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.

Carl-Michael Nathanson

Research output: ThesisDoctoral Thesis (compilation)

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.
Original languageEnglish
QualificationDoctor
Awarding Institution
  • Division of Clinical Chemistry and Pharmacology
Supervisors/Advisors
  • [unknown], [unknown], Supervisor, External person
Award date2002 Nov 28
Publisher
ISBN (Print)91-628-5491-7
Publication statusPublished - 2002

Bibliographical note

Defence details

Date: 2002-11-28
Time: 10:15
Place: Segerfalkssalen, Wallenberg Neurocentrum, Lund

External reviewer(s)

Name: Johansen, Harald Thideman
Title: Professor
Affiliation: Dept. of Pharmacology, School of Pharmacy, University of Oslo, P.O. Box 1068 Blindern, 0316 Oslo, Norway

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Article: 1. Huh C, Håkansson K, Nathanson C-M, Thorgeirsson UP, Jonsson N, Grubb A, Abrahamson M, Karlsson S. Decreased metastatic spread in mice homozygous for a null allele of the cystatin C protease inhibitor gene. Mol Pathol 1999; 52: 332-40

Article: 2. Werle B, Sauckel K, Nathanson C-M, Bjarnadottir M, Spiess E, Ebert W, Abrahamson M. Cystatins C, E/M and F in human pleural fluids of patients with neoplastic and inflammatory lung disorders. Biol Chem 2002 (in press)

Article: 3. Nathanson C-M, Wassélius J, Wallin H, Abrahamson M. Regulated expression and intracellular localisation of cystatin F in U937 cells. Eur J Biochem 2002 (in press)

Article: 4. Nathanson C-M, Wallin H, Egesten A, Håkansson K, Wassélius J, Abrahamson M. Cystatins F and C are differentially expressed and localised in native human blood cells: cystatin F is distinctly localised in eosinophilic granulocytes. Manuscript

Article: 5. Nathanson C-M, Ni J, Abrahamson M. Cystatin G, the product of one of the cystatin-related genes expressed in human testis, is a potent cysteine peptidase inhibitor: Cloning of cystatin G, H and I cDNA; expression and characterization of cystatin G. Manuscript

Subject classification (UKÄ)

  • Medicinal Chemistry
  • Pharmacology and Toxicology

Keywords

  • Clinical chemistry
  • Klinisk kemi
  • enzyme kinetics
  • eosinophilic granulocytes
  • expression pattern
  • pleural effusion
  • knockout mouse
  • Cysteine peptidase
  • cystatin

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