scholarly journals Identification of fibronectin fragments that bind to carboxy-group-modified proteins

1983 ◽  
Vol 215 (3) ◽  
pp. 613-616 ◽  
Author(s):  
M Vuento ◽  
K Sekiguchi ◽  
M Korkolainen
Keyword(s):  
Binding Site ◽  
Human Plasma ◽  
Carboxy Group ◽  
Binding Sites ◽  
Limited Proteolysis ◽  
Plasma Fibronectin ◽  
C Terminus ◽  
Fibronectin Fragments ◽  
Modified Proteins ◽  
Polypeptide Chains

Limited proteolysis of human plasma fibronectin with chymotrypsin, trypsin or thermolysin has been used to localize binding sites responsible for binding [Vuento, Korkolainen & Stenman (1982) Biochem. J. 205, 303-311] of fibronectin to carboxy-group-modified proteins. These bindings sites are different from those mediating binding of fibronectin to gelatin or heparin. They are located close to the C-terminus of the polypeptide chains of fibronectin, and apparently overlap with the C-terminal fibrin binding site.

scholarly journals Primary structure of human plasma fibronectin. Characterization of a 38 kDa domain containing the C-terminal heparin-binding site (Hep III site) and a region of molecular heterogeneity

1987 ◽  
Vol 241 (3) ◽  
pp. 923-928 ◽  
Author(s):  
A Garcia-Pardo ◽  
A Rostagno ◽  
B Frangione
Keyword(s):  
Human Plasma ◽  
Primary Structure ◽  
Terminal Segment ◽  
Molecular Heterogeneity ◽  
Heparin Binding ◽  
Plasma Fibronectin ◽  
Heparin Binding Site ◽  
Heparin Binding Domain ◽  
Polypeptide Chains

The primary structure of a 38 kDa heparin-binding domain from human plasma fibronectin has been determined. This domain contains 380 residues arranged in three type-III homology regions of approx. 90 residues each, and a 67-amino-acid C-terminal segment. This segment has been shown to be encoded by certain mRNA species only, due to alternative splicing [Kornblihtt, Vibe-Pedersen & Baralle (1984) Nucleic Acids Research 12, 5853-5868], and therefore represents a region of heterogeneity in fibronectin. Our data indicate that at least one of the constituent polypeptide chains contains this region.

scholarly journals The actin filament-severing domain of plasma gelsolin.

1986 ◽  
Vol 103 (4) ◽  
pp. 1473-1481 ◽  
Author(s):  
C Chaponnier ◽  
P A Janmey ◽  
H L Yin
Keyword(s):  
Binding Site ◽  
Human Plasma ◽  
Actin Filament ◽  
Binding Sites ◽  
Actin Filaments ◽  
The Other ◽  
Actin Binding ◽  
Plasma Gelsolin ◽  
Chymotryptic Peptide ◽  
Native Plasma

Gelsolin, a multifunctional actin-modulating protein, has two actin-binding sites which may interact cooperatively. Native gelsolin requires micromolar Ca2+ for optimal binding of actin to both sites, and for expression of its actin filament-severing function. Recent work has shown that an NH2-terminal chymotryptic 17-kD fragment of human plasma gelsolin contains one of the actin-binding sites, and that this fragment binds to and severs actin filaments weakly irrespective of whether Ca2+ is present. The other binding site is Ca2+ sensitive, and is found in a chymotryptic peptide derived from the COOH-terminal two-thirds of plasma gelsolin; this fragment does not sever F-actin or accelerate the polymerization of actin. This paper documents that larger thermolysin-derived fragments encompassing the NH2-terminal half of gelsolin sever actin filaments as effectively as native plasma gelsolin, although in a Ca2+-insensitive manner. This result indicates that the NH2-terminal half of gelsolin is the actin-severing domain. The stringent Ca2+ requirement for actin severing found in intact gelsolin is not due to a direct effect of Ca2+ on the severing domain, but indirectly through an effect on domains in the COOH-terminal half of the molecule to allow exposure of both actin-binding sites.

scholarly journals Calmodulin Binding to Connexin 35: Specializations to Function as an Electrical Synapse

2020 ◽  
Vol 21 (17) ◽  
pp. 6346
Author(s):  
Jaya Aseervatham ◽  
Xiaofan Li ◽  
Cheryl K. Mitchell ◽  
Ya-Ping Lin ◽  
Ruth Heidelberger ◽  
...  
Keyword(s):  
Binding Site ◽  
Binding Sites ◽  
Electrical Synapse ◽  
Cytoplasmic Calcium ◽  
Calcium Dependent ◽  
Calmodulin Binding ◽  
Connexin 36 ◽  
Calcium Calmodulin Dependent ◽  
C Terminus ◽  
A Site

Calmodulin binding is a nearly universal property of gap junction proteins, imparting a calcium-dependent uncoupling behavior that can serve in an emergency to decouple a stressed cell from its neighbors. However, gap junctions that function as electrical synapses within networks of neurons routinely encounter large fluctuations in local cytoplasmic calcium concentration; frequent uncoupling would be impractical and counterproductive. We have studied the properties and functional consequences of calmodulin binding to the electrical synapse protein Connexin 35 (Cx35 or gjd2b), homologous to mammalian Connexin 36 (Cx36 or gjd2). We find that specializations in Cx35 calmodulin binding sites make it relatively impervious to moderately high levels of cytoplasmic calcium. Calmodulin binding to a site in the C-terminus causes uncoupling when calcium reaches low micromolar concentrations, a behavior prevented by mutations that eliminate calmodulin binding. However, milder stimuli promote calcium/calmodulin-dependent protein kinase II activity that potentiates coupling without interference from calmodulin binding. A second calmodulin binding site in the end of the Cx35 cytoplasmic loop, homologous to a calmodulin binding site present in many connexins, binds calmodulin with very low affinity and stoichiometry. Together, the calmodulin binding sites cause Cx35 to uncouple only at extreme levels of intracellular calcium.

scholarly journals Expression of human plasma gelsolin in Escherichia coli and dissection of actin binding sites by segmental deletion mutagenesis.

1989 ◽  
Vol 109 (2) ◽  
pp. 593-605 ◽  
Author(s):  
M Way ◽  
J Gooch ◽  
B Pope ◽  
A G Weeds
Keyword(s):  
Escherichia Coli ◽  
Human Plasma ◽  
Binding Sites ◽  
Current Knowledge ◽  
Limited Proteolysis ◽  
Actin Binding ◽  
High Yield ◽  
Soluble Form ◽  
Proteolytic Fragment ◽  
Plasma Gelsolin

Human plasma gelsolin has been expressed in high yield and soluble form in Escherichia coli. The protein has nucleating and severing activities identical to those of plasma gelsolin and is fully calcium sensitive in its interactions with monomeric actin. A number of deletion mutants have been expressed to explore the function of the three actin binding sites. Their design is based on the sixfold segmental repeat in the protein sequence. (These sites are located in segment 1, segments 2-3, and segments 4-6). Two mutants, S1-3 and S4-6, are equivalent to the NH2- and COOH-terminal halves of the molecule obtained by limited proteolysis. S1-3 binds two actin monomers in the presence or absence of calcium, it severs and caps filaments but does not nucleate polymerization. S4-6 binds a single actin monomer but only in calcium. These observations confirm and extend current knowledge on the properties of the two halves of gelsolin. Two novel constructs have also been studied that provide a different pairwise juxtaposition of the three sites. S2-6, which lacks the high affinity site of segment 1 (equivalent to the 14,000-Mr proteolytic fragment) and S1,4-6, which lacks segments 2-3 (the actin filament binding domain previously identified using the 28,000-Mr proteolytic fragment). S2-6 binds two actin monomers in calcium and nucleates polymerization; it associates laterally with filaments in the presence or absence of calcium and has a weak calcium-dependent fragmenting activity. S1,4-6 also binds two actin monomers in calcium and one in EGTA, has weak severing activity but does not nucleate polymerization. A model is presented for the involvement of the three binding sites in the various activities of gelsolin.

scholarly journals Fibronectin binding to complement subcomponent C1q. Localization of their respective binding sites

1985 ◽  
Vol 226 (1) ◽  
pp. 207-215 ◽  
Author(s):  
J Sorvillo ◽  
I Gigli ◽  
E Pearlstein
Keyword(s):  
Amino Acids ◽  
Binding Site ◽  
Binding Sites ◽  
Solid Phase ◽  
Fluid Phase ◽  
Globular Domain ◽  
Plasma Fibronectin ◽  
Fibronectin Binding ◽  
Inhibition Studies ◽  
Radiobinding Assay

The interaction of purified human plasma fibronectin with the C1q subcomponent of complement was investigated by using a solid-phase radiobinding assay. 125I-fibronectin binding to native C1q, purified collagen domain (C1q-c) or globular domain (C1q-g) was compared. When the purified domains were insolubilized by binding to plastic, the C1q-c exhibited 59% of the binding demonstrated with intact C1q, whereas the C1q-g exhibited 35% of the binding. N-Terminal sequencing of the globular domain showed that a sequence of seven collagen-like amino acids was retained on each chain of the C1q-g fragment. 125I-fibronectin binding to C1q could be inhibited equally well by fluid-phase C1q and C1q-c, but not by fluid-phase C1q-g, implying that the collagen-like region retained on the C1q-g is masked in the fluid phase. In addition, studies were performed to determine which subunit(s) of C1q bind(s) fibronectin. The percentages of fibronectin bound by the A, B, and C chain of C1q were found to be 38, 21 and 41% respectively. Inhibition studies with purified 200-180 kDa, 50 kDa or 29 kDa fragments of fibronectin show that the binding site on fibronectin for C1q is the 50 kDa gelatin-binding domain.

Binding Site in Human Plasma Fibronectin to HL-60 Cells Localizes in the C-Terminal Heparin-Binding Region Independently of RGD and CS1

1995 ◽  
Vol 217 (2) ◽  
pp. 484-489 ◽  
Author(s):  
Hiroyuki Fujita ◽  
Hiroshi Mohri ◽  
Heiwa Kanamori ◽  
Akihiro Iwamatsu ◽  
Takao Okubo
Keyword(s):  
Binding Site ◽  
Human Plasma ◽  
Heparin Binding ◽  
Plasma Fibronectin ◽  
Binding Region

scholarly journals Probing the Structure of Rotavirus NSP4: a Short Sequence at the Extreme C Terminus Mediates Binding to the Inner Capsid Particle

2000 ◽  
Vol 74 (11) ◽  
pp. 5388-5394 ◽  
Author(s):  
Judith A. O'Brien ◽  
John A. Taylor ◽  
A. R. Bellamy
Keyword(s):  
Amino Acids ◽  
Binding Sites ◽  
Limited Proteolysis ◽  
Coiled Coil ◽  
Surface Binding ◽  
Virus Maturation ◽  
C Terminus ◽  
Tetramerization Domain ◽  
Necessary And Sufficient ◽  
Sequence Requirements

ABSTRACT The rotavirus nonstructural glycoprotein NSP4 functions as the receptor for the inner capsid particle (ICP) which buds into the lumen of the endoplasmic reticulum during virus maturation. The structure of the cytoplasmic domain of NSP4 from rotavirus strain SA11 has been investigated by using limited proteolysis and mass spectrometry. Digestion with trypsin and V8 protease reveals a C-terminal protease-sensitive region that is 28 amino acids long. The minimal sequence requirements for receptor function have been defined by constructing fusions with glutathione S-transferase and assessing their ability to bind ICPs. These experiments demonstrate that 17 to 20 amino acids from the extreme C terminus are necessary and sufficient for ICP binding and that this binding is cooperative. These observations are consistent with a model for the structure of the NSP4 cytoplasmic region in which four flexible regions of 28 amino acids are presented by a protease-resistant coiled-coil tetramerization domain, with only the last ∼20 amino acids of each peptide interacting with the surface binding sites on the ICP.

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