©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
COOH-terminal Substitutions in the Serpin C1 Inhibitor That Cause Loop Overinsertion and Subsequent Multimerization (*)

(Received for publication, March 3, 1994; and in revised form, November 17, 1994)

Eric Eldering (§) Elisabeth Verpy Dorina Roem (1) Tommaso Meo Mario Tosi

From the Unité d'Immunogénétique, Institut Pasteur, INSERM Unit 276, 25 rue du Dr Roux, 75724 Paris Cedex 15, France Department of Autoimmunediseases, Central Laboratory of the Red Cross Blood Transfusion Service, Plesmanlaan 125, Amsterdam 1066 CX, The Netherlands

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The region COOH-terminal to the reactive center loop is highly conserved in the serine protease inhibitor (serpin) family. We have studied the structural consequences of three substitutions (Val Met, Phe Ser, and Pro Ser) found in this region of C1 inhibitor in patients suffering from hereditary angioedema. Equivalent substitutions have been described in alpha1-antitrypsin and antithrombin III. The mutant C1 inhibitor proteins were only partially secreted upon transient transfection into COS-7 cells and were found to be dysfunctional. Immunoprecipitation of conditioned media demonstrated that in the intact, uncleaved form they all bind to a monoclonal antibody which recognizes specifically the protease-complexed or reactive center-cleaved normal C1 inhibitor. A second indication for an intrinsic conformational change was the increased thermostability compared to the normal protein. Furthermore, gel filtration studies showed that the Val Met and Pro Ser mutant proteins, and to a lesser extent Phe Ser, were prone to spontaneous multimerization. Finally, a reduced susceptibility to reactive center cleavage by trypsin was observed for all three mutants, and the cleaved Val Met and Pro Ser mutants failed to adopt the conformation recognized by a cleavage-specific monoclonal antibody. Investigation of plasmas of patients with the Val Met or Pro Ser substitutions showed that these dysfunctional proteins circulate at low levels and are recognized by the complex-specific antibody. These results strongly indicate a conformational change as a result of these carboxyl-terminal substitutions, such that anchoring of the reactive center loop at the COOH-terminal side is not achieved properly. We propose that this results in overinsertion of the loop into beta-sheet A, which subsequently leads to multimerization.


INTRODUCTION

C1 inhibitor is a member of the serine protease inhibitor (serpin) (^1)family. The inhibitory members of the serpins in human plasma are important regulators of the coagulation, fibrinolytic and complement cascades, and of proteases released from activated neutrophils (see (1, 2, 3) for reviews). They share a common structure, and the exact structural and kinetic mechanism of inhibition is the subject of considerable investigation and continuing debate. Attempted proteolytic attack on the so-called P1-P1` peptidyl-bond (Schechter and Berger nomenclature, (4) ) in the reactive center results in the formation of very stable complexes between inhibitor and target protease. The reactive center is located in the COOH-terminal part on an exposed loop(5) , which upon complete reactive center cleavage recedes into the central beta-sheet A as the fourth of six strands(6) . This structural transition induces an irreversible change to a more stable form, the so-called S R transition, which can be monitored by an increase in thermostability (5) , conformation-specific monoclonal antibodies(7, 8) , CD spectra, and resistance to unfolding in denaturants(9) . The ability of the reactive center loop to incorporate is an essential requirement for inhibitory function. Experimental evidence for the proposal (10) that complex formation of a serpin with target proteases involves partial insertion of the loop into beta-sheet A has been obtained recently for antithrombin III(11) .

Studies with serpin mutants have focused on the P14-P10 hinge region at the base of the loop, which is highly conserved and contains predominantly alanines in inhibitory serpins(12) . Substitutions are found in this region in the non-inhibitory serpins ovalbumin and angiotensinogen (13) and also in dysfunctional mutants of antithrombin III (ATIII) and C1 inhibitor in patients with thrombosis (14, 15) and hereditary angioedema (HAE) type II(10, 16) , respectively. Mutations at P14 or P12, which were presumed to obstruct correct fitting into underlying structures during incorporation of the loop (10) , turn the serpin into a substrate for target proteases. Recent evidence (17) indicates that such mutations may not necessarily prevent loop incorporation, but cause a shift in the branched reaction pathway (18) from inhibitory to substrate-type reaction. The effect of mutations at P10 can be diverse; an Ala Ser substitution in ATIII results in dysfunction toward thrombin while factor Xa is still inhibited(19) . An engineered P10 Ala Pro mutation in alpha1-antitrypsin (alpha1-AT) does also result in substrate-type behavior, but again it does not prevent the S R transition, and consequently, insertion of the loop(20) . Finally, a C1 inhibitor P10 Ala Thr mutant does not inhibit, nor is cleaved by, target proteases C1s, kallikrein and factor XIIa(16) . This mutant was recently shown to be in a conformation recognized by a complex-specific monoclonal antibody and to be prone to multimer formation(21) .

The potential of serpins to form multimers by non-covalent intermolecular loop to sheet bonding has been recognized recently. This phenomenon occurs upon heating in intact, normal serpins (22) and is in fact responsible for the observed thermal ``instability,'' as the multimers escape immunological detection (see e.g.(23) ), or are removed upon centrifugation. Multimerization can also occur after mild treatment with denaturants (24, 25) or after cleavage at P8 or P9 in alpha1-AT(22) . Furthermore, multimerization of intact alpha1-AT occurs in certain mutants at 37 °C, notably the common alpha1-AT-Z variant with a Glu Lys mutation at the turn leading into strand 4A(25) , and in the rare Siiyama (Ser Phe) and Malton (Phe deleted) variants(26) . The latter mutations are located in or near helix B, which underlies the central beta-sheet A. The affected residues were predicted to play a role in opening of the A-sheet (27) by providing a ridge along which sheet 3A can slide.

The present study is concerned with three substitutions in the region COOH-terminal to the reactive site in C1 inhibitor; Val Met, Phe Ser, and Pro Ser. These residues are equivalent to Phe, Phe, and Pro in alpha1-AT, respectively (12) . The substitutions were identified in a screening for mutations in exon 8 of the C1 inhibitor gene in HAE patients and are located at generally highly conserved residues in serpins. Similar mutations at equivalent positions have been found in alpha1-AT and ATIII(28, 29, 30, 31) . In general, they affect secretion of the mutant protein, and this was also found for the three C1 inhibitor mutants, as illustrated by the reduced levels of C1 inhibitor in the patients plasma. The observation that they are, however, secreted to a certain extent in vitro(32) allowed investigation of the structural consequences of each substitution. Evidence will be presented that, although they are not in the direct vicinity of the reactive center loop or other parts proposed to be involved in conformational changes(27) , these substitutions affect loop insertion and also favor multimerization.


MATERIALS AND METHODS

Enzymes and Reagents

Trypsin (type XIII, Tos-Phe-CH(2)Cl treated), soybean trypsin inhibitor (SBTI), phenylmethanesulfonyl fluoride (PMSF), and 3,3`,5,5`-tetramethyl benzidine were purchased from Sigma. Aliquots of trypsin stock (1 mg/ml) were frozen in liquid N(2) and used only once. 4-(Amidinophenyl)-methanesulfonyl fluoride (APMSF) was from Boehringer (Mannheim, Germany). [S]Methionine and ^14C-methylated protein markers were obtained from the Radiochemical center (Amersham, United Kingdom). Purified human plasma C1 inhibitor was a gift from Behringwerke (Marburg, Germany) and activated C1s was obtained from a Cohn I fraction of human plasma by anion exchange chromatography on a DEAE-Sephacel column, as described before(7, 33) .

Patient Plasmas

Blood samples were collected in siliconized vacutainer tubes (Becton Dickinson, Plymouth, U.K.). SBTI (100 µg/ml), benzamidine (10 mM), and EDTA (10 mM) were added to prevent in vitro activation of the complement and contact systems. Tubes were centrifuged at 1,300 times g for 10 min, and plasma was aliquoted and stored at -70 °C until testing.

COS-7 Transfection, Metabolic Labeling, Immunoprecipitation, and SDS-PAGE

The expression plasmid for transient expression of C1 inhibitor has been described(34) . Vectors containing the mutations coding for the substitutions Val Met, Phe Ser, and Pro Ser were constructed as described by Verpy et al.(32) . The standard abbreviation for these substitutions will be used: the one-letter code for the substituted amino acid followed by the residue number and the new amino acid. C1 inhibitor mutants will be designated by the suffix r for recombinant, followed by the amino acid substitution, e.g. rV451M. COS-7 transfection was done with the DEAE-dextran method(34, 35) . Metabolic labeling was done after the first harvest of conditioned medium, i.e. 72 h post-transfection, with 50 µCi/ml of [S]methionine for 4 h and an overnight chase, as described(33) . After harvesting, Tween-20 (0.02% v/v) and NaN(3) (0.02% w/v) were added. To compensate for the lower levels of secreted mutant proteins, labeled conditioned media of rV451M and rP476S were twice, and that of rF455S four times the volume of C1 inhibitor wild-type in immunoprecipitation experiments. Samples were precleared with nonspecific rabbit immunoglobulins adsorbed onto protein A-Sepharose (Pharmacia, Uppsala, Sweden) for 2-4 h at 4 °C. Specific immunoprecipitations were performed overnight at 4 °C with monoclonal antibodies (mAbs) coupled to Sepharose 4B (Pharmacia). mAb RII binds all forms of C1 inhibitor (native, complexed, and cleaved), mAb KOK12 reacts with complexed and cleaved normal C1 inhibitor, and mAb KII is specific for cleaved, inactivated C1 inhibitor(7, 36, 37) . SDS-PAGE was performed on 7.5% gels under non-reducing conditions, followed by impregnation with Enhance (DuPont NEN) and fluorography.

Temperature Stability Assays, Gel Filtration, and ELISA

Heat stability tests were done as described(13, 21) . After heating for 2 h at different temperatures, samples containing recombinant C1 inhibitor were centrifuged at 16,000 times g for 20 min and subsequently tested for C1 inhibitor antigen by ELISA (see below).

Gel filtration was performed on TSK G3000 SW and G4000SW columns (TosoHaas, Montgomeryville, PA) coupled to an Applied Biosystems HPLC system (Warrington, U.K.). The buffer used was phosphate-buffered saline, pH 7.4 (PBS), 0.02% (v/v) Tween-20, 0.02% (w/v) NaN(3), and the flow rate was 0.5 ml/min. Marker proteins were thyroglobulin (667 kDa), apoferritin (443 kDa), beta-amylase (200 kDa), albumin (68 kDa), and ovalbumin (46 kDa); the void volume was monitored with dextran blue (>2000 kDa). Unlabeled transfection media were concentrated 10-20-fold by repeated centrifugation (4500 times g) at 4 °C in Centricon-10 devices (Amicon, Beverly, MA) and stored at -20 °C. Concentration was necessary to obtain a measurable response in ELISA upon gel filtration. When analyzing recombinant C1 inhibitor, 50 µl of wild-type (rC1 inh-wt) concentrates was injected onto the gel filtration column, and 100 µl of recombinant C1 inhibitor mutants. With whole plasma, 10 µl of normal plasma and 25 µl of HAE plasma were diluted to 100 µl in running buffer and injected. Collection of fractions was started at 9 min after injection, just before elution of the void volume. Fractions were collected with an E-6000c dropcounter/collector (Ismatec, Zürich, Switzerland) and were stored in microtiter plates at 4 °C until testing the following day.

ELISAs using mAb RII or KOK12 as catching antibody were done as described(38) . Briefly, microtiter plates were coated with 2 µg/ml mAb in PBS overnight at 4 °C. All subsequent incubations were done at room temperature. Blocking was done with 3% (w/v) bovine serum albumin in PBS for 30-45 min. Aliquots of HPLC fractions up to 100 µl were incubated for 1.5 h followed by four washes with PBS, 0.1% (v/v) Tween-20. Detection of adsorbed C1 inhibitor antigen was with biotinylated rabbit polyclonal anti-C1 inhibitor antibodies (1 h), followed by a 30-min incubation with streptavidin-coupled horseradish peroxidase (Amersham), both diluted 1:1000 in PBS, 0.1% (v/v) Tween-20, 0.2% (w/v) gelatin. Development was with 3,3`,5,5`-tetramethylbenzidine and reactions were stopped after 5 to 10 min with 2 M H(2)SO(4).


RESULTS

Expression

Secreted rC1 inh species were quantitated 72-h post-transfection by ELISA using mAb RII. The substitutions did not affect binding to mAb RII, which was confirmed by comparing the results obtained with RII with those of an assay with only polyclonal antibodies. Levels of rV451M and rP476S were generally 30-50% of the wild-type protein, which was present at 1-1.5 µg/ml (not shown). Secretion of rF455S was 5-10% compared to the normal protein.

Binding to Complex-specific Monoclonal Antibody KOK12

Metabolically labeled normal C1 inhibitor and the three COOH-terminal mutants were incubated with or without activated C1s, and immunoprecipitated with mAb KOK12, which recognizes the complexed and cleaved forms of the normal protein(36) . As expected, rC1 inh-wt was only recognized by mAb KOK12 after interacting with C1s (Fig. 1, lanes 1 and 2), which yielded predominantly C1s-C1 inhibitor complexes of 180 kDa, and a minor fraction of cleaved C1 inhibitor of 97 kDa. In contrast, the mutant C1 inhibitor proteins bound efficiently to mAb KOK12 and were dysfunctional toward C1s (Fig. 1, lanes 3-8). Only very faint complex bands could be observed with rV451M and rP476S, while rF455S displayed some complex formation, but this still represented only a small fraction of the total. The possibility that rF455S formed SDS-labile complexes could be excluded by immunoprecipitation with a mAb specific for C1s (not shown). Thus, all three C1 inhibitor mutants are intrinsically in a conformation recognized by a complex-specific mAb (but not by a cleavage-specific mAb, see below). Since in the case of ATIII immunological evidence has linked the protease-complexed state with insertion of the reactive center loop(11) , this suggested that the loop of the C1 inhibitor mutants might also be in a partially incorporated state.


Figure 1: Incubation of recombinant C1 inhibitor mutants with C1s and immunoprecipitation with mAb KOK12. [S]Methionine-labeled transfection media were incubated with or without C1s (1.5-2 µg/ml final concentration) for 2.5 h at 37 °C. PMSF was then added to a final concentration of 5 mM and, after preclearing, specific immunoprecipitation was done with mAb KOK12-Sepharose. After washing and heating at 95 °C for 3 min, samples were subjected to SDS-PAGE on 7.5% gels under non-reducing conditions. Recombinant C1 inhibitor wild-type (wt) or mutant species are indicated at the top of the figure. Lane M contains ^14C marker proteins, and molecular mass is indicated in kDa on the left. Df denotes the dye front. The lanes containing rF455S derive from a separate experiment and are relatively overexposed to visualize the complex with C1s.



Thermostability

Treatment of ATIII with approx1 M guanidine-HCl at 4 °C induces a latent or so-called L-state, but at 37 °C results predominantly in the formation of multimers,(25, 39) . In the L-state the reactive center loop is proposed to be overinserted into beta-sheet A(11, 40) , and the molecule displays intermediate thermostability compared to the intact and reactive center cleaved forms. We therefore tested whether the conformational change of the C1 inhibitor mutants coincided with increased thermostability. In Fig. 2, it can be seen that this is indeed the case. When incubated at 60 °C for 2 h, the recombinant normal C1 inhibitor is no longer detectable by ELISA. The mutant proteins consistently remained detectable up to 60 °C and disappeared from solution only at 70 °C. Since an increase in stability of a serpin is typically observed upon loop or peptide insertion into beta-sheet A(22, 40, 41) , the moderate but reproducible increase in thermostability of the intact rC1 inhibitor mutants pointed to an inherent partial insertion of the reactive center loop beyond that of the normal protein.


Figure 2: Thermostability of recombinant C1 inhibitor species. Aliquots of transfection media (100 µl of rC1 inh-F455S, 50 µl of other C1 inhibitor species) were incubated at the indicated temperatures for 2 h. After centrifugation at 16,000 times g for 20 min, total C1 inhibitor antigen in the supernatant was determined by ELISA using mAb RII as catching antibody. Results are expressed as percentage antigen remaining in solution relative to a sample incubated at 37 °C. Symbols represent normal C1 inh (wt, circles), rV451M (plus), rP476S (squares), and rF455S (triangles).



Gel Filtration and Native PAGE

The findings described above for the COOH-terminal mutants were similar to those reported for a C1 inhibitor P10 Ala Thr mutant (A436T)(21) . This particular reactive center loop mutant is prone to multimerization, and this prompted us to investigate the COOH-terminal C1 inhibitor mutant proteins on gel filtration columns. Purified plasma C1 inhibitor eluted as a sharp peak on TSK G3000SW and G4000SW columns, between the 443- and 200-kDa marker proteins (see also Fig. 5A). Semilogarithmic plots of various runs indicated an apparent mass of approximately 315 kDa, much larger than the currently accepted value of 75 kDa(42) . This anomalous behavior of C1 inhibitor on gel filtration columns has been noted before (43) and is most probably the result of the elongated shape of C1 inhibitor(42, 44) , causing it to appear in the effluent considerably sooner than globular proteins. On both TSK G3000SW and G4000SW, rC1 inh-wt gave an ELISA profile of a single broad peak at the position corresponding with the purified plasma protein. In Fig. 3, the gel filtration profile on the TSK G3000SW column of the three C1 inhibitor mutants shortly after concentration of unlabeled transfection media is shown. Both the rV451M and rP476S preparations contained a fraction of high molecular weight which eluted at the void volume position. The majority of the molecules eluted at the same position as rC1 inh-wt and purified plasma C1 inhibitor. In contrast, rF455S appeared almost exclusively as a single peak at monomer position. Significantly, the elution profile as analyzed by mAb KOK12 ELISA (Fig. 3B) reproduced that of RII, indicating that the conformation recognized by KOK12 was present in both the low and high molecular weight forms of the mutants. In subsequent gel filtration experiments, a G4000SW column was used, in order to evaluate better the size of the high molecular weight fraction. It was observed that the ratio of multimers to monomers varied between different batches of transfection media and tended to increase upon storage and repeated freeze-thaw cycles. In general, rV451M and rP476S behaved comparably, while rF455S displayed a distinctly lower tendency to form multimers. The shift from monomeric to multimeric forms, as well as the relative size of the multimers, is depicted in Fig. 4. In this experiment, where concentrated preparations were subjected to gel filtration after repeated freeze-thaw cycles, rP476S appeared predominantly in multimeric form, with a shoulder at monomer position. The use of the G4000SW column allowed an estimate of the size of the multimers as being in the 750 to 1000 kDa range. The rF455S mutant was still predominantly in monomeric form, but multimers of similar size as with rP476S were now also present. The multimer peaks of the mutant rC1 inh preparations consistently eluted in fraction 9, i.e. 13.5-13.7 min after injection, indicating that the size of the multimers does not increase beyond a certain point. When tested in the KOK12-ELISA at a 1:4 dilution, rC1 inh-wt fractions showed no reaction (Fig. 4B). The multimeric fractions of rP476S and rF455S reacted to the same extent as in the RII-ELISA. At the monomer position, a response was observed, but it was slightly lower than with mAb RII, indicating that not all mutant monomers were in the conformation recognized by mAb KOK12 (see also below).


Figure 5: Gel filtration and native PAGE of heat-induced multimers of purified plasma C1 inhibitor. Purified plasma C1 inhibitor in PBS was incubated at 50 or 55 °C for 1 h at a concentration of 10 or 1 mg/ml. Aliquots were subsequently injected onto the G4000SW column (A), or subjected to native PAGE on 4-15% gradient gels (B). Gel filtration profile of samples containing 20 µg of C1 inhibitor protein incubated at 55 °C at original concentration of 10 mg/ml is indicated by a broken line, at 1 mg/ml by a stippled line, and of 1 mg/ml C1 inhibitor incubated at 37 °C by an unbroken line. The scale on the left indicates minutes after injection. Elution times (in min) of markers during a preceding run were as follows: dextran blue, 10.0; thyroglobulin, 15.07; apoferritin, 16.9; beta-amylase, 18.5; ovalbumin, 20.26. Samples subjected to native PAGE contained 4 µg of C1 inhibitor protein and were visualized by Coomassie staining. Lane 1, 1 mg/ml C1 inhibitor incubated at 37 °C. Lane 2, 1 mg/ml C1 inhibitor incubated at 50 °C. Lane 3, 1 mg/ml C1 inhibitor incubated at 55 °C. Lane 4, 10 mg/ml C1 inhibitor incubated at 55 °C. Lane M contains marker proteins.




Figure 3: Gel filtration of recombinant C1 inhibitor species on G3000SW column. Transfection media were concentrated 15-fold (rC1 inh-wt, rV451M, and rP476S) or 10-fold (rF455S) by ultrafiltration, and separated over a TSK G3000SW column at a flow rate of 0.5 ml/min. Fractions of 4 drops (82-85 µl; one fraction equivalent to approx10 s) were collected, starting at 9 min after injection. After addition of 20 µl of PBS, 0.1% (v/v) Tween-20, 100 µl was tested in ELISA for total C1 inhibitor antigen using mAb RII (A), and subsequently in ELISA with the complex-specific mAb KOK12 (B). Position of the void volume (>667 kDa) and elution of marker proteins is indicated at the top. rC1 inh-wt eluted as a single peak at the same position as the purified plasma protein (not shown for clarity, see also Fig. 4). Response of rP476S is indicated by closed squares, rV451M by plus symbols, and rF455S by closed triangles. Color reaction was overdeveloped, which caused high responses to reach a maximum and display significant tailing.




Figure 4: Gel filtration of recombinant C1 inhibitor species on G4000SW column. Transfection media were concentrated 15-fold (rC1 inh-wt and rP476S), or 20-fold (rF455S), and separated over a G4000SW column. Fractions of 12 drops (250-260 µl; one fractions equalled approximately 31 s) were collected, and tested in RII-ELISA (A), or KOK12-ELISA (B). Fractions of rC1 inh-wt were tested at a 1:4 dilution in a 100 µl volume, and rC1 inh mutants were tested at 100 µl undiluted. The position of the void volume (>2000 kDa) and elution of marker proteins is indicated at the top. Response of rC1 inh-wt is indicated by open circles, rP476S by closed squares, and rF455S by closed triangles.



Attempts to purify and visualize S-labeled C1 inh mutants by native gel electrophoresis in separate monomeric and multimeric forms were unsuccessful, due to the low amounts of these secretion deficient proteins available, and, more importantly, their tendency to multimerize during manipulation. Therefore, heat-induced multimers of purified plasma C1 inhibitor were used as a reference to estimate the degree of multimerization. Upon incubation for 1 h at 55 °C at a concentration of 10 mg/ml, purified plasma protein eluted from the G4000SW column mainly at the void volume position (geq2000 kDa), and at 1 mg/ml a broad multimer peak at 1000-2000 kDa position was obtained (Fig. 5A). The majority of multimers obtained at 10 mg/ml C1 inhibitor were too large to enter the separating gel when subjected to native PAGE (Fig. 5B, lane 4). Based on the migration of marker proteins, the smallest C1 inhibitor multimer that was clearly visible appeared to be a trimer. The size of the multimers obtained at 1 mg/ml appeared to range from 4 to 10 C1 inhibitor molecules (Fig. 5B, lane 3), and these eluted from the gel filtration column with a broad peak at 12.07 min. It could be deduced, therefore, that the average size of the mutant rC1 inhibitor multimers is smaller than this because they eluted at approximately 13.5 min (see Fig. 4, fraction 9).

Comparison of Reactivity with mAbs RII and KOK12

To address the question to which extent the C1 inhibitor species are recognized by mAb KOK12, fresh transfection media were tested immediately upon harvesting in parallel ELISAs with mAbs RII or KOK12. In Fig. 6A, it can be seen that rC1 inh-wt yielded a typical dilution curve with mAb RII. With mAb KOK12, a low response was observed at higher levels of medium tested. This response represented approximately 5% of the total amount as detected with mAb RII and was not due to complexed or cleaved forms present. It most probably represents a fraction of molecules which, due to the mobility of the reactive center loop, adopts a conformation with a partially inserted loop which is subsequently recognized by mAb KOK12. This monomeric fraction can indeed sometimes be observed on SDS-gels upon immunoprecipitation with mAb KOK12(21) , or testing of gel filtration fractions at low dilution. (^2)In contrast with the normal protein, the dilution curves in mAb RII and KOK12 ELISA of the C1 inhibitor mutants were almost superimposable (Fig. 6, B and C), or only moderately shifted (Fig. 6D), indicating that in fresh, unconcentrated media which can be expected to contain a minimal amount of multimers (cf.Fig. 3), the great majority of molecules had a conformation recognized by mAb KOK12. Averaged over several experiments, the fraction of C1 inhibitor species recognized by mAb KOK12 was 5-7% for the normal protein, 70-90% for rV451M, 90-100% for rP476S, and 50-75% for rF455S. In addition, these results and ratios were confirmed upon parallel immunoprecipitations of fresh S-labeled media, which showed that binding of the mutants to both RII and KOK12 is virtually quantitative (not shown). Thus, these experiments demonstrated that even a fraction of rC1 inh-wt, which was never in multimer form upon gel filtration, can adopt a conformation recognized by mAb KOK12. In the mutant proteins this conformation is intrinsically present in the majority of molecules before the onset of extensive multimerization.


Figure 6: Quantitative comparison of rC1 inhibitor species in RII and KOK12 ELISA. Fresh transfection media were tested in 2-fold serial dilutions in ELISA with mAb RII (bullet) or mAb KOK12 (times). To ensure identical conditions, the top half of the microtiter plates was coated with RII, and the bottom half with KOK12. The outcome of a similar experiment where Tween-20 was omitted from the media or the sample incubation step of the ELISA was identical (not shown).



Trypsin Cleavage and Immunoprecipitation with mAb KII

Based on the previous experiments, the properties of rF455S appeared to be slightly different from rV451M and rP476S. This was further investigated by trypsin cleavage and immunoprecipitation with mAb KII. Trypsin is a non-target protease which readily cleaves and thereby inactivates normal C1 inhibitor at the P1-Arg residue(45, 46) . The resulting complete insertion of the reactive center loop in beta-sheet A is specifically recognized by mAb KII(7) , as can be seen in Fig. 7, lanes 1-3. At a trypsin concentration of 0.05 µg/ml, rC1 inh-wt was completely converted to the 97 kDa cleaved form. Increasing the concentration to 5 µg/ml yielded a further reduction to approximately 86 kDa, which is due to secondary cleavage at the NH(2)-terminal side of the protein(46) . For all three mutants, the susceptibility to trypsin cleavage was reduced, as evidenced by the presence of uncleaved material at 0.05 µg/ml trypsin. Complete cleavage, resulting in a mixture of 97 and 86 kDa forms, was observed at 5 µg/ml trypsin. Significantly, binding of the mutant proteins to mAb KII was also different; rV451M and rP476S did hardly bind at all (bottom panel, lanes 5, 6, 8, and 9). rF455S appeared to expose the epitope for mAb KII partially upon cleavage (lanes 11 and 12), as the bands immunoprecipitated by mAb KII were weaker than those seen with RII. In addition, with rF455S only the lower band of the 97-86-kDa doublet was immunoprecipitated by KII at 5 µg/ml trypsin (compare lanes 11 and 12 of the upper and lower panels), indicating that the 97 kDa band in lane 11 represented a mixture of NH(2) terminally and reactive center cleaved material, and that the 97 kDa band in lane 12, upper panel, represents residual NH(2) terminally cleaved C1 inhibitor. This suggested that the order of cleavage by trypsin of the mutant proteins may be different from the normal protein. Thus, although all three mutants contain a P1-Arg which was less accessible to trypsin than in the normal protein, there was a clear divergence in the recognition by the cleavage-specific mAb KII, indicative of structural differences between rF455S and the other two mutants.


Figure 7: Incubation of recombinant C1 inhibitor mutants with trypsin and immunoprecipitation with mAbs RII and KII. Metabolically labeled transfection media were incubated with or without trypsin at a final concentration of 0.05 or 5 µg/ml for 2 h at 37 °C. SBTI and APMSF were then added to final concentrations of 10 µg/ml and 100 µM, respectively. After preclearing, samples were split in two and immunoprecipitated with mAb RII-Sepharose (top panel) or mAb KII-Sepharose (bottom panel). C1 inhibitor species and trypsin concentrations are indicated at the top. Additional bands in lane 10, top panel, apart from the C1 inhibitor band at 104 kDa, are ^14C marker proteins accidentally carried over from the marker lane M at the left.



Studies with HAE Plasmas

In order to validate the results obtained with the recombinant C1 inhibitor mutants, the plasmas of the HAE patients from which they derived were re-examined. Plasmas from the families containing the P476S and the V451M mutations were available for study. The results of relevant C1 inhibitor parameters are summarized in Table 1. Both families had low functional and antigenic levels of C1 inhibitor, symptomatic of type I HAE(47, 48) . However, all three patients displayed a modest discrepancy between functional and antigenic levels; the latter were 25-35% higher. This was not due to increased levels of circulating cleaved or complexed C1 inhibitor, which were well within the range found in normal individuals (49) . Furthermore, in a radioimmunoassy with mAb KOK12 as catching antibody and polyclonal anti-C1 inhibitor as detecting reagent(33) , the patient plasmas showed a significantly higher response compared to a healthy subject. The lack of a well-defined reference of known concentration precluded exact calculation of the levels of KOK12-reactive C1 inhibitor. The averaged response in this assay was 3.2-3.8 times higher compared to normal plasma. This difference gains in significance when it is considered that the total amount of C1 inhibitor antigen present was at least 3-fold lower. When fractionated over the G4000SW column immediately after thawing, and tested with mAb RII, both normal and HAE plasma showed a predominant peak at monomeric position (Fig. 8A). A shoulder at higher molecular mass was visible in all three plasmas, which may represent the low amount of circulating C1s-C1 inh complexes (Table 1), as the position corresponded with the elution of complexes between purified C1 inhibitor and C1s (results not shown). The same fractions were assayed with mAb KOK12, and the HAE plasmas showed prominent peaks at monomer position, in contrast to plasma from a healthy sibling (Fig. 8B). Collectively, these results indicated strongly that in vivo the dysfunctional mutant C1 inhibitor proteins are also partially secreted and that they circulate in plasma mainly in monomeric form. Next, the normal and HAE plasmas were incubated for 1 h at 37 °C and subjected to gel filtration. A pronounced increase in high molecular weight C1 inhibitor was observed in the HAE plasmas, yielding in the case of plasma containing C1 inhibitor-V451M a broad distribution ranging from the void volume to monomeric C1 inhibitor (Fig. 8C). In the KOK12-ELISA, the elution profile of the HAE plasmas incubated at 37 °C mirrored that of the RII-ELISA, while the normal plasma, as expected, showed a low response (not shown). The majority of the multimers that accumulated in HAE plasma were smaller than those obtained with the recombinant proteins, suggesting that the presence of the normal C1 inhibitor protein or an unknown factor may be limiting. In support of this, the size of the multimers formed in plasma did not increase when plasma was incubated at 37 °C for 5 h.




Figure 8: Gel filtration of normal and HAE plasma. Plasma samples were injected onto the G4000SW column immediately after thawing (A and B) or after incubation at 37 °C for 1 h (C). 48 fractions of 125-130 µl (one fraction equivalent to 15-15.5 s) were collected and tested in 1:5 dilution in the RII-ELISA (A and C), or KOK12-ELISA (B). Results with plasma from a healthy member of family 30 are indicated by open circles, a HAE patient from family 30 (P476S substitution) by closed squares, and a HAE patient from family 10 (V451M) by plus symbols. Note that color development in the different assays was not arrested at identical times, and responses in the different panels cannot quantitatively be compared.




DISCUSSION

Of the approximately 30 residues forming the COOH-terminal region in serpins, consisting of s1C, s4B, and s5B (P362-P391 in alpha1-AT), 17 are strictly or highly conserved in the entire protein family(12) . F455 and P476 in C1 inhibitor correspond to residues F370 and P391 in alpha1-AT and are strictly conserved, V451 is less well conserved and corresponds to F366 in alpha1AT and an alanine in ovalbumin. These residues are located in close proximity in the three-dimensional structure, forming a hydrophobic stack in the core of the protein (see Fig. 9, and below). The importance of this region has already been inferred from studies on stop- and frameshift mutations in alpha1-AT(50, 51) , and substitutions in ATIII(29, 31) , which prevent or significantly reduce secretion. More recently, clues that substitutions in this region produce conformational changes emerged from studies with ATIII mutants(30, 52) . However, a direct role for the conserved COOH terminus of serpins in determining reactive center loop conformation or in inducing multimerization has not been proposed before. The somewhat unexpected findings reported here with three different C1 inhibitor mutants may provide further insight into the nature of the structural changes involved.


Figure 9: Local environment of the substituted residues. Depicted is a Calpha-trace of the COOH-terminal region of cleaved alpha1-AT(6) , showing predominantly beta-sheet B in the plane of the picture and beta-sheet C in perpendicular view to the left. The side chains and van der Waals surfaces of the residues which correspond with the substitutions in C1 inhibitor studied here are shown (F366, F370, and P391), as are the side chains of F208 and V388 which contribute to the conserved hydrophobic pocket. The position of the P1` residue S359 is indicated. For clarity, beta-sheet A and helices A, B, and D are removed. Coordinates of cleaved alpha1-AT were obtained from the Protein Data Bank (entry 7API) and were visualized with InsightII software (Biosym, San Diego, CA).



The results with conformation-specific mAbs and the thermostability assay are strong indications for an intrinsic conformational change in the C1 inhibitor mutants. When taken separately, neither binding to mAb KOK12 nor the increase in thermostability might be considered unequivocal proof for loop overinsertion, since unknown changes affecting other regions of the protein could in principle result in the same properties. The combination of these two observations, however, is highly suggestive of insertion of the reactive center loop as the underlying event, since it has been postulated and experimentally demonstrated that loop insertion is linked to both complex formation and increased stability(10, 11, 22, 40, 53) . Similarly, the presence of high molecular weight material in the C1 inhibitor mutant preparations could be explained by nonspecific aggregation resulting from misfolding and subsequent exposure of hydrophobic regions, rather than by the more ordered process of loop to sheet multimerization. Such misfolded proteins are, however, unlikely to be secreted(54) , and aggregated material would not be expected to be limited in size, as was observed with these C1 inhibitor mutants. Based on these considerations, and current concepts of serpin structure and function (2, 3, 11) , we favor the explanation that the substitutions in C1 inhibitor cause overinsertion of the reactive center loop, which subsequently renders the protein prone to intermolecular multimerization. Conceivably, the equivalent substitutions which have been found in ATIII and alpha1-AT mutants (28, 29, 30, 31) may have the same effect. In support of this is the decreased heparin affinity of several ATIII mutants(31) , which is also observed when the reactive center loop is inserted(53) .

Since the mutant C1 inhibitor proteins tended to multimerize, it could be put forward that intrinsic binding to mAb KOK12 and the increased thermostability are properties that merely reflect the presence of pre-existing multimers. The available data argue against this and instead point to a direct relationship between loop overinsertion and multimer formation. First, intrinsic binding to mAb KOK12 is an important indicator of a conformation resembling the complexed state. The notion of a conformational change during complex formation which involves a partially inserted loop is at the center of current models of serpin structure and function(10, 11) . Second, recognition by mAb KOK12 of the C1 inhibitor mutants preceded multimerization, as demonstrated by the monomer fractions on gel filtration of both recombinant and natural C1 inhibitor mutants (Fig. 3, Fig. 4, and Fig. 8). Furthermore, in ELISA and immunoprecipitation experiments of fresh media, binding to mAb KOK12 is virtually quantitative, and even a fraction of native normal rC1 inhibitor, which does not spontaneously multimerize, can be detected. Lastly, no mechanism other than loop or exogenous peptide insertion into beta-sheet A is known at present which would explain the increase in thermostability (Fig. 2). The concept of thermal instability as solely representing a structural disintegration at elevated temperatures has been refined recently. In the case of alpha1-AT, it is now known that as temperature increases, the process of multimerization itself occurs simultaneously with a loss of detectable antigen(25) . Significantly, alpha1-AT-Z (E342K), which spontaneously multimerizes at 37 °C, has a similar thermal transition point as the normal alpha1-AT-M(25) , indicating that pre-existing multimers do not obligatory alter the thermostability. Therefore, a shift in stability profile most probably results from an intrinsic conformational change which is in principle independent of multimerization. How this change might lead to multimerization, and the difference between these multimers and heat-induced multimers of the normal protein, is addressed below in more detail.

The C1 inhibitor mutant proteins were predominantly retained intracellularly. Pulse-chase experiments demonstrated that rV451M and rP476S accumulated in the cell during an 8-h chase(32) , but that the intracellular fraction of rF455S was degraded.^2 Clearly, correct folding of C1 inhibitor is dependent on the affected residues, and this is achieved only when this cluster of conserved residues is able to anchor the reactive center loop at the COOH-terminal side, and to counteract the tendency of the loop to insert into beta-sheet A. The secreted mutant proteins apparently are liable to loop insertion, resulting in exposure of the KOK12 epitope. The overinsertion of the loop would subsequently cause weakening of the bonds between sheet 3A and 5A, which in turn renders the molecule prone to intermolecular loop to sheet polymerization(21, 25) . This suggested mechanism of multimer formation is slightly different than that proposed for the alpha1-AT-Z and Siiyama (S53F) mutants(25, 26) , for which locking of beta-sheet A in an open state has been postulated to be the primary event.

The recent elucidation of the crystallographic structure of intact ATIII by two separate groups (39, 55) has provided yet another mechanism by which serpins may multimerize. Both ATIII structures consist of dimers of an intact inhibitory molecule of which part of the reactive center loop (P3-P7) replaces sheet 1C of either a cleaved (55) or a latent (39) second ATIII molecule. As discussed in more detail in (39) , this novel mode of loop-sheet 1C bonding may contribute to the process of multimerization, instead of exclusive loop-sheet A bonding. Moreover, we postulate that loop-sheet 1C bonding may play a role in the C1 inhibitor mutants described here, since these probably already have weakened tethering of sheet 1C to the body of the molecule as a result of the substitutions. This novel mechanism also explains the puzzling observation that heating of normal C1 inhibitor yields very large multimers (see Fig. 5), while certain mutants spontaneously form multimers of a distinctly smaller size. In addition, with two C1 inhibitor mutants tested, rP476S (this study) as well as the A436T mutant reported earlier(21) , multimers are formed that do not grow larger upon heating.^2 Indeed, the C1 inhibitor mutants display the predicted small size (dimer-tetramer) of multimers formed by loop-sheet 1C bonding.

A partial view of the Calpha-backbone of cleaved alpha1-AT, with the region surrounding the residues which are substituted in C1 inhibitor highlighted, is shown in Fig. 9. The local structure in intact ovalbumin is essentially identical. It is evident that the affected residues are part of a hydrophobic pocket, which is highly conserved among the serpins. The three substitutions studied here most probably disrupt formation of this pocket. Other conserved residues which are likely to contribute to the local structural integrity are F208 in s4C, and V388 in s5B(12) . Interestingly, several of the residues involved (F208, F370, and P391) have also been implicated in the transition to the latent form in plasminogen activator inhibitor-1 (PAI-1)(56) . In addition, we note that although the residue corresponding to F366 in alpha1-AT is generally phenylalanine or an aliphatic residue, PAI-1 is the only serpin with a methionine at this position, as in the case of the C1 inh-V451M mutant studied here. It may therefore be possible that the typical features of latent PAI-1, i.e. almost complete loop insertion, pivoting of s1C around F370, and distortion of s3C and s4C(56) , also play a role in the conformational changes observed in the C1 inhibitor mutants. Such structural features would also agree with the occurrence of loop-sheet 1C bonding mentioned above.

The mutant C1 inhibitor proteins were all three quantitatively immunoprecipitated by mAb KOK12 and had moderately increased thermostability in the intact form. With respect to complex formation with C1s and binding to mAb KII, however, rF455S behaved differently from rV451M and rP476S and instead resembled the normal protein to a certain extent. Binding to mAb KII upon trypsin cleavage is linked to the structural transition of complete loop incorporation into sheet A. Since trypsin-cleaved mutants rV451M and rP476S were unable to bind to mAb KII (Fig. 7), this indicates that complete loop insertion is somehow perturbed. This observation implies that subtle structural differences exist between C1 inhibitor F455S and the other two mutants. One possibility for such differences might be the configuration of beta-sheet A, where strands 3 and 5 normally should be able to separate completely in order to accommodate the reactive center loop as strand 4 upon cleavage.

Investigation of plasmas of two HAE families demonstrated that in vivo the C1 inhibitor V451M and P476S mutant proteins are also partially secreted. Again, this is similar to findings with ATIII and alpha1-AT, where low levels of circulating dysfunctional mutants are sometimes found(31, 50) . Thus, the phenotype of HAE in these C1 inhibitor-deficient patients falls between the classically defined type I and II(47) . Accurate quantification of both functional and antigenic C1 inhibitor levels, in combination with the telltale binding to mAb KOK12, may lead to early recognition of this phenotype in future screening. The C1 inhibitor mutant proteins appeared to circulate in monomeric form in plasma, but multimerized upon incubation at 37 °C in vitro. This was also found for alpha1-AT-Z, where slow multimerization of the purified protein from a homozygous patient took place in vitro at 37 °C(24, 25) . In the case of C1 inhibitor, the absence of detectable multimers in circulation might be due to the presence of the normal protein, other factors that may be limiting, or increased clearance of multimers. In addition, the tendency of the recombinant C1 inhibitor mutants to multimerize could possibly be increased compared to the plasma protein, due to the addition of Tween-20 to the transfection media. Non-ionic detergents like Nonidet P-40 and Tween-20 increase multimerization in normal plasma alpha1-AT (22) and C1 inhibitor.^2 Multimers can however sometimes be observed directly in plasma, as in the case of alpha1-AT-Siiyama (26) and C1 inhibitor A436T(21) .

In conclusion, we have shown that substitutions at conserved COOH-terminal positions in C1 inhibitor result in a protein configuration which is increasingly recognized among members of the serpin family. Since mutations leading to overinsertion of the loop and/or multimerization can apparently be located in diverse regions in serpins (helix B, the turn between s5A and sS4A, the reactive center loop itself, and the extreme COOH terminus), it may be expected that novel sites which produce this configuration will be identified.


FOOTNOTES

*
This work was supported by grants from the Caisse Nationale de l'Assurance Maladie des Travailleurs Salariés (CNAMTS), the Ministère de la Recherche et de la Technologie, the Groupement de Recherches et d'Etudes sur les Génomes (GREG), and the Mutuelle Générale de l'Education Nationale (MGEN). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Recipient of a Poste Vert fellowship awarded by INSERM and of a short term fellowship from the Fondation pour la Recherche Médicale. To whom correspondence should be addressed: Dept. of Autoimmunediseases, Central Laboratory of the Red Cross Blood Transfusion Service, Plesmanlaan 125, 1066 CX, Amsterdam, The Netherlands. Tel.: 31-20-5123171; Fax: 31-20-5123170.

(^1)
The abbreviations used are: serpin, serine protease inhibitor; alpha1-AT, alpha1-antitrypsin; APMSF, 4-(amidinophenyl)-methanesulfonyl fluoride; ATIII, antithrombin III; HAE, hereditary angioedema; HPLC, high performance liquid chromatography; mAb, monoclonal antibody; PAI-1, plasminogen activator inhibitor-1; PBS, phosphate-buffered saline pH 7.4; PMSF, phenylmethanesulfonyl fluoride; rC1 inh, recombinant C1 inhibitor; SBTI, soybean trypsin inhibitor; wt, wild-type; PAGE, polyacrylamide gel electrophoresis; ELISA, enzyme-linked immunosorbent assay.

(^2)
E. Eldering, E. Verpy, D. Roem, T. Meo, and M. Tosi, unpublished observations.


ACKNOWLEDGEMENTS

We thank Drs. Jerôme Laurent (Paris) and Margarita Lopez-Trascara (Madrid) for collection of blood samples. Thanh Tong Nguyen contributed expert technical assistance with the HPLC system. We are grateful to Ada Prochnicka-Chalufour for her kind help with the modeling program and to Erik Hack for critical evaluation of the manuscript.


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