Molecular cloning, gene structure and expression profile of mouse C1 inhibitor

Abstract

The gene encoding C1 inhibitor, the major control element of activation of the classical pathway of complement and a major inhibitor of several plasma serine proteases, has been studied only in man, where deficiency of C1 inhibitor results in the dominantly transmitted disease hereditary angioedema. Full-length mouse C1 inhibitor cDNA and genomic clones were isolated and characterized as a first step towards the complete characterization of the pattern of C1 inhibitor expression and the production of an animal model of C1 inhibitor deficiency. Restriction-enzyme and sequence analyses of a full-length genomic clone demonstrated that the mouse gene has the same structure as the human homologue, but differs in size (9 kb versus 17 kb), mostly due to the presence of repetitive Alu elements in the human gene. Sequence comparisons in the promoter region indicate important similarities, i.e. the absence of a TATA box, the presence of an initiator sequence encompassing the transcription-start site and of a γ-interferonactivated sequence (GAS) element at position Ϫ124 of the human sequence. A stretch of about 100 nucleotides in intron 1 reveals an unusually high degree of conservation for non-coding sequences and contains non-canonical but conserved tandemly arranged GAS elements at positions 369 and 388 of the human sequence. This finding supports the conclusions of functional studies on the human C1INH gene indicating a role of this region in modulation of transcription by interferons. The profile of C1 inhibitor expression in mouse liver, lung, heart, kidney, spleen and brain was determined by quantitative northern blot analysis.Keywords : complement ; C1 inhibitor ; serpin ; mouse complement ; interferon response.The plasma protein C1 inhibitor (C1inh) belongs to the su-cells have been shown to be the major sites of C1inh synthesis [7, 8]. However, C1inh synthesis has been described in several perfamily of serine-protease inhibitors (serpins) [1, 2] and is the only physiological inhibitor of the activated proteases C1r and extrahepatic cells, such as monocytes, skin fibroblasts, umbelical-vein endothelial cells, platelets and several types of brain C1s of the complement system. Moreover, C1inh is the major inhibitor of plasma kallikrein and coagulation factor XIIa. Like cells [9Ϫ13]. In cell cultures C1inh synthesis is upregulated by a number of cytokines. While interferon γ has the strongest efother serpins, it inactivates target proteases by forming an SDSresistant heat-resistant protease · inhibitor complex through an fect in most of the cell types mentioned above [14Ϫ15], C1inh synthesis is also increased to different extents by interferon A, exposed peptide loop. The sequence of the reactive center contained in this loop determines the specificity toward proteases. interleukin 6 and tumor-necrosis factor A in different cell lines and primary cultures [16Ϫ17]. C1inh homologues have been In humans, genetic and acquired C1inh deficiencies lead to hereditary and acquired angioedema respectively, characterized by pu...

In Vivo Biosynthesis of Endogenous and of Human C1 Inhibitor in Transgenic Mice: Tissue Distribution and Colocalization of Their Expression

“…1). We have previously reported the mouse sequence (accession AF052039) corresponding to the region from Ϫ813 (upstream of the transcription start site) to ϩ650 (intron 2) of the human C1NH gene, and have discussed the striking conservation of regulatory elements in the promoter region and within intron 1 (35). Functional studies also support the important role of intron 1 sequences in determining the transcriptional regulation and particularly the responses to IFNs (47) (our unpublished data).…”

“…We describe in this study the in vivo biosynthesis of C1 inh in transgenic mice that carry all assigned regulatory elements of the human C1NH gene (33)(34)(35).…”

“…Human and mouse C1 inh mRNAs were localized by in situ hybridization using 35 S-labeled antisense RNA probes. In vitro transcription and in situ hybridization were performed according to the methods of Melton et al (45) and Schäfer et al (46), respectively.…”

“…Low-resolution in situ hybridization of spleen and brain sections from transgenic mice using C1 inh probes. The 20-m-thick cryostat sections of spleen (A and B) and forebrain (C and D) from transgenic mice were hybridized with 35 S-labeled cRNA probes specific for mouse (A and C) and human (B and D) C1 inh mRNAs. The control in E shows a transgenic mouse brain hybridized with a sense RNA probe for mouse C1 inh, and the control in F shows brain from a nontransgenic mouse, hybridized with an antisense probe for human C1 inh RNA.…”

“…To reveal human C1 inh, we used a rabbit polyclonal Ab, which does not cross-react with mouse C1 inh (35).…”