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John E. Volanakis - One of the best experts on this subject based on the ideXlab platform.
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the structure of C2b a fragment of Complement Component C2 produced during c3 convertase formation
Acta Crystallographica Section D-biological Crystallography, 2009Co-Authors: Vengadesan Krishnan, Kevin Macon, John E. Volanakis, Yuanyuan Xu, Sthanam V. L. NarayanaAbstract:The second Component of Complement (C2) is a multi-domain serine protease that provides catalytic activity for the C3 and C5 convertases of the classical and lectin pathways of human Complement. The formation of these convertases requires the Mg2+-dependent binding of C2 to C4b and the subsequent cleavage of C2 by C1s or MASP2, respectively. The crystal structure of full-length C2 is not yet available, although the structure of its C-terminal catalytic segment C2a has been determined. The crystal structure of the N-terminal segment C2b of C2 determined to 1.8 A resolution presented here reveals the arrangement of its three CCP domains. The domains are arranged differently compared with most other CCP-domain assemblies, but their arrangement is similar to that found in the Ba part of the full-length factor B structure. The crystal structures of C2a, C2b and full-length factor B are used to generate a model for C2 and a discussion of the domain association and possible interactions with C4b during formation of the C4b–C2 complex is presented. The results of this study also suggest that upon cleavage by C1s, C2a domains undergo conformational rotation while bound to C4b and the released C2b domains may remain folded together similar to as observed in the intact protein.
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down regulation of secretion of human Complement Component C2 by the product of an alternatively spliced C2 messenger rna
Journal of Immunology, 1996Co-Authors: Hidekazu Tsukamoto, Albert Tousson, Richard B Marchase, John E. VolanakisAbstract:We have previously described alternatively spliced transcripts of the human C2 gene. Among those, C2delta(17), in which exon 17 has been spliced out, encodes a polypeptide that contains the C4b binding and the CIs cleavage sites of C2, but lacks the serine protease active center. To study the possible function of this variant, we constructed C2delta(17) cDNA by deletional mutagenesis and expressed it transiently in COS cells. Transfected COS cells secreted only trace amounts of the C2delta(17) polypeptide, which had no detectable hemolytic activity and could not be cleaved by CIs. Pulse-chase experiments using [35S]methionine demonstrated that the majority of the 88-kDa C2delta(17) remained intracellular. Control wild-type (wt) C2 in the intracellular compartment consisted of two bands of 93 and 99 kDa, the latter corresponding to mature secreted C2. Intracellular C2delta(17) and only the 93-kDa wt C2 were sensitive to endoglycosidase H, a marker for transport from the endoplasmic reticulum (ER) to the Golgi. Experiments using brefeldin A and double label immunofluorescence staining indicated that C2delta(17) exhibited a typical ER distribution pattern, while wt C2 accumulated in the ER-Golgi intermediate compartment. Secretion of C2 by COS cells cotransfected with wt C2 and C2delta(17) cDNA was significantly decreased compared with that by cells transfected with wt C2 alone. These combined results indicate that C2delta(17) is retained in the ER probably because it is incorrectly folded and that it could down-regulate the expression of the wt C2 gene.
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production and interferon γ mediated regulation of Complement Component C2 and factors b and d by the astroglioma cell line u105 mg
Biochemical Journal, 1992Co-Authors: Scott R Barnum, Y Ishii, A Agrawal, John E. VolanakisAbstract:In this paper, we demonstrate the synthesis of the Complement Component C2 and factors B and D by the human astroglioma cell line U105-MG. All three Components were structurally and antigenically similar to their serum counterparts, as determined by biosynthetic labelling studies or Western blot analysis. Northern blot analysis demonstrated that the mRNAs of all three Components had the same apparent sizes as the equivalent mRNAs from hepatocyte and monocyte cell lines. Interestingly, U105-MG cells produce two C2 transcripts with sizes of approximately 2.8 and 2.3 kb. Interferon-gamma (IFN-gamma) enhanced the expression of C2 and factor B mRNA and protein in a dose- and time-dependent fashion, while factor D expression was refractory to IFN-gamma. IFN-gamma appeared to predominantly enhance the expression of the large (2.8 kb) C2 transcript. Kinetic studies demonstrated peak C2 and factor B expression in 48 h in response to IFN-gamma, similar to the acute-phase response of factor B in serum. These data are the first to demonstrate the synthesis of C2 and factor D by astroglioma cells. Combined with previous reports documenting the synthesis of C3 by astrocytes, our data suggest that endogenous synthesis of Complement proteins, and particularly of alternative pathway activation Components (C3, factors B and D), may play an important role in host defence in the central nervous system.
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Production and interferon-gamma-mediated regulation of Complement Component C2 and factors B and D by the astroglioma cell line U105-MG.
The Biochemical journal, 1992Co-Authors: Scott R Barnum, Y Ishii, A Agrawal, John E. VolanakisAbstract:In this paper, we demonstrate the synthesis of the Complement Component C2 and factors B and D by the human astroglioma cell line U105-MG. All three Components were structurally and antigenically similar to their serum counterparts, as determined by biosynthetic labelling studies or Western blot analysis. Northern blot analysis demonstrated that the mRNAs of all three Components had the same apparent sizes as the equivalent mRNAs from hepatocyte and monocyte cell lines. Interestingly, U105-MG cells produce two C2 transcripts with sizes of approximately 2.8 and 2.3 kb. Interferon-gamma (IFN-gamma) enhanced the expression of C2 and factor B mRNA and protein in a dose- and time-dependent fashion, while factor D expression was refractory to IFN-gamma. IFN-gamma appeared to predominantly enhance the expression of the large (2.8 kb) C2 transcript. Kinetic studies demonstrated peak C2 and factor B expression in 48 h in response to IFN-gamma, similar to the acute-phase response of factor B in serum. These data are the first to demonstrate the synthesis of C2 and factor D by astroglioma cells. Combined with previous reports documenting the synthesis of C3 by astrocytes, our data suggest that endogenous synthesis of Complement proteins, and particularly of alternative pathway activation Components (C3, factors B and D), may play an important role in host defence in the central nervous system.
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site directed mutagenesis of the region around cys 241 of Complement Component C2 evidence for a c4b binding site
Journal of Immunology, 1991Co-Authors: Takahiko Horiuchi, Kevin Macon, Jeffrey A Engler, John E. VolanakisAbstract:We probed the functional significance of the region around Cys-241 in human C2 by testing the hemolytic activity of a series of mutant rC2. Mutant C2 cDNA were constructed by oligonucleotide-directed site-specific mutagenesis and expressed transiently in COS cells. Wild-type rC2 had threefold higher specific hemolytic activity than native serum C2. Substitution of Gly, Ala, or Ser for Cys-241 resulted in a slightly, but significantly, increased activity. In addition, I2 had no effect on the activity of these mutant C2. Substitution of Lys for Gln-243 increased the hemolytic activity by more than two-fold. Increased activity in all cases was due to slower decay rates of the C3 convertase. Finally, substitution of Leu or Ala for Asp-240 or Ser-244, respectively, resulted in more than 100-fold decrease of hemolytic activity. The results suggest that residues 240 to 244 of human C2 represent an important structural determinant of the C4b binding site of C2a. They also confirm that Cys-241 is the residue responsible for the increased activity of C2 reacted with I2.
Scott R Barnum - One of the best experts on this subject based on the ideXlab platform.
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production and interferon γ mediated regulation of Complement Component C2 and factors b and d by the astroglioma cell line u105 mg
Biochemical Journal, 1992Co-Authors: Scott R Barnum, Y Ishii, A Agrawal, John E. VolanakisAbstract:In this paper, we demonstrate the synthesis of the Complement Component C2 and factors B and D by the human astroglioma cell line U105-MG. All three Components were structurally and antigenically similar to their serum counterparts, as determined by biosynthetic labelling studies or Western blot analysis. Northern blot analysis demonstrated that the mRNAs of all three Components had the same apparent sizes as the equivalent mRNAs from hepatocyte and monocyte cell lines. Interestingly, U105-MG cells produce two C2 transcripts with sizes of approximately 2.8 and 2.3 kb. Interferon-gamma (IFN-gamma) enhanced the expression of C2 and factor B mRNA and protein in a dose- and time-dependent fashion, while factor D expression was refractory to IFN-gamma. IFN-gamma appeared to predominantly enhance the expression of the large (2.8 kb) C2 transcript. Kinetic studies demonstrated peak C2 and factor B expression in 48 h in response to IFN-gamma, similar to the acute-phase response of factor B in serum. These data are the first to demonstrate the synthesis of C2 and factor D by astroglioma cells. Combined with previous reports documenting the synthesis of C3 by astrocytes, our data suggest that endogenous synthesis of Complement proteins, and particularly of alternative pathway activation Components (C3, factors B and D), may play an important role in host defence in the central nervous system.
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Production and interferon-gamma-mediated regulation of Complement Component C2 and factors B and D by the astroglioma cell line U105-MG.
The Biochemical journal, 1992Co-Authors: Scott R Barnum, Y Ishii, A Agrawal, John E. VolanakisAbstract:In this paper, we demonstrate the synthesis of the Complement Component C2 and factors B and D by the human astroglioma cell line U105-MG. All three Components were structurally and antigenically similar to their serum counterparts, as determined by biosynthetic labelling studies or Western blot analysis. Northern blot analysis demonstrated that the mRNAs of all three Components had the same apparent sizes as the equivalent mRNAs from hepatocyte and monocyte cell lines. Interestingly, U105-MG cells produce two C2 transcripts with sizes of approximately 2.8 and 2.3 kb. Interferon-gamma (IFN-gamma) enhanced the expression of C2 and factor B mRNA and protein in a dose- and time-dependent fashion, while factor D expression was refractory to IFN-gamma. IFN-gamma appeared to predominantly enhance the expression of the large (2.8 kb) C2 transcript. Kinetic studies demonstrated peak C2 and factor B expression in 48 h in response to IFN-gamma, similar to the acute-phase response of factor B in serum. These data are the first to demonstrate the synthesis of C2 and factor D by astroglioma cells. Combined with previous reports documenting the synthesis of C3 by astrocytes, our data suggest that endogenous synthesis of Complement proteins, and particularly of alternative pathway activation Components (C3, factors B and D), may play an important role in host defence in the central nervous system.
A Agrawal - One of the best experts on this subject based on the ideXlab platform.
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production and interferon γ mediated regulation of Complement Component C2 and factors b and d by the astroglioma cell line u105 mg
Biochemical Journal, 1992Co-Authors: Scott R Barnum, Y Ishii, A Agrawal, John E. VolanakisAbstract:In this paper, we demonstrate the synthesis of the Complement Component C2 and factors B and D by the human astroglioma cell line U105-MG. All three Components were structurally and antigenically similar to their serum counterparts, as determined by biosynthetic labelling studies or Western blot analysis. Northern blot analysis demonstrated that the mRNAs of all three Components had the same apparent sizes as the equivalent mRNAs from hepatocyte and monocyte cell lines. Interestingly, U105-MG cells produce two C2 transcripts with sizes of approximately 2.8 and 2.3 kb. Interferon-gamma (IFN-gamma) enhanced the expression of C2 and factor B mRNA and protein in a dose- and time-dependent fashion, while factor D expression was refractory to IFN-gamma. IFN-gamma appeared to predominantly enhance the expression of the large (2.8 kb) C2 transcript. Kinetic studies demonstrated peak C2 and factor B expression in 48 h in response to IFN-gamma, similar to the acute-phase response of factor B in serum. These data are the first to demonstrate the synthesis of C2 and factor D by astroglioma cells. Combined with previous reports documenting the synthesis of C3 by astrocytes, our data suggest that endogenous synthesis of Complement proteins, and particularly of alternative pathway activation Components (C3, factors B and D), may play an important role in host defence in the central nervous system.
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Production and interferon-gamma-mediated regulation of Complement Component C2 and factors B and D by the astroglioma cell line U105-MG.
The Biochemical journal, 1992Co-Authors: Scott R Barnum, Y Ishii, A Agrawal, John E. VolanakisAbstract:In this paper, we demonstrate the synthesis of the Complement Component C2 and factors B and D by the human astroglioma cell line U105-MG. All three Components were structurally and antigenically similar to their serum counterparts, as determined by biosynthetic labelling studies or Western blot analysis. Northern blot analysis demonstrated that the mRNAs of all three Components had the same apparent sizes as the equivalent mRNAs from hepatocyte and monocyte cell lines. Interestingly, U105-MG cells produce two C2 transcripts with sizes of approximately 2.8 and 2.3 kb. Interferon-gamma (IFN-gamma) enhanced the expression of C2 and factor B mRNA and protein in a dose- and time-dependent fashion, while factor D expression was refractory to IFN-gamma. IFN-gamma appeared to predominantly enhance the expression of the large (2.8 kb) C2 transcript. Kinetic studies demonstrated peak C2 and factor B expression in 48 h in response to IFN-gamma, similar to the acute-phase response of factor B in serum. These data are the first to demonstrate the synthesis of C2 and factor D by astroglioma cells. Combined with previous reports documenting the synthesis of C3 by astrocytes, our data suggest that endogenous synthesis of Complement proteins, and particularly of alternative pathway activation Components (C3, factors B and D), may play an important role in host defence in the central nervous system.
Y Ishii - One of the best experts on this subject based on the ideXlab platform.
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production and interferon γ mediated regulation of Complement Component C2 and factors b and d by the astroglioma cell line u105 mg
Biochemical Journal, 1992Co-Authors: Scott R Barnum, Y Ishii, A Agrawal, John E. VolanakisAbstract:In this paper, we demonstrate the synthesis of the Complement Component C2 and factors B and D by the human astroglioma cell line U105-MG. All three Components were structurally and antigenically similar to their serum counterparts, as determined by biosynthetic labelling studies or Western blot analysis. Northern blot analysis demonstrated that the mRNAs of all three Components had the same apparent sizes as the equivalent mRNAs from hepatocyte and monocyte cell lines. Interestingly, U105-MG cells produce two C2 transcripts with sizes of approximately 2.8 and 2.3 kb. Interferon-gamma (IFN-gamma) enhanced the expression of C2 and factor B mRNA and protein in a dose- and time-dependent fashion, while factor D expression was refractory to IFN-gamma. IFN-gamma appeared to predominantly enhance the expression of the large (2.8 kb) C2 transcript. Kinetic studies demonstrated peak C2 and factor B expression in 48 h in response to IFN-gamma, similar to the acute-phase response of factor B in serum. These data are the first to demonstrate the synthesis of C2 and factor D by astroglioma cells. Combined with previous reports documenting the synthesis of C3 by astrocytes, our data suggest that endogenous synthesis of Complement proteins, and particularly of alternative pathway activation Components (C3, factors B and D), may play an important role in host defence in the central nervous system.
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Production and interferon-gamma-mediated regulation of Complement Component C2 and factors B and D by the astroglioma cell line U105-MG.
The Biochemical journal, 1992Co-Authors: Scott R Barnum, Y Ishii, A Agrawal, John E. VolanakisAbstract:In this paper, we demonstrate the synthesis of the Complement Component C2 and factors B and D by the human astroglioma cell line U105-MG. All three Components were structurally and antigenically similar to their serum counterparts, as determined by biosynthetic labelling studies or Western blot analysis. Northern blot analysis demonstrated that the mRNAs of all three Components had the same apparent sizes as the equivalent mRNAs from hepatocyte and monocyte cell lines. Interestingly, U105-MG cells produce two C2 transcripts with sizes of approximately 2.8 and 2.3 kb. Interferon-gamma (IFN-gamma) enhanced the expression of C2 and factor B mRNA and protein in a dose- and time-dependent fashion, while factor D expression was refractory to IFN-gamma. IFN-gamma appeared to predominantly enhance the expression of the large (2.8 kb) C2 transcript. Kinetic studies demonstrated peak C2 and factor B expression in 48 h in response to IFN-gamma, similar to the acute-phase response of factor B in serum. These data are the first to demonstrate the synthesis of C2 and factor D by astroglioma cells. Combined with previous reports documenting the synthesis of C3 by astrocytes, our data suggest that endogenous synthesis of Complement proteins, and particularly of alternative pathway activation Components (C3, factors B and D), may play an important role in host defence in the central nervous system.
Paolo G V Martini - One of the best experts on this subject based on the ideXlab platform.
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expression and purification methods for the production of recombinant human Complement Component C2
Methods of Molecular Biology, 2014Co-Authors: Angela W Norton, Paolo G V Martini, Lynette C Cook, Scott Alderucci, Dianna Lundberg, Susan M Fish, Charles Bedard, John Gill, Arthur O Tzianabos, Michael F ConcinoAbstract:Abstract Human Complement Component C2 is a critical factor of the classical Complement pathway. Here we provide a method for the production of recombinant human C2 (rhC2) protein for research purposes. The human Complement Component C2 (hC2) is cloned from a human cDNA library by polymerase chain reaction and inserted in a mammalian expression vector (Martini et al., BMC Immunol 11:43, 2010). Transient transfection is utilized to express hC2 in a mammalian cell line, and the expressed C2 is harvested from the conditioned media. rhC2 is purified from the conditioned media by sequential steps of cation exchange and affinity column chromatography. The purified hC2 is characterized for protein purity, stability, and enzymatic activity. The recombinant hC2 activity is tested in a Complement activation ELISA assay that measures classical, alternative, and lectin Complement pathway activity in C2-depleted serum.
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Recombinant human Complement Component C2 produced in a human cell line restores the classical Complement pathway activity in-vitro: an alternative treatment for C2 deficiency diseases
BMC Immunology, 2010Co-Authors: Paolo G V Martini, Angela W Norton, Lynette C Cook, Scott Alderucci, Susan M Fish, Dianna M Lundberg, Knut Langsetmo, Göran Jönsson, Christian Lood, Birgitta GullstrandAbstract:Background Complement C2 deficiency is the most common genetically determined complete Complement deficiency and is associated with a number of diseases. Most prominent are the associations with recurrent serious infections in young children and the development of systemic lupus erythematosus (SLE) in adults. The links with these diseases reflect the important role Complement C2 plays in both innate immunity and immune tolerance. Infusions with normal fresh frozen plasma for the treatment of associated disease have demonstrated therapeutic effects but so far protein replacement therapy has not been evaluated. Results Human Complement C2 was cloned and expressed in a mammalian cell line. The purity of recombinant human C2 (rhC2) was greater than 95% and it was characterized for stability and activity. It was sensitive to C1s cleavage and restored classical Complement pathway activity in C2-deficient serum both in a Complement activation ELISA and a hemolytic assay. Furthermore, rhC2 could increase C3 fragment deposition on the human pathogen Streptococcus pneumoniae in C2-deficient serum to levels equal to those with normal serum. Conclusions Taken together these data suggest that recombinant human C2 can restore classical Complement pathway activity and may serve as a potential therapeutic for recurring bacterial infections or SLE in C2-deficient patients.