Structural and functional studies on C4b-binding protein, a regulatory component of the human complement system

1985 ◽  
Vol 5 (10-11) ◽  
pp. 855-865 ◽  
Author(s):  
L. Ping Chung ◽  
Kenneth B. M. Reid
Keyword(s):  
Amino Acid ◽  
Binding Protein ◽  
Protein A ◽  
Limited Proteolysis ◽  
Binding Activity ◽  
Amino Acid Sequencing ◽  
Functional Studies ◽  
Before And After ◽  
Cofactor Activity ◽  
Long Chain

The binding and cofactor activities of C4b-binding protein were examined before and after limited proteolysis by pepsin, trypsin and chymotrypsin. The major fragments generated were characterized by amino acid sequencing, thus establishing the precise points of limited proteolysis. These studies allow a tentative assignment of the cofactor activity site to the residues 177–322 of the 549 amino acid long chain of C4b-binding protein but indicated that residues in the region 332–395 are important in the binding activity.

scholarly journals A novel DNA-binding motif in the nuclear matrix attachment DNA-binding protein SATB1

1994 ◽  
Vol 14 (3) ◽  
pp. 1852-1860
Author(s):  
K Nakagomi ◽  
Y Kohwi ◽  
L A Dickinson ◽  
T Kohwi-Shigematsu
Keyword(s):  
Amino Acid ◽  
Dna Binding ◽  
Direct Contact ◽  
Binding Protein ◽  
Binding Activity ◽  
Binding Domain ◽  
Binding Motif ◽  
Dna Binding Domain ◽  
Sequence Context ◽  
Dna Binding Activity

The nuclear matrix attachment DNA (MAR) binding protein SATB1 is a sequence context-specific binding protein that binds in the minor groove, making virtually no contact with the DNA bases. The SATB1 binding sites consist of a special AT-rich sequence context in which one strand is well-mixed A's, T's, and C's, excluding G's (ATC sequences), which is typically found in clusters within different MARs. To determine the extent of conservation of the SATB1 gene among different species, we cloned a mouse homolog of the human STAB1 cDNA from a cDNA expression library of the mouse thymus, the tissue in which this protein is predominantly expressed. This mouse cDNA encodes a 764-amino-acid protein with a 98% homology in amino acid sequence to the human SATB1 originally cloned from testis. To characterize the DNA binding domain of this novel class of protein, we used the mouse SATB1 cDNA and delineated a 150-amino-acid polypeptide as the binding domain. This region confers full DNA binding activity, recognizes the specific sequence context, and makes direct contact with DNA at the same nucleotides as the whole protein. This DNA binding domain contains a novel DNA binding motif: when no more than 21 amino acids at either the N- or C-terminal end of the binding domain are deleted, the majority of the DNA binding activity is lost. The concomitant presence of both terminal sequences is mandatory for binding. These two terminal regions consist of hydrophilic amino acids and share homologous sequences that are different from those of any known DNA binding motifs. We propose that the DNA binding region of SATB1 extends its two terminal regions toward DNA to make direct contact with DNA.

scholarly journals Characterization of a glutathione S-transferase and a related glutathione-binding protein from gill of the blue mussel, Mytilus edulis

1995 ◽  
Vol 305 (1) ◽  
pp. 145-150 ◽  
Author(s):  
P J Fitzpatrick ◽  
T O B Krag ◽  
P Højrup ◽  
D Sheehan
Keyword(s):  
Amino Acid ◽  
Mytilus Edulis ◽  
Blue Mussel ◽  
Binding Protein ◽  
Protein A ◽  
Gel Filtration ◽  
Cumene Hydroperoxide ◽  
Gill Tissue ◽  
Glutathione S Transferase ◽  
Sequencing Studies

The major isoenzyme of glutathione S-transferase (GST 1) was purified to homogeneity from cytosolic extracts of Mytilus edulis gill tissue by GSH-agarose affinity chromatography followed by Mono Q ion-exchange f.p.l.c. This enzyme was particularly active with 1-chloro-2,4-dinitrobenzene, ethacrynic acid and cumene hydroperoxide as substrates. Immunoblotting and amino acid sequencing studies indicate that the enzyme belongs to the Pi class of GSTs. A related protein which binds to GSH-agarose was also purified. This GSH-binding protein did not immunoblot with GST antisera and showed no detectable catalytic activity with GST substrates although its N-terminal sequence was similar to Mu-class GSTs. Gel-filtration chromatography indicated that GST 1 is a dimer and the GSH-binding protein a monomer. Mass spectrometry and SDS/PAGE indicate subunit molecular masses of 24 kDa (GST 1) and 25 kDa (GSH-binding protein), respectively. Both proteins have amino acid compositions typical of GSTs.

Bacterial lactoferrin receptors: insights from characterizing theMoraxella bovisreceptors

2002 ◽  
Vol 80 (1) ◽  
pp. 81-90 ◽  
Author(s):  
Rong-Hua Yu ◽  
Anthony B Schryvers
Keyword(s):  
Amino Acid ◽  
Binding Protein ◽  
Protein A ◽  
Bovine Lactoferrin ◽  
Predicted Amino Acid Sequence ◽  
Amino Acid Homology ◽  
Affinity Ligand ◽  
Affinity Isolation ◽  
Moraxella Bovis ◽  
Isogenic Mutants

Moraxella bovis is the causative agent of infectious conjunctivitis in cattle. Moraxella bovis isolates were shown to specifically bind bovine lactoferrin (bLf) and bovine transferrin (bTf) and to use these proteins as a source of iron to support the growth of iron-limited cells. Affinity isolation experiments with immobilized bTf yielded two proteins readily resolved by SDS-PAGE analysis, whereas only a single band of approximately 100 kDa was detected when immobilized bLf was used as the affinity ligand. Using a novel cloning strategy, regions containing the genes encoding the lactoferrin (Lf) and transferrin (Tf) receptor proteins were isolated and sequenced, demonstrating that they both consisted of two genes, with the tbpB or lbpB gene preceding the tbpA or lbpA gene. The cloned lbp genes were used to generate isogenic mutants deficient in lactoferrin binding protein A and (or) B, and the resulting strains were tested in growth and binding assays. The isogenic mutants were deficient in their use of bLf for growth and had substantially diminished bLf binding capability. The predicted amino acid sequence from the segment encoding Lf binding protein B revealed an internal amino acid homology suggesting it is a bi-lobed protein, with a C-lobe enriched in acidic amino acids, but without the evident clustering observed in Lf-binding proteins from other species.Key words: outer membrane protein, iron-binding protein, lactoferrin, receptor, iron, transport, specificity.

scholarly journals A novel DNA-binding motif in the nuclear matrix attachment DNA-binding protein SATB1.

1994 ◽  
Vol 14 (3) ◽  
pp. 1852-1860 ◽  
Author(s):  
K Nakagomi ◽  
Y Kohwi ◽  
L A Dickinson ◽  
T Kohwi-Shigematsu
Keyword(s):  
Amino Acid ◽  
Dna Binding ◽  
Direct Contact ◽  
Binding Protein ◽  
Binding Activity ◽  
Binding Domain ◽  
Binding Motif ◽  
Dna Binding Domain ◽  
Sequence Context ◽  
Dna Binding Activity

The nuclear matrix attachment DNA (MAR) binding protein SATB1 is a sequence context-specific binding protein that binds in the minor groove, making virtually no contact with the DNA bases. The SATB1 binding sites consist of a special AT-rich sequence context in which one strand is well-mixed A's, T's, and C's, excluding G's (ATC sequences), which is typically found in clusters within different MARs. To determine the extent of conservation of the SATB1 gene among different species, we cloned a mouse homolog of the human STAB1 cDNA from a cDNA expression library of the mouse thymus, the tissue in which this protein is predominantly expressed. This mouse cDNA encodes a 764-amino-acid protein with a 98% homology in amino acid sequence to the human SATB1 originally cloned from testis. To characterize the DNA binding domain of this novel class of protein, we used the mouse SATB1 cDNA and delineated a 150-amino-acid polypeptide as the binding domain. This region confers full DNA binding activity, recognizes the specific sequence context, and makes direct contact with DNA at the same nucleotides as the whole protein. This DNA binding domain contains a novel DNA binding motif: when no more than 21 amino acids at either the N- or C-terminal end of the binding domain are deleted, the majority of the DNA binding activity is lost. The concomitant presence of both terminal sequences is mandatory for binding. These two terminal regions consist of hydrophilic amino acids and share homologous sequences that are different from those of any known DNA binding motifs. We propose that the DNA binding region of SATB1 extends its two terminal regions toward DNA to make direct contact with DNA.

scholarly journals Molecular cloning and characterization of the cDNA coding for C4b-binding protein, a regulatory protein of the classical pathway of the human complement system

1985 ◽  
Vol 230 (1) ◽  
pp. 133-141 ◽  
Author(s):  
L P Chung ◽  
D R Bentley ◽  
K B Reid
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Cdna Sequence ◽  
Binding Protein ◽  
Regulatory Protein ◽  
Protein A ◽  
Classical Pathway ◽  
Amino Acid Residues ◽  
Cloned Cdna

By using synthetic oligonucleotides as probes, plasmid clones containing portions of cDNA coding for human C4b-binding protein were isolated from a liver cDNA library. The entire amino acid sequence of the C4b-binding protein can be predicted from this study of the cloned cDNA when allied to a previous sequence study at the protein level [Chung, Gagnon & Reid (1985) Mol. Immunol. 22, 427-435], in which over 55% of the amino acid sequence, including the N-terminal 62 residues, was obtained. The plasmid clones isolated allowed the unambiguous determination of 1717 nucleotides of cDNA sequence between the codon for the 32nd amino acid in the sequence of C4b-binding protein and the 164th nucleotide in the 3′ non-translated region. The sequence studies show that the secreted form of C4b-binding protein, found in plasma, is composed of chains of apparent Mr 70 000 that contains 549 amino acid residues. Examination of the protein and cDNA sequence results show that there are at least two polymorphic sites in the molecule. One is at position 44, which can be glutamine or threonine, and the other is at position 309, which can be tyrosine or histidine. Northern-blot analysis indicated that the mRNA for C4b-binding protein is approx. 2.5 kilobases long. The N-terminal 491 amino acids of C4b-binding protein can be divided into eight internal homologous regions, each approx. 60 amino acids long, which can be aligned by the presence in each region of four half-cystine, one tryptophan and several other conserved residues. These regions in C4b-binding protein are homologous with the three internal-homology regions that have been reported to be present within the Ba region of the complement enzyme factor B and also to the internal-homology regions found in the non-complement beta 2-glycoprotein I.

scholarly journals Long-chain polyphosphates impair SARS-CoV-2 infection and replication

2021 ◽  
Vol 14 (690) ◽  
pp. eabe5040
Author(s):  
Veronica Ferrucci ◽  
Dae-Young Kong ◽  
Fatemeh Asadzadeh ◽  
Laura Marrone ◽  
Angelo Boccia ◽  
...  
Keyword(s):  
Amino Acid ◽  
Limited Proteolysis ◽  
High Energy ◽  
Site Directed Mutagenesis ◽  
Linear Polymers ◽  
Amino Acid Residues ◽  
Human Nasal Epithelial Cells ◽  
Antiviral Activities ◽  
Long Chain

Inorganic polyphosphates (polyPs) are linear polymers composed of repeated phosphate (PO43−) units linked together by multiple high-energy phosphoanhydride bonds. In addition to being a source of energy, polyPs have cytoprotective and antiviral activities. Here, we investigated the antiviral activities of long-chain polyPs against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. In molecular docking analyses, polyPs interacted with several conserved amino acid residues in angiotensin-converting enzyme 2 (ACE2), the host receptor that facilitates virus entry, and in viral RNA-dependent RNA polymerase (RdRp). ELISA and limited proteolysis assays using nano– LC-MS/MS mapped polyP120 binding to ACE2, and site-directed mutagenesis confirmed interactions between ACE2 and SARS-CoV-2 RdRp and identified the specific amino acid residues involved. PolyP120 enhanced the proteasomal degradation of both ACE2 and RdRp, thus impairing replication of the British B.1.1.7 SARS-CoV-2 variant. We thus tested polyPs for functional interactions with the virus in SARS-CoV-2–infected Vero E6 and Caco2 cells and in primary human nasal epithelial cells. Delivery of a nebulized form of polyP120 reduced the amounts of viral positive-sense genomic and subgenomic RNAs, of RNA transcripts encoding proinflammatory cytokines, and of viral structural proteins, thereby presenting SARS-CoV-2 infection in cells in vitro.

scholarly journals Specificity of human tissue kallikrein towards substrates containing Phe–Phe pair of amino acids

1999 ◽  
Vol 339 (2) ◽  
pp. 473-479 ◽  
Author(s):  
Daniel C. PIMENTA ◽  
Julie CHAO ◽  
Lee CHAO ◽  
Maria A. JULIANO ◽  
Luiz JULIANO
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Human Tissue ◽  
Binding Protein ◽  
General Structure ◽  
Protein A ◽  
Tissue Kallikrein ◽  
Hybrid Peptides ◽  
Loop Sequence ◽  
Reactive Centre Loop

We have explored in detail the determinants of specificity for the hydrolysis by human tissue kallikrein (hK1) of substrates containing the Phe–Phe amino acid pair, after which hK1 cleaves kallistatin (human kallikrein-binding protein), a specific serpin for this protease, as well as somatostatin 1–14. Internally quenched fluorogenic peptides were synthesized with the general structure Abz-peptidyl-EDDnp [Abz, o-aminobenzoic acid; EDDnp, N-(2,4-dinitrophenyl)ethylenediamine], based on the natural reactive-centre loop sequence of kallistatin from P9 to P´13, and the kinetic parameters of their hydrolysis by hK1 were determined. All these peptides were cleaved after the Phe–Phe pair. For comparison, we have also examined peptides containing the reactive-centre loop sequences of human protein-C inhibitor (PCI) and rat kallikrein-binding protein, which were hydrolysed after Phe–Arg and Leu–Lys bonds, respectively. Hybrid peptides containing kallistatin–PCI sequences showed that the efficiency of hK1 activity on the peptides containing kallistatin and PCI sequences depended on both the nature of the P1 amino acid as well as on residues at the P- and P´-sides. Moreover, we have made systematic modifications on the hydrophobic pair Phe–Phe, and on Lys and Ile at the P3 and P4 positions according to the peptide substrate, Abz-AIKFFSRQ-EDDnp. All together, we concluded that tissue kallikrein was very effective on short substrates that are cleaved after the Phe–Arg pair; however, hydrolysis after Phe–Phe or other hydrophobic pairs of amino acids was more restrictive, requiring additional enzyme–substrate interaction and/or particular substrate conformations.

scholarly journals Cloning and Functional Characterization of an NAD+-Dependent DNA Ligase from Staphylococcus aureus

2001 ◽  
Vol 183 (10) ◽  
pp. 3016-3024 ◽  
Author(s):  
Frank S. Kaczmarek ◽  
Richard P. Zaniewski ◽  
Thomas D. Gootz ◽  
Dennis E. Danley ◽  
Mahmoud N. Mansour ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Amino Acid ◽  
Dna Binding ◽  
Functional Characterization ◽  
Limited Proteolysis ◽  
Binding Activity ◽  
Dna Ligase ◽  
E Coli ◽  
Dna Binding Activity ◽  
C Terminus

ABSTRACT A Staphylococcus aureus mutant conditionally defective in DNA ligase was identified by isolation of complementing plasmid clones that encode the S. aureus ligA gene. Orthologues of the putative S. aureus NAD+-dependent DNA ligase could be identified in the genomes of Bacillus stearothermophilus and other gram-positive bacteria and confirmed the presence of four conserved amino acid motifs, including motif I, KXDG with lysine 112, which is believed to be the proposed site of adenylation. DNA sequence comparison of the ligA genes from wild type and temperature-sensitive S. aureus strain NT64 identified a single base alteration that is predicted to result in the amino acid substitution E46G. The S. aureus ligA gene was cloned and overexpressed in Escherichia coli, and the enzyme was purified to near homogeneity. NAD+-dependent DNA ligase activity was demonstrated with the purified enzyme by measuring ligation of 32P-labeled 30-mer and 29-mer oligonucleotides annealed to a complementary strand of DNA. Limited proteolysis of purified S. aureus DNA ligase by thermolysin produced products with apparent molecular masses of 40, 22, and 21 kDa. The fragments were purified and characterized by N-terminal sequencing and mass analysis. The N-terminal fragment (40 kDa) was found to be fully adenylated. A fragment from residues 1 to 315 was expressed as a His-tagged fusion in E. coli and purified for functional analysis. Following deadenylation with nicotinamide mononucleotide, the purified fragment could self-adenylate but lacked detectable DNA binding activity. The 21- and 22-kDa C-terminal fragments, which lacked the last 76 amino acids of the DNA ligase, had no adenylation activity or DNA binding activity. The intact 30-kDa C terminus of the S. aureus LigA protein expressed in E. coli did demonstrate DNA binding activity. These observations suggest that, as in the case with the NAD+-dependent DNA ligase fromB. stearothermophilus, two independent functional domains exist in S. aureus DNA ligase, consisting of separate adenylation and DNA binding activities. They also demonstrate a role for the extreme C terminus of the ligase in DNA binding. As there is much evidence to suggest that DNA ligase is essential for bacterial survival, its discovery in the important human pathogen S. aureus indicates its potential as a broad-spectrum antibacterial target for the identification of novel antibiotics.

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