domains and six domains in PIWF1 and PIWF4, respectively, were extended. The inter-domain linkers in anosmin-1 could not all be extended at the same time, and there was evidence for inter-domain flexibility. Models with folded-back domain arrangements do not fit the data. These solution structures account for the known biological function of anosmin-1, in particular its ability to interact with its three macromolecular ligands.     

The layered fold of the TSR domain of P. falciparum TRAP contains a heparin binding site

Thrombospondin-related anonymous protein, TRAP, has a critical role in the hepatocyte invasion step of Plasmodium sporozoites, the transmissible form of the parasite causing malaria. The extracellular domains of this sporozoite surface protein interact with hepatocyte surface receptors whereas its intracellular domain acts as a link to the sporozoite actomyosin motor system. Liver heparan sulfate proteoglycans have been identified as potential ligands for TRAP. Proteoglycan binding has been associated with the A- and TSR domains of TRAP. We present the solution NMR structure of the TSR domain of TRAP and a chemical shift mapping study of its heparin binding epitope. The domain has an elongated structure stabilized by an array of tryptophan and arginine residues as well as disulfide bonds. The fold is very similar to those of thrombospondin type-1 (TSP-1) and F-spondin TSRs. The heparin binding site of TRAP-TSR is located in the N-terminal half of the structure, the layered side chains forming an integral part of the site. The smallest heparin fragment capable of binding to TRAP-TSR is a tetrasaccharide.     

Properdin binds to sulfatide [Gal(3-SO4)beta 1-1 Cer] and has a sequence homology with other proteins that bind sulfated glycoconjugates

Properdin, which stabilizes the C3 convertase during the activation of the alternate complement pathway, contains amino acid sequence homologies with several proteins that bind sulfated glycoconjugates, including the adhesive protein thrombospondin and the leech salivary protein antistasin. This homology is based around the sequence Cys-Ser-Val-Thr-Cys-Gly-X-Gly-X-X-X-Arg-X-Arg. To determine if these homologous amino acid sequences are sulfated glycoconjugate-binding domains, purified native properdin, as well as activated properdin (a high molecular weight form of properdin), were examined for binding to various lipids in solid phase radioimmunoassays. Of the lipids tested, both native and activated properdin bind with high affinity only to sulfatide [Gal(3-SO4)beta 1-1 Cer], but not to comparable levels of cholesterol-3-SO4, or several neutral glycolipids, gangliosides, and phospholipids. Sulfatide binding by both forms of properdin is inhibited by dextran sulfate (Mr = 500,000) or fucoidan, whereas only the activated form is inhibited by dextran sulfate (Mr = 5,000) or heparin. Comparable levels of chondroitin sulfates A, B, and C, keratan sulfate, dextran (Mr = 90,000), or hyaluronic acid do not inhibit binding. Taken together, these data suggest that properdin, like antistasin and thrombospondin, binds sulfated glycoconjugates and supports the conclusion that the homologous sequences are sulfated glycoconjugate-binding domains.     

Expression and characterisation of the thrombospondin type I repeats of human properdin

Properdin, an upregulator of the alternative complement pathway, is central to deposition of the activated complement fragment C3b on the surfaces of the pathogens, which it achieves by preventing the dissociation of the Bb catalytic subunit from the inherently labile C3bBb complexes. It is also known to bind sulphated glycoconjugates, such as sulphatides. Properdin has an unusual structure formed by oligomerisation of a rod-like monomer into cyclic dimers, trimers and tetramers. The monomer (approximately 53 kDa) contains an N-terminal region of no known homology, followed by six non-identical repeats of 60 amino acids (based on exon/intron boundaries), called 'thrombospondin type I repeats' or TSR modules. We have expressed and purified the N-terminal region and each of the individual TSR repeats in Escherichia coli. Although the individual recombinant TSRs, after a denaturation-renaturation cycle, appeared to be correctly folded modules, as judged by the one-dimensional (1D)- and 2D-nuclear magnetic resonance spectra of TSR3, they did not show binding to either C3b or sulphatide. Polyclonal antibodies were raised against each TSR and were found to be module-specific. The anti-TSR5 polyclonal antibody was found to inhibit binding of native human properdin to solid-phase C3b, or sulphatides. It could also block properdin-dependent haemolysis of rabbit erythrocytes. These results are consistent with the view that the TSR5 contains the major site in properdin which is involved in both C3b and sulphatide binding. It also suggests that a co-operative intramolecular interaction between TSRs, as found in the native molecule, is required for TSR5 to bind either C3b or sulphatides.     

Molecular modeling of the domain structure of C9 of human complement by neutron and X-ray solution scattering.

C9 is the most abundant component of the membrane attack complex of the complement system of immune defense. This is a typical mosaic protein with thrombospondin (TSR) and low density lipoprotein receptor (LDLr) domains at its N-terminus and an epidermal growth factor-like (EGF) domain at its C-terminus. Between these lies a perforin-like sequence. In order to define the arrangement in solution of these four moieties in C9, high-flux neutron and synchrotron X-ray solution scattering studies were carried out. The neutron radius of gyration RG at infinite contrast is 3.33 nm, and its cross-sectional RG (RXS) is 1.66 nm. Similar values were obtained by synchrotron X-ray scattering after allowance for radiation effects. Stuhrmann analyses showed that the neutron radial inhomogeneity of scattering density alpha is 35 X 10(-5) from the RG data and 16 X 10(-5) from the RXS data. These values are typical for soluble glycoproteins and show no evidence for the existence of any large hydrophobic surface patches on free C9 that might form contacts with lipids. Indirect transformation of the neutron and X-ray scattering curves into real space showed that C9 had a maximum dimension estimated at 12 +/- 2 nm, and this suggests that the lengths of 7-8 nm deduced from previous electron microscopy studies in vacuo are underestimated. Molecular modeling of the C9 scattering curves utilized small spheres in the Debye equation, in which the analyses were constrained by the known volumes of the four moieties of C9 and the known sizes of the TSR and EGF-like domains. The most likely models for C9 suggest that these four regions of C9 are arranged in a V-shaped structure, with an angle of 10 degrees between the two arms, each of length 11.1 nm. This structure has a more hydrophobic character between the two arms. The scattering model is fully consistent with hydrodynamic sedimentation data on C9. Similar V-shaped hydrodynamic models could be developed for C6, C7, C8, and C9 of complement. Such a compact structure is atypical of other multidomain complement proteins so far studied by solution scattering and is fully compatible with mechanisms in which C9 is postulated, on activation, to undergo a drastic unfolding of its domain structure and to expose a more hydrophobic surface which can be embedded into lipid bilayers.     

Expression, purification and structural characterization of functionally replete thrombospondin-1 type 1 repeats in a bacterial expression system

The matrix glycoprotein thrombospondin-1 (TSP-1) is a prominent regulator of endothelial cells and angiogenesis. The anti-angiogenic and anti-tumorigenic properties of TSP-1 are in part mediated by the TSP-1 type 1 repeat domains 2 and 3, TSR(2,3). Here, we describe the expression and purification of human TSR(2,3) in milligram quantities from an Escherichia coli expression system. Microvascular endothelial cell migration assays and binding assays with a canonical TSP-1 ligand, histidine-rich glycoprotein (HRGP), indicate that recombinant TSR(2,3) exhibits anti-chemotactic and ligand binding properties similar to full length TSP-1. Furthermore, we determined the structure of E. coli expressed TSR(2,3) by X-ray crystallography at 2.4? and found the structure to be identical to the existing TSR(2,3) crystal structure determined from a Drosophila expression system. The TSR(2,3) expression and purification protocol developed in this study allows for facile expression of TSR(2,3) for biochemical and biophysical studies, and will aid in the elucidation of the molecular mechanisms of TSP-1 anti-angiogenic and anti-tumorigenic activities.     

Structure of the F‐spondin domain of mindin,an integrin ligand and pattern recognition molecule

Mindin (spondin‐2) is an extracellular matrix protein of unknown structure that is required for efficient T‐cell priming by dendritic cells. Additionally, mindin functions as a pattern recognition molecule for initiating innate immune responses. These dual functions are mediated by interactions with integrins and microbial pathogens, respectively. Mindin comprises an N‐terminal F‐spondin (FS) domain and C‐terminal thrombospondin type 1 repeat (TSR). We determined the structure of the FS domain at 1.8‐Å resolution. The structure revealed an eight‐stranded antiparallel β‐sandwich motif resembling that of membrane‐targeting C2 domains, including a bound calcium ion. We demonstrated that the FS domain mediates integrin binding and identified the binding site by mutagenesis. The mindin FS domain therefore represents a new integrin ligand. We further showed that mindin recognizes lipopolysaccharide (LPS) through its TSR domain, and obtained evidence that C‐mannosylation of the TSR influences LPS binding. Through these dual interactions, the FS and TSR domains of mindin promote activation of both adaptive and innate immune responses.     

Solution structure of human von Willebrand factor studied using small angle neutron scattering

von Willebrand factor (VWF) binding to platelets under high fluid shear is an important step regulating atherothrombosis. We applied light and small angle neutron scattering to study the solution structure of human VWF multimers and protomer. Results suggest that these proteins resemble prolate ellipsoids with radius of gyration (R(g)) of approximately 75 and approximately 30 nm for multimer and protomer, respectively. The ellipsoid dimensions/radii are 175 x 28 nm for multimers and 70 x 9.1 nm for protomers. Substructural repeat domains are evident within multimeric VWF that are indicative of elements of the protomer quarternary structure (16 nm) and individual functional domains (4.5 nm). Amino acids occupy only approximately 2% of the multimer and protomer volume, compared with 98% for serum albumin and 35% for fibrinogen. VWF treatment with guanidine.HCl, which increases VWF susceptibility to proteolysis by ADAMTS-13, causes local structural changes at length scales <10 nm without altering protein R(g). Treatment of multimer but not protomer VWF with random homobifunctional linker BS(3) prior to reduction of intermonomer disulfide linkages and Western blotting reveals a pattern of dimer and trimer units that indicate the presence of stable intermonomer non-covalent interactions within the multimer. Overall, multimeric VWF appears to be a loosely packed ellipsoidal protein with non-covalent interactions between different monomer units stabilizing its solution structure. Local, and not large scale, changes in multimer conformation are sufficient for ADAMTS-13-mediated proteolysis.     

Glycosaminoglycan-binding properties and aggrecanase activities of truncated ADAMTSs: comparative analyses with ADAMTS-5, -9, -16 and -18

Aggrecanases are ADAMTS (a disintegrin and metalloproteinase with thrombospondin type I motifs) proteases capable of primary (patho)physiological cleavage at specific Glu-Xaa bonds within the core protein of the hyaluronan-binding proteoglycan aggrecan. Accumulating evidence suggests that regulation of the activity of one such aggrecanase, ADAMTS-4 (or Aggrecanase-1), involves post-translational C-terminal processing (truncation) which modulates both glycosaminoglycan (GAG)-binding affinity and enzymatic activity. In the present study, we compared the effects of C-terminal truncation on the GAG-binding properties and aggrecanase activity of ADAMTS-5 (Aggrecanase-2) relative to three other ADAMTS family members, ADAMTS-9, ADAMTS-16 and ADAMTS-18. Full-length recombinant human ADAMTS-5 (M(r) approximately 85 kDa; ADAMTS-5p85) underwent autolytic cleavage during expression by CHO/A2 cells, and co-purified with C-terminally truncated (tr) isoforms of M(r) approximately 60 kDa (ADAMTS-5p60 and M(r) approximately 45 kDa (ADAMTS-5p45). All three ADAMTS-5 isoforms bound to sulfated GAGs (heparin and chondroitin sulfate (CS)). An ADAMTS-5p45 structural mimetic, terminating at Phe628 and comprising the catalytic domain, disintegrin-like domain and thrombospondin type I repeat (TSR)-1 domain (designated trADAMTS-5F628), also bound to heparin, and exhibited potent aggrecanase activity toward cleavage sites both in the aggrecan CS-2-attachment region (at Glu1771-Ala1772) and in the interglobular domain (at Glu373-Ala374). Further truncation (deletion of the TSR-1 domain) of ADAMTS-5 significantly reduced aggrecanase activity, although appreciable GAG (heparin)-binding affinity was maintained. Other TSR-1 domain-bearing truncated ADAMTS constructs demonstrating either positive GAG-binding ability (trADAMTS-9F649) or negligible GAG-affinity (trADAMTS-16F647 and trADAMTS-18F650) displayed comparably low aggrecanase activities. Thus, the presence of TSR-1 on truncated ADAMTSs appears to be necessary, but not sufficient, for effective aggrecanase-mediated catalysis of target Glu-Xaa bonds. Similarly, GAG-binding ability, irrespective of the presence of a TSR-1 domain, does not necessarily empower truncated ADAMTSs with proficient aggrecanase activity.     

Folded-back solution structure of monomeric factor H of human complement by synchrotron X-ray and neutron scattering, analytical ultracentrifugation and constrained molecular modelling

Factor H (FH) is a regulatory cofactor for the protease factor I in the breakdown of C3b in the complement system of immune defence, and binds to heparin and other polyanionic substrates. FH is composed of 20 short consensus/complement repeat (SCR) domains, for which the overall arrangement in solution is unknown. As previous studies had shown that FH can form monomeric or dimeric structures, X-ray and neutron scattering was accordingly performed with FH in the concentration range between 0.7 and 14 mg ml(-1). The radius of gyration of FH was determined to be 11.1-11.3 nm by both methods, and the radii of gyration of the cross-section were 4.4 nm and 1.7 nm. The distance distribution function P(r) showed that the overall length of FH was 38 nm. The neutron data showed that FH was monomeric with a molecular mass of 165,000(+/-17,000) Da. Analytical ultracentrifugation data confirmed this, where sedimentation equilibrium curve fits gave a mean molecular mass of 155,000(+/-3,000) Da. Sedimentation velocity experiments using the g*(s) derivative method showed that FH was monodisperse and had a sedimentation coefficient of 5.3(+/-0.1) S. In order to construct a full model of FH for scattering curve and sedimentation coefficient fits, homology models were constructed for 17 of the 20 SCR domains using knowledge of the NMR structures for FH SCR-5, SCR-15 and SCR-16, and vaccinia coat protein SCR-3 and SCR-4. Molecular dynamics simulations were used to generate a large conformational library for each of the 19 SCR-SCR linker peptides. Peptides from these libraries were combined with the 20 SCR structures in order to generate stereochemically complete models for the FH structure. Using an automated constrained fit procedure, the analysis of 16,752 possible FH models showed that only those models in which the 20 SCR domains were bent back upon themselves were able to account for the scattering and sedimentation data. The best-fit models showed that FH had an overall length of 38 nm and is flexible. This length is significantly less than a predicted length of 73 nm if the 20 SCR structures had been arranged in an extended arrangement. This outcome is attributed to several long linker sequences. These bent-back domain structures may correspond to conformational flexibility in FH and enable the multiple FH binding sites for C3 and heparin to come into close proximity.     

Structure and function insights garnered from in silico modeling of the thrombospondin type-1 domain-containing 7A antigen

The thrombospondin type-1 domain containing 7A (THSD7A) protein is known to be one of the antigens responsible for the autoimmune disorder idiopathic membranous nephropathy. The structure of this antigen is currently unsolved experimentally. Here we present a homology model of the extracellular portion of the THSD7A antigen. The structure was evaluated for folding patterns, epitope site prediction, and function was predicted. Results show that this protein contains 21 extracellular domains and with the exception of the first two domains, has a regular repeating pattern of TSP-1-like followed by F-spondin-like domains. Our results indicate the presence of a novel Trp-ladder sequence of WxxxxW in the TSP-1-like domains. Of the 21 domains, 18 were shown to have epitope binding sites as predicted by epitopia. Several of the F-spondin-like domains have insertions in the canonical TSP fold, most notably the coiled coil region in domain 4, which may be utilized in protein-protein binding interactions, suggesting that this protein functions as a heparan sulfate binding site.     

Heparin-binding growth-associated molecule contains two heparin-binding beta -sheet domains that are homologous to the thrombospondin type I repeat

Heparin-binding growth-associated molecule (HB-GAM) is an extracellular matrix-associated protein implicated in the development and plasticity of neuronal connections of brain. Binding to cell surface heparan sulfate is indispensable for the biological activity of HB-GAM. In the present paper we have studied the structure of recombinant HB-GAM using heteronuclear NMR. These studies show that HB-GAM contains two beta-sheet domains connected by a flexible linker. Both of these domains contain three antiparallel beta-strands. In addition to this domain structure, HB-GAM contains the N- and C-terminal lysine-rich sequences that lack a detectable structure and appear to form random coils. Studies using CD and NMR spectroscopy suggest that HB-GAM undergoes a conformational change upon binding to heparin, and that the binding occurs primarily to the beta-sheet domains of the protein. Search of sequence data bases shows that the beta-sheet domains of HB-GAM are homologous to the thrombospondin type I repeat (TSR). Sequence comparisions show that the beta-sheet structures found previously in midkine, a protein homologous with HB-GAM, also correspond to the TSR motif. We suggest that the TSR sequence motif found in various extracellular proteins defines a beta-sheet structure similar to that found in HB-GAM and midkine. In addition to the apparent structural similarity, a similarity in biological functions is suggested by the occurrence of the TSR sequence motif in a wide variety of proteins that mediate cell-to-extracellular matrix and cell-to-cell interactions, in which the TSR domain mediates specific cell surface binding.     

Molecular cloning and characterization of a novel human beta1,3-glucosyltransferase, which is localized at the endoplasmic reticulum and glucosylates O-linked fucosylglycan on thrombospondin type 1 repeat domain

Protein O-linked fucosylation is an unusual glycosylation associated with many important biological functions such as Notch signaling. Two fucosylation pathways synthesizing O-fucosylglycans have been reported on cystein-knotted proteins, that is, on epidermal growth factor-like (EGF-like) domains and on thrombospondin Type 1 repeat (TSR) domains. We report here the molecular cloning and characterization of a novel beta1,3-glucosyltransferase (beta3Glc-T) that synthesizes a Glcbeta1,3Fucalpha- structure on the TSR domain. We found a novel glycosyltransferase gene with beta1,3-glycosyltransferase (beta3GT) motifs in databases. The recombinant enzyme expressed in human embryonic kidney 293T (HEK293T) cells exhibited glucosyltransferase activity toward fucose-alpha-para-nitrophenyl (Fucalpha-pNp). Thin-layer chromatography (TLC) analysis revealed that the product of the recombinant enzyme migrated to the same position as did the product of endogenous beta3Glc-T of Chinese hamster ovary (CHO) cells. The two products could be digested by beta-glucosidase from almond and by exo-1,3-beta-glucanase from Trichoderma sp. These results strongly suggested that the product has the structure of Glcbeta1-3Fuc. Therefore, we named this novel enzyme beta3Glc-T. Immunostaining revealed that FLAG-tagged beta3Glc-T is an enzyme residing in the endoplasmic reticulum (ER) via retention signal, "REEL," which is a KDEL-like sequence, at the C-terminus. The TSR domain expressed in Escherichia coli was first fucosylated by the recombinant protein O-fucosyltransferase 2 (POFUT2), after which it became an acceptor substrate for the recombinant beta3Glc-T, which could apparently transfer Glc to the fucosylated TSR domain. Our results suggest that a novel glycosyltransferase, beta3Glc-T, contributes to the elongation of O-fucosylglycan and that this occurs specifically on TSR domains.     

Properdin, the positive regulator of complement, is highly C-mannosylated

Properdin is the positive regulator of the alternative pathway of complement activation. The 53-kDa protein is essentially composed of six thrombospondin type 1 repeats, all of which contain the WXXW motif, the recognition sequence for C-mannosylation. C-Mannosylation is a post-translational modification of tryptophan residues in which, in contrast to the well known N- and O-glycosylation, the carbohydrate is attached via a C-C bond to C-2 of the indole moiety of tryptophan. C-Mannosylation was first found in human RNase 2 and interleukin-12. The terminal complement proteins C6-C9 also carry this modification as part of their thrombospondin type 1 repeats. We studied the C-mannosylation pattern of human properdin by mass spectrometry and Edman degradation. Properdin contains 20 tryptophans of which 17 are part of a WXXW motif. Fourteen tryptophans were found to be modified 100%. This is the first example of a protein in which the majority of tryptophan residues occurs in the C-mannosylated form. These results show that C-mannosylated proteins occur at several steps along the complement activation cascade. Therefore, this system would be ideal to investigate the function of C-mannosylation.     

Extended flexible linker structures in the complement chimaeric conjugate CR2-Ig by scattering, analytical ultracentrifugation and constrained modelling: implications for function and therapy

Complement receptor 2 (CR2; CD21) is a membrane-bound regulator of complement activation, being comprised of 15 or 16 short complement repeat (SCR) domains. A recombinant glycosylated human CR2 SCR 1-2 domain pair was engineered with the Fc fragment of a mouse IgG1 antibody to create a chimaera CR2-Ig containing the major ligand binding domains. Such a chimaera has therapeutic potential as a complement inhibitor or immune modulator. X-ray and neutron scattering and analytical ultracentrifugation identified its domain structure in solution, and provided a comparison with controversial folded-back crystal structures for deglycosylated CR2 SCR 1-2. The radius of gyration R(G) of CR2-Ig was determined to be 5.39(+/-0.14) nm and 5.29(+/-0.01) nm by X-ray and neutron scattering, respectively. The maximum dimension of CR2-Ig was determined to be 17 nm. The molecular mass of CR2-Ig ranged between 101,000 Da and 107,000 Da as determined by neutron scattering and sedimentation equilibrium, in good agreement with the sequence-derived value of 106,600 Da. Sedimentation velocity gave a sedimentation coefficient of 4.49(+/-0.11) S. Stereochemically complete models for CR2-Ig were constructed from crystal structures for the CR2 SCR 1-2 and mouse IgG1 Fc fragments. The two SCR domains and the Fc fragment were joined by randomised conformational peptides. The analysis of 35,000 possible CR2-Ig models showed that only those models in which the two SCR domains were arranged in an open V-shape in random orientations about the Fc fragment accounted for the scattering and sedimentation data. It was not possible to define one single conformational family of Fab-like fragment relative to the Fc fragment. This flexibility is attributed to the relatively long linker sequence and the absence of the antibody light chain from CR2-Ig. The modelling also confirmed that the structure of CR2 SCR 1-2 is more extended in solution than in its crystal structure.     

Crystal structure of human filamin C domain 23 and small angle scattering model for filamin C 23-24 dimer

Filamin C is a dimeric, actin-binding protein involved in organization of cortical cytoskeleton and of the sarcomere. We performed crystallographic, small-angle X-ray scattering and analytical ultracentrifugation experiments on the constructs containing carboxy-terminal domains of the protein (domains 23-24 and 19-21). The crystal structure of domain 23 of filamin C showed that the protein adopts the expected immunoglobulin (Ig)-like fold. Small-angle X-ray scattering experiments performed on filamin C tandem Ig-like domains 23 and 24 reveal a dimer that is formed by domain 24 and that domain 23 has little interactions with itself or with domain 24, while the analytical ultracentrifugation experiments showed that the filamin C domains 19-21 form elongated monomers in diluted solutions.     

Identification of tubular heparan sulfate as a docking platform for the alternative complement component properdin in proteinuric renal disease

Properdin binds to proximal tubular epithelial cells (PTEC) and activates the complement system via the alternative pathway in vitro. Cellular ligands for properdin in the kidney have not yet been identified. Because properdin interacts with solid-phase heparin, we investigated whether heparan sulfate proteoglycans (HSPG) could be the physiological ligands of properdin. Kidneys from proteinuric rats showed colocalization of syndecan-1, a major epithelial HSPG, and properdin in the apical membranes of PTEC, which was not seen in control renal tissue. In vitro, PTEC did not constitutively express properdin. However, exogenous properdin binds to these cells in a dose-dependent fashion. Properdin binding was prevented by heparitinase pretreatment of the cells and was dose-dependently inhibited by exogenous heparin. ELISA and surface plasmon resonance spectroscopy (BIAcore) showed a strong dose-dependent interaction between heparan sulfate (HS) and properdin (K(d) = 128 nm). Pretreatment of HSPG with heparitinase abolished this interaction in ELISA. Competition assays, using a library of HS-like polysaccharides, showed that sulfation pattern, chain length, and backbone composition determine the interaction of properdin with glycosaminoglycans. Interestingly, two nonanticoagulant heparin derivatives inhibited properdin-HS interaction in ELISA and BIAcore. Incubation of PTEC with human serum as complement source led to complement activation and deposition of C3 on the cells. This C3 deposition is dependent on the binding of properdin to HS as shown by heparitinase pretreatment of the cells. Our data identify tubular HS as a novel docking platform for alternative pathway activation via properdin, which might play a role in proteinuric renal damage. Our study also suggests nonanticoagulant heparinoids may provide renoprotection in complement-dependent renal diseases.     

Interactions among Stalk Modules of Thrombospondin-1

Thrombospondin-1 is a trimeric, modular calcium-binding glycoprotein. The subunit is composed of an N-terminal module; oligomerization domain; stalk modules including a von Willebrand factor type C module, three properdin or thrombospondin type 1 repeat (TSR) modules, and two thrombospondin-type EGF-like modules; and a C-terminal signature domain comprising single copies of the epidermal growth factor (EGF)-like, wire, and lectin-like modules. Conformational changes in the signature domain influence ligand binding to the N-terminal modules. Interactions have been demonstrated among the modules of the signature domain and the thrombospondin-type EGF-like modules. We have extended this analysis to the rest of the stalk modules. Differential scanning calorimetry revealed interactions between the most C-terminal TSR module and the EGF-like modules. Calorimetry and differences in expression levels of single versus tandem modules indicated that the three TSRs interact with each other as well. No evidence of interactions between the von Willebrand factor type C and TSR modules were detected by differential scanning calorimetry, circular dichroism, or intrinsic fluorescence. These results indicate that the TSR and thrombospondin-type EGF-like stalk modules act as a unit that may relay conformational information between the N-terminal and C-terminal parts of the protein.Thrombospondin-1 (TSP-1)² is a major secreted protein of platelets that plays multiple roles after vascular injury (1, 2). TSPs are a family of multimodular, calcium-binding, extracellular glycoproteins. There are five family members in tetropods, each of which has a specific pattern of expression in embryonic and adult tissues (3). TSPs have two unique features, a signature domain comprising single copies of EGF-like, Ca²⁺-binding wire, and lectin-like modules and the TSP-type EGF-like module in which Cys⁴ and Cys⁵ are separated by two rather than one residue (3, 4). The family falls into two groups: A or trimeric TSPs, TSP-1 and TSP-2; and B or pentameric TSPs, TSP-3, TSP-4, and TSP-5. As depicted in Fig. 1, a subunit of the group A TSPs is composed of an N-terminal module tethered to an oligomerization domain, a von Willebrand Factor type C (vWF-C) module, three properdin or TSP type 1 repeat (TSR) modules, two TSP-type EGF-like modules, and the signature domain (3, 4). Subunits of group B TSPs lack vWF-C and TSR modules and have an extra TSP-type EGF-like module (4). Multiple interactions have been demonstrated among the modules of the signature domain of Ca²⁺-replete TSP-2 and TSP-5 (5, 6) and between the signature domain wire and second TSP-type EGF-like module of Ca²⁺-replete TSP-2 (5, 7).Open in a separate windowFIGURE 1.Schematics of (A) TSP-1 stalk modules studied in this paper, (B) TSP-1 in its Ca²⁺-depleted conformation, and (C) TSP-1 in its Ca²⁺-replete formation. Parts of TSP-1 in panels A and B are labeled as follows: N, N-terminal module; T, tether; C, vWF-C module; P, properdin or TSR module, E, EGF-like module; wire, Ca²⁺-binding repeats with 26 Ca²⁺-binding sites; and L, lectin-like module. The TSP-type EGF-like modules, E1 and E2, contain central shading. Sites of binding to heparin sulfate proteoglycan (HSPG), latent transforming growth factor-β (TGF), and CD36 are indicated in panel C. The schematics have been drawn based on structures described in the text. Sites of fucosylation of TSRs are indicated by open diamonds, and inter-module CPIXG sequences between P2 and P3 and between P3 and E1 are indicated with dots. As per the “Discussion,” changes in conformation and charge density of the signature domain due to gain or loss of Ca²⁺ are proposed to be propagated throughout trimeric TSP-1 by the stalk modules.TSP-1 has a distinctive appearance when examined by rotary shadowing electron microscopy: three bunched globules, which are thought to be the N-terminal modules, are connected by three stalks to three larger globules thought to be the C-terminal signature domains (4). Rotary shadowing electron microscopy demonstrates a striking conformational change upon removal of Ca²⁺ from the C-terminal signature domain with apparent lengthening of the stalk and loss of size of the C-terminal globules (8–10). Considerations of structures of the parts of TSP-1 indicate that the vWF-C, TSR, and TSP-type EGF-like modules form the stalk in Ca²⁺-replete TSP-1 (4), as depicted in Fig. 1. Immunochemical studies suggest that lengthening of the stalk is due, at least in part, to unraveling of two of the 13 Ca²⁺-binding repeats of the wire module (11).Removal of Ca²⁺ from binding sites on the C-terminal signature domain impacts binding of ligands or antibodies to the N-terminal modules of TSP-1 (12). The N700S polymorphism in TSP-1 that alters coordination of Ca²⁺ by the first Ca²⁺-binding wire repeat (13) also impacts interactions of the N-terminal modules with ligands (14). These observations indicate that TSP-1 possesses an allosteric mechanism whereby changes in the C-terminal signature domain are transmitted to the N-terminal modules. We have reported that the two TSP-type EGF-like modules and the signature domain EGF-like module interact with each other, suggesting a mechanism by which conformational changes in the signature domain can be propagated N-terminal as far as the first TSP-type EGF-like module (15). We have now explored the potential of EGF-like modules to work with TSR and vWF-C modules to transmit conformational information between the two ends of TSP-1.