Redefinition of the Carbohydrate Specificity ofErythrina corallodendronLectin Based on Solid-Phase Binding Assays and Molecular Modeling of Native and Recombinant Forms Obtained by Site-Directed Mutagenesis†

Biochemistry ◽  
1997 ◽  
Vol 36 (15) ◽  
pp. 4429-4437 ◽  
Author(s):  
Ernesto Moreno ◽  
Susann Teneberg ◽  
Rivka Adar ◽  
Nathan Sharon ◽  
Karl-Anders Karlsson ◽  
...  
Keyword(s):  
Molecular Modeling ◽  
Solid Phase ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Carbohydrate Specificity ◽  
Binding Assays ◽  
Solid Phase Binding

scholarly journals Identification of Residues within Human Glycoprotein VI Involved in the Binding to Collagen

2004 ◽  
Vol 279 (50) ◽  
pp. 52293-52299 ◽  
Author(s):  
Christelle Lecut ◽  
Véronique Arocas ◽  
Hans Ulrichts ◽  
Anthony Elbaz ◽  
Jean-Luc Villeval ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Monoclonal Antibodies ◽  
Molecular Modeling ◽  
Binding Sites ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Binding Assays ◽  
Glycoprotein Vi ◽  
Ligand Binding Sites ◽  
Human Receptor

Glycoprotein VI (GPVI) has a crucial role in platelet responses to collagen. Still, little is known about its interaction with its ligands. In binding assays using soluble or cell-expressed human GPVI, we observed that (i) collagen, and the GPVI-specific ligands collagen-related peptides (CRP) and convulxin, competed with one another for the binding to GPVI and (ii) monoclonal antibodies directed against the extracellular part of the human receptor displayed selective inhibitory properties on GPVI interaction with its ligands. Monoclonal antibody 9E18 strongly reduced the binding of GPVI to collagen/CRP, 3F8 inhibited its interaction with convulxin, whereas 9O12 prevented all three interactions. These observations suggest that ligand-binding sites are distinct, exhibiting specific features but at the same time also sharing some common residues participating in the recognition of these ligands. The epitope of 9O12 was mapped by phage display, along with molecular modeling of human GPVI, which allowed the identification of residues within GPVI potentially involved in ligand recognition. Site-directed mutagenesis revealed that valine 34 and leucine 36 are critical for GPVI interaction with collagen and CRP. The loop might thus be part of a collagen/CRP-binding site.

scholarly journals Streptococcus pneumoniae Choline-Binding Protein E Interaction with Plasminogen/Plasmin Stimulates Migration across the Extracellular Matrix

2007 ◽  
Vol 76 (2) ◽  
pp. 466-476 ◽  
Author(s):  
Cécile Attali ◽  
Cécile Frolet ◽  
Claire Durmort ◽  
Julien Offant ◽  
Thierry Vernet ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Streptococcus Pneumoniae ◽  
Solid Phase ◽  
Binding Protein ◽  
Physiological Role ◽  
Site Directed Mutagenesis ◽  
Binding Assays ◽  
Solid Phase Binding ◽  
Vitro Model

ABSTRACT The virulence mechanisms leading Streptococcus pneumoniae to convert from nasopharyngeal colonization to a tissue-invasive phenotype are still largely unknown. Proliferation of infection requires penetration of the extracellular matrix, which occurs by recruitment of host proteases to the bacterial cell surface. We present evidence supporting the role of choline-binding protein E (CBPE) (a member of the surface-exposed choline-binding protein family) as an important receptor for human plasminogen, the precursor of plasmin. The results of ligand overlay blot analyses, solid-phase binding assays, and surface plasmon resonance experiments support the idea of an interaction between CBPE and plasminogen. We have shown that the phosphorylcholine esterase (Pce) domain of CBPE interacts with the plasminogen kringle domains. Analysis of the crystal structure of the Pce domain, followed by site-directed mutagenesis, allowed the identification of the plasminogen-binding region composed in part by lysine residues, some of which map in a linear fashion on the surface of the Pce domain. The biological relevance of the CBPE-plasminogen interaction is supported by the fact that, compared to the wild-type strain, a mutant of pneumococcus with the cbpE gene deleted (i) displays a reduced level of plasminogen binding and plasmin activation and (ii) shows reduced ability to cross the extracellular matrix in an in vitro model. These results support the idea of a physiological role for the CBPE-plasminogen interaction in pneumococcal dissemination into human tissue.

Ability of PknA, a mycobacterial eukaryotic-type serine/threonine kinase, to transphosphorylate MurD, a ligase involved in the process of peptidoglycan biosynthesis

2008 ◽  
Vol 415 (1) ◽  
pp. 27-33 ◽  
Author(s):  
Meghna Thakur ◽  
Pradip K. Chakraborti
Keyword(s):  
Protein Kinases ◽  
Solid Phase ◽  
Morphological Changes ◽  
Binding Assays ◽  
Serine Threonine Kinase ◽  
Threonine Kinase ◽  
Peptidoglycan Biosynthesis ◽  
Vitro Binding ◽  
Solid Phase Binding

Eukaryotic-type serine/threonine protein kinases in bacteria have been implicated in controlling a host of cellular activities. PknA is one of eleven such protein kinases from Mycobacterium tuberculosis which regulates morphological changes associated with cell division. In the present study we provide the evidence for the ability of PknA to transphosphorylate mMurD (mycobacterial UDP-N-acetylmuramoyl-L-alanine:D-glutamate-ligase), the enzyme involved in peptidoglycan biosynthesis. Its co-expression in Escherichia coli along with PknA resulted in phosphorylation of mMurD. Consistent with these observations, results of the solid-phase binding assays revealed a high-affinity in vitro binding between the two proteins. Furthermore, overexpression of m-murD in Mycobacterium smegmatis yielded a phosphorylated protein. The results of the present study therefore point towards the possibility of mMurD being a substrate of PknA.

Ultrastructural Evidence for Proteoglycans Mediating an Attachment of Type VI Collagen to Banded Collagen Fibrils

1997 ◽  
Vol 3 (S2) ◽  
pp. 153-154
Author(s):  
Douglas R. Keene ◽  
Catherine C. Ridgway ◽  
Renato V. Iozzo
Keyword(s):  
Dermatan Sulfate ◽  
Core Protein ◽  
Solid Phase ◽  
Collagen Fibrils ◽  
Binding Assays ◽  
Connective Tissues ◽  
Type Vi Collagen ◽  
Dermatan Sulfate Proteoglycan ◽  
Individual Type ◽  
Solid Phase Binding

Immunolocalizaton studies of type VI collagen in skin have previously demonstrated that type VI collagen forms a flexible network that anchors large interstitial structures such as nerves, blood vessels, and collagen fibers into the surrounding connective tissues matrix. The purpose of this study is to determine if individual type VI collagen microfilaments might be connected to banded collagen fibrils, thereby stabilizing the network.Solid phase binding assays suggest a specific, high affinity interaction between the core protein of the dermatan sulfate proteoglycan decorin and type VI collagen, and immunocytochemical studies in fetal and neonate rabbit cornea suggest an association of decorin with type VI microfilaments. Other studies in skin and perichondrium have localized decorin to a region between the d and e bands of banded collagen fibrils. However, no direct documentation has demonstrated a specific structural interaction between type VI microfilaments and banded collagen fibrils. We, therefore, sought to determine if type VI microfilaments cross banded collagen fibrils between the “d” and “e” bands.

scholarly journals Fibulin-1 mediates platelet adhesion via a bridge of fibrinogen

Blood ◽  
1996 ◽  
Vol 88 (7) ◽  
pp. 2569-2577 ◽  
Author(s):  
S Godyna ◽  
M Diaz-Ricart ◽  
WS Argraves
Keyword(s):  
Extracellular Matrix ◽  
Vascular Injury ◽  
Whole Blood ◽  
Platelet Adhesion ◽  
Solid Phase ◽  
Blood Perfusion ◽  
General Mechanism ◽  
Binding Assays ◽  
Solid Phase Binding ◽  
Coated Surfaces

Fibulin-1 is a component of the extracellular matrix that surrounds vascular smooth muscle. This observation, along with the recent finding that fibulin-1 can bind fibrinogen (J Biol Chem 270:19458, 1995), prompted investigation into the potential role of fibulin-1 as a thrombogenic agent. In perfusion chamber assays, platelets in whole blood under flow conditions attached and spread on surfaces coated with fibulin-1. This adhesion was completely blocked by fibulin-1 antibodies. Platelets free of plasma did not attach to fibulin-1 coated surfaces; however, with the addition of fibrinogen, platelet adhesion to fibulin-1 took place. When detergent extracts of platelets were subjected to fibulin-1-Sepharose affinity chromatography, the integrin alpha IIb beta 3 was selected. Solid phase binding assays using purified components showed that integrin alpha IIb beta 3 could not bind directly to fibulin-1 but in the presence of fibrinogen the integrin bound to fibulin-1-coated surfaces. Monoclonal alpha IIb beta 3 antibodies capable of blocking its interaction with fibrinogen completely blocked platelet adhesion to fibulin-1 in both whole blood perfusion and static adhesion assays. The results show that fibulin-1 can support platelet attachment via a bridge of fibrinogen to the platelet integrin alpha IIb beta 3. When fibroblast monolayers containing extracellular matrix-incorporated fibulin-1 were used as adhesion substrates, platelet adhesion in the presence of fibrinogen could be inhibited by 30% using antibodies to fibulin-1. Following vascular injury, fibulin-1 present in the extracellular matrix of the vessel wall may therefore interact with plasma fibrinogen and promote platelet adhesion, leading to the formation of a platelet plug. Thus, fibulin-1 joins the list of matrix proteins including collagens I and IV and fibronectin that mediate platelet adhesion via a plasma protein bridge. This bridging phenomenon may represent a general mechanism by which platelets interact with exposed subendothelial matrices following vascular injury.

Immobilized whole algal cells for solid-phase binding assays

1986 ◽  
Vol 86 (2) ◽  
pp. 171-177 ◽  
Author(s):  
Paul Bubrick ◽  
Leon Goldstein ◽  
Asher Frensdorff
Keyword(s):  
Solid Phase ◽  
Binding Assays ◽  
Solid Phase Binding

scholarly journals Identification of essential residues for binding and activation in the human 5-HT7(a) serotonin receptor by molecular modeling and site-directed mutagenesis

2015 ◽  
Vol 9 ◽  
Author(s):  
Agata Antonina Rita Impellizzeri ◽  
Matteo Pappalardo ◽  
Livia Basile ◽  
Ornella Manfra ◽  
Kjetil Wessel Andressen ◽  
...  
Keyword(s):  
Molecular Modeling ◽  
Serotonin Receptor ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis

Combination of Molecular Modeling, Site-Directed Mutagenesis, and SAR Studies To Delineate the Binding Site of Pyridopyrimidine Antagonists on the Human CCK1 Receptor

2005 ◽  
Vol 48 (15) ◽  
pp. 4842-4850 ◽  
Author(s):  
Mercedes Martín-Martínez ◽  
Anne Marty ◽  
Maud Jourdan ◽  
Chantal Escrieut ◽  
Elodie Archer ◽  
...  
Keyword(s):  
Molecular Modeling ◽  
Binding Site ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Sar Studies ◽  
Cck1 Receptor

Production of transferrin receptors byHistophilus ovis: three of five strains require two signals

2001 ◽  
Vol 47 (5) ◽  
pp. 417-423 ◽  
Author(s):  
Andrew Ekins ◽  
Donald F Niven
Keyword(s):  
Solid Phase ◽  
Iron Acquisition ◽  
Isolation Procedure ◽  
Transferrin Receptors ◽  
Binding Assays ◽  
Surface Receptors ◽  
Iron Restriction ◽  
Affinity Isolation ◽  
Solid Phase Binding ◽  
Bovine Transferrin

Five strains of Histophilus ovis (9L, 642A, 714, 5688T, and 3384Y) were investigated with respect to iron acquisition. All strains used ovine, bovine, and goat transferrins (Tfs), but not porcine or human Tfs, as iron sources for growth. In solid phase binding assays, total membranes from only two (9L and 642A) of the five strains, grown under iron-restricted conditions, were able to bind Tfs (ovine, bovine, and goat, but not porcine or human). However, when the organisms were grown under iron-restricted conditions in the presence of bovine transferrin (Tf), total membranes from all strains exhibited Tf binding (as above); competition experiments demonstrated that all three Tfs (ovine, bovine, and goat) were bound by the same receptor(s). Membranes from organisms grown under iron-replete conditions in the presence or absence of bovine Tf failed to bind any of the test Tfs. An affinity-isolation procedure allowed the isolation of two putative Tf-binding polypeptides (78 and 66 kDa) from total membranes of strains 9L and 642A grown under iron-restricted conditions, and from membranes of all strains if the growth medium also contained Tf. It is concluded that all strains tested acquire Tf-bound iron by means of siderophore-independent mechanisms involving surface receptors analogous to the Tf-binding proteins (TbpA and TbpB) found in comparable organisms; although iron restriction alone is sufficient to promote the expression of these proteins by strains 9L and 642A, their production by strains 714, 5688T, and 3384Y appears to require two signals, iron restriction and the presence of Tf.Key words: Histophilus ovis, iron acquisition, transferrins, receptors, regulation.

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