Magnus Abrahamson

Molecular cloning and sequence analysis of cDNA coding for the precursor of the human cysteine proteinase inhibitor cystatin C

Recombinant cystatin C producing clones were isolated from a human placenta λgt11 cDNA library. The cDNA insert of one of the clones, containing 777 base pairs, encodes the complete mature cystatin C (120 amino acids) and a hydrophobic leader sequence of 26 amino acids, indicating an extracellular function of the inhibitor. The deduced protein sequence confirms the protein sequence of cystatin C isolated from human urine, but differs in one position from the sequence of the cystatin C fragment deposited as amyloid in hereditary cerebral hemorrhage with amyloidosis.

Mutation in cystatin C gene causes hereditary brain haemorrhage

Hereditary cystatin C amyloid angiopathy (HCCAA) is an autosomal dominant disorder in which a cysteine proteinase inhibitor, cystatin C, is deposited as amyloid fibrils in the cerebral arteries of patients and leads to massive brain haemorrhage and death in young adults. A full length cystatin C cDNA probe revealed a mutation in the codon for leucine at position 68 which abolishes an Alu I restriction site in the cystatin C gene of HCCAA patients. The Alu I marker has been used to show that this mutation is transmitted only in affected members of all eight families investigated, and that the mutated cystatin C gene causes HCCAA.

Efficient production of native, biologically active human cystatin C by Escherichia coli

A cDNA encoding the mature human cysteine proteinase inhibitor cystatin C was fused to the coding sequence for the Escherichia coli outer membrane protein A signal peptide, and the recombinant gene was expressed in E. coli under the control of the λ PR promoter, an optimized Shine‐Dalgarno sequence and the λ cI 857 repressor. When induced at 42°C, such cells expressed large amounts of recombinant cystatin C. The recombinant protein was isolated in high yield and characterized. All physicochemical properties investigated, including the positions of disulfide bonds, indicated that the E. coli derived cystatin C was identical to cystatin C isolated from human biological fluids, except that the proline residue in position three was not hydroxylated. The recombinant protein displayed full biological activity against papain, cathepsin B and dipeptidyl peptidase I.

Folding-related Dimerization of Human Cystatin C

With the aim to improve our understanding of the structural basis for protein self-association and aggregation, in particular in relationship to protein refolding and amyloid formation, folding-related processes for human cystatin C have been studied. Using NMR spectroscopy together with chromatographic and electrophoretic methods, a self-association process resulting in dimer formation for protein samples treated with denaturing agents as well as for samples subjected to low pH or high temperature conditions could be studied with amino acid resolution. In all three cases, the dimerization involves properly folded molecules and proceeds via the reactive site of the inhibitor, which leads to complete loss of its biological activity. This dimerization process has potential relevance for amyloid formation by the brain hemorrhage-causing Leu-Gln variant of cystatin C. The results also indicate that cystatin C dimerization and inactivation may occur in acidified compartments in vivo, which could be relevant for the physiological regulation of cysteine proteinase activity.

Modulation of invasive properties of murine squamous carcinoma cells by heterologous expression of cathepsin B and cystatin C

Murine SCC‐VII squamous carcinoma cells have the capacity to penetrate reconstituted basement membranes (Matrigel) in vitro. The invasion of Matrigel layers by SCC‐VII cells was significantly reduced by E‐64, a specific inhibitor of lysosomal cysteine proteinases. The cathepsin‐B‐selective E‐64 derivative, CA‐074, inhibited penetration of Matrigel by SCC‐VII cells to the same extent, indicating a major role for this particular lysosomal enzyme in extracellular‐matrix degradation during squamous‐carcinoma‐cell invasion. SCC‐VII cells were stably transfected with a cDNA encoding human procathepsin B, in an attempt to modulate the invasive properties of the cell line. The transfected cells expressed the heterologous gene, secreted increased amounts of procathepsin B and displayed enhanced invasive potential. In vivo, the activity of cathepsin B is strictly regulated by endogenous inhibitors. SCC‐VII cells were therefore also stably transfected with a cDNA encoding human cystatin C, the most potent cysteine‐proteinase inhibitor in mammalian tissues. The expression of this transgene resulted in the production of active recombinant cystatin C and a pronounced reduction in Matrigel invasion. These studies demonstrate that the invasive properties of squamous‐cell carcinomas can be changed by modulation of the balance between cathepsin B and its endogenous inhibitors, and provide further evidence for the involvement of this lysosomal cysteine proteinase in tumour invasion and metastasis. Int. J. Cancer. 83:526–531, 1999.

Decreased metastatic spread in mice homozygous for a null allele of the cystatin C protease inhibitor gene

AIMS: Increased or altered activities of cysteine proteases have been implicated in serious human disorders such as cancer, rheumatoid arthritis, sepsis, and osteoporosis. To improve the current knowledge of the regulatory role of a major mammalian cysteine protease inhibitor, cystatin C, in such disease processes, a cystatin C deficient mouse was generated and characterized. METHODS: The mouse cystatin C gene was inactivated by insertion of a bacterial neo gene through homologous recombination in 129/Sv embryonic stem cells. Embryonic stem cell clones were injected into C57BL/6J blastocysts followed by injection of the blastocysts into pseudopregnant female mice. F1 offspring with agouti coat colour after mating of chimaeric males with C57BL/6J females were examined by DNA analysis, and mice carrying the targeted mutation were intercrossed to obtain homozygous cystatin C deficient (CysC-/-) mice. To study the role of cysteine proteases and their inhibitors in metastasis, the spread of B16-F10 melanoma cells in CysC-/- and wild-type mice was compared. Analysis of the formation of remote metastases was carried out by intravenous injection of beta-galactosidase transfected B16-F10 cells and subsequent determination of cancer cell colonies in the lungs. RESULTS: Cystatin C deficient mice were fertile and showed no gross pathological abnormality up to 6 months of age. Compared with wild-type mice, seven times fewer large metastatic colonies were counted by means of a dissecting microscope in CysC-/- mice two weeks after tail vein injection of B16-F10 cells. At all of eight time points from 15 minutes to two weeks after intravenous injection of tumour cells, the CysC-/- mice had significantly fewer lung metastases. The observed differences were smaller when beta-galactosidase transfected cells were used to allow counting of small colonies. Subcutaneous and intracerebral tumour growth was not different in the CysC-/- mice. CONCLUSIONS: Cystatin C concentrations in vivo might influence metastasis in some tissues. The decreased metastatic spread of B16-F10 cells in CysC-/- mice is the result of both reduced seeding and reduced growth of tumour cells in their lungs.

Levels of neutrophil elastase and cathepsin B activities, and cystatins in human sputum: relationship to inflammation

Sputum samples from 25 patients with bronchiectasis were assayed enzymatically for myeloperoxidase, neutrophil elastase and cathepsin B, and immunologically for cystatin A, cystatin B, cystatin C, cystatin S and kininogen. High myeloperoxidase and neutrophil elastase levels were found in those sputum samples that were assessed visually to be purulent. These samples were also found to contain high levels of cathepsin B activity and cystatin A, but low levels of cystatin S and of the most effective cathepsin B inhibitor, cystatin C. In contrast, sputum samples that were low in myeloperoxidase and neutrophil elastase activities had low levels of cathepsin B and cystatin A, but high cystatin C and S levels. It is concluded that cathepsin B activity in sputum is positively correlated with the degree of inflammation and neutrophil recruitment. Although this may be due in part to reduced amounts of cathepsin B inhibitors, particularly cystatin C, theoretical considerations suggest that factors other than the gross level of inhibitors must be involved in the control of cathepsin B activity.

3D domain-swapped human cystatin C with amyloidlike intermolecular beta-sheets.

Oligomerization of human cystatin C (HCC) leads to amyloid deposits in brain arteries, and this process is greatly accelerated with a naturally occurring L68Q variant. The crystal structures of N‐truncated and full‐length HCC (cubic form) showed dimer formation via three‐dimensional (3D) domain swapping, and this observation has led to the suggestion that an analogous domain‐swapping mechanism, but propagated in an open‐ended fashion, could be the basis of HCC fibril formation. Here we report that full‐length HCC, when crystallized in a new, tetragonal form, dimerizes by swapping the same secondary structure elements but with a very different overall structure generated by the flexibility of the hinge linking the moveable elements. The β‐strands of the β‐cores of the two folding units of the present dimer are roughly parallel, while they formed an angle of about 100° in the previous two structures. The dimers pack around a crystallographic dyad by extending their molecular β‐sheets in an intermolecular context. At the other edge of the molecular β‐sheet, side‐chain–side‐chain hydrogen bonds propagate the β‐structure in the same direction. In consequence, a supramolecular crystal structure is generated, with all the β‐strands of the domain‐swapped dimers being perpendicular to one crystallographic direction. This observation is relevant to amyloid aggregation of HCC, as X‐ray diffraction studies of amyloid fibrils show them to have ordered, repeating structure, consistent with the so‐called cross‐β structure, in which extended polypeptide chains are perpendicular to the fiber axis and form infinite β‐sheets that are parallel to this axis. Proteins 2005.

High-level expression of active human cystatin C in Escherichia coli

Expression of the human cysteine proteinase inhibitor, cystatin C (CysC) in the cytoplasm of Escherichia coli was studied using a cDNA fragment encoding the cysteine proteinase inhibitor controlled by the phage lambda pR/cI857 system. The yield of CysC was low, probably due to proteolytic degradation. By fusing the cysC cDNA to a DNA fragment encoding the signal peptide of the E. coli outer membrane protein A, it was possible to produce a substantial amount of CysC in the periplasm. The processing of the signal peptide was shown to be quantitative and to result in CysC with the correct N-terminal amino acid. Yields higher than 1000 micrograms CysC/ml can be obtained by initiating the product formation at a moderate temperature (40 degrees C) late in an optimized fermentation process. A method that gives selective extraction of the periplasmic proteins and at the same time stabilizes CysC has been used.

Production, characterization and use of monoclonal antibodies against the major extracellular human cysteine proteinase inhibitors cystatin C and kininogen

Murine monoclonal antibodies against the major cysteine proteinase inhibitors of human biological fluids, cystatin C and kininogen, were produced. The cystatin C antibody, HCC3, with a Ka of 2 x 10(7) l/mol, increased the inhibition of papain by cystatin C and was suitable for use in immunoblotting, immunohistochemistry and in the construction of a sensitive sandwich enzyme immunoassay for quantification of cystatin C. It recognized not only free cystatin C but also cystatin C in complexes with cysteine proteinases. The kininogen antibody, HK4, was directed against the third, cysteine proteinase inhibitory domain of the heavy chain of kininogen (Ka = 1 X 10(7) l/mol), but did not influence the papain inhibitory activity of kininogen. It reacted with free kininogen as well as kininogen in complex with cysteine proteinases. Both antibodies could be used for the production of specific immunosorbents.