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. The two-step anion- buffer but also containing 6 M guanidinium chloride. Frac- exchange/size-exclusion chromatography procedure we rou- tions displaying cystatin C immunoreactivity were pooled, tinely use for isolation ofrecombinant cystatin C variants (13, concentrated to -1 ml by ultrafiltration at 4°C, and applied to 24) could, therefore, not be used for L68Q-cystatin C. Thus, a column (1 x 30 cm) of Superdex HR 75 equilibrated in 0.05 immunoaffinity chromatography using a Sepharose-immo- M ammonium bicarbonate (pH 8.0) containing 1 mM benz- bilized monoclonal antibody was used as an alternative amidinium chloride, at room temperature (flow rate, 0.5 isolation procedure. However, although all immunoreactive ml/min). The protein populations in each fraction were tested material bound to the column, the standard elution condition for cystatin C immunoreactivity by single radial immunodif- (0.1 M glycine buffer, pH 2.2) resulted in slow elution and low fusion and for charge and size homogeneity by agarose and yield of the recombinant protein. But a distinct peak of SDS/polyacrylamide gel electrophoreses, respectively. immunoreactivity and a high yield of L68Q-cystatin C were Assays for Cysteine Proteinase Inhibitory Activity. Concen- obtained by use of a neutral elution buffer containing 6 M trations of inhibitorily active cystatin were determined by guanidinium chloride. After concentration of the eluate, gel titration of papain, standardized with E-64 (26). Continuous filtration at room temperature in 0.1 M ammonium bicarbon- rate assays (27) were used to determine the equilibrium ate buffer (pH 8.0) was used as the final purification step. The constants for dissociation (Kj) of wild-type or L68Q-cystatin main peak of cystatin C immunoreactivity was eluted under C complexes with human cathepsin B, exactly as described these conditions in a position corresponding to that of mo- elsewhere (24, 28-30). nomeric cystatin C. Structural and Functional Characterization of L68Q- Cystatin C. Material of the main peak obtained at the gel RESULTS filtration was analyzed by agarose and SDS/polyacrylamide Production of Recombinant L68Q-Cystatin C. A recombi- gel electrophoreses immediately after completion of the nant cystatin C gene, containing the coding sequence for chromatography. The results demonstrated that it consisted wild-type human cystatin C and the E. coli OmpA signal of a pure protein, indistinguishable in charge and size from peptide (22), was subjected to oligonucleotide-directed mu- recombinant wild-type cystatin C (Fig. 1). Ten steps of tagenesis in phage M13 to replace the Leu codon (CTG) for automated Edman degradation produced the single N-termi- residue 68 with a GOn codon (CAG). DNA sequencing was nal sequence Ser-Ser-Pro-Lys-Pro-Pro-Gly-Arg-Leu-Val-, used to verify that the DNA of a selected phage subclone which is identical to that of wild-type cystatin C and dem- contained the predetermined codon. The mutated cystatin C onstrates correct cleavage of the recombinant protein at the gene fragment was ligated into an expression vector that has expected signal peptide processing site. Quantitative amino earlier been used for efficient production of wild-type cys- acid analysis of the hydrolyzed protein gave a result consis- tatin C with full biological activity in E. coli (13). The tent with that expected for full-length L68Q-cystatin C (data construct, which contains the phage A PR promoter under not shown). In addition, we sequenced the entire cystatin C control of the phage A cI temperature-sensitive repressor gene insert of the expression vector in a sample of the gene, was introduced in E. coli MC1061 cells. Expression bacterial culture used to produce L68Q-cystatin C, with a was induced in a subclone of the transformed bacteria under result demonstrating that no unintentional mutations had conditions that are optimal for wild-type cystatin C produc- occurred in the coding sequence due to accidental mutagen- tion-i.e., induction of expression from a culture of half the esis during the course of our study. Thus, the protocol maximal bacterial density by a temperature shift from 30 to described allows production and isolation of the cystatin C 42°C followed by a 3-h incubation at the higher temperature. variant that is deposited as amyloid in patients with HCCAA. Analysis of the periplasmic fraction revealed that very low The inhibitory activity of freshly isolated L68Q-cystatin C amounts (<0.1 mg/liter of culture) of cystatin C immunore- was studied by titration experiments using a papain solution active material was produced compared to the amounts (-30 of known enzyme concentration and specific activity. A mg/liter of culture) of wild-type cystatin C or five other preparation offreshly isolated L68Q-cystatin C in ammonium single-residue-substituted cystatin C variants produced un- bicarbonate buffer, containing 24.8 ,uM protein according to Downloaded by guest on October 1, 2021 1418 Medical Sciences: Abrahamson and Grubb Proc. Natl. Acad Sci. USA 91 (1994) A + A 'p so .r`

:e',' ': *: f" _- so.,. 20.1

.- :-I .-: -..; 5 X 4 4 2.

I -:Z.( Z. ( M 1 2 1 2 P FIG. 1. Agarose and SDS/polyacrylamide gel electrophoreses of B freshly isolated recombinant L68Q-cystatin C. (A) SDS/polyacryl- amide gel electrophoresis of reduced samples in a 17% gel. (B) *- 0.8 Agarose gel electrophoresis at pH 8.6. The point of application and the anode are marked by an arrow and a plus sign, respectively. Lanes: 1, isolated recombinant wild-type cystatin C; 2, freshly o 0.6 isolated recombinant L68Q-cystatin C; M, molecular mass marker proteins with positions of relevant proteins indicated to the left in 0.4 kDa; P, human blood plasma. .E

a quantitative amino acid analysis, displayed an inhibitorily m< 0.2. active concentration of 20.7 ,AM. This molar inhibitory ac- tivity (83%) is even higher than that determined for native human cystatin C isolated from body fluids using the same 20 40 60 80 100 experimental system (13) and demonstrates that the isolated Temperature, °C recombinant protein had been properly refolded. To further characterize the cysteine proteinase inhibitory FIG. 2. Temperature stability of L68Q-cystatin C. Samples of activity of L68Q-cystatin C, we decided to investigate its isolated L68Q- and wild-type (wt) cystatin C were incubated for 30 interaction with human cathepsin B, since the equilibrium min at various temperatures. (A) Agarose gel electrophoresis at pH constant for dissociation (Kj) ofthe complex between recom- 8.6 of selected samples. The point of application and the anode are binant wild-type cystatin C and this enzyme is relatively high marked by an arrow and a plus sign, respectively. (B) Remaining C of supernatants after (Ki, 0.50 nM; ref. 13), allowing a fast and reliable affinity L68Q-cystatin immunoreactivity sample incubation and centrifugation as determined by single radial immu- determination in continuous-rate assays by use offluorogenic nodiffusion. cathepsin B substrates. The mean estimated K, value for the interaction at pH 6.0 and 37°C of two freshly prepared, formed using wild-type cystatin C dissolved in such a dena- independently isolated L68Q-cystatin C preparations with turing buffer and then desalted. No difference in temperature cathepsin B was 0.4 nM, i.e., marginally lower than the stability for the two preparations of wild-type cystatin C corresponding value for wild-type recombinant cystatin C. It could be seen. Gel filtration of the L68Q-cystatin C sample was observed in the continuous-rate assays, however, that incubated for 30 min at 55°C, which resulted in an almost the steady-state rate of cathepsin B activity reached jinitially complete conversion to the molecular species with a more after addition of L68Q-cystatin C slowly increased at longer anodal mobility, demonstrated that this species represented incubation periods. The enzyme rate was increased by -z5o0 dimerized L68Q-cystatin C (data not shown). Papain titration after incubation for 1 h at 37°C, indicating a successive with the same sample, minimally diluted in the enzyme assay, decrease in inhibitory activity of the L68Q-cystatin C in the revealed that the cysteine proteinase inhibitory activity ofthe assay system. Temperature Stability of L68Q-Cystatin C. The stability of dimerized protein had dropped to below 10%1 of the original that the dimerization process results in freshly isolated recombinant L68Q-cystatin C was compared activity, indicating to that of thp¢ wild-type inhibitor by incubation of samples of complete loss of biological activity. both proteins for 30 min at several temperatures between 25 Quantitation of the cystatin C immunoreactive material in and 96°C with protein concentratioQp Qf 0.2 mg/ml and an the sample supernatants demonstrated a decreased concen- incubation medium of 0.05 M amp onium bicarbonate buffer tration ofboth L68Q-cystatin C and wild-type cystatin C after (pH 8.0). The samples were centrifuged for 2 min at 12,000 x incubation at high temperatures, but whereas the concentra- g immediately after the incubation and the supernatants were tion of wild-type cystatin C was virtually unchanged up to a analyzed by agarose gel electrophoresis and quantitatively temperatlire of 70°C, that of L68Q-cystatin C had dropped tested for cystatin C-immunoreactive material by single ra- significantly at 35°C (Fig. 2B). Thus, the considera le differ- dial immunodiffusion. Agarose gel electrophoresis demon- ence in dimerization tendency between L68Q-cystatin C and strated that L68Q-cystatin C displayed a temperature- wild-type cystatin C seemed to be paralleled by a significantly dependent transformation to a molecular species with a more different tendency of the two protein variants to forpn larger anodal electrophoretic mobility as a result of all incubations insoluble molecular aggregates. above 30°C. This was in contrast to wild-type cystatin C, Kinetics of Dimerization and Aggregation of L68Q-Cystatin which did not display this transformation at any incubation C Under Physiological Conditions. The sigjfcant difference temperature (Fig. 2A). Since the isolation procedure for observed for the dimerization of L68Q-cy~statin C at 35 and wild-type cystatin C, in contrast to that for L68Q-cystatin C, 40°C (Fig. 2A) prompted a detailed study of the dimerization did not involve any step employing a denaturing buffer with and aggregation tendency of this disease-associated cystatin 6 M guanidinium chloride, a control experiment was per- C variant at normal and elevated body temperatures. Samples Downloaded by guest on October 1, 2021 Medical Sciences: Abrahamson and Grubb Proc. Natl. Acad. Sci. USA 91 (1994) 1419 offreshly prepared L68Q-cystatin C and wild-type cystatin C DISCUSSION (0.2 mg/ml) were, therefore, simultaneously incubated at 37 and 40'C in a physiological buffer [0.05 M ammonium bicar- A mutation in the cystatin C gene produces the dominantly bonate (pH 8.0) containing 0.1 M NaCl]. Aliquots were taken inherited disease HCCAA (8, 11, 12). However, the patho- at timed intervals and immediately subjected to agarose gel physiological events relating this mutation to the salient electrophoresis. Densitometric scanning ofthe electrophero- features of the disease (systemic amyloid deposition of cys- grams was used to determine the ratio between monomers tatin C and cerebral hemorrhage) are unknown. Moreover, and dimers in the samples. The tlq2 for transformation of the mechanism by which this mutation results in a dramatic monomeric L68Q-cystatin C into dimers was decreased from and specific decrease in the cerebrospinal fluid level of 33 to 13 min (i.e., by 154%) when the incubation temperature cystatin C (31) is also obscure. As the disease-causing was raised from 37 to 40'C (Fig. 3A). In contrast, no dimer- mutation specifies a variant ofcystatin C with a Leu-68-* Gln ization of wild-type cystatin C could be demonstrated, not replacement (L68Q-cystatin C), a logical step in the elucida- even after an incubation period of 1100 min at 40'C. tion ofthe pathophysiology ofthe disease was to produce the To investigate the rate of formation of large insoluble cystatin C variant and investigate its physicochemical and aggregates of L68Q-cystatin C and wild-type cystatin C, the functional properties. However, although an E. coli expres- disappearance rate ofthe cystatin C immunoreactive material sion system for efficient production of several other cystatin of solutions of these cystatin C variants was determined after C variants as well as wild-type cystatin C has been estab- various incubation periods at 37 and 40'C. A physiological lished, our attempts to use this system for the production of concentration (4 pg/ml) of the cystatin C variants and an the HCCAA-associated cystatin C variant were unsuccessful incubation medium of 0.05 M ammonium bicarbonate buffer until we discovered that the temperature regularly used to (pH 8.0) containing 0.1 M NaCl and bovine albumin (40 induce protein synthesis in the system (420C) resulted in g/liter) were selected for the experiments. Aliquots were aggregation ofthe variant. This observation not only allowed removed daily from the incubation mixtures and centrifuged the production of L68Q-cystatin C by a modified expression for 2 min at 12,000 x g, and the cystatin C immunoreactive system but also indicated that the variant had physicochem- material in the supernatants was quantitated. As calculated ical properties that might be crucial to the pathophysiological from the results (Fig. 3B), the periods required for a 50% process of the disease. Further investigations of freshly reduction ofthe soluble L68Q-cystatin C immunoreactivity at refolded and isolated L68Q-cystatin C demonstrated a pro- 37 and 40°C were 160 and 100 h, respectively, indicating that nounced tendency of the variant to polymerize and precipi- the aggregation rate of L68Q-cystatin C increased by -60% tate even at refrigeration temperatures. Moreover, the pro- as a result of the temperature elevation. No reduction of the cess leading to precipitation displayed a marked temperature soluble wild-type cystatin C immunoreactivity could be dem- dependence resulting in significant aggregation of the variant onstrated, not even after incubation for 1000 h at 40°C. at normal body temperature. It is thus possible that HCCAA belongs to the class of disorders in which temperature- dependent abnormal protein deposition might be a pivotal 0 1 pathophysiological event. This seems, for example, to be the 0r. o 0.8 case for the most common form of cystic fibrosis (32) and for a1-antitrypsin deficiency (33). In analogy with the proposed 0 C4-+ 0.6 pathophysiological processes in these disorders, the cystatin 0 C variant in HCCAA might, because of its tendency to 0 E 0.4 aggregate spontaneously to a significant extent at 370C, be trapped intracellularly and not secreted from the cell as efficiently as wild-type cystatin C. Continuous intracellular U 0.2 accumulation of cystatin C aggregates would eventually lead at to cell damage and death. The impaired secretion of the cystatin C variant would also explain the decreased cerebro- spinal fluid level of cystatin C in HCCAA. Indeed, it has been Time, min observed that monocytes isolated from HCCAA patients secrete significantly less cystatin C than monocytes from healthy individuals (34). Although HCCAA is a systemic .2 disease with deposition of cystatin C in many body tissues (5, CZC) 6), the deposition in the central nervous system vasculture is 0 particularly pronounced and probably causes the iterated 0 cerebral hemorrhages of the disorder. The high degree of cystatin C deposition in the cerebral vasculture could be explained by a particularly high rate of cystatin C synthesis E; by cells in this tissue or by a less-efficient system for removal a- of abnormal insoluble aggregates. An additional pathogenetic mechanism for the cerebral hemorrhages in HCCAA has been offered (35). It suggests 0 50 100 150 200 250 300 350 that the hemorrhages are caused by a local excess of cysteine Time, h proteinases that might result from the low cerebrospinal fluid concentration of cystatin C in HCCAA patients. The ob- FIG. 3. Kinetics of L68Q-cystatin C dimerization and aggregation served functional properties of L68Q-cystatin C are also at body temperatures. Samples from incubations of L68Q-cystatin C compatible with this hypothesis since the apparent first step in physiological buffer at 37 and 40'C were analyzed by agarose gel of the aggregation process, the dimerization, rapidly and electrophoresis, with densitometric scanning of electropherograms the used to determine the amount of remaining monomeric cystatin C (A) completely abolishes the inhibitory activity displayed by and immunochemical analysis by ELISA of the remaining soluble variant in monomeric form. They also indicate that the low cystatin C (B), at each time point. The incubation mixtures in A total cysteine proteinase inhibitory capacity of cerebrospinal contained L68Q-cystatin C at 0.2 g/liter and those in B had L68Q- fluid of HCCAA patients (35) might be due not only to its low cystatin C at 4 mg/liter. total concentration of cystatin C (31) but also to the fact that Downloaded by guest on October 1, 2021 1420 Medical Sciences: Abrahamson and Grubb Proc. Nad. Acad Sci. USA 91 (1994)

a large proportion of the secreted L68Q-cystatin C is present 7. Grubb, A. & Lofberg, H. (1982) Proc. Natd. Acad. Sci. USA 79, as nonmonomeric inhibitorily inactive forms in the fluid. 3024-3027. A particularly interesting observation concerning the tem- 8. Ghiso, J., Jensson, 0. & Frangione, B. (1986) Proc. Natl. Acad. perature-dependent spontaneous aggregation ofthe HCCAA Sci. USA 83, 2974-2978. cystatin C variant is that a temperature elevation from 37 to 9. Abrahamson, M., Grubb, A., Olafsson, I. & Lundwall, A. rate the variant (1987) FEBS Lett. 216, 229-233. 40°C increases the aggregation of by =150%o. 10. Abrahamson, M., Olafsson, I., Palsdottir, A., Ulvsback, M., This strongly suggests that efforts to avoid extended periods Lundwall, A., Jensson, 0. & Grubb, A. (1990) Biochem. J. 268, offever might reduce the velocity ofthe aggregation process, 287-294. which might be the crucial pathophysiological event in the 11. Palsdottir, A., Abrahamson, M., Thorsteinsson, L., Arnason, development of HCCAA. Since carriers of the allele for A., Olafsson, I., Grubb, A. & Jensson, 0. (1988) Lancet H, L68Q-cystatin C can easily, and with a sensitivity and 603-604. specificity of 100%o, be identified (12), medical intervention to 12. Abrahamson, M., Jonsdottir, S., Olafsson, I., Jensson, 0. & reduce body temperature seems to be an immediately avail- Grubb, A. (1992) Hum. Genet. 189, 377-380. able measure that might be applied in an effort to delay the 13. Abrahamson, M., Dalb0ge, H., Olafsson, I., Carisen, S. & HCCAA disease process. Grubb, A. (1988) FEBS Lett. 236, 14-18. 14. Rich, D. H., Brown, M. A. & Barrett, A. J. (1986) Biochem. J. A recent report describes a similar accelerated aggregation 235, 731-734. as the result of a slight temperature increase from 37 to 41°C 15. Funk, M. O., Nakagawa, Y., Skochdopole, J. & Kaiser, E. T. for the Z variant of ai-antitrypsin (33). By in vitro experi- (1979) Int. J. Peptide Protein Res. 13, 296-303. ments, the mechanism for this aggregation was demonstrated 16. Jeppsson, J.-O., Laurell, C.-B. & Franzdn, B. (1979) Clin. to be an ordered polymerization process. The corresponding Chem. 25, 629-638. in vivo consequence was intracellular formation of large 17. Scheidegger, J. J. (1955) Int. Arch. Allergy Appl. Immunol. 7, ar-antitrypsin aggregates and impeded transport out from 103-110. hepatocytes, leading to a markedly increased incidence of 18. SchAgger, H. & von Jagow, G. (1987) Anal. Biochem. 166, liver disease in individuals homozygous for the a1-antitrypsin 368-379. 19. Mancini, G., Carbonara, A. 0. & Heremans, J. F. (1965) Z variant encoding allele. The molecular mechanism of the Immunochemistry 2, 235-254. L68Q-cystatin C aggregation is most likely different from that 20. Lofberg, H. & Grubb, A. 0. (1979) Scand. J. Clin. Lab. Invest. of Z a1-antitrypsin, however, since the two proteins display 39, 619-626. gross structural differences, although both are proteinase 21. Olafsson, I., Ldfberg, H., Abrahamson, M. & Grubb, A. (1988) inhibitors. Scand. J. Clin. Lab. Invest. 48, 573-582. Ifthe crucial pathophysiological process in HCCAA is the 22. Dalb0ge, H., Bech Jensen, E., T0ttrup, H., Grubb, A., Abra- aggregation of L68Q-cystatin C, as the present investigation hamson, M., Olafsson, I. & Carlsen, S. (1989) Gene 79, implies, the logical treatment modality is to inhibit or slow 325-332. down this process. To design effective treatments, 23. Zoller, M. J. & Smith, M. (1982) Nucleic Acids Res. 10, including 6487-6500. the use of antipyretic drugs, the aggregation process must be 24. Hall, A., Dalb0ge, H., Grubb, A. & Abrahamson, M. (1993) further characterized at the molecular, cellular, and organ- Biochem. J. 291, 123-129. ismic levels. 25. Sambrook, J., Fritsch, E. F. & Maniatis, T. 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