Identification of disulfide bonds in the ninth component (C9) of human complement

Lengweiler, Stephan; Schaller, Johann; Rickli, Egon E.

doi:10.1016/0014-5793(95)01541-8

Cited by 18 publications

(18 citation statements)

References 35 publications

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“…The pattern of disulfide bridges is the same in C8␣ and C8␤ and is analogous to that reported for F-spondin (34). Surprisingly, this pattern (9 -44, 20 -54, and 28 -60 in C8␣; 11-46, 22-56, and 25-62 in C8␤) is different from that reported for C9 (35). The four cysteines with altered disulfide bond connectivity are conserved between C8␣, C8␤, and C9; however, they are fairly close to one another, and the connectivity proposed for C9 may not require an unlikely change of the overall TSP1 fold.…”

Section: Resultscontrasting

confidence: 83%

Structure of Human C8 Protein Provides Mechanistic Insight into Membrane Pore Formation by Complement

Lovelace

Cooper

Sodetz

et al. 2011

Journal of Biological Chemistry

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C8 is one of five complement proteins that assemble on bacterial membranes to form the lethal pore-like "membrane attack complex" (MAC) of complement. The MAC consists of one C5b, C6, C7, and C8 and 12-18 molecules of C9. C8 is composed of three genetically distinct subunits, C8␣, C8␤, and C8␥. The C6, C7, C8␣, C8␤, and C9 proteins are homologous and together comprise the MAC family of proteins. All contain N-and C-terminal modules and a central 40-kDa membrane attack complex perforin (MACPF) domain that has a key role in forming the MAC pore. Here, we report the 2.5 Å resolution crystal structure of human C8 purified from blood. This is the first structure of a MAC family member and of a human MACPF-containing protein. The structure shows the modules in C8␣ and C8␤ are located on the periphery of C8 and not likely to interact with the target membrane. The C8␥ subunit, a member of the lipocalin family of proteins that bind and transport small lipophilic molecules, shows no occupancy of its putative ligand-binding site. C8␣ and C8␤ are related by a rotation of ϳ22°with only a small translational component along the rotation axis. Evolutionary arguments suggest the geometry of binding between these two subunits is similar to the arrangement of C9 molecules within the MAC pore. This leads to a model of the MAC that explains how C8-C9 and C9-C9 interactions could facilitate refolding and insertion of putative MACPF transmembrane ␤-hairpins to form a circular pore. Assembly of the "membrane attack complex" (MAC)3 of complement on the surface of Gram-negative bacteria and other pathogenic organisms involves the sequential interaction of complement proteins C5b, C6, C7, C8, and C9 (1-3). Association of the first four components produces a membranebound tetrameric C5b-8 complex, which then initiates the recruitment and sequential binding of 12-18 C9 molecules to form a cylindrical transmembrane pore (supplemental Fig. S1). Pore formation leads to loss of membrane integrity and lysis of the cell under attack.The sequence of interactions leading to MAC formation is well defined; however, the mechanism by which the MAC disrupts membrane organization is poorly understood. C6, C7, the C8␣ and C8␤ subunits, and C9 are homologous and together comprise the "MAC family" of proteins (4, 5). Until now, structures have not been determined for any of these proteins. All contain N-and C-terminal modules and a central 40-kDa "membrane attack complex/perforin" (MACPF) domain. The MACPF domain was named as such because of sequence similarity between the MAC family proteins and perforin. The modules range in number from three to eight; all are small domains of 40 -60 amino acids that contain multiple disulfide bonds (supplemental Fig. S2). One type of module, thrombospondin type 1 (TSP1), contains several mannosylated tryptophans (6).Several hundred MACPF-containing proteins have been identified; however, functions are known for only a few. MACPF proteins exhibit limited sequence similarity, but all contain the MACPF signature motif ((Y/...

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Section: Resultscontrasting

confidence: 83%

Structure of Human C8 Protein Provides Mechanistic Insight into Membrane Pore Formation by Complement

Lovelace

Cooper

Sodetz

et al. 2011

Journal of Biological Chemistry

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“…A, location of disulfide bonds in human C9 as published by Lengweiler et al (27). E. coli dsbA mutant GJ73 transformed with pYW60 (q) or pYW61 (‚) (B) and dsbB mutants NLM100, transformed with pYW60 (q) (C), and YW10 transformed with pYW57 (ࡗ) or pYW58 (‚) (D) were induced at Ϫ40 min with either anhydrotetracycline or isopropyl ␤-D-thiogalactoside to express periplasmic C9 (closed symbols) or cytoplasmic C9 (open symbols), and at 0 min NaC was added to promote disulfide bond formation.…”

Section: Fig 3 Expression Of C9 In Dsb Mutants and Effect Of N-acetmentioning

confidence: 99%

Molecular Aspects of Complement-mediated Bacterial Killing

Bjes

Esser

2000

Journal of Biological Chemistry

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As part of the membrane attack complex complement protein C9 is responsible for direct killing of bacteria. Here we show that in the periplasmic space of an Escherichia coli cell C9 is converted from a protoxin to a toxin by periplasmic conditions missing in spheroplasts. This conversion is independent of the pathway by which C9 enters the periplasm. Both, C9 shocked into the periplasm and plasmid-expressed C9 targeted to the periplasm via a signal sequence are toxic. Toxicity requires disulfide-linked C9 because export into the periplasm of cells defective in disulfide bond synthesis (dsbA and dsbB mutants) is not toxic unless N-acetylcysteine is added externally to promote cystines. A N-terminal fragment, C9[1-144], is not toxic nor is cytoplasmically expressed C9, even in trxB mutants that are able to form disulfide bonds in the cytoplasm. Importantly, expression of full-length C9 in complement-resistant cells has no effect on their viability. Expression and translocation into the periplasm may provide a novel model to identify molecular mechanisms of other bactericidal disulfide-linked proteins and to investigate the nature of bacterial complement resistance.

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“…a disulphide bridge. Similar bonds have been previously described and the sequence CXXC is found in the active site of To explain the proteolytic susceptibility of H2 which is preferentially cleaved near its C-terminal extremity when IAI was in thiol-disulphide oxydoreductases [19,20]. Using amino acid sequencing and MS-analysis, we demonstrate that Cys215 and vitro incubated in the presence of neutrophil proteinases [5,6], each heavy chain and IAI were separately digested by V-8 proCys218 in H1 and Cys at positions 207 and 210 in H2 are disulphide linked.…”

Section: Peptidesmentioning

confidence: 79%

“…This enzyme was chosen for several reasons: (a) its specificity is very different from that of neutrophil proteinases Disulphide cross-links between adjacent cysteine residues are rarely encountered because the residues involved have a since it hydrolyzes highly specifically peptide bonds at the carboxylic side of the glutamate residues in the conditions used; highly constrained conformation. Such a structure is found in regions implicated in local conformational changes [19,21]. (b) it is less cationic than human leucocyte elastase and thus its possible affinity for the glycosaminoglycan could be less imporConcerning the H2 heavy chain, the vicinal cysteine residues are linked by a disulphide bond.…”

Section: Peptidesmentioning

confidence: 99%

Disulphide bonds assignment in the inter‐α‐inhibitor heavy chains

Flahaut¹,

Mizon²,

Aumercier-Maes³

et al. 1998

European Journal of Biochemistry

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Human inter-A-inhibitor (IAI) is a plasma serine-proteinase inhibitor. It consists of three polypeptide chains covalently linked by a glycosaminoglycan : a light one named bikunin, carrying the antiproteinase activity and two heavy chains H1 and H2. The amino acid sequences of these heavy chains are highly similar; however when IAI is digested by neutrophil proteinases, their proteolytic susceptibility strongly differs [Balduyck, M., Piva, F., Mizon, C., Maes, P., Malki, N., Gressier, B., Michalski, C. & Mizon, J. (1993) Human leucocyte elastase (HLE) preferentially cleaves the heavy chain H2 of inter-A-trypsin inhibitor (ITI), Biol. Chem. 895Ϫ901].We mapped the disulphide topology of the IAI heavy chains in order to investigate whether or not disulphide bonds might be responsible for their differential susceptibility to proteolysis. Using amino acid sequencing and mass spectrometry analysis, we demonstrate that the H1 heavy chain contains one free thiol group and two disulphide bridges of which one links two largely spaced cysteine residues (Cys239 and Cys511). Thus H1 is clearly different from H2 which contains two disulphide bonds between closely located cysteine residues. However, using immunoprint analysis, we show that, when IAI is subjected to a limited digestion by Staphylococcus aureus V-8 proteinase, the two polypeptide chains are similarly susceptible to proteolysis. This enzyme preferentially cleaves the IAI heavy chains from their N-terminal extremity. These results are consistent with the circular dichroism (CD) analysis, suggesting that the conformation of the polypeptide backbone of H1 is not very different from that of H2, with calculated A-helicities of 24% and 28%, respectively. The CD measurements reveal that the aromatic amino acids of H1 and H2 are in a different asymmetrical environment.Inside the IAI molecule, the heavy chains are linked to the glycosaminoglycan chain via their Cterminal aspartic acid residue. Thus we suggest that the affinity of cationic neutrophil proteinases for the anionic glycosaminoglycan is responsible for the cleavage of the heavy chains (mainly H2) near their Cterminal end and the high susceptibility of IAI to these proteinases.Keywords : inter-A-inhibitor; heavy chain; disulfide bond; proteolysis.Inter-A-inhibitor (IAI) is a serine-proteinase inhibitor found that this molecule which is unusually sensitive to proteinases released by triggered neutrophils, would be partially cleaved in in mammalian plasma. IAI is a complex of three different polyacquired diseases in which there is a systemic proteolysis. Thus peptide chains : the first one named bikunin (m ϭ 26000) is a IAI would be converted into smaller size derivatives able to double-headed Künitz-type inhibitor carrying the antiproteinase reach various tissues where they exert their physiological activactivity of IAI. The two other polypeptide chains exhibit higher ity: proteinase inhibition but also for instance modulation of cell molecular masses, near 80 000 Da. For human IAI, they are desproliferation [3]...

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Identification of disulfide bonds in the ninth component (C9) of human complement

Cited by 18 publications

References 35 publications

Structure of Human C8 Protein Provides Mechanistic Insight into Membrane Pore Formation by Complement

Structure of Human C8 Protein Provides Mechanistic Insight into Membrane Pore Formation by Complement

Molecular Aspects of Complement-mediated Bacterial Killing

Disulphide bonds assignment in the inter‐α‐inhibitor heavy chains

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