Complement C9 binding site and the anti-microbial activity of caprine vitronectin are localized in close proximity in the N-terminal region of the protein

Abstract Abstract 1550 Background: We previously reported that mutations of CD20 gene were found in patients with B-cell non-Hodgkin's lymphoma, and we proposed that C-terminal deletion mutations of CD20 might be involved in relapse/resistance after rituximab containing therapy. Most of the patients that had mutation in the C-terminal leagion were diagnosed as CD20 negative by immunohistochemistry using L26 monoclonal antibody. L26 recognizes the cytoplasmic region of CD20 molecules, but no more detailed information about its epitope had been reported. So at first we determined the binding site of L26 antibody on CD20 protein. Then we developed novel diagnostic antibodies that recognize wide variety of CD20 molecular subtypes including those having mutations. Methods: To determine the epitope of L26 antibody, we established six sub-lines expressing various length of C-terminal truncated CD20 using an originally CD20 negative myeloma cell line. Then we carried out epitope-mapping using these cell lines. To detect comprehensive CD20 molecules including that having mutation in C-terminal region, we developed antibodies that recognize near the amino terminus of CD20 molecules (CD20N antibody). CD20N antibody is the only monoclonal antibody that recognizes N-terminal region of CD20 so far. Using these antibodies, we screened the specimens of the cases diagnosed as CD20 negative determined by L26-based immunohistochemistry. Results: The epitope-mapping revealed that L26 recognizes near the C-terminus of CD20. This suggested that most of CD20 molecules with the C-terminal deletion mutation or frame-shift mutation could not be recognized by L26. Then we screened previously diagnosed specimens and found several cases that having the cells stained by our novel antibody but not by L26. Genetic analysis revealed that all these cells had a mutation in the C-terminal cytoplasmic region of CD20. One of these cases, we successfully analyzed the phenotype of lymphoma cells with mutated CD20 in detail using cryopreserves living specimens. In this case, a frame shift mutation occurred due to one base nucleotide deletion, resulting in the translation of peptide of another reading frame of 41 amino acids with premature stop at the amino acid position 250. Interestingly, mutant CD20 molecule expressed adjacent to the plasma membrane, but rituximab could not bind to these cells. DNA sequencing study about genome and mRNA of CD20 gene suggested that the lymphoma cells of this patient had one normal and one mutated CD20 allele. Discussions: The C-terminal region of CD20 may undertake a pivotal role in presentation of the large loop where the rituximab binding site locates. Thus, deletion or frame-shift mutation of CD20 in C-terminal cytoplasmic region impairs the antigenicity against rituximab and it may cause resistance to rituximab therapy. The resistance caused by gene mutation thought to be irreversible. And it should be discriminated from transient downregulation of antigen expression. We propose here that immunohistochemical screening using CD20N antibody is very rapid and effective screening stategy that find out irreversible rituximab resistant cases. Disclosures: No relevant conflicts of interest to declare.

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Interaction of Ras with phosphoinositide 3-kinase γ

Biochemical Journal ◽

10.1042/bj3260891 ◽

1997 ◽

Vol 326 (3) ◽

pp. 891-895 ◽

Cited By ~ 56

Author(s):

Ignacio RUBIO ◽

Pablo RODRIGUEZ-VICIANA ◽

Julian DOWNWARD ◽

Reinhard WETZKER

Keyword(s):

Binding Site ◽

G Proteins ◽

Terminal Region ◽

Regulatory Subunit ◽

Heterotrimeric G Proteins ◽

Modest Increase ◽

Pi3k Activity ◽

Phosphoinositide 3 Kinase ◽

Stimulation Of

Phosphoinositide 3-kinase γ (PI3Kγ) can be activated in vitro by both α and βγ subunits of heterotrimeric G-proteins and does not interact with p85, the regulatory subunit of PI3Kα. Here we demonstrate the binding of Ras to PI3Kγ in vitro. An N-terminal region of PI3Kγ was identified as a binding site for Ras. After co-expression with PI3Kγ in COS-7 cells, Ras induced only a modest increase in PI3K activity compared with the stimulation of PI3Kα by Ras in the same cells.

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Alignment of caldesmon on the actin-tropomyosin filaments

Biochemical Journal ◽

10.1042/bj3090951 ◽

1995 ◽

Vol 309 (3) ◽

pp. 951-957 ◽

Cited By ~ 9

Author(s):

T S Tsuruda ◽

M H Watson ◽

D B Foster ◽

J J J C Lin ◽

A S Mak

Keyword(s):

Escherichia Coli ◽

Smooth Muscle ◽

Binding Site ◽

Actin Filament ◽

Binding Sites ◽

Human Fibroblast ◽

Thin Filament ◽

Terminal Region ◽

Terminal Domain ◽

Terminal Fragment

We have reported previously that each smooth-muscle caldesmon binds predominantly to a region within residues 142-227 of tropomyosin, but a weaker binding site also exists at the N-terminal region of tropomyosin [Watson, Kuhn, Novy, Lin and Mak (1990) J. Biol. Chem. 265, 18860-18866]. In view of recent evidence for the presence of tropomyosin-binding sites at both the N- and C-terminal domains of caldesmon, we have studied the binding of the N- and C-terminal fragments of human fibroblast caldesmon expressed in Escherichia coli to tropomyosin and its CNBr fragments. The N-terminal fragment, CaD40 (residues 1-152), binds tropomyosin, but the interaction is mostly abolished in the presence of actin. CaD40 binds strongly to Cn1B(142-281) of tropomyosin, but weakly to Cn1A(11-127). The C-terminal fragment, CaD39, which corresponds to residues 443-736 of gizzard caldesmon, binds tropomyosin, and the interaction is enhanced by actin. CaD39 binds to both Cn1A(11-127) and Cn1B(142-281) of tropomyosin. Our results suggest that the N-terminal domain of caldesmon interacts with the C-terminal half of one tropomyosin molecule, whereas the C-terminal domain binds to both N- and C-terminal regions of the adjacent tropomyosin molecule along the actin filament. In addition, the binding of the N-terminal domain of caldesmon to the actin-tropomyosin filament is weak, which may allow this domain to project off the thin filament to interact with myosin.

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The heparin-binding site in tetranectin is located in the N-terminal region and binding does not involve the carbohydrate recognition domain

Biochemical Journal ◽

10.1042/0264-6021:3470083 ◽

2000 ◽

Vol 347 (1) ◽

pp. 83 ◽

Cited By ~ 6

Author(s):

Rikke H. LORENTSEN ◽

Jonas H. GRAVERSEN ◽

Nigel R. CATERER ◽

Hans C. THØGERSEN ◽

Michael ETZERODT

Keyword(s):

Binding Site ◽

Terminal Region ◽

Carbohydrate Recognition Domain ◽

Heparin Binding ◽

Carbohydrate Recognition ◽

Heparin Binding Site

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Anticoagulant peptides. Nature of the interaction of the C-terminal region of hirudin with a noncatalytic binding site on thrombin

Journal of Medicinal Chemistry ◽

10.1021/jm00392a030 ◽

1987 ◽

Vol 30 (9) ◽

pp. 1688-1691 ◽

Cited By ~ 43

Author(s):

John L. Krstenansky ◽

Thomas J. Owen ◽

Mark T. Yates ◽

Simon J. T. Mao

Keyword(s):

Binding Site ◽

Terminal Region

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Molecular analysis of the interaction of LCMV with its cellular receptor α-dystroglycan

The Journal of Cell Biology ◽

10.1083/jcb.200104103 ◽

2001 ◽

Vol 155 (2) ◽

pp. 301-310 ◽

Cited By ~ 114

Author(s):

Stefan Kunz ◽

Noemí Sevilla ◽

Dorian B. McGavern ◽

Kevin P. Campbell ◽

Michael B.A. Oldstone

Keyword(s):

Binding Site ◽

Binding Affinity ◽

Receptor Binding ◽

Divalent Cations ◽

Terminal Region ◽

Cellular Receptor ◽

Globular Domain ◽

Cell Surface Molecule ◽

Laminin 1

α-Dystroglycan (DG) has been identified as the cellular receptor for lymphocytic choriomeningitis virus (LCMV) and Lassa fever virus (LFV). This subunit of DG is a highly versatile cell surface molecule that provides a molecular link between the extracellular matrix (ECM) and a β-DG transmembrane component, which interacts with the actin-based cytoskeleton. In addition, DG exhibits a complex pattern of interaction with a wide variety of ECM and cellular proteins. In the present study, we characterized the binding of LCMV to α-DG and addressed the role of α-DG–associated host-derived proteins in virus infection. We found that the COOH-terminal region of α-DG's first globular domain and the NH2-terminal region of the mucin-related structures of α-DG together form the binding site for LCMV. The virus–α-DG binding unlike ECM α-DG interactions was not dependent on divalent cations. Despite such differences in binding, LCMV and laminin-1 use, in part, an overlapping binding site on α-DG, and the ability of an LCMV isolate to compete with laminin-1 for receptor binding is determined by its binding affinity to α-DG. This competition of the virus with ECM molecules for receptor binding likely explains the recently found correlation between the affinity of LCMV binding to α-DG, tissue tropism, and pathological potential. LCMV strains and variants with high binding affinity to α-DG but not low affinity binders are able to infect CD11c+ dendritic cells, which express α-DG at their surface. Infection followed by dysfunction of these antigen-presenting cells contributes to immunosuppression and persistent viral infection in vivo.

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Characterisation of the paxillin-binding site and the C-terminal focal adhesion targeting sequence in vinculin

Journal of Cell Science ◽

10.1242/jcs.107.2.709 ◽

1994 ◽

Vol 107 (2) ◽

pp. 709-717 ◽

Cited By ~ 16

Author(s):

C.K. Wood ◽

C.E. Turner ◽

P. Jackson ◽

D.R. Critchley

Keyword(s):

Amino Acids ◽

Fusion Protein ◽

Binding Site ◽

Focal Adhesion ◽

Focal Adhesions ◽

Cytoskeletal Proteins ◽

Terminal Region ◽

Nih3t3 Cells ◽

Cell Biol

Paxillin and vinculin are cytoskeletal proteins that colocalise to focal adhesions, specialised regions of the cell involved in attachment to the extracellular matrix. These two molecules form part of a complex of proteins that link the actin network to the plasma membrane. Paxillin has been shown to bind directly in vitro to the C-terminal region of vinculin (Turner et al. (1990). J. Cell Biol. 111, 1059–1068), which also contains a focal adhesion targeting sequence (Bendori et al. (1989). J. Cell Biol. 108, 2383–2393). In the present study, we have used a series of vinculin deletion mutants to map more precisely the sites in vinculin responsible for paxillin binding and focal adhesion localisation. A glutathione-S-transferase fusion protein spanning vinculin residues 881–1000 was sufficient to support 125I-paxillin binding in a gel-blot assay while no detectable binding was observed to a fusion protein spanning residues 881–978. Transfection experiments using cDNAs encoding chick vinculin residues 398–1066 and 398–1028 demonstrated that amino acids C-terminal to residue 1028 were not necessary for targeting to focal adhesions. In contrast, a vinculin polypeptide expressed from a cDNA encoding residues 398–1000 failed to localise to focal adhesions in stably transfected NIH3T3 cells. We have therefore identified a region of 50 amino acids (residues 979–1028) within the C-terminal region of vinculin that contains both the paxillin-binding site and the focal adhesion targeting sequence. This region is highly conserved in human and chicken vinculin and is likely to be important in regulation of the assembly of focal adhesions.

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TatB Functions as an Oligomeric Binding Site for Folded Tat Precursor Proteins

Molecular Biology of the Cell ◽

10.1091/mbc.e10-07-0585 ◽

2010 ◽

Vol 21 (23) ◽

pp. 4151-4161 ◽

Cited By ~ 45

Author(s):

Carlo Maurer ◽

Sascha Panahandeh ◽

Anna-Carina Jungkamp ◽

Michael Moser ◽

Matthias Müller

Keyword(s):

Binding Site ◽

Binding Sites ◽

Membrane Vesicles ◽

Twin Arginine Translocation ◽

Amphipathic Helices ◽

Collective Data ◽

Close Proximity ◽

Precursor Proteins ◽

Cross Linker ◽

Folded Proteins

Twin-arginine-containing signal sequences mediate the transmembrane transport of folded proteins. The cognate twin-arginine translocation (Tat) machinery of Escherichia coli consists of the membrane proteins TatA, TatB, and TatC. Whereas Tat signal peptides are recognized by TatB and TatC, little is known about molecular contacts of the mature, folded part of Tat precursor proteins. We have placed a photo-cross-linker into Tat substrates at sites predicted to be either surface-exposed or hidden in the core of the folded proteins. On targeting of these variants to the Tat machinery of membrane vesicles, all surface-exposed sites were found in close proximity to TatB. Correspondingly, incorporation of the cross-linker into TatB revealed multiple precursor-binding sites in the predicted transmembrane and amphipathic helices of TatB. Large adducts indicative of TatB oligomers contacting one precursor molecule were also obtained. Cross-linking of Tat substrates to TatB required an intact twin-arginine signal peptide and disappeared upon transmembrane translocation. Our collective data are consistent with TatB forming an oligomeric binding site that transiently accommodates folded Tat precursors.

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