title: The functional integrity of the serpin domain of C1-inhibitor depends on the unique N-terminal domain, as revealed by a pathological mutant.
abstract: C1-inhibitor (C1-Inh) is a serine protease inhibitor (serpin) with a unique, non-conserved N-terminal domain of unknown function. Genetic deficiency of C1-Inh causes hereditary angioedema. A novel type of mutation (Delta 3) in exon 3 of the C1-Inh gene, resulting in deletion of Asp62-Thr116 in this unique domain, was encountered in a hereditary angioedema pedigree. Because the domain is supposedly not essential for inhibitory activity, the unexpected loss-of-function of this deletion mutant was further investigated. The Delta 3 mutant and three additional mutants starting at Pro76, Gly98, and Ser115, lacking increasing parts of the N-terminal domain, were produced recombinantly. C1-Inh76 and C1-Inh98 retained normal conformation and interaction kinetics with target proteases. In contrast, C1-Inh115 and Delta 3, which both lack the connection between the serpin and the non-serpin domain via two disulfide bridges, were completely non-functional because of a complex-like and multimeric conformation, as demonstrated by several criteria. The Delta 3 mutant also circulated in multimeric form in plasma from affected family members. The C1-Inh mutant reported here is unique in that deletion of an entire amino acid stretch from a domain not shared by other serpins leads to a loss-of-function. The deletion in the unique N-terminal domain results in a "multimerization phenotype" of C1-Inh, because of diminished stability of the central beta-sheet. This phenotype, as well as the location of the disulfide bridges between the serpin and the non-serpin domain of C1-Inh, suggests that the function of the N-terminal region may be similar to one of the effects of heparin in antithrombin III, maintenance of the metastable serpin conformation.
authors.affiliation: Department of Immunopathology, Sanquin Research at CLB, and Landsteiner Laboratory, Academic Medical Center, University of Amsterdam, 1066 CX Amsterdam, The Netherlands. i.bos@sanquin.nl
authors.forename: Ineke G A
authors.initials: IG
authors.lastname: Bos
authors.forename: Yvonne T P
authors.initials: YT
authors.lastname: Lubbers
authors.forename: Dorina
authors.initials: D
authors.lastname: Roem
authors.forename: Jan Pieter
authors.initials: JP
authors.lastname: Abrahams
authors.forename: C Erik
authors.initials: CE
authors.lastname: Hack
authors.forename: Eric
authors.initials: E
authors.lastname: Eldering
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D015153:Blotting, Western; D002499:Centrifugation, Density Gradient; D003174:Complement C1 Inactivator Proteins; D050718:Complement C1 Inhibitor Protein; D018360:Crystallography, X-Ray; D003545:Cysteine / Q000737:chemistry; D004247:DNA / Q000378:metabolism; D004220:Disulfides / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D005091:Exons; D017353:Gene Deletion; D005998:Glycine / Q000737:chemistry; D006493:Heparin / Q000737:chemistry; D006358:Hot Temperature; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D010641:Phenotype; D010957:Plasmids / Q000378:metabolism; D016133:Polymerase Chain Reaction; D011392:Proline / Q000737:chemistry; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry; D012694:Serine / Q000737:chemistry; D015843:Serpins / Q000737:chemistry*; D013395:Sucrose / Q000494:pharmacology; D013696:Temperature
chemical_list: D003174:Complement C1 Inactivator Proteins; D050718:Complement C1 Inhibitor Protein; D004220:Disulfides; D011994:Recombinant Proteins; C469952:SERPING1 protein, human; D015843:Serpins; D012694:Serine; D013395:Sucrose; D006493:Heparin; D004247:DNA; D011392:Proline; D003545:Cysteine; D005998:Glycine