The N-Terminal Part of the Enzyme Component (C2I) of the BinaryClostridium botulinum C2 Toxin Interacts with the Binding Component C2II and Functions as a Carrier System for a Rho ADP-Ribosylating C3-Like Fusion Toxin

ABSTRACT The binary actin–ADP-ribosylating Clostridium botulinum C2 toxin consists of the enzyme component C2I and the binding component C2II, which are separate proteins. The active component C2I enters cells through C2II by receptor-mediated endocytosis and membrane translocation. The N-terminal part of C2I (C2IN), which consists of 225 amino acid residues but lacks ADP-ribosyltransferase activity, was identified as the C2II contact site. A fusion protein (C2IN-C3) of C2IN and the full-length C3-like ADP-ribosyltransferase from Clostridium limosum was constructed. The fusion protein C2IN-C3 ADP-ribosylated Rho but not actin in CHO cell lysates. Together with C2II, C2IN-C3 induced complete rounding up of CHO and HeLa cells after incubation for 3 h. No cell rounding was observed without C2II or with the original C3-like transferase from C. limosum. The data indicate that the N-terminal 225 amino acid residues of C2I are sufficient to cause the cellular uptake of C. limosum transferase via the binding component of C2II, thereby increasing the cytotoxicity of the C3-like exoenzyme several hundred-fold.

Download Full-text

Characterization of the Enzymatic Component of the ADP-Ribosyltransferase Toxin CDTa from Clostridium difficile

Infection and Immunity ◽

10.1128/iai.69.10.6004-6011.2001 ◽

2001 ◽

Vol 69 (10) ◽

pp. 6004-6011 ◽

Cited By ~ 92

Author(s):

Irene Gülke ◽

Gunther Pfeifer ◽

Jan Liese ◽

Michaela Fritz ◽

Fred Hofmann ◽

...

Keyword(s):

Enzyme Activity ◽

Clostridium Difficile ◽

Full Length ◽

Amino Acid Residues ◽

Terminal Part ◽

Structural Requirement ◽

Toxin Binding ◽

Adp Ribosyltransferase ◽

C2 Toxin ◽

Binding Component

ABSTRACT Certain strains of Clostridium difficile produce the ADP-ribosyltransferase CDT, which is a binary actin ADP-ribosylating toxin. The toxin consists of the binding component CDTb, which mediates receptor binding and cellular uptake, and the enzyme component CDTa. Here we studied the enzyme component (CDTa) of the toxin using the binding component of Clostridium perfringens iota toxin (Ib), which is interchangeable with CDTb as a transport component. Ib was used because CDTb was not expressed as a recombinant protein inEscherichia coli. Similar to iota toxin, CDTa ADP-ribosylates nonmuscle and skeletal muscle actin. The N-terminal part of CDTa (CDTa1–240) competes with full-length CDTa for binding to the iota toxin binding component. The C-terminal part (CDTa244–263) harbors the enzyme activity but was much less active than the full-length CDTa. Changes of Glu428 and Glu430 to glutamine, Ser388 to alanine, and Arg345 to lysine blocked ADP-ribosyltransferase activity. Comparison of CDTa with C. perfringens iota toxin and Clostridium botulinumC2 toxin revealed full enzyme activity of the fragment Ia208–413 but loss of activity of several N-terminally deleted C2I proteins including C2I103–431, C2I190–431, and C2I30–431. The data indicate that CDTa belongs to the iota toxin subfamily of binary actin ADP-ribosylating toxins with respect to interaction with the binding component and substrate specificity. It shares typical conserved amino acid residues with iota toxin and C2 toxin that are suggested to be involved in NAD-binding and/or catalytic activity. The enzyme components of CDT, iota toxin, and C2 toxin differ with respect to the minimal structural requirement for full enzyme activity.

Download Full-text

Expression and partial purification of human pro-opiomelanocortin in Escherichia coli

Journal of Molecular Endocrinology ◽

10.1677/jme.0.0030105 ◽

1989 ◽

Vol 3 (2) ◽

pp. 105-112 ◽

Cited By ~ 5

Author(s):

T. S. Grewal ◽

P. J. Lowry ◽

D. Savva

Keyword(s):

Escherichia Coli ◽

Amino Acid ◽

Fusion Protein ◽

Western Blot ◽

Blot Analysis ◽

Expression Vectors ◽

Partial Purification ◽

Amino Acid Residues ◽

E Coli ◽

Dna Fragment

ABSTRACT A large portion of the human pro-opiomelanocortin (POMC) peptide corresponding to amino acid residues 59–241 has been cloned and expressed in Escherichia coli. A 1·0 kb DNA fragment encoding this peptide was cloned into the expression vectors pUC8 and pUR291. Plasmid pJMBG51 (a pUC8 recombinant) was found to direct the expression of a 24 kDa peptide. The recombinant pUR291 (pJMBG52) was shown to produce a β-galactosidase fusion protein of 140 kDa. Western blot analysis showed that both the 24 kDa and 140 kDa peptides are recognized by antibodies raised against POMC-derived peptides. The β-galactosidase fusion protein has been partially purified from crude E. coli cell lysates using affinity chromatography on p-aminobenzyl-1-thio-β-d-galactopyranoside agarose.

Download Full-text

ADP-Ribosylation of Actin by the Clostridium botulinum C2 Toxin in Mammalian Cells Results in Delayed Caspase-Dependent Apoptotic Cell Death

Infection and Immunity ◽

10.1128/iai.00651-08 ◽

2008 ◽

Vol 76 (10) ◽

pp. 4600-4608 ◽

Cited By ~ 40

Author(s):

Karin Heine ◽

Sascha Pust ◽

Stefanie Enzenmüller ◽

Holger Barth

Keyword(s):

Cell Death ◽

Cell Lines ◽

Mammalian Cells ◽

Clostridium Botulinum ◽

Apoptotic Cell ◽

Apoptotic Cell Death ◽

Adp Ribosylation ◽

Mammalian Cell Lines ◽

Adp Ribosyltransferase ◽

C2 Toxin

ABSTRACT The binary C2 toxin from Clostridium botulinum mono-ADP-ribosylates G-actin in the cytosol of eukaryotic cells. This modification leads to depolymerization of actin filaments accompanied by cell rounding within 3 h of incubation but does not immediately induce cell death. Here we investigated the long-term responses of mammalian cell lines (HeLa and Vero) following C2 toxin treatment. Cells stayed round even though the toxin was removed from the medium after its internalization into the cells. No unmodified actin reappeared in the C2 toxin-treated cells within 48 h. Despite actin being completely ADP-ribosylated after about 7 h, no obvious decrease in the overall amount of actin was observed for at least 48 h. Therefore, ADP-ribosylation was not a signal for an accelerated degradation of actin in the tested cell lines. C2 toxin treatment resulted in delayed apoptotic cell death that became detectable about 15 to 24 h after toxin application in a portion of the cells. Poly(ADP)-ribosyltransferase 1 (PARP-1) was cleaved in C2 toxin-treated cells, an indication of caspase 3 activation and a hallmark of apoptosis. Furthermore, specific caspase inhibitors prevented C2 toxin-induced apoptosis, implying that caspases 8 and 9 were activated in C2 toxin-treated cells. C2I, the ADP-ribosyltransferase component of the C2 toxin, remained active in the cytosol for at least 48 h, and no extensive degradation of C2I was observed. From our data, we conclude that the long-lived nature of C2I in the host cell cytosol was essential for the nonreversible cytotoxic effect of C2 toxin, resulting in delayed apoptosis of the tested mammalian cells.

Download Full-text

Amino acid residues involved in the heparin-binding activity of murine IL-12 in the context of an antibody-cytokine fusion protein

Cytokine ◽

10.1016/j.cyto.2019.04.004 ◽

2019 ◽

Vol 120 ◽

pp. 220-226 ◽

Cited By ~ 1

Author(s):

Rosendo Luria-Pérez ◽

Pierre V. Candelaria ◽

Tracy R. Daniels-Wells ◽

José A. Rodríguez ◽

Gustavo Helguera ◽

...

Keyword(s):

Amino Acid ◽

Fusion Protein ◽

Binding Activity ◽

Amino Acid Residues ◽

Heparin Binding

Download Full-text

Transmembrane signaling in the sensor kinase DcuS ofEscherichia coli: A long-range piston-type displacement of transmembrane helix 2

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1507217112 ◽

2015 ◽

Vol 112 (35) ◽

pp. 11042-11047 ◽

Cited By ~ 15

Author(s):

Christian Monzel ◽

Gottfried Unden

Keyword(s):

Amino Acid ◽

Ligand Binding ◽

Transmembrane Helix ◽

Binding Domain ◽

Cross Linking ◽

Sensor Kinase ◽

Amino Acid Residues ◽

Terminal Part ◽

Type Shift

The C4-dicarboxylate sensor kinase DcuS is membrane integral because of the transmembrane (TM) helices TM1 and TM2. Fumarate-induced movement of the helices was probed in vivo by Cys accessibility scanning at the membrane–water interfaces after activation of DcuS by fumarate at the periplasmic binding site. TM1 was inserted with amino acid residues 21–41 in the membrane in both the fumarate-activated (ON) and inactive (OFF) states. In contrast, TM2 was inserted with residues 181–201 in the OFF state and residues 185–205 in the ON state. Replacement of Trp 185 by an Arg residue caused displacement of TM2 toward the outside of the membrane and a concomitant induction of the ON state. Results from Cys cross-linking of TM2/TM2′ in the DcuS homodimer excluded rotation; thus, data from accessibility changes of TM2 upon activation, either by ligand binding or by mutation of TM2, and cross-linking of TM2 and the connected region in the periplasm suggest a piston-type shift of TM2 by four residues to the periplasm upon activation (or fumarate binding). This mode of function is supported by the suggestion from energetic calculations of two preferred positions for TM2 insertion in the membrane. The shift of TM2 by four residues (or 4–6 Å) toward the periplasm upon activation is complementary to the periplasmic displacement of 3–4 Å of the C-terminal part of the periplasmic ligand-binding domain upon ligand occupancy in the citrate-binding domain in the homologous CitA sensor kinase.

Download Full-text

Optimizing amino acid composition of CHO cell culture media for a fusion protein production

Process Biochemistry ◽

10.1016/j.procbio.2011.03.014 ◽

2011 ◽

Vol 46 (7) ◽

pp. 1423-1429 ◽

Cited By ~ 54

Author(s):

Zizhuo Xing ◽

Brian Kenty ◽

Inna Koyrakh ◽

Michael Borys ◽

Shih-Hsie Pan ◽

...

Keyword(s):

Cell Culture ◽

Amino Acid ◽

Fusion Protein ◽

Amino Acid Composition ◽

Acid Composition ◽

Protein Production ◽

Culture Media ◽

Cho Cell ◽

Cell Culture Media ◽

Cho Cell Culture

Download Full-text

Oligomerization of Hepatitis C Virus Core Protein Is Crucial for Interaction with the Cytoplasmic Domain of E1 Envelope Protein

Journal of Virology ◽

10.1128/jvi.01203-06 ◽

2006 ◽

Vol 80 (22) ◽

pp. 11265-11273 ◽

Cited By ~ 40

Author(s):

Kousuke Nakai ◽

Toru Okamoto ◽

Tomomi Kimura-Someya ◽

Koji Ishii ◽

Chang Kweng Lim ◽

...

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Amino Acid ◽

Fusion Protein ◽

Core Protein ◽

Type I ◽

Amino Acid Residues ◽

Cytoplasmic Loop ◽

The Core ◽

Er Membrane

ABSTRACT Hepatitis C virus (HCV) contains two membrane-associated envelope glycoproteins, E1 and E2, which assemble as a heterodimer in the endoplasmic reticulum (ER). In this study, predictive algorithms and genetic analyses of deletion mutants and glycosylation site variants of the E1 glycoprotein were used to suggest that the glycoprotein can adopt two topologies in the ER membrane: the conventional type I membrane topology and a polytopic topology in which the protein spans the ER membrane twice with an intervening cytoplasmic loop (amino acid residues 288 to 360). We also demonstrate that the E1 glycoprotein is able to associate with the HCV core protein, but only upon oligomerization of the core protein in the presence of tRNA to form capsid-like structures. Yeast two-hybrid and immunoprecipitation analyses reveal that oligomerization of the core protein is promoted by amino acid residues 72 to 91 in the core. Furthermore, the association between the E1 glycoprotein and the assembled core can be recapitulated using a fusion protein containing the putative cytoplasmic loop of the E1 glycoprotein. This fusion protein is also able to compete with the intact E1 glycoprotein for binding to the core. Mutagenesis of the cytoplasmic loop of E1 was used to define a region of four amino acids (residues 312 to 315) that is important for interaction with the assembled HCV core. Taken together, our studies suggest that interaction between the self-oligomerized HCV core and the E1 glycoprotein is mediated through the cytoplasmic loop present in a polytopic form of the E1 glycoprotein.

Download Full-text

Binding and Internalization of Clostridium botulinum C2 Toxin

Infection and Immunity ◽

10.1128/iai.00638-09 ◽

2009 ◽

Vol 77 (11) ◽

pp. 5139-5148 ◽

Cited By ~ 33

Author(s):

Masahiro Nagahama ◽

Tohko Hagiyama ◽

Takashi Kojima ◽

Kouhei Aoyanagi ◽

Chihiro Takahashi ◽

...

Keyword(s):

Clostridium Botulinum ◽

Mdck Cells ◽

Binary Toxin ◽

Akt Inhibitor ◽

Surface Plasmon Resonance Analysis ◽

Akt Phosphorylation ◽

Resonance Analysis ◽

Clostridium Botulinum C2 Toxin ◽

C2 Toxin ◽

Binding Component

ABSTRACT Clostridium botulinum C2 toxin is a binary toxin composed of an enzymatic component (C2I) and a binding component (C2II). The activated binding component (C2IIa) forms heptamers, and the oligomer with C2I is taken up by receptor-mediated endocytosis. We investigated the binding and internalization of C2IIa in cells. The C2IIa monomer formed oligomers on lipid rafts in membranes of MDCK cells. Methyl-beta-cyclodextrin inhibited the binding of C2IIa and the rounding of the cells induced by C2I plus C2IIa. C2I was localized to the rafts in the presence, but not the absence, of C2IIa. Surface plasmon resonance analysis revealed that C2I bound to the oligomer of C2IIa, but not the monomer of C2IIa. C2I and C2IIa were rapidly internalized in the cells. LY294002, a phosphatidylinositol 3-kinase (PI3K) inhibitor, inhibited the internalization of C2IIa in the cells and the rounding activity in the presence of C2I plus C2IIa. Incubation of the cells with C2I plus C2IIa resulted in the activation of PI3K and in phosphorylation of phosphoinositide-dependent kinase 1 and protein kinase B/Akt (Akt), but that with C2IIa alone did not. Akt inhibitor X, an Akt phosphorylation inhibitor, inhibited the rounding activity but not the internalization of C2IIa. The results suggest that the binding of C2I to the oligomer of C2IIa on rafts triggers the activation of the PI3K-Akt signaling pathway and, in turn, the initiation of endocytosis.

Download Full-text

Localization of a collagen-interactive domain of human von Willebrand factor between amino acid residues Gly 911 and Glu 1,365

Blood ◽

10.1182/blood.v70.5.1577.bloodjournal7051577 ◽

1987 ◽

Vol 70 (5) ◽

pp. 1577-1583 ◽

Cited By ~ 2

Author(s):

M Kalafatis ◽

Y Takahashi ◽

JP Girma ◽

D Meyer

Keyword(s):

Amino Acid ◽

Von Willebrand Factor ◽

Binding Sites ◽

Type Iii Collagen ◽

Amino Acid Residues ◽

Terminal Part ◽

Human Type ◽

Von Willebrand ◽

Willebrand Factor ◽

Scatchard Plots

A collagen-binding domain of von Willebrand factor (vWF) has been identified in the central part of the molecule by comparing the binding properties of vWF and Staphylococcus aureus V-8 protease-generated vWF fragments with collagen. The binding of purified human vWF to human type III collagen was found to be specific. At saturation, 38 to 50.2 micrograms of vWF bound per milligram of collagen. Scatchard plots derived from binding isotherms demonstrated the presence of at least two classes of binding sites. Purified vWF was digested with S aureus V- 8 protease into two complementary fragments (SpIII and SpII). SpII, the C-terminal end of vWF (amino acid residues 1,366 to 2,050), was totally devoid of affinity for collagen. Contrarily, purified SpIII, the N- terminal part of vWF (residues 1 to 1,365), totally displaced vWF binding and specifically bound to collagen. At saturation, 25 to 45 micrograms of SpIII bound per milligram of collagen. Scatchard plots demonstrated the presence of a single class of binding sites. SpIII was further digested with the same enzyme to generate SpI, a 52-kilodalton fragment from the C-terminal part of SpIII (residues 911 to 1,365). Spl induced a dose-dependent inhibition of both vWF and SpIII binding to collagen. A series of six monoclonal antibodies against SpIII that completely abolished vWF and SpIII interaction with collagen also bound to SpI. In conclusion, SpI extending between amino acid residues 911 and 1,365 of vWF contains a specific site that interacts with human type III collagen.

Download Full-text

The C Terminus of Component C2II ofClostridium botulinum C2 Toxin Is Essential for Receptor Binding

Infection and Immunity ◽

10.1128/iai.68.8.4566-4573.2000 ◽

2000 ◽

Vol 68 (8) ◽

pp. 4566-4573 ◽

Cited By ~ 53

Author(s):

Dagmar Blöcker ◽

Holger Barth ◽

Elke Maier ◽

Roland Benz ◽

Joseph T. Barbieri ◽

...

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Receptor Binding ◽

Sodium Dodecyl ◽

Cytotoxic Action ◽

Amino Acid Residues ◽

C Terminus ◽

Terminal Amino ◽

Function Relationship ◽

C2 Toxin

ABSTRACT The binary Clostridium botulinum C2 toxin consists of two separate proteins, the binding component C2II (80.5 kDa) and the actin-ADP-ribosylating enzyme component C2I (49.4 kDa). For its cytotoxic action, C2II binds to a cell membrane receptor and induces cell entry of C2I via receptor-mediated endocytosis. Here we studied the structure-function relationship of C2II by constructing truncated C2II proteins and producing polyclonal antisera against selective regions of C2II. An antibody raised against the C terminus (amino acids 592 to 721) of C2II inhibited binding of C2II to cells. The antibody prevented pore formation by C2II oligomers in artificial membranes but did not influence the properties of existing channels. To further define the region responsible for receptor binding, we constructed proteins with deletions in C2II; specifically, they lacked amino acid residues 592 to 721 and the 7 C-terminal amino acid residues. The truncated proteins still formed sodium dodecyl sulfate-stable oligomers but were unable to bind to cells. Our data indicate that the C terminus of C2II mediates binding of the protein to cells and that the 7 C-terminal amino acids are structurally important for receptor binding.

Download Full-text