title: Expression of recombinant human complement C1q allows identification of the C1r/C1s-binding sites.
abstract: Complement C1q is a hexameric molecule assembled from 18 polypeptide chains of three different types encoded by three genes. This versatile recognition protein senses a wide variety of immune and nonimmune ligands, including pathogens and altered self components, and triggers the classical complement pathway through activation of its associated proteases C1r and C1s. We report a method for expression of recombinant full-length human C1q involving stable transfection of HEK 293-F mammalian cells and fusion of an affinity tag to the C-terminal end of the C chain. The resulting recombinant (r) C1q molecule is similar to serum C1q as judged from biochemical and structural analyses and exhibits the characteristic shape of a bunch of flowers. Analysis of its interaction properties by surface plasmon resonance shows that rC1q retains the ability of serum C1q to associate with the C1s-C1r-C1r-C1s tetramer, to recognize physiological C1q ligands such as IgG and pentraxin 3, and to trigger C1r and C1s activation. Functional analysis of rC1q variants carrying mutations of LysA59, LysB61, and/or LysC58, in the collagen-like stems, demonstrates that LysB61 and LysC58 each play a key role in the interaction with C1s-C1r-C1r-C1s, with LysA59 being involved to a lesser degree. We propose that LysB61 and LysC58 both form salt bridges with outer acidic Ca(2+) ligands of the C1r and C1s CUB (complement C1r/C1s, Uegf, bone morphogenetic protein) domains. The expression method reported here opens the way for deciphering the molecular basis of the unusual binding versatility of C1q by mapping the residues involved in the sensing of its targets and the binding of its receptors.
authors: Commissariat à l'Energie Atomique et aux Energies Alternatives, Direction des Sciences du Vivant, Institut de Biologie Structurale, F-38027 Grenoble, France.
authors: Isabelle
authors: I
authors: Bally
authors: Sarah
authors: S
authors: Ancelet
authors: Christine
authors: C
authors: Moriscot
authors: Florence
authors: F
authors: Gonnet
authors: Alberto
authors: A
authors: Mantovani
authors: Régis
authors: R
authors: Daniel
authors: Guy
authors: G
authors: Schoehn
authors: Gérard J
authors: GJ
authors: Arlaud
authors: Nicole M
authors: NM
authors: Thielens
mesh_terms: D019943:Amino Acid Substitution; D001665:Binding Sites; D002097:C-Reactive Protein / Q000737:chemistry / Q000378:metabolism; D002118:Calcium / Q000378:metabolism; D003167:Complement Activation / Q000502:physiology*; D015922:Complement C1q / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015923:Complement C1r / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D003173:Complement C1s / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015870:Gene Expression; D057809:HEK293 Cells; D006801:Humans; D007074:Immunoglobulin G / Q000737:chemistry / Q000378:metabolism; D020125:Mutation, Missense; D011485:Protein Binding / Q000502:physiology; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D000683:Serum Amyloid P-Component / Q000737:chemistry / Q000378:metabolism; D020349:Surface Plasmon Resonance; D000099153:Pentraxins
chemical_list: D002097:C-Reactive Protein; D002118:Calcium; D015922:Complement C1q; D015923:Complement C1r; D003173:Complement C1s; D007074:Immunoglobulin G; D011994:Recombinant Proteins; D000683:Serum Amyloid P-Component; D000099153:Pentraxins
keywords: C1 complex; innate immunity; protein engineering
references: Behring Inst Mitt. 1989 Jul;(84):8-19
references: 2679537
references: Mol Immunol. 1979 Jul;16(7):445-50
references: 40870
references: J Mol Biol. 1981 May 15;148(2):191-7
references: 6975380
references: Mol Cell Proteomics. 2010 Apr;9(4):593-610
references: 20008834
references: Biochem J. 1978 Sep 1;173(3):863-8
references: 708376
references: Biochem J. 2011 Oct 15;439(2):185-93
references: 21954942
references: J Biol Chem. 2009 Jul 17;284(29):19340-8
references: 19473974
references: J Biol Chem. 2010 Oct 15;285(42):32251-63
references: 20592021
references: Biochemistry. 2005 Feb 22;44(7):2602-9
references: 15709773
references: J Biol Chem. 1997 Dec 26;272(52):32817-23
references: 9407058
references: Structure. 2011 Nov 9;19(11):1635-43
references: 22078562
references: J Biol Chem. 2010 Jun 4;285(23):17681-92
references: 20363749
references: Trends Immunol. 2004 Jul;25(7):368-73
references: 15207504
references: Nature. 2010 Mar 18;464(7287):445-8
references: 20237569
references: Biochem Soc Trans. 2002 Nov;30(Pt 6):1010-4
references: 12440963
references: Eur J Immunol. 2003 Feb;33(2):465-73
references: 12645945
references: J Immunol. 2009 Jun 15;182(12):7708-17
references: 19494295
references: J Biol Chem. 2003 Nov 21;278(47):46974-82
references: 12960167
references: J Immunol. 2001 Dec 1;167(11):6374-81
references: 11714802
references: J Immunol. 2013 Apr 1;190(7):3721-31
references: 23460739
references: J Immunol. 2005 Mar 1;174(5):2870-7
references: 15728497
references: Adv Immunol. 1985;37:151-216
references: 3890478
references: Immunol Lett. 2010 Jul 8;131(2):139-50
references: 20381531
references: Biochem J. 1979 May 1;179(2):367-71
references: 486087
references: Nat Immunol. 2010 Sep;11(9):785-97
references: 20720586
references: Mol Immunol. 2007 Jan;44(1-3):33-43
references: 16908067