Type 2 Cystatins. Studies on the role of the major cystatin, cystatin C, in mice and on properties and distribution of the novel human cystatins F, G, H and I

Abstract: The aim the thesis work was to study the human cystatins C, E/M, F, G, H and I both in vivo and in vitro. A mouse devoid of cystatin C was used for the cystatin C studies. The levels of cystatins C, E/M and F were measured in pleural effusion samples of patients with various lung disorders. Cystatin F protein and RNA levels were measured in the human haematopoietic cell line U937 as well as in cells isolated from human whole blood. The testis specific cystatin-like mRNA:s cystatin G, H and I were cloned and sequenced and cystatin G was expressed in a Pichia pastoris expression system. The recombinant protein was used to determine the inhibition of the cysteine peptidases papain, cathepsin B, H, L and S as well as mammalian legumain. Mice devoid of cystatin C appear phenotypically normal and are fertile. Injection of a metastatic melanoma cell line gives fewer and smaller metastases in the lungs of the cystatin C-/- mice than in normal control mice. Cystatin E/M is significantly elevated in pleural fluid from patients with pleural mesotehliomas compared to both patients with secondary pleural tumours and patients with benign diseases. The cystatin F levels in pleural fluid are significantly elevated in patients with parapneumonia and tuberculosis compared to patients with malign and other benign disorders. The cystatin locus, covering ~1.7 Mb on chromosome 20p11.21, constitutes 13 cystatin genes of which three are pseudogenes. Cystatin F is an inhibitor specifically expressed in haematopoietic cells and its expression is readily regulated with ATRA or TPA. Both substances down-regulate the expression of the inhibitor. A comparably high portion of the protein is retained intracellularly. The inhibitor does not co-localize with ER in immunocytochemical staining but co-elutes with fractions containing elevated levels of the lysosomal enzyme, ß-hexosaminidase, after gradient ultracentrifugation. Among eight tested isolated blood cell types all but B-cells contained cystatin F but only eosinophilic granulocytes, monocytes and neutrophilic granulocytes contained cystatin C in significant amounts. Immunocytochemical staining of cells in whole blood shows that eosinophilic granulocytes gives the strongest cystatin F signal. ELISA quantitation demonstrates that eosinophilic granulocytes contain by far the highest levels of both inhibitors of all tested blood cell types. Recombinant cystatin G can be expressed in Pichia pastoris. The protein is secreted in one long and one short form. The long form was isolated and shown to be a tight-binding inhibitor of papain, the lysosomal cathepsins H, L and S, and of mammalian legumain but not of the lysosomal cathepsin B.