Mapping of epitopes for monoclonal antibodies against human platelet thrombospondin with electron microscopy and high sensitivity amino acid sequencing

A panel of monoclonal antibodies (Mab's) has been raised against human platelet thrombospondin (TSP). One Mab, designated A2.5, inhibits the hemagglutinating activity of TSP and immunoprecipitates the NH2 terminal 25 kD heparin binding domain of TSP (Dixit, V.M., D. M. Haverstick, K. M. O'Rourke, S. W. Hennessy, G. A. Grant, S. A. Santoro, and W. A. Frazier, 1985, Biochemistry, in press). Another Mab, C6.7, blocks the thrombin-stimulated aggregation of live platelets and immunoprecipitates an 18-kD fragment distinct from the heparin binding domain (Dixit, V. M., D. M. Haverstick, K. M. O'Rourke, S. W. Hennessy, G. A. Grant, S. A. Santoro, and W. A. Frazier, 1985, Proc. Natl. Acad. Sci. 82: 3472-3476). To determine the relative locations of the epitopes for these Mabs in the three-dimensional structure of TSP, we have examined TSP-Mab complexes by electron microscopy of rotary-shadowed proteins. The TSP molecule is composed of three 180-kD subunits, each of which consists of a small globular domain (approximately 8 nm diam) and a larger globular domain (approximately 16 nm diam) connected by a thin, flexible strand. The subunit interaction site is on the thin connecting strands, nearer the small globular domains. Mab A2.5 binds to the cluster of three small domains, indicating that this region contains the heparin binding domain and thus represents the NH2 termini of the TSP peptide chains. Mab C6.7 binds to the large globular domains on the side opposite the point at which the connecting strand enters the domain, essentially the maximum possible distance from the A2.5 epitope. Using high sensitivity automated NH2 terminal sequencing of TSP chymotryptic peptides we have ordered these fragments within the TSP peptide chain and have confirmed that the epitope for C6.7 in fact lies near the extreme COOH terminus of the peptide chain. In combination with other data, we have been able to construct a map of the linear order of the identified domains of TSP that indicates that to a large extent, the domains are arranged co-linearly with the peptide chain.     

The platelet glycoprotein thrombospondin binds specifically to sulfated glycolipids

The human platelet glycoprotein thrombospondin (TSP) binds specifically and with high affinity to sulfatides (galactosylceramide-I3-sulfate). Binding of 125I-TSP to lipids from sheep and human erythrocytes and human platelets resolved on thin layer chromatograms indicates that sulfatides are the only lipids in the membrane which bind TSP. Binding to less than 2 ng of sulfatide could be detected. TSP failed to bind to other purified lipids including cholesterol 3-sulfate, phospholipids, neutral glycolipids, and gangliosides. Binding of 125I-TSP was inhibited by unlabeled TSP, by low pH, and by reduction of intersubunit disulfide bonds with dithiothreitol. A monoclonal antibody against TSP (A2.5), which inhibits hemagglutination and agglutination of fixed activated platelets by TSP, strongly inhibited TSP binding to sulfatides. A second monoclonal antibody (C6.7), which inhibits hemagglutination and aggregation of thrombin-activated live platelets, weakly inhibited sulfatide binding. Binding was inhibited by high ionic strength and by some monosaccharide sulfates including methyl-alpha-D-GlcNAc-3-sulfate. Neutral sugars did not inhibit. Fucoidan, a sulfated fucan, strongly inhibited binding with 50% inhibition at 0.3 micrograms/ml fucoidan. Other sulfated polysaccharides including heparin and dextran sulfates were good inhibitors, whereas hyaluronic acid and keratan sulfate were very weak.     

Thrombospondin 1 and thrombospondin 2 are expressed as both homo- and heterotrimers.

There exist two distinct thrombospondin molecules (designated TSP1 and TSP2) which are encoded by separate genes. TSP1 is a trimeric cell surface and extracellular matrix molecule. Sequence comparison reveals that the 2 cysteines involved in interchain disulfide linkage and trimer assembly in TSP1 are conserved in TSP2 (Laherty, C. D., O'Rourke, K., Wolf, F. W., Katz, R., Seldin, M. F., and Dixit, V. M. (1992) J. Biol. Chem. 267, 3274-3281). Swiss 3T3 fibroblasts express both TSP1 and TSP2, and, therefore, an important question is whether TSP in such cells is expressed as homotrimers or as heterotrimers. We find that Swiss 3T3 cells and epithelial cells transfected with TSP expression vectors express both homo- and heterotrimeric forms of TSP. In addition, homotrimeric TSP2 has a lower affinity for heparin than homotrimeric TSP1. Thus, the heparin affinity of TSP can be modulated by the expression of TSP as homo- or heterotrimers.     

Monoclonal antibodies that recognize calcium-dependent structures of human thrombospondin. Characterization and mapping of their epitopes

Monoclonal antibodies (mAbs) raised against reduced and alkylated thrombospondin (TSP) were screened for the ability to react with Ca2+-replete TSP versus EDTA-treated TSP. Two mAbs designated A6.1 and D4.6 were found to react much more strongly with TSP after EDTA treatment. The dissociation constants for these mAbs were measured in 5 mM EDTA and found to be 6 X 10(-10) M for A6.1 and 7 X 10(-9) M for D4.6. Binding to A6.1 was undetectable in the presence of 1 mM Ca2+ while binding of D4.6 occurred with about 100-fold lower affinity. The Ca2+ concentration dependence of A6.1 binding was broad with a midpoint near 50 microM free Ca2+ while that of D4.6 showed a sharp transition below 0.1 microM. Upon dialysis of EDTA-treated TSP into Ca2+ containing buffer, the binding of the mAbs was prevented or decreased, indicating reversibility of the conformational transition induced by the initial removal of Ca2+ . Mg2+ can compete with the Ca2+ binding sites involved in mAb binding, but TSP dialyzed from Ca2+ into Mg2+ binds the two mAbs as well as EDTA-treated TSP, indicating that Mg2+ cannot maintain the Ca2+-replete structure of TSP. The proteolytic fragments of TSP with which the two mAbs react were determined by probing Western blots of digests of TSP with the mAbs. A6.1 reacts with the 70-kDa fragment generated by chymotrypsin in EDTA which contains the interchain disulfide bonds of TSP and the binding site(s) for type V collagen (Mumby, S. M., Raugi, G. J., and Bornstein, P. (1984) J. Cell Biol. 98, 646-652). D4.6 reacts with fragments of 140 and 120 kDa found in digests of Ca2+-replete TSP which are absent from digests in EDTA. Electron microscopy of rotary shadowed, carbon-coated replicas of TSP mAb complexes confirms the Ca2+ sensitivity of mAb binding and has been used to localize the epitopes for both mAbs on the three-dimensional structure of TSP.     

Characterization of the platelet agglutinating activity of thrombospondin

Thrombospondin (TSP) is a glycoprotein secreted from the alpha-granules of platelets upon activation. In the presence of divalent cations, the secreted protein binds to the surface of the activated platelets and is responsible for the endogenous lectin-like activity associated with activated platelets. Platelets fixed with formaldehyde following activation by thrombin are agglutinated by exogenously added TSP. Fixed, nonactivated platelets are not agglutinated. The platelet agglutinating activity of TSP is optimally expressed in the presence of 2 mM each of Mg2+ and Ca2+. Reduction of the disulfide bonds within the TSP molecule inhibits its platelet agglutinating activity. TSP bound to the surface of fixed, activated platelets can be eluted by the addition of disodium ethylenediaminetetraacetate. This approach was exploited to identify the region of the TSP molecule containing the platelet binding site. The binding site resides within a thermolytic fragment of TSP with Mr 140 000 but is not present in the Mr 120 000 fragment derived from the polypeptide of Mr 140 000. Since both the Mr 140 000 and 120 000 fragments contain fibrinogen binding sites, this finding suggests that the binding of TSP to the platelet surface requires interaction with other platelet surface components in addition to fibrinogen. The observation that fibrinogen only partially inhibits the TSP-mediated agglutination of fixed, activated platelets is consistent with this interpretation.     

The A20 cDNA induced by tumor necrosis factor alpha encodes a novel type of zinc finger protein

We have previously identified in endothelial cells a gene product designated A20, that is rapidly and profoundly induced by tumor necrosis factor alpha (Dixit, V. M., Green, S., Sarma, V., Holzman, L. B., Wolf, F. W., O'Rourke, K., Ward, P. A., Prochownik, E. V., and Marks, R. M. (1990) J. Biol. Chem. 265, 2973-2978). Isolation of the full-length cDNA followed by sequence analysis has revealed that A20 codes for a novel zinc finger protein. Structural features suggest that this putative protein, which contains multiple Cys2/Cys2 finger motifs, defines a novel class of zinc finger proteins. Southern analysis supports the existence of other members of the class.     

Cooperation between thrombospondin-1 type 1 repeat peptides and alpha(v)beta(3) integrin ligands to promote melanoma cell spreading and focal adhesion kinase phosphorylation.

CD47-binding sequences from the carboxyl-terminal domain of thrombospondin-1 (TSP1) are known to regulate activity of the alpha(v)beta(3) integrin (Gao, G., Lindberg, F. P., Dimitry, J. M., Brown, E. J., and Frazier, W. A. (1996) J. Cell Biol. 135, 533-544). Here we show that peptides from the type 1 repeats of TSP1 also stimulate alpha(v)beta(3) integrin function in melanoma cells. Addition of soluble peptide 246 (KRFKQDGGWSHWSPWSS) enhances spreading of A2058 melanoma cells on several alpha(v)beta(3) integrin ligands, including vitronectin, recombinant TSP1 fragments containing the Arg-Gly-Asp sequence, and native TSP1. This activity requires the Trp residues and is independent of CD36-binding sequences in the type 1 repeats. Recombinant type 1 repeats expressed as a glutathione S-transferase fusion protein also enhance spreading on vitronectin and TSP1. Activation of alpha(v)beta(3) integrin by the soluble peptide 246 stimulates organization of F-actin and increases tyrosine phosphorylation of focal adhesion kinase. In contrast, direct adhesion of melanoma cells on immobilized peptide 246 inhibits tyrosine phosphorylation of focal adhesion kinase. Stimulation of alpha(v)beta(3) integrin function by the type 1 repeat peptide differs from that induced by CD47-binding TSP1 peptides in that heparan sulfate proteoglycans are required and pertussis toxin does not inhibit the former activity. Thus, the type 1 repeats contain a second sequence of TSP1 that can enhance alpha(v)beta(3) integrin signaling, and these two sequences stimulate recognition of both vitronectin and TSP1 by the alpha(v)beta(3) integrin.     

Isolation and characterization of a heparin-binding domain from the amino terminus of platelet thrombospondin

Calcium-replete thrombospondin has been purified from outdated platelets using heparin-Sepharose affinity chromatography, gelatin-Sepharose to remove fibronectin, and gel filtration to eliminate low-molecular-weight heparin-binding proteins. Edman degradation of six different preparations revealed the amino-terminal sequence of thrombospondin (TSP) to be Asn-Arg-Ile-Pro-Glu-Ser-Gly-Gly-Asp-Asn-Ser-Val-Phe-. This sequence was obtained in initial yields as high as 85%, indicating that no blocked chains are present. Cleavage of calcium-replete TSP with thermolysin or plasmin results in the production of relatively stable fragments. Chromatography of these digests on heparin-Sepharose followed by elution with 0.6 M NaCl affords purification of an Mr 25,000 fragment from the thermolysin digest and an Mr 35,000 fragment from the plasmin digest. The binding of these fragments to heparin-Sepharose does not require divalent metal ions. Neither fragment is disulfide-bonded to other fragments present in the digests. The heparin-binding domains from both digests have similar amino acid compositions and their tryptic peptide maps on high performance liquid chromatography are identical with the exception of one peptide unique to each fragment. Automated Edman degradation in a vapor-phase sequenator of the thermolytic heparin-binding domain electroeluted from sodium dodecyl sulfate-gels indicates that the heparin-binding domain resides at the amino terminus of the Mr 180,000 TSP peptide chain.     

Characterization of the calmodulin-binding and catalytic domains in skeletal muscle myosin light chain kinase

Limited proteolysis has been utilized to study the structural organization of rabbit skeletal muscle myosin light chain kinase. The enzyme (Mr approximately 89,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis) consists of an amino-terminal, protease-susceptible region of unidentified function and a carboxyl-terminal, protease-resistant region of Mr approximately 40,000 containing the catalytic and calmodulin-binding domains. Partial digestion with trypsin produced an intermediate 56,000-dalton fragment and a stable 38,000-dalton fragment, both of which were catalytically active and calmodulin-dependent. Chymotryptic digestion yielded three catalytically active fragments of about 37,000, 36,000, and 35,000 daltons. The Mr = 37,000 fragment was calmodulin-dependent with an apparent affinity equivalent to that of the native enzyme (approximately 1 nM). The 36,000-dalton fragment was also calmodulin-dependent but had a approximately 200-fold lower apparent affinity. The Mr = 35,000 fragment was calmodulin-independent. These three chymotryptic fragments, had identical amino termini. Nineteen residues were missing from the carboxyl terminus of the calmodulin-independent chymotryptic fragment whereas only 8 or 9 carboxyl-terminal residues were missing from the calmodulin-dependent tryptic fragments. These results suggest that the 11-residue sequence (IAVSAANRFKK) in the carboxyl-terminal region of myosin light chain kinase contributes directly to the binding of calmodulin. This conclusion is in accord with data (Blumenthal, D. K., Takio, K., Edelman, A. M., Charbonneau, H., Titani, K., Walsh, K. A., and Krebs, E. G. (1985) Proc. Natl. Acad. Sci. U. S. A. 82, 3187-3191) that the carboxyl-terminal, 27-residue CNBr peptide of the native enzyme shows Ca2+-dependent, high affinity binding to calmodulin and that similar calmodulin-binding activity, although detectable in unfractionated CNBr digests of calmodulin-dependent enzyme forms, is much reduced in a CNBr digest of the calmodulin-independent, Mr = 35,000 chymotryptic fragment.     

The N-terminal module of thrombospondin-1 interacts with the link domain of TSG-6 and enhances its covalent association with the heavy chains of inter-alpha-trypsin inhibitor

We recently found that leukocytes from thrombospondin-1 (TSP1)-deficient mice exhibit significant reductions in cell surface CD44 relative to those from wild type mice. Because TSG-6 modulates CD44-mediated cellular interactions with hyaluronan, we examined the possibility that TSP1 interacts with TSG-6. We showed that recombinant full-length human TSG-6 (TSG-6Q) and the Link module of TSG-6 (Link_TSG6) bind 125I-TSP1 with comparable affinities. Trimeric recombinant constructs containing the N-modules of TSP1 or TSP2 inhibit binding of TSP1 to TSG-6Q and Link_TSG6, but other recombinant regions of TSP1 do not. Therefore, the N-modules of both TSP1 and TSP2 specifically recognize the Link module of TSG-6. Heparin, which binds to these domains of both proteins, strongly inhibits binding of TSP1 to Link_TSG6 and TSG-6Q, but hyaluronan does not. Inhibition by heparin results from its binding to TSP1, because heparin also inhibits TSP1 binding to Link_TSG6 mutants deficient in heparin binding. Removal of bound Ca2+ from TSP1 reduces its binding to full-length TSG-6. Binding of TSP1 to Link_TSG6, however, is enhanced by chelating divalent cations. In contrast, divalent cations do not influence binding of the N-terminal region of TSP1 to TSG-6Q. This implies that divalent cation dependence is due to conformational effects of calcium-binding to the C-terminal domains of TSP1. TSP1 enhances covalent modification of the inter-alpha-trypsin inhibitor by TSG-6 and transfer of its heavy chains to hyaluronan, suggesting a physiological function of TSP1 binding to TSG-6 in regulation of hyaluronan metabolism at sites of inflammation.     

Polylactosamine glycosylation on human fetal placental fibronectin weakens the binding affinity of fibronectin to gelatin

Gelatin-binding chymotryptic fragments from placental fibronectin contain polylactosamine carbohydrates (Zhu, B.C.R., Fisher, S.R., Pande, H., Calaycay, J. Shively, J.E., and Laine, R.A. (1984) J. Biol. Chem. 259, 3962-3970). We have separated polylactosamine-containing gelatin-binding fragments of placental fibronectin from their counterparts containing smaller "complex" N-linked saccharides using Sephadex G-200 gel permeation chromatography. The peptide portions of both fragments have similar amino acid composition and N-terminal sequence (see reference above). The strength of binding of these two glycosylation types of chymotryptic fragments to gelatin differs as shown by the following experiments. 1) Upon urea gradient elution of affinity-bound fibronectin fragments from gelatin-Sepharose chromatography, the apex of the elution peak for polylactosamine-containing fragments occurs at 2.0 M urea while the peak for complex N-linked carbohydrate-containing fragments maximized at 2.5 M urea indicating a tighter binding. Removal of polylactosamine sequences from the former glycopeptide by endo-beta-galactosidase digestion caused the elution peak for this fraction to change from 2.0 to 2.5 M, the same as for the complex N-linked carbohydrate-containing glycopeptide. 2) Competitive displacement experiments give an apparent dissociation constant of polylactosamine-containing fragments at 3 X 10(-9) M whereas this constant for complex carbohydrate-containing fragments is 1 X 10(-9) M. These results indicate that the binding of placental fibronectin to gelatin is weakened by the presence of high molecular weight polylactosamine carbohydrate. To our knowledge this is the first report that the type and extent of glycosylation of a glycoprotein can affect its binding affinity to a proteinacious ligand. Thus, fetal placental fibronectin may have different biological properties than fibronectins containing only the smaller N-linked complex carbohydrate.     

Identification of aspartic acid 514 through glutamic acid 542 as a glycoprotein Ib-IX complex receptor recognition sequence in von Willebrand factor. Mechanism of modulation of von Willebrand factor by ristocetin and botrocetin.

As the first step in hemostasis, the binding of von Willebrand factor (vWF) to the platelet membrane glycoprotein (GP) Ib-IX complex is essential for platelet adhesion at high-shear blood flow. This interaction in vivo requires the prior binding of vWF to the subendothelial matrix, a process which exposes a normally cryptic binding site on vWF for the GP Ib-IX complex. This process can be mimicked in vitro by modulators such as ristocetin or the snake venom protein botrocetin or by desialation of vWF. We have previously localized the GP Ib binding site on vWF to a monomeric dispase fragment which extends from Leu-480/Val-481 to Gly-718 in the primary sequence of mature vWF [Andrews, R. K., Gorman, J. J., Booth, W. J., Corino, G. L., Castaldi, P. A., & Berndt, M. C. (1989) Biochemistry 28, 8326-8336]. This fragment also contains a distinct binding site for botrocetin. Analysis of synthetic peptides corresponding to hydrophilic stretches of sequence within this fragment indicated that the sequence Asp-514-Glu-542 represents a major adhesive sequence involved in receptor recognition. This peptide inhibited both the ristocetin- and botrocetin-mediated binding of vWF to either platelets or purified GP Ib-IX complex (IC50 approximately 50-200 microM) as well as the asialo-vWF- and bovine vWF-dependent agglutination of platelets. Both the N- and C-terminal halves of the peptide were inhibitory but less so than the intact peptide. This peptide also inhibited botrocetin binding to vWF, suggesting that botrocetin modulates vWF-GP Ib interaction by binding in close proximity to the vWF adhesion sequence.(ABSTRACT TRUNCATED AT 250 WORDS)     

Salicylihalamide A inhibits the V0 sector of the V-ATPase through a mechanism distinct from bafilomycin A1

The newly identified specific V-ATPase inhibitor, salicylihalamide A, is distinct from any previously identified V-ATPase inhibitors in that it inhibits only mammalian V-ATPases, but not those from yeast or other fungi (Boyd, M. R., Farina, C., Belfiore, P., Gagliardi, S., Kim, J. W., Hayakawa, Y., Beutler, J. A., McKee, T. C., Bowman, B. J., and Bowman, E. J. (2001) J. Pharmacol. Exp. Ther. 297, 114-120). In addition, salicylihalamide A does not compete with concanamycin or bafilomycin for binding to V-ATPase, indicating that it has a different binding site from those classic V-ATPase inhibitors (Huss, M., Ingenhorst, G., Konig, S., Gassel, M., Drose, S., Zeeck, A., Altendorf, K., and Wieczorek, H. (2002) J. Biol. Chem. 277, 40544-40548). By using purified bovine brain V-pump and its dissociated V(1) and V(0) sectors, we identified the recognition and binding site for salicylihalamide to be within the V(0) domain. Salicylihalamide does not inhibit the ATP hydrolysis activity of the dissociated V(1)-ATPase but inhibits the ATPase activity of the holoenzyme by inhibiting the V(0) domain. Salicylihalamide causes a dramatic redistribution of cytosolic V(1) from soluble to membrane-associated form, a change not observed in cells treated with either bafilomycin or NH(4)Cl. By synthesizing and characterizing a series of salicylihalamide derivatives, we investigated the structural determinants of salicylihalamide inhibition in terms of potency and reversibility, and used this information to suggest a possible binding mechanism.     

The heparin/heparan sulfate-binding site on apo-serum amyloid A. Implications for the therapeutic intervention of amyloidosis

Serum amyloid A isoforms, apoSAA1 and apoSAA2, are apolipoproteins of unknown function that become major components of high density lipoprotein (HDL) during the acute phase of an inflammatory response. ApoSAA is also the precursor of inflammation-associated amyloid, and there is strong evidence that the formation of inflammation-associated and other types of amyloid is promoted by heparan sulfate (HS). Data presented herein demonstrate that both mouse and human apoSAA contain binding sites that are specific for heparin and HS, with no binding for the other major glycosaminoglycans detected. Cyanogen bromide-generated peptides of mouse apoSAA1 and apoSAA2 were screened for heparin binding activity. Two peptides, an apoSAA1-derived 80-mer (residues 24-103) and a smaller carboxyl-terminal 27-mer peptide of apoSAA2 (residues 77-103), were retained by a heparin column. A synthetic peptide corresponding to the CNBr-generated 27-mer also bound heparin, and by substituting or deleting one or more of its six basic residues (Arg-83, His-84, Arg-86, Lys-89, Arg-95, and Lys-102), their relative importance for heparin and HS binding was determined. The Lys-102 residue appeared to be required only for HS binding. The residues Arg-86, Lys-89, Arg-95, and Lys-102 are phylogenetically conserved suggesting that the heparin/HS binding activity may be an important aspect of the function of apoSAA. HS linked by its carboxyl groups to an Affi-Gel column or treated with carbodiimide to block its carboxyl groups lost the ability to bind apoSAA. HDL-apoSAA did not bind to heparin; however, it did bind to HS, an interaction to which apoA-I contributed. Results from binding experiments with Congo Red-Sepharose 4B columns support the conclusions of a recent structural study which found that heparin binding domains have a common spatial distance of about 20 A between their two outer basic residues. Our present work provides direct evidence that apoSAA can associate with HS (and heparin) and that the occupation of its binding site by HS, and HS analogs, likely caused the previously reported increase in amyloidogenic conformation (beta-sheet) of apoSAA2 (McCubbin, W. D., Kay, C. M., Narindrasorasak, S., and Kisilevsky, R. (1988) Biochem. J. 256, 775-783) and their amyloid-suppressing effects in vivo (Kisilevsky, R., Lemieux, L. J., Fraser, P. E., Kong, X., Hultin, P. G., and Szarek, W. A. (1995) Nat. Med. 1, 143-147), respectively.     

Evidence that the hormone-binding domain of the mouse glucocorticoid receptor directly represses DNA binding activity in a major portion of receptors that are "misfolded" after removal of hsp90.

After dissociation of cytosolic heteromeric glucocorticoid receptor complexes by steroid, salt, and other methods, only 35-60% of the dissociated receptors can bind to DNA-cellulose. The DNA-binding and non-DNA-binding forms of the dissociated receptors have the same Mr and are phosphorylated to the same extent (Tienrungroj, W., Sanchez, E. R., Housley, P. R., Harrison, R. W., and Pratt, W. B. (1987) J. Biol. Chem. 262, 17347-17349). The basis for the different DNA-binding activities is unknown, but the DNA-binding fraction of the receptor has a more basic pI than the non-DNA-binding fraction (Smith, A. C., Elsasser, M. S., and Harmon, J. M. (1986) J. Biol. Chem. 261, 13285-13292). We have separated the non-DNA-binding state of the receptor from the DNA-binding state and then cleaved it with trypsin and chymotrypsin. We find that the 15-kDa tryptic fragment derived from the non-DNA-binding state of the dissociated receptor is fully competent in binding DNA, whereas the 42-kDa chymotryptic fragment containing both the hormone-binding and DNA-binding domains does not bind DNA. Trypsin cleavage of the molybdate-stabilized untransformed receptor also yields a 15-kDa fragment that is fully competent in binding DNA. Reducing agents do not restore DNA-binding to the non-DNA-binding fraction of the receptor and the hormone-binding domain can be separated from the DNA-binding domain on nonreducing gel electrophoresis. These results argue that the two domains are not linked by disulfide bridges, and they are consistent with the proposal that there are two least energy states of folding after dissociation of hsp90. A significant portion of the receptors is "misfolded" in such a manner that the steroid binding domain is directly preventing DNA-binding activity.     

A heparin-binding domain from N-CAM is involved in neural cell- substratum adhesion

Cell-substratum adhesion in the embryonic chicken nervous system has been shown to be mediated in part by a 170,000-mol-wt polypeptide that is a component of adherons. Attachment of retinal cells to the 170,000-mol-wt protein is inhibited by the C1H3 monoclonal antibody and by heparan sulfate (Cole, G. J., D. Schubert, and L. Glaser, 1985, J. Cell Biol., 100:1192-1199). In the present study we have demonstrated that the 170,000-mol-wt C1H3 polypeptide is immunologically identical to the neural cell adhesion molecule N-CAM, and that the 170,000-mol-wt component of N-CAM is preferentially secreted by cells as a component of adherons. We have identified a monoclonal antibody, designated B1A3, that inhibits heparin binding to N-CAM and cell-to-substratum adhesion. A 25,000-mol-wt heparin (heparan sulfate)-binding domain of N-CAM has been identified by limited proteolysis, and this fragment promotes cell attachment when bound to glass surfaces. The fragment also partially inhibits cell binding to adherons when bound to retinal cells, and the B1A3 monoclonal antibody inhibits retinal cell attachment to substrata composed of intact N-CAM or the heparin-binding domain. These data are the first evidence that N-CAM is a multifunctional protein that contains both cell-and heparin (heparan sulfate)-binding domains.     

Purification and molecular characterization of a sialic acid specific lectin from the phytopathogenic fungus Macrophomina phaseolina

A lectin was isolated and purified from the culture filtrate of the plant pathogenic fungus Macrophomina phaseolina by a combination of ammonium sulfate precipitation, affinity chromatography on fetuin-Sepharose 4B and ion-exchange chromatography on DEAE-A 50. The lectin designated MPL was homogeneous by PAGE and HPLC and a monomeric protein with a molecular weight of approximately 34 kDa as demonstrated by SDS-PAGE. It is a glycoprotein and agglutinated human erythrocytes regardless of the human blood type. Neuraminidase treatment of erythrocytes reduced the agglutination activity of the lectin. It is thermally stable and exhibits maximum activity between pH 6 and 7.2. Its carbohydrate binding specificity was investigated both by hapten inhibition of hemagglutination and by enzyme-conjugated lectin inhibition assay. Although, M. phaseolina lectin bound sialic acid, it exhibited binding affinity towards neuraminyl oligosaccharides of N-linked glycoproteins, alpha-Neu5Ac-(2-->3)-beta-Gal-(1-->4)-GlcNAc being maximum.     

Localization and chemical synthesis of fibronectin peptides with melanoma adhesion and heparin binding activities

Tumor cell adhesion to the extracellular matrix is an important consideration in tumor metastasis. Recent results show that multiple adhesion-promoting domains for melanoma cells can be purified from proteolytic digests of fibronectin [McCarthy, J. B., Hagen, S. T., & Furcht, L. T. (1986) J. Cell Biol. 102, 179-188]. Monoclonal antibodies were generated against a tryptic/catheptic 33K heparin binding fragment of fibronectin derived from the carboxyl terminal of the A chain. This region contains a tumor cell adhesion-promoting domain(s). The amino-terminal sequence was determined for this fragment, as well as a tryptic 31K fragment which is located to the carboxyl-terminal side of the 33K heparin binding fragment in A chains of fibronectin. The partial sequence data demonstrate that arginyl-glycyl-aspartyl-serine (RGDS) or the related arginyl-glutamyl-aspartyl-valine (REDV) is not present in the 33K heparin binding fragment, confirming earlier results which demonstrated that cells adhere to this fragment by an RGDS-independent mechanism. Two monoclonal antibodies, termed AHB-1 and AHB-2, recognized epitopes common to heparin binding fragments derived from the carboxyl terminus of both the A and B chains of fibronectin. Monoclonal antibody AHB-2 inhibited melanoma adhesion to the 33K heparin binding fragment of fibronectin in a concentration-dependent manner, whereas monoclonal antibody AHB-1 had no effect on adhesion to this fragment. Neither monoclonal antibody inhibited adhesion to intact fibronectin. However, monoclonal AHB-2 potentiated the inhibitory effect of suboptimal levels of exogenous RGDS on cell adhesion to intact fibronectin. AHB-2 recognized an epitope common to both the A- and B-chain carboxyl-terminal heparin binding region of fibronectin.(ABSTRACT TRUNCATED AT 250 WORDS)     

The collagen-binding A-domains of integrins alpha(1)beta(1) and alpha(2)beta(1) recognize the same specific amino acid sequence, GFOGER, in native (triple-helical) collagens

We have previously assigned an integrin alpha(2)beta(1)-recognition site in collagen I to the sequence, GFOGERGVEGPOGPA (O = Hyp), corresponding to residues 502-516 of the alpha(1)(I) chain and located in the fragment alpha(1)(I)CB3 (Knight, C. G., Morton, L. F., Onley, D. J., Peachey, A. R., Messent, A. J., Smethurst, P. A., Tuckwell, D. S., Farndale, R. W., and Barnes, M. J. (1998) J. Biol. Chem. 273, 33287-33294). In this study, we show that recognition is entirely contained within the six-residue sequence GFOGER. This sequence, when in triple-helical conformation, readily supports alpha(2)beta(1)-dependent cell adhesion and exhibits divalent cation-dependent binding of isolated alpha(2)beta(1) and recombinant alpha(2) A-domain, being at least as active as the parent collagen. Replacement of E by D causes loss of recognition. The same sequence binds integrin alpha(1) A-domain and supports integrin alpha(1)beta(1)-mediated cell adhesion. Triple-helical GFOGER completely inhibits alpha(2) A-domain binding to collagens I and IV and alpha(2)beta(1)-dependent adhesion of platelets and HT 1080 cells to these collagens. It also fully inhibits alpha(1) A-domain binding to collagen I and strongly inhibits alpha(1)beta(1)-mediated adhesion of Rugli cells to this collagen but has little effect on either alpha1 A-domain binding or adhesion of Rugli cells to collagen IV. We conclude that the sequence GFOGER represents a high-affinity binding site in collagens I and IV for alpha(2)beta(1) and in collagen I for alpha(1)beta(1). Other high-affinity sites in collagen IV mediate its recognition of alpha(1)beta(1).