scholarly journals Interaction of human thrombospondin with types I-V collagen: direct binding and electron microscopy.

1987 ◽  
Vol 104 (5) ◽  
pp. 1413-1422 ◽  
Author(s):  
N J Galvin ◽  
P M Vance ◽  
V M Dixit ◽  
B Fink ◽  
W A Frazier
Keyword(s):  
Electron Microscopy ◽  
Binding Site ◽  
Binding Domain ◽  
Collagen Molecule ◽  
Heparin Binding ◽  
Binding Assays ◽  
Collagen Binding ◽  
Type V Collagen ◽  
Direct Binding ◽  
Type V

Binding of thrombospondin (TSP) to types I-V collagen was examined by direct binding assays using 125I-TSP and by visualization of rotary-shadowed intermolecular complexes in the electron microscope. The binding of TSP was highest to type V collagen in the absence of Ca, while lower but significant levels of binding were observed to all other collagen types in the presence or absence of Ca. Unlike intact TSP, the trimeric collagen-binding domain of TSP composed of 70-kD chains showed no Ca dependence in its binding to type V collagen. Further evidence for binding of TSP to types I and III collagen was obtained by competition studies in which these soluble collagens effectively inhibited binding of 125I-TSP to immobilized type V collagen. The binding of TSP to type V collagen was inhibited by heparin and fucoidin, both high-affinity ligands of TSP's heparin-binding domain. mAb A6.1, which binds to the 70-kD domain of TSP, is also the best of a panel of anti-TSP mAbs at inhibiting the TSP-collagen interaction. Electron microscopy of rotary-shadowed replicas of TSP-collagen complexes revealed that all five types of collagen examined had a binding site for TSP at one end of the pepsinized, triple helical molecule. The specificity of this site was tested by examining the ability of BSA to form a complex with the end of the pepsinized collagens. Rotary-shadowed replicas revealed a low frequency of apparent BSA-collagen complexes, and histograms of these data showed no evidence for the preferential association of BSA with the end of the collagen molecules. In addition to the specific end site, type V collagen had an internal binding site for TSP located about two-thirds of the distance along the length of the collagen molecule from the end site. The internal binding site for TSP on type V collagen is apparently the site responsible for the higher affinity binding of TSP to that protein observed in direct binding assays. The trimeric 70-kD collagen-binding domain of TSP bound to the same sites on the collagens as did intact TSP.

Primary structure of the heparin-binding site of type V collagen

1990 ◽  
Vol 1035 (2) ◽  
pp. 139-145 ◽  
Author(s):  
Yoshihito Yaoi ◽  
Kahoko Hashimoto ◽  
Hiroyuki Koitabashi ◽  
Kazuhiko Takahara ◽  
Manabu Ito ◽  
...  
Keyword(s):  
Binding Site ◽  
Primary Structure ◽  
Heparin Binding ◽  
Type V Collagen ◽  
Heparin Binding Site ◽  
Type V

scholarly journals Monoclonal antibodies against chicken type IV and V collagens: electron microscopic mapping of the epitopes after rotary shadowing.

1984 ◽  
Vol 98 (5) ◽  
pp. 1637-1644 ◽  
Author(s):  
R Mayne ◽  
H Wiedemann ◽  
M H Irwin ◽  
R D Sanderson ◽  
J M Fitch ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Monoclonal Antibodies ◽  
Cleavage Site ◽  
Type Iv Collagen ◽  
Collagen Molecule ◽  
Electron Microscopic ◽  
Type V Collagen ◽  
Type Iv ◽  
Type V ◽  
Rotary Shadowing

The location of the epitopes for monoclonal antibodies against chicken type IV and type V collagens were directly determined in the electron microscope after rotary shadowing of antibody/collagen mixtures. Three monoclonal antibodies against type IV collagen were examined, each one of which was previously demonstrated to be specific for only one of the three pepsin-resistant fragments of the molecule. The three native fragments were designated (F1)2F2, F3, and 7S, and the antibodies that specifically recognize each fragment were called, respectively, IA8 , IIB12 , and ID2 . By electron microscopy, monoclonal antibody IA8 recognized an epitope located in the center of fragment (F1)2F2 and in tetramers of type IV collagen at a distance of 288 nm from the 7S domain, the region of overlap of four type IV molecules. Monoclonal antibody IIB12 , in contrast, recognized an epitope located only 73 nm from the 7S domain. This result therefore provides direct visual evidence that the F3 fragment is located closest to the 7S domain and the order of the fragments must be 7S-F3-(F1)2F2. The epitope for antibody ID2 was located in the overlap region of the 7S domain, and often several antibody molecules were observed to binding to a single 7S domain. The high frequency with which antibody molecules were observed to bind to fragments of type IV collagen suggests that there is a single population of type IV molecules of chain organization [alpha 1(IV)]2 alpha 2(IV), and that four identical molecules must form a tetramer that is joined in an antiparallel manner at the 7S domain. The monoclonal antibodies against type V collagen, called AB12 and DH2 , were both found to recognize epitopes close to one another, the epitopes being located 45-48 nm from one end of the type V collagen molecule. The significance of this result still remains uncertain, but suggests that this site is probably highly immunoreactive. It may also be related to the specific cleavage site of type V collagen by selected metalloproteinases and by alpha-thrombin. This cleavage site is also known to be located close to one end of the type V molecule.

Overlap of IGF- and heparin-binding sites in rat IGF-binding protein-5

2000 ◽  
Vol 24 (1) ◽  
pp. 43-51 ◽  
Author(s):  
H Song ◽  
J Beattie ◽  
IW Campbell ◽  
GJ Allan
Keyword(s):  
Binding Site ◽  
Conformational Changes ◽  
Binding Protein ◽  
Binding Activity ◽  
Binding Domain ◽  
Site Directed Mutagenesis ◽  
Heparin Binding ◽  
Igf I ◽  
Heparin Binding Domain ◽  
Igf Binding

Using site-directed mutagenesis, we have undertaken a study of a potential IGF-binding site in the C-terminal domain of rat IGFBP-5, lying close to or within a previously described heparin-binding domain (residues 201-218) in this protein. After analysis of binding activity using three different methods - ligand blotting, solution phase equilibrium binding and biosensor measurement of real-time on- and off-rates - we report that the mutation of two highly conserved residues within this region (glycine 203 and glutamine 209) reduces the affinity of the binding protein for both IGF-I and IGF-II, while having no effect on heparin binding. In addition, we confirm that mutation of basic residues within the heparin-binding domain (R201L, K202E, K206Q and R214A) results in a protein that has attenuated heparin binding but shows only a small reduction in affinity for IGF-I and -II. Previous findings have described the reduction in affinity of IGFBP-5 for IGFs that occurs after complexation of the binding protein with heparin or other components of the extracellular matrix (ECM) and have postulated that such an interaction may result in conformational changes in protein structure, affecting subsequent IGF interaction. Our data suggesting potential overlap of heparin- and IGF-binding domains argue for a more direct effect of ECM modulation of the affinity of IGFBP-5 for ligand by partial occlusion of the IGF-binding site after interaction with ECM.

Collagen fibrillogenesis in vitro: interaction of types I and V collagen regulates fibril diameter

1990 ◽  
Vol 95 (4) ◽  
pp. 649-657 ◽  
Author(s):  
D.E. Birk ◽  
J.M. Fitch ◽  
J.P. Babiarz ◽  
K.J. Doane ◽  
T.F. Linsenmayer
Keyword(s):  
Type I Collagen ◽  
Small Diameter ◽  
Collagen Molecule ◽  
Type I ◽  
Collagen Types ◽  
Type V Collagen ◽  
Amino Terminal ◽  
Type V ◽  
Fibril Diameter

The small-diameter fibrils of the chick corneal stroma are heterotypic, composed of both collagen types I and V. This tissue has a high concentration of type V collagen relative to other type I-containing tissues with larger-diameter fibrils, suggesting that heterotypic interactions may have a regulatory role in the control of fibril diameter. The interactions of collagen types I and V were studied using an in vitro self-assembly system. Collagens were purified from lathyritic chick embryos in the presence of protease inhibitors. The type V collagen preparations contained higher molecular weight forms of the alpha 1(V) and alpha 2(V) chains constituting 60–70% of the total. Rotary-shadow electron micrographs showed a persistence of a small, pepsin-sensitive terminal region in an amount consistent with that seen by electrophoresis. In vitro, this purified type V collagen formed thin fibrils with no apparent periodicity, while type I collagen fibrils had a broad distribution of large diameters. However, when type I collagen was mixed with increasing amounts of type V collagen a progressive and significant decrease in both the mean fibril diameter and the variance was observed for D periodic fibrils. The amino-terminal domain of the type V collagen molecule was required for this regulatory effect and in its absence little diameter reducing activity was observed. Electron microscopy using collagen type-specific monoclonal antibodies demonstrated that the fibrils formed were heterotypic, containing both collagen types I and V. These data indicate that the interaction of type V with type I collagen is one mechanism modulating fibril diameter and is at least partially responsible for the regulation of collagen fibril formation.

scholarly journals Studies of the interaction between titin and myosin.

1995 ◽  
Vol 131 (6) ◽  
pp. 1471-1481 ◽  
Author(s):  
A Houmeida ◽  
J Holt ◽  
L Tskhovrebova ◽  
J Trinick
Keyword(s):  
Electron Microscopy ◽  
Binding Site ◽  
Thick Filament ◽  
Protein Sequencing ◽  
Binding Assays ◽  
Apparent Dissociation Constant ◽  
Filament Assembly ◽  
Filament Structure ◽  
Binding Data ◽  
20 Nm

The interaction of titin with myosin has been studied by binding assays and electron microscopy. Electron micrographs of the titin-myosin complex suggest a binding site near the tip of the tail of the myosin molecule. The distance from the myosin head-tail junction to titin indicates binding 20-30 nm from the myosin COOH terminus. Consistent with this, micrographs of titin-light meromyosin (LMM) show binding near the end of the LMM molecule. Plots of myosin- and LMM-attachment positions along the titin molecule show binding predominantly in the region located in the A band in situ, which is consistent with the proposal that titin regulates thick filament assembly. Estimates of the apparent dissociation constant of the titin-LMM complex were approximately 20 nM. Assays of LMM cyanogen bromide fragments also suggested a strong binding site near the COOH terminus. Proteolysis of a COOH-terminal 17.6-kD CNBr fragment isolated from whole myosin resulted in eight peptides of which only one, comprising 17 residues, bound strongly to titin. Two isoforms of this peptide were detected by protein sequencing. Similar binding data were obtained using synthetic versions of both isoforms. The peptide is located immediately COOH-terminal of the fourth "skip" residue in the myosin tail, which is consistent with the electron microscopy. Skip-4 may have a role in determining thick filament structure, by allowing abrupt bending of the myosin tail close to the titin-binding site.

scholarly journals Binding of human α-thrombin to platelet GpIb: energetics and functional effects

1998 ◽  
Vol 332 (3) ◽  
pp. 643-650 ◽  
Author(s):  
Raimondo de CRISTOFARO ◽  
Erica de CANDIA ◽  
Giovanni CROCE ◽  
Roberta MOROSETTI ◽  
Raffaele LANDOLFI
Keyword(s):  
Monoclonal Antibody ◽  
Binding Site ◽  
Competitive Inhibition ◽  
Enzyme Inhibitor ◽  
Solid Phase ◽  
Hydrophobic Effect ◽  
Binding Domain ◽  
Heat Capacity Change ◽  
Heparin Binding ◽  
Solid Phase Assay

Thrombin interaction with platelet glycocalicin (GC), the 140 kDa extracytoplasmic fragment of the membrane glycoprotein Ib, was investigated by using a solid-phase assay. Thrombin bound to GC-coated polystyrene wells was detected by measuring the hydrolysis of a chromogenic substrate. The monoclonal antibody LJ-Ib10, which specifically binds to the thrombin-binding site of GC, could displace thrombin from immobilized GC, whereas the monoclonal antibody LJ-Ib1, which interacts with the von Willebrand factor-binding domain of GC, did not affect thrombin binding to GC. Competitive inhibition of thrombin binding to immobilized GC was also observed using GC in solution or ligands that bind to the thrombin heparin-binding site, such as heparin and prothrombin fragment 2. Furthermore functional experiments demonstrated that GC binding to thrombin competes with heparin for thrombin inactivation by the antithrombin III–heparin complex as well. Thrombin–GC interaction was also studied as a function of temperature over the range 4–37 °C. A large negative heat capacity change (ΔCp), of -4.14±0.8 kJ·mol-1·K-1, was demonstrated to dominate the thermodynamics of thrombin–GC complex-formation. Finally it was demonstrated that GC binding to thrombin can allosterically decrease the enzyme affinity for hirudin via a simultaneous decrease in association rate and increase in the dissociation velocity of the enzyme–inhibitor adduct. Together these observations indicate the GC binding to the heparin-binding domain of thrombin is largely driven by a hydrophobic effect and that such interaction can protect the enzyme from inhibition by the heparin–anti-thrombin III complex.

scholarly journals Mapping the heparin-binding sites on type I collagen monomers and fibrils.

1994 ◽  
Vol 125 (5) ◽  
pp. 1179-1188 ◽  
Author(s):  
J D San Antonio ◽  
A D Lander ◽  
M J Karnovsky ◽  
H S Slayter
Keyword(s):  
Electron Microscopy ◽  
Extracellular Matrix ◽  
Cell Adhesion ◽  
Heparan Sulfate ◽  
Binding Site ◽  
Binding Sites ◽  
Type I Collagen ◽  
Type I ◽  
Heparin Binding ◽  
Heparin Binding Site

The glycosaminoglycan chains of cell surface heparan sulfate proteoglycans are believed to regulate cell adhesion, proliferation, and extracellular matrix assembly, through their interactions with heparin-binding proteins (for review see Ruoslahti, E. 1988. Annu. Rev. Cell Biol. 4:229-255; and Bernfield, M., R. Kokenyesi, M. Kato, M. T. Hinkes, J. Spring, R. L. Gallo, and E. J. Lose. 1992. Annu. Rev. Cell Biol. 8:365-393). Heparin-binding sites on many extracellular matrix proteins have been described; however, the heparin-binding site on type I collagen, a ubiquitous heparin-binding protein of the extracellular matrix, remains undescribed. Here we used heparin, a structural and functional analogue of heparan sulfate, as a probe to study the nature of the heparan sulfate proteoglycan-binding site on type I collagen. We used affinity coelectrophoresis to study the binding of heparin to various forms of type I collagen, and electron microscopy to visualize the site(s) of interaction of heparin with type I collagen monomers and fibrils. Using affinity coelectrophoresis it was found that heparin has similar affinities for both procollagen and collagen fibrils (Kd's approximately 60-80 nM), suggesting that functionally similar heparin-binding sites exist in type I collagen independent of its aggregation state. Complexes of heparin-albumin-gold particles and procollagen were visualized by rotary shadowing and electron microscopy, and a preferred site of heparin binding was observed near the NH2 terminus of procollagen. Native or reconstituted type I collagen fibrils showed one region of significant heparin-gold binding within each 67-nm period, present near the division between the overlap and gap zones, within the "a" bands region. According to an accepted model of collagen fibril structure, our data are consistent with the presence of a single preferred heparin-binding site near the NH2 terminus of the collagen monomer. Correlating these data with known type I collagen sequences, we suggest that the heparin-binding site in type I collagen may consist of a highly basic triple helical domain, including several amino acids known sometimes to function as disaccharide acceptor sites. We propose that the heparin-binding site of type I collagen may play a key role in cell adhesion and migration within connective tissues, or in the cell-directed assembly or restructuring of the collagenous extracellular matrix.

scholarly journals Schwann Cell Adhesion to a Novel Heparan Sulfate Binding Site in the N-terminal Domain of α4 Type V Collagen Is Mediated by Syndecan-3

2001 ◽  
Vol 277 (9) ◽  
pp. 7619-7625 ◽  
Author(s):  
Robert Erdman ◽  
Richard C. Stahl ◽  
Katrina Rothblum ◽  
Michael A. Chernousov ◽  
David J. Carey
Keyword(s):  
Cell Adhesion ◽  
Schwann Cell ◽  
Heparan Sulfate ◽  
Binding Site ◽  
Type V Collagen ◽  
Terminal Domain ◽  
Type V
Sign in / Sign up

Export Citation Format

Share Document