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- Amano, Mariane T, et al.
(författare)
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Genetic analysis of complement C1s deficiency associated with systemic lupus erythernatosus highlights alternative splicing of normal C1s gene
- 2008
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Ingår i: Molecular Immunology. - : Elsevier BV. - 1872-9142 .- 0161-5890. ; 45:6, s. 1693-1702
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Tidskriftsartikel (refereegranskat)abstract
- Deficiencies of complement proteins of the classical pathway are strongly associated with the development of autoimmune diseases. Deficiency of Clr has been observed to occur concomitantly with deficiency in Cls and 9 out of 15 reported cases presented systemic lupus erythernatosus (SLE). Here, we describe a family in which all four children are deficient in Cls but only two of them developed SLE. Hemolytic activity mediated by the alternative and the lectin pathways were normal, but classical pathway activation was absent in all children's sera. Cls was undetectable, while in the parents' sera it was lower than in the normal controls. The levels of Clr observed in the siblings and parents sera were lower than in the control, while the concentrations of other complement proteins (C3, C4, MBL and MASP-2) were normal in all family members. Impairment of Cls synthesis was observed in the patients' fibroblasts when analyzed by confocal microscopy. We show that all four siblings are homozygous for a mutation at position 938 in exon 6 of the Cls cDNA that creates a premature stop codon. Our investigations led us to reveal the presence of previously uncharacterized splice variants of Cls mRNA transcripts in normal human cells. These variants are derived from the skipping of exon 3 and from the use of an alternative 3' splice site within intron I which increases the size of exon 2 by 87 nucleotides.
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- Andersson, Mattias K., et al.
(författare)
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Extended cleavage specificity of mMCP-1, the major mucosal mast cell protease in mouse - High specificity indicates high substrate selectivity
- 2008
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Ingår i: Molecular Immunology. - : Elsevier BV. - 0161-5890 .- 1872-9142. ; 45:9, s. 2548-2558
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Tidskriftsartikel (refereegranskat)abstract
- Mucosal mast cells are in the mouse predominantly found in the epithelium of the gastrointestinal tract. They express the beta-chymases mMCP-1 and mMCP-2. During nematode infections these intraepithelial mast cells increase in numbers and high amounts of mMCP-1 appear in the jejunal lumen and in the circulation. A targeted deletion of this enzyme leads to decreased ability to expel the intraepithelial nematode Trichinella spiralis. A suggested role for mMCP-1 is alteration of epithelial permeability by direct or indirect degradation of epithelial and endothelial targets, however, no such substrates have yet been identified. To enable a screening for natural substrates we performed a detailed analysis of the extended cleavage specificity of mMCP-1, using substrate phage display technology. In positions P1 and P1' distinct preferences for Phe and Ser, respectively, were observed. In position P2 a high selectivity for large hydrophobic amino acids Phe, Trp and Leu was detected, and in position P2' aliphatic amino acids Leu, Val and Ala was preferred. In positions P3 and P4, N-terminal of the cleaved bond, mMCP-1 showed specificity for aliphatic amino acids. The high selectivity in the P2, P1, P1' and P2' positions indicate that mMCP-1 has a relatively narrow set of in vivo substrates. The consensus sequence was used to screen the mouse protein database for potential substrates. A number of mouse extracellular or membrane proteins were identified and cell adhesion and connective tissue components were a dominating subfamily. This information, including the exact position of potential cleavage sites, can now be used in a more focused screening to identify which of these target molecules is/are responsible for the increased intestinal permeability observed in parasite infected mice.
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- Andersson, Mattias K., et al.
(författare)
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The extended cleavage specificity of the rodent β-chymases rMCP-1 and mMCP-4 reveal major fumctional similarities to the human mast cell chymase
- 2008
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Ingår i: Molecular Immunology. - : Elsevier BV. - 0161-5890 .- 1872-9142. ; 45:3, s. 766-775
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Tidskriftsartikel (refereegranskat)abstract
- In rat and mouse the phylogenetic homologues of the human mast cell alpha-chymase (rMCP-5 and mMCP-5) have lost their chymase activity and instead become elastases. To investigate whether rodents hold enzymes with equivalent function as the primate alpha-chymases, we have determined the extended cleavage specificity of the major connective tissue mast cell beta-chymases in rat and mouse, rMCP-1 and mMCP-4. By using a phage display approach we determined the enzyme/substrate interaction in seven positions, both N- and C-terminal of the cleaved bond. The two proteases were found to display rather similar specificities. Both enzymes prefer Phe in position P1, and aliphatic amino acids are favoured N-terminal of the cleaved bond, i.e. Leu in P2 and Val in P3 and P4. Val and Leu are overrepresented also in positions P1' and P3'. The two enzymes differ clearly only in one position, the P2' residue, where mMCP-4 strongly prefers negatively charged amino acids while rMCP-1 favours Ser. Interestingly, Asp and Glu are often present in position P2' of known substrates for the human chymase. Overall, these two rodent beta-chymases have very similar amino acid preferences as the human chymase, particularly mMCP-4, which most likely have a very similar function as the human chymase. This finding indicates that rodent and primate connective tissue mast cells seem to have relatively similar proteolytic repertoires, although they express different sets of serine proteases.
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- Barrios del Pino, Yvelise, et al.
(författare)
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Clonal repertoire diversification of a neutralizing cytomegalovirus glycoprotein B-specific antibody results in variants with diverse anti-viral properties.
- 2007
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Ingår i: Molecular Immunology. - : Elsevier BV. - 1872-9142 .- 0161-5890. ; 44:5, s. 680-690
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Tidskriftsartikel (refereegranskat)abstract
- Cytomegalovirus induces a chronic infection that in normal individuals is controlled by the immune system. In the case of humoral immunity, epitopes, in particular antigenic domain-1, in glycoprotein B have proven to be important for the induction of virus-neutralizing activity. Such antibodies can exert potent virus-neutralizing activity but can also block neutralizing antibodies from binding. Furthermore, these antibodies differ in their fine recognition of antigenic domain-1 as determined by epitope mapping. By using combinatorial library and phage display technologies we have now isolated a large array of clonally related antibody fragments to understand the origin of this diversity. This procedure allowed us to demonstrate that much of the diversity in functional activity (virus neutralization) and epitope recognition can arise from a single parental molecule through somatic mutation processes. We have thus demonstrated that the clonal diversification of a single antigen-specific clone can account for much of the diversity in antibody anti-viral activity. These findings have implications on the development of a gB-based subunit vaccine, as an effective vaccine preparation need not only to recruit appropriate clones into the immune response but also to evolve them properly so as to maintain an appropriate biological function.
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