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Gln Mutant Cystatin C, the Amyloid-Forming Protein

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Gln Mutant Cystatin C, the Amyloid-Forming Protein Proc. Nati. Acad. Sci. USA Vol. 91, pp. 1416-1420, February 1994 Medical Sciences Increased body temperature accelerates aggregation of the Leu-68 -- Gln mutant cystatin C, the amyloid-forming protein in hereditary cystatin C amyloid angiopathy (amyloidosis/brain hemorrhage/cysteine proteinase inhibitor/Escherchia coli expression/site-directed mutagenesis) MAGNUS ABRAHAMSON AND ANDERS GRUBB Department of Clinical Chemistry, University of Lund, University Hospital, S-221 85 Lund, Sweden Communicated by Jan Waldenstrom, September 30, 1993 (receivedfor review June 9, 1993) ABSTRACT Hereditary cystatin C amyloid angiopathy is polypeptide chain of normal cystatin C (7-10). Furthermore, a dominantly inherited disorder, characterized by dementia, genetic studies have proven that the substitution demon- paralysis, and death from cerebral hemorrhage in early adult strated, Leu-68 -- Gin (L68Q), corresponds to a mutation life. A variant of the cysteine proteinase inhibitor, cystatin C, exclusively found in HCCAA patients' DNA (11, 12). It is is deposited as amyloid in the tissues of the patients and their therefore likely that some intrinsic property ofL68Q-cystatin spinal-fluid level of cystatin C is abnormally low. The disease- C is directly responsible for the fatal consequences of the associated Leu-68 -* Gin mutant (L68Q) cystatin C has been disease. The aim of the present investigation was to produce produced in an Escherichia coi expression system and isolated recombinant L68Q-cystatin C and investigate some of its by use of denaturing buffers, immunosorption, and gel fitra- physicochemical and functional properties to elucidate the tion. Parallel physicochemical and functional investigations of molecular pathophysiology of HCCAA. L68Q-cystatin C and wild-type cystatin C revealed that both proteins effectively inhibit the cysteine proteinase cathepsin B (equilibrium constants for dissociation, 0.4 and 0.5 nM, re- EXPERBIENTAL PROCEDURES spectively) but differ considerably in their tendency to dimerize Materials. Wild-type human cystatin C was produced by and form aggregates. While wild-type cystatin C is monomeric expression in Escherichia coli and purified as described (13). and functionally active even after prolonged storage at elevated Affinity-purified human cathepsin B (EC 3.4.22.1) (14) was temperatures, L68Q-cystatin C starts to dimerize and lose purchased from Calbiochem. Papain (EC 3.4.22.2) of high biological activity immediately after it is transferred to a specific activity (activatable to at least 80%) was prepared nondenaturing buffer. The dimerization of L68Q-cystatin C is from the Sigma type III preparation by affinity chromatog- highiy temperature-dependent, with a rise in incubation tem- raphy using Gly-Gly-Tyr-Arg-Sepharose (15). Superdex HR perature from 37 to 40C resulting in a 150% increase in 75 and CNBr-activated Sepharose 4B were purchased from dimerization rate. The aggregation at physiological concentra- Pharmacia LKB Biotechnology AB, centrifugal microcon- tions is likewise increased at 40 compared to 37C, by =60%. centrators (Microsep concentrators with a nominal cutoff- These properties of L68Q-cystatin C have bearing upon our limit of3 kDa) were from Filtron Technology (Northborough, understanding of the pathophysiological process of hereditary MA), and proteinase substrates and enzymes for DNA ex- cystatin C amyloid angiopathy. They might also be of clinical periments were from Bachem and Bethesda Research Lab- relevance, since medical intervention to abort febrile periods of oratories. All other chemicals used were of analytical grade carriers ofthe disease trait may reduce the in vivo formation of and obtained from Sigma. L68Q-cystatin C aggregates. Protein Analyses. Analytical agarose gel electrophoresis and immunoelectrophoresis were performed as described by Hereditary cystatin C amyloid angiopathy (HCCAA), also Jeppsson et al. (16) and Scheidegger (17), respectively. known as "hereditary cerebral hemorrhage with amyloido- SDS/polyacrylamide gel electrophoresis was done as de- sis, Icelandic type," is a dominantly inherited disorder char- scribed by Schagger and von Jagow (18), with separation gels acterized by tissue deposition ofamyloid. The disease results containing 17% (wt/vol) acrylamide. Cystatin C in solution in paralysis and development of dementia due to multiple was quantitated by original (19), or enzyme-amplified (20), strokes, and generally, death from cerebral hemorrhage single radial immunodiffusion or by ELISA (21). Automated before 40 years of age (1). After extraction of the amyloid amino acid sequence analysis and quantitative amino acid material from Icelandic HCCAA patients' brain vessels, it analysis after hydrolysis in 6 M HCl were done by standard was originally demonstrated by protein sequencing that cys- methods (2). Quantitative densitometric scanning of agarose tatin C, a potent cysteine proteinase inhibitor ubiquitous in gel electropherograms was performed using a ScanMaker body fluids (2), is the amyloid-forming protein (3). This has IIXE color scanner from Microtek, a Macintosh Quadra 700 been verified by immunohistochemical studies, which in computer equipped with the Scan Analysis program from addition have demonstrated that the disease is systemic, Biosoft, and standard solutions of recombinant wild-type since cystatin C amyloid deposits are present not only in the cystatin C. brain but also in lymph nodes, spleen, salivary glands, Production of L68Q-Cystatin C. A modified cDNA encod- seminal vesicles, and skin (4-6). ing human cystatin C and the E. coli OmpA signal peptide has Complete sequencing ofthe cystatin C amyloid from a pool been used to construct an expression vector for production of offour HCCAA patients displayed one amino acid difference active cystatin C in E. coli (13, 22). A HindIII-EcoRI between the sequence obtained and that determined at the fiagment containing the entire recombinant cystatin C gene protein, mRNA, or gene level for the 120-residue single was excised from the vector, subcloned in M13mpl8, and subjected to site-directed mutagenesis (23) by using the The publication costs ofthis article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" Abbreviations: HCCAA, hereditary cystatin C amyloid angiopathy; in accordance with 18 U.S.C. §1734 solely to indicate this fact. L68Q-cystatin C, Leu-68 -- Gln mutant cystatin C. 1416 Downloaded by guest on October 1, 2021 Medical Sciences: Abrahamson and Grubb Proc. Natl. Acad. Sci. USA 91 (1994) 1417 oligonucleotide 5'-GACGTGGAGCAGGGCCGAACC-3' der the same conditions (13, 24). When several parameters of (where the underlined residue is the mismatched nucleotide). the expression system were varied to improve the yield, we The correctly mutated fragment, verified by complete nucle- observed that a significantly increased level of periplasmic otide sequencing, was transferred back to the expression cystatin C immunoreactivity could be obtained by decreasing plasmid and introduced into E. coli MC1061 as described in the time of expression at 420C from 3 h to 1 h, indicating that detail elsewhere (24). the recombinant protein was unstable. Induction of expres- To optimize the expression system, its culture density, sion at lower temperatures was, therefore, systematically induction temperature, and incubation time were varied. tested. The optimal result, with an approximate yield of 0.5 Cultures of bacteria containing the L68Q-cystatin C expres- mg of periplasmic L68Q-cystatin C per liter of culture, was sion vector at various optical densities (555 nm) between 1.0 obtained by induction at 38TC for 1 h. and 7.0 were initiated in 0.5 liter of TB medium (25). Isolation of Recombinant L68Q-Cystatin C. Initial attempts Expression was induced at 42, 41, 40, 39, 38, or 370C. The to isolate recombinant L68Q-cystatin C demonstrated that incubation time for the expression-induced cultures was the cystatin C immunoreactivity was distributed in numerous varied from 45 min to 3 h. After expression, the cells were fractions on gel filtration ofperiplasmic extracts stored at 40C subjected to osmotic shock (22), resulting in a 20-ml peri- for a few days. When freshly prepared extracts were ana- plasmic fraction extract from each culture. A protease inhib- lyzed by gel filtration, the main peak of cystatin C immuno- itor mixture concentrate was added to the extracts to 5 mM reactivity was eluted at a position corresponding to that of benzamidinium chloride, 10 mM EDTA, and 0.1% NaN3. dimeric cystatin C. In contrast, virtually all wild-type cystatin Isolation of L68Q-Cystatin C. Monoclonal antibodies C in similar extracts eluted in a position corresponding to that (HCC3) raised against wild-type cystatin C (21) were cova- of monomeric cystatin C. In addition, immunoelectrophore- lently coupled to CNBr-activated Sepharose. Five milliliters sis at pH 8.6 of periplasmic extracts containing recombinant of immunosorbent beads in 0.05 M Tris-HCl (pH 7.4) con- L68Q-cystatin C revealed an apparent charge heterogeneity taining 0.5 M NaCl and 1 mM benzamidinium chloride was of the immunoreactive material, which was distributed in the mixed with 20 ml of periplasmic extract, and the suspension entire ,8 and 'y zones, whereas the immunoreactive material was gently rocked at 40C for 16 h. The immunosorbent beads of similar extracts containing wild-type cystatin C displayed were extensively washed and then eluted with the same the distinct post-y mobility expected.
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