- Email: [email protected]
[5] Preparation and properties of human C1 inhibitor
[5]
HUMAN C1 INHIBITOR
[5] P r e p a r a t i o n
and Properties
of Human
43
C1 Inhibitor
By R. B~ SlM and A. REBOCL Introduction C1 inhibitor (Cl-Inh) is a serum glycoprotein responsible for regulating the activities of the c o m p l e m e n t system serine proteases C l r and Cls. The presence of an inhibitor of C l s in h u m a n plasma was initially observed by Ratnoff and L e p o w / and substantial purification of the inhibitor was first reported by Pensky e t al." C l - I n h is in m a n y respects a typical plasma protease inhibitor, and appears to act in a similar way to, for example antithrombin III, a2-antiplasmin, and c~l-antitrypsin. C l - I n h is, however, the only p l a s m a - p r o t e a s e inhibitor that interacts with C l r and C l s under physiological conditions? Methods for the purification and assay of C l - I n h have been described previously in this series? Using information derived from earlier metho d s / a simplified, higher-yield purification method has since been developed,'~ and further information is now available on the reaction of C l - I n h with C l r and C l s . Assay Determination of activity of C l - I n h in serum or plasma is of clinical importance for diagnosis o f hereditary angioedema, a relatively frequent genetic defect characterized by total or partial absence of C l - I n h activity. (For discussion, see Ref. 4.) C l - I n h is most conveniently assayed by determining the extent to which it inhibits the hydrolysis of amino acid esters by a standard preparation of Cls. A n u m b e r of different amino acid esters are routinely used for this purpose, including N-acetyl-L-tyrosine ethyl ester (ATEE), 0 N ~acetyl-L-lysine methyl ester ( A L M E ) , 4 N'~-tosyl-L-arginine methyl ester (TAME), '~ and N~-carbobenzoxy-L-lysine p-nitrophenol ester ( Z L N E ) . 7 o. D. Ratnoff and I. H. Lepow, J. Exp. Med. 106, 327 (1957). J. Pensky, L. R. Levy, and I. H. Lepow, J. Biol. Chem. 236, 1674 (1961). :~ R. B. Sim, A. Reboul, G. J. Arlaud, C. L. Villiers, and M. G. Colomb, FEBS Lett. 97, lll (1979). 4 p. C. Harpel, this series Vol. 45, p. 751. :' A. Reboul, G. J. Arlaud, R. B. Sim, and M. G. Colomb, FEBS Lett. 79, 45 (1977). '; F. P. Schena, C. Manno, R. D'Agostino, G. Bruno, F. Cramarossa, and L. Bonomo, J. Clin. Chem. Clin. Biochem. 18, 17 (1980). 7 R. B. Sim, G. J. Arlaud, and M. G. Colomb, Biochim. Biophys. Acta 612, 433 (1980).
METHODS IN ENZYMOLOGY, VOL. 80
Copyright © 1981 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-181980-9
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COMPLEMENT
[5]
All four methods cited provide similar sensitivity in that approximately 1 /xg of Cl-Inh can be detected, but spectrophotometric assays using TAME and ZLNE are more rapid and require fewer reagents. An assay using ZLNE is described in detail here. A standard preparation of Cls is required for assaying Cl-Inh. For simple comparative assays, a neutral euglobulin precipitate from human serum, prepared as described by Linscott, ~ may be used as a crude Cls preparation. This material should be incubated for 1 hr at 37°C and pH 7.5 after preparation to ensure complete activation of Cls. To obtain higher sensitivity, however, it is necessary to use highly purified and fully activated Cls. This is most easily obtained by using precipitation and DEAE-cellulose purification steps as described by Arlaud and colleagues. 9 For the purpose described here, a final affinity-chromatography step described by these authors, which removes traces of contaminant Clr, may be omitted. The concentration of active Cls in a crude or highly purified preparation can be estimated by direct assay with ZLNE. "~
Reagents Cls. The stock Cls preparation is dialyzed against 10 mM Tris-HC1100 mM NaCI-1 mM EDTA, pH 8.0, and adjusted to a final concentration of 75-125/xg of active Cls/ml. Substrate. ZLNE (Sigma Chemical Co.) is dissolved to a final concentration of 3 mg/ml in a mixture of 9 parts by volume acetonitrile and 1 part by volume water. The stock substrate solution should be made up each day and kept on ice. Assay buffer. Hydrolysis of ZLNE must be monitored at pH 6.0, as the substrate is unstable near neutral pH. The assay is done in 100 mM sodium phosphate-100 mM NaC1, 15 mM EDTA, pH 6.0. Cl-Inh sample. The Cl-Inh samples to be tested should be at pH 7.0-8.0. The assay is suitable for titrating Cl-Inh in plasma or serum, as well as at various stages of purification. Plasma and serum samples should be diluted 5- to 10-fold with 10 mM TrisHCI-100 mM NaCI-1 mM EDTA, pH 8.0.
Procedure The Cls preparation (50 ~tl) and Cl-Inh (up to 500 ~1, 0-20 ~zg) are mixed and incubated for 15 min at 37°C. If the Cl-Inh samples are very dilute (<2/~g/ml), incubation should be performed for 30 min at 37°C. For W. D. Linscott, Immunochemistry 5, 311 (1968). ~ G. J. Arlaud, A. Reboul, C. M. Meyer, and M. G. Colomb, Biochim. Biophys. Acta 485, 215 (1977). ~0 R. B. Sim, this volume [41.
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HUMAN CT INHIBITOR
45
each sample of Cl-Inh to be tested, a range of 3-5 different volumes should be incubated with Cls. An appropriate control, containing 50 ~1 10 mM Tris-HCl-100 mM NaCI-1 mM EDTA, pH 8.0, instead of Cls, is set up for each volume of Cl-Inh used. The controls are treated throughout in the same way as test samples. At the end of the incubation period, each Cls + Cl-Inh mixture and each control is diluted to a final volume of 3.2 ml with the pH 6.0 assay buffer. Each 3.2-ml mixture is then transferred to a spectrophotometer cuvette and 50 tzl of the stock substrate solution added. The contents of the cuvette are mixed by inversion, and the increase in absorbance at 340 nm due to p-nitrophenol release is then monitored for 2-5 min. Substrate hydrolysis may be determined at any temperature between 20°C and 37°C. Greater sensitivity and speed are obtained at higher temperatures. Increase in absorbance is linear up to an absorbance value of 0.35. The rate of change of absorbance per minute (-.XA340/min) is calculated for each sample. The -XA340/min value for each Cls + Cl-Inh mixture is corrected by subtracting from it the -XA340/min value of the corresponding control. This correction takes account of spontaneous substrate hydrolysis and the possible presence of esterases in the Cl-Inh sample. ~ For each set of assays, the activity of the uninhibited Cls must be determined by measuring the .-XA340/min for a mixture of 50 gl Cls, 3.15 ml of pH 6.0 assay buffer, and 50/xl of substrate. This value is corrected for spontaneous hydrolysis of ZLNE by subtracting from it the AA340/min developed in a mixture of 3.2 ml of pH 6.0 assay buffer and 50/sl substrate. Corrected -XAa40/min values for each Cls + Cl-Inh mixture are expressed as a percentage of the corrected -XA340/min for Cls alone, and finally the concentration of Cl-Inh in the sample is calculated from a graph of the type shown in Fig. 1. Cl-Inh concentration may be calculated in absolute terms provided that highly purified Cls of known concentration is used. Alternatively, the concentration of both Cls and Cl-Inh may be expressed as activity units or arbitrary units, as in Ref. 4. Cl-Inh may also be titrated by any of several methods in which inhibition of Cls or C1 hemolytic activity is determined. Such methods may be up to 100-fold more sensitive than the esterase assays described previously, but they require relatively complex reagents. An assay of this type has been described by Gigli and colleagues. ~2
~ Use of a split-beam spectrophotometer for these assays permits direct reading of test samples against controls, thus halving the number of individual assays to be performed. With experience in the assay method, a large number of similar Cl-Inh samples (e.g., plasmas) can be compared using only a single Cl-lnh dilution. ~ 1. Gigli, S. Ruddy, and K. F. Austen, d. Immltpto[. 10(I, 1154 (1968).
46
COMPLEMENT
[5]
100¢
C rg
#
_> ,~
~
-<3_
(~
100
200
300
v o l u m e diluted plasma (pl)
FIG. 1. Titration of Cl-Inh. Cls (5 tzg in 50 •1) was incubated as described in the assay procedure with varying volumes of two diluted human plasmas, A and B. Plasma dilution factor was 1 volume plasma: 9 volumes buffer. The titer of Cl-lnh is calculated from the volume of diluted plasma required to give 50% inhibition ($). For plasma A, this volume is 150 ~1, and for B, 96 #1. The Cl-lnh concentration may be expressed in arbitrary units, where for example 1 unit the quantity of Cl-Inh required to cause 50% inhibition of the Cls present. Undiluted plasma A therefore contains [(1000/150) × plasma dilution] = 66.7 units/ml, and undiluted plasma B, 104.2 units/ml. Alternatively, from the known molecular weights of Cls and Cl-Inh, and the 1 : 1 stoichiometry of inhibition, it can be calculated that inhibition of half the Cls present requires 2.94-3.16 /xg of Cl-Inh. Undiluted plasma A contains this quantity of Cl-lnh in 15 ~tl, and therefore contains 196-211 tzg Cl-Inh/ml. Similarly, undiluted plasma B contains 306-329 #g/ml.
Purification Cl-Inh can be purified from human plasma in three successive steps: (1) polyethylene glycol precipitation; (2) DEAE-cellulose chromatography; (3) concanavalin A-Sepharose chromatography? Since Cl-Inh participates in inhibition of several proteases and can be degraded by others (e.g., plasmin4), it is essential to avoid activation of plasma proteases. To minimize protease activation and activity, coagulation contact-activation inhibitors and protease inhibitors are used during the preparation. The plasma used for purification of Cl-Inh should not have been in contact with glass, and contact with unsiliconized glass should be avoided during steps 1 and 2 of the preparation. Plasma should be kept frozen (-20°C) until it is required. Storage at 4°C for more than 3-4 hr should be avoided to limit cold-promoted activation of Factor VII.~3 Fresh plasma, containing acid citrate-dextrose (ACD) or phosphate-citrate-dextrose (PCD) anticoagulants is ideal for the preparation, but outdated ACD- or PCD-plasma is an adequate starting material, provided it has been stored at 20°Cwith no glass contact. All purification procedures are carried out at 2-4°C unless stated otherwise. ~:~E. A. van Royen, S. Lohman, M. Voss, and K. W. Pondman, J. Lab. Clin. Med. 92, 152 (1978).
[5]
HUMAN C]- INHIBITOR
47
Protease Inhibitors Diisopropyl phosphoroflurodate (DFP; Sigma or Aldrich Chemical Co.) is made up as a 2.5 M stock solution by adding 1 g DFP to 1.2 ml anhydrous isopropanol." Polybrene. Polybrene (Aldrich Chemical Co.) is made up as a 24 mg/ml solution in 0.4 M trisodium EDTA, pH 7.5.
Procedure Human plasma (250 ml) is thawed at 37°C and centrifuged for 30 min at 10,000g to remove debris. The plasma is then made 4 mM with DFP by addition of 400/zl of the DFP stock solution. The stock polybrene solution (13.2 ml) is then added to give final polybrene and EDTA concentrations of 1.2 mg/ml and 20 mM, respectively. The plasma is then stirred gently for 30 min at 25°C, and a further 400 ~l of stock DFP solution is added. Step 1. Polyethylene Glycol Precipitation. The plasma is now cooled to 4°C, and made 6% w/v polyethylene glycol (PEG) by addition of 36.4 ml of a 50% w/v solution of PEG 6000 (Sigma). The PEG solution is added slowly over a period of 20-25 min, with constant stirring. The plasma is stirred for a further 30 rain at 4°C, then centrifuged (30 min at 10,000 g). The heavy yellow precipitate is discarded. A further 400/zl of stock DFP solution is added to the supernatant, and the supernatant is dialyzed to equilibrium against 5 liters of 20 mM sodium phosphate-50 mM NaCI-5 mM EDTA, pH 7.0. The precipitation step results in removal of 35-40% of the total plasma protein with 75-85% recovery of Cl-Inh activity, representing about 1.3fold purification. Step 2. DEAE-Celhdose Chromatography. The dialyzed supernatant is centrifuged (30 min, 10,000g). A small gellike precipitate usually forms at this stage, and is discarded. The supernatant is loaded onto a column (20 × 5 cm diameter) of DEAE-cellulose (DE-32 or DE-52, Whatman), equilibrated in the dialysis buffer. Flow rate should be 80-100 ml/hr. Protein that does not bind to the column is discarded and the column is washed with the starting buffer until the absorbance at 280 nm of the eluate is <0.05. Cl-Inh is then eluted in a linear gradient of NaCI formed by mixing 700 ml of the starting buffer with 700 ml of 20 mM sodium phosphate-300 mM NaCI-5 mM EDTA, pH 7.0. A typical elution profile ~ Since DFP is volatile and highly toxic, some laboratories may prefer to use the less toxic inhibitor phenyl methyl sulphonyl fluoride (PMSF; Sigma Chemical Co.). This material is prepared as a stock 110 m M solution in ethanol and added to aqueous solutions to a final concentration of about 1 raM. It is less effective than DFP as a general serine-protease inhibitor.
48
COMPLEMENT
[5]
2c
100 r
I <~
7o4
I
L)
~6
i
so S
18 O2 I
~
C12 0
10 20 v o l u m e e l u t e d (liters)
30
FIG. 2. DEAE-cellulose chromatography. Conditions are as described in the text. - - , A280;.... , Cl-Inh activity, expressed as % inhibition of Cls in a standard assay by 100 ~tl of eluant fraction; • •, relative salt concentration (RSC) (a solution with RSC = 0.1 M has the same conductivity as 0.1 M NaC1). An arrow marks the start of gradient elution, and a bar indicates the fractions pooled for the next purification step. is shown in Fig. 2. This step results in elimination of >99% of the total protein, with 50-55% recovery of C l - I n h activity, representing 120- to 170-fold purification. Step 3. Concanavalin A-Sepharose Chromatography: The pooled material from D E A E - c e l l u l o s e chromatography is again made 4 mM with D F P and dialyzed against 5 liters of 20 mM Tris-HCl-100 mM NaCI, pH 8.0, and then applied to a column (8 x 5 cm diameter) of concanavalin A - S e p h a r o s e (Pharmacia), equilibrated in the same buffer. Flow rate should be about 20 ml/hr. The column is washed with the starting buffer until the absorbance at 280 nm of the eluate is <0.02. C l - I n h is then eluted as a sharp peak (Fig. 3) by washing the column with 450 ml of the same buffer made 0.5% w/v with a-methylmannoside (Sigma). This purification step results in elimination of 30-50% of the remaining protein, with over 90% recovery of C l - I n h activity, and therefore represents a 1.3- to 1.8fold purification. The pool of C l - I n h is then dialyzed against 2 liters of 20 mM TrisH C I - 1 0 0 mM N a C I - 1 mM EDTA, pH 8.0, to remove c~-methylmannoside, and finally concentrated to about 1 mg/ml by ultrafiltration on a PM-10 membrane (Amicon). The purified protein is stable on storage at - 2 0 ° C for more than 1 year. The yield of C l - I n h by this method is 19-36 mg of C l - I n h (average over 14 preparations = 24 mg) from the 250 ml of starting material. The yield of activity is in the range 33-42%. The concentration of C l - I n h in
[5]
HUMAN C1 INHIBITOR
49
Cl-lnh I
<(
\ 0
~00
800
1200
v o l u m e eluted (ml
FIG. 3. Concanavalin A - S e p h a r o s e chromatography. Conditions are as described in the text. - - , A2s0. An arrow marks the start of elution with ~-methylmannoside, and a bar indicates the fractions pooled.
individual plasmas shows a relatively wide variation, and other investigators have suggested mean plasma concentrations of 235,1~ 185,1" and about 130 mg/liter.17 We have not systematically titrated Cl-Inh in plasma from single donors, but single radial immunodiffusion and functional assays performed on pooled plasmas (8-15 donors) used for preparing Cl-Inh consistently show an average Cl-Inh concentration greater than 200 mg/liter. Examination of Cl-Inh purified by the above method on sodium dodecyl sulfate (SDS)-polyacrylamide gels reveals a single major component of 100,000 apparent molecular weight, with traces (generally <5%) of two other components of apparent MW 60,000 and 30,000. These are likely to be degradation products of Cl-Inh. '~ Prolonged (over 1 month) storage of the isolated protein at 4°C in the absence of protease inhibitors leads to an increase in the quantity of the two minor components present. The final preparations are therefore likely to contain trace contamination with protease(s). Occasional contamination with transferrin and albumin is observed. Contaminating albumin can be removed by repeating step 3 of the preparation. Cl-Inh isolated by cther methods 4"~ exhibited two major bands on Coomassie blue-stained SDS-polyacrylamide gels. One species had an apparent MW 9,000-10,000 lower than Cl-Inh, and appeared to corre~:' N. Heimburger, Cold Spring Harbor Con f~ Cell Proliferation 2, 367 (1975). "; F. S. Rosen, C. A. Alper, J. Pensky, M. R. Klemperer, and V. H. Donaldson, J. Clin. Invest. 50, 2143 (1971). ~7 R. J. Ziccardi and N. R. Cooper, Clin. lmmunol. Immunopathol. 15, 465 (1980). ~ D. P. Man and J. O. Minta, Fed. Proe., Fed. A m . Soc. Exp. Biol. 37, 591 (abstr. 1991) (1978).
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COMPLEMENT
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spond to a major fragment produced by plasmin or trypsin digestion of Cl-Inh. ~9This degradation product is also observed in our preparations if protease inhibitors are not used during purification. C1--Inh in Other Species Cl-Inh has been isolated from rabbit serum in low yield,z° and has been shown to be very similar in molecular weight, temperature, and pH stability to the human protein. Rabbit Cl-Inh inhibits rabbit Clr, Cls, and plasmin, and also human Cls. "° Guinea pig Cl-Inh has also been purified, and although similar to human Cl-Inh in activity, was reported to have a much higher sedimentation coefficient (6.1 S). '~ Inhibitory activity against human Cls has been detected in the sera of numerous mammals, birds, and fish. 2'''3 The sera of several primates, but not of lower mammals, contain protein that is antigenically related to human Cl-Inh. 2'~ Partial purifications of Cl-Inh from other mammalian species have been summarized by Gigli and Austen. "4 Structural Properties Isolated human Cl-Inh is a single polypeptide chain monomer of MW 100,000-105,000 as estimated by equilibrium centrifugation and by SDSpolyacrylamide gel electrophoresis. 4'5'23 It has a sedimentation coefficient of 3.T5-4.0 S? The isoelectric point is 2.7-2.8. 2~ The amino acid and carbohydrate compositions of the protein have been determined by several groups and have been presented earlier in this series? The amino acid composition is unremarkable, except for an unusually low content of glycine and sulfur-containing amino acids. The tyrosine and tryptophan content is low, consistent with the low extinction coefficient of the protein (a~% Z X l c r n 280 nm = 4.525). Cl-Inh is, however, very heavily glycosylated, containing 69 tool hexose, 47 mol hexosamine, and 51 mol sialic acid per mole of protein. 2~ Consistent with the high carbohydrate content, the partial specific volume is low (0.667 ml/g2~). No sequence data are available for Cl-Inh. ' ' P. C. Harpel and N. R. Cooper, J. Clin. Invest. 55, 593 (1975). ~" E. Ishizaki, Y. Mori, and J. K o y a m a , J. Biochem. (Tokyo) 82, 1155 (1977). ~ M. Loos, W. Opferkuch, and R. Ringelmann, Z. Med. Microbiol. lmmunol. 156, 194 (1971). 22 L. R. L e v y and I. H. Lepow, Proc. Soc. Exp. Biol. Med. !01, 608 (1959). ~:~ V. H. Donaldson and J. Pensky, J. hnrnunol. 104, 1388 (1970). '-,4 I. Gigli and K. F. Austen, Annu. Rev. Microbiol. 25, 309 (1971). '-'~ H. Haupt, N. Heimburger, T. Kranz, and H. G. Schwick, Eur. J. Biochem. ! 7, 254 (1970).
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HUMAN C1 INHIBITOR
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Activity and Stability Isolated C l - I n h loses activity on exposure to pH values outside the range 6.0-9.5. 2 The activity is totally destroyed by heating at 60°C for 30 rain, but is stable to temperatures below 48°C. ~ Inhibitory activity is unaffected by neuraminidase treatment ~s but has been reported to be destroyed by exposure to 1 mM 2-mercaptoethanol. ~ C l - I n h has been shown to inhibit, in vitro, plasma kallikrein, ='"'~7plasrain, ~r activated Hageman Factor ( F X I I J s and its fragments, 29 and activated Factor XI (FXI~02s as well as C l r 27 and Cls. ~ It has no significant inhibitory activity against trypsin or chymotrypsin. ~~:' It is degraded by trypsin and by excess plasmin. 1'~ Inhibition of plasmin by C l - I n h does not appear to be of physiological significance since, in whole plasma, a2antiplasmin and a2-macroglobulin are more effective plasmin inhibitors, a° C l - I n h may, however, be a major physiological inhibitor of plasma kallikrein, :~':1'-' of FXII~ fragments, ~a'29":~':~3 and FXI,, a2 It is the only plasma inhibitor of C l r and Cls. a Inhibition of Clr, Cls, and plasmin by C l - I n h correlates with the formation of tightly bound complexes consisting of one molecule of inhibitor per molecule of protease, r'~:~'a4 These protease-(protease inhibitor) complexes are not dissociated by strong denaturants such as guanidine hydrochloride, urea, and SDS, or by low pH, and can be observed on S D S - p o l y a c r y l a m i d e gels after staining with Coomassie blue. a'a'r'l:''a4 The complexes are also resistant to reduction and alkylation. Patterns obtained on S D S - p o l y a c r y l a m i d e gel electrophoresis of reduced and alkylated complexes of C l - I n h with Clr, Cls, or plasmin show that the inhibitor binds to the protease through the light " B " chains, which contain the active site. a'~9 Prior inactivation of C l r or C l s with D F P prevents subsequent binding of C l - l n h 3~'aa and vice versa. :~4Thus it is likely that binding of C l - I n h to these proteins occurs via the catalytic active site. Similar formation of denaturation-resistant equimolar complexes has been described for other proteases with plasma protease inhibitors, for e,; I. Gigli, J. W. Mason, R. W. Colman, and K. F. Austen, J. Immured. 104, 574 (1970). 27 O. D. Ratnoff, J. Pensky, D. Ogston, and G. B. Naff, J. Exp. Med. 129, 315 (1969). ~ C. D. Forbes, J. Pensky, and O. D. Ratnoff, J. Lab. Clin. Med. 76, 809 (19711). z~'A. D. Schreiber, A. P. Kaplan, and K. F. Austen, J. Clin. Invest. 52, 1402 11973). a, p. C. Harpel, J. E.vp. Med. 146, 1033 (1977). :~ H. Fritz, E. Fink, and E. Truscheit, Fed. Proc.. Feel. A m . Soc. Exp. Biol. 38, 2753 (1979). :~2H. Saito, G. H. Goldsmith, M. Moroi, and N. Aoki, Proc. Natl. Acad. Sci. U.S.A. 76, 2013 (1979). a:~p. S. Damus, M. Hicks, and R. D. Rosenberg, Nature (London) 246, 355 11973). :~ G. J. Arlaud, A. Reboul, R. B. Sire, and M. G. C o l o m b , B i o c h i m . Bit, phys. Acta 576, 151 (1979). aa R. B. Sim, G. J. Arlaud, and M. G. Colomb, Biochem. J. 179, 449 (1979).
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COMPLEMENT
[5]
example, Factor X~,-antithrombin III, 3'; t h r o m b i n - a n t i t h r o m b i n III, :~" trypsin-oq-antitrypsin, 37 trypsin-antithrombin III, 37 and plasmin-azantiplasmin. 3° Jesty 3" and Mahoney and colleagues 37 have recently shown that complexes of this type dissociate slowly on exposure to p H >9. Preliminary evidence (R. B. Sim, unpublished results; A. W. Dodds, unpublished results) indicates that this is also the case for p l a s m i n - C l - I n h and C l s - C l - I n h complexes. It is likely that the protease and inhibitor in such c o m p l e x e s are covalently associated, via formation of a tetrahedral intermediate? 7 Interaction with C l r and C l s The kinetics of interaction of C l - I n h with isolated C l r and C l s have been studied. 7 Both proteases have a similar affinity for C l - I n h (functional dissociation constant = 10 7 M).7 At 37°C, in the presence of EDTA, C l r reacts 4- to 5-fold slower with C-l-Inh (second-order rate constant kl = 2.8 × 103 M -~ sec ') than does C l s (kl - 1.2 × 104 M 1 sec ,).7 In buffers containing Ca 2+ ions, the rate of reaction of C l r is reduced 2-fold, but the reactivity of C l s toward C l - I n h is unaffected. 7 The activation energy for interaction of C l r and C l - I n h (44.3 kcal/mol) is much higher than that for the C l s - C l - I n h interaction (11.7 kcal/mol), and thus at temperatures below 37°C the disparity in the reaction rates with C l r and C l s b e c o m e s much greater. 7 Isolated C l r , which exists normally as a dimer, '° forms large complexes with C l - I n h , which are likely to have the composition Clr2-Cl-Inh2.:~ Under certain conditions, however, the C l r dimer appears to dissociate on interaction with C l - I n h , and C l r , - C l - I n h i complexes are formed. 3s Interaction of C l r that is present in a n t i b o d y - a n t i g e n - C 1 complexes, with C l - I n h , also results in dissociation of the C l r dimer. 39 Inhibition of isolated C l r and C l s by C l - I n h shows a p H o p t i m u m of 7.5-8.2 (R. B. Sim, unpublished results). The reaction of C l s with C l - I n h is insensitive to alteration in ionic strength, but the corresponding reaction with C l r proceeds optimally at an ionic strength corresponding to 110-300 m M NaCl. ~ Inhibition of C1 by C l - I n h is greatly enhanced in the presence of low concentrations of heparin. 7'4°'4~ This enhancement is due principally to an :~'~J. Jesty, J. Biol. Chem. 254, 1044 (1979). :~rW. C. Mahoney, K. Kurachi, and M. A. Hermodson, Eur. J. Biochem. 105, 545 (1980). ..~sS. Chesne, G. J. Arlaud, C. L. Villiers, and M. G. Colomb, Abstr.. Int. Congr. hnmunol.. 4th, 1980 Abstract 15.1.05 (1980). :~' R. J. Ziccardi and N. R. Cooper, J. lmmunol. 123, 788 (1979). '" R. Rent, R. Myrrmann, B. A. Fiedel, and H. Gewurz, Clin. Exp. lmmunol. 23, 264 (1976). H K. Nagaki and S. lnai, Int. Arch. Allergy Appl. lmmunol. 511, 172 (1976).
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HUMAN C 1 INHIBITOR
53
acceleration of the rate of reaction of Cl-Inh with Cls. 7'4~ Heparin also increases the rate of Cl-Inh inactivation of Clr 7 and plasmin (R. B. Sim and P. E. Gasson, unpublished results), but the enhancement is much smaller than that seen with Cls. As discussed in Ref. 7, this enhancement is very similar to the effect of heparin on antithrombin III. The physiological significance of this effect is uncertain, as heparin has many inhibitory actions in the complement system?" As discussed above, there is evidence that Cl-Inh interacts directly with the active sites of the proteases that it inhibits. It is therefore to be expected that Cl-Inh inhibits in parallel all of the esterolytic and proteolytic activities of each enzyme. Parallel inhibition by Cl-Inh of Clsdependent cleavage of C2, C4, and amino acid esters has been reported? 3 However other investigators have suggested differences in the sensitivities to Cl-Inh of the C2-cleaving, C4-cleaving, and various esterase activities of Cls. 44'45 These discrepancies may be partly attributable to the diversity and complexity of the assay systems used, or to the use of insufficiently pure reagents. Nevertheless, this point has not been investigated systematically and requires further study. Interaction with C1 In the course of complement activation, Clr and Cls do not exist as free proteases in solution, but rather as Ca2+-dependent macromolecular Clr2-Cls2 complexes bound to Clq, which is in turn bound to the activating surface (e.g., antibody-antigen complexes). Cls bound within antibody-antigen-C1 reacts with C-l-Inh at the same rate as does isolated Cls, 7:~'~but the reactivity of Clr toward Cl-Inh is enhanced up to 4-fold when Clr is bound within C1. 7'35 Thus the reactivities of Clr and Cls toward Cl-Inh are much closer when Clr and Cls are within antibodyantigen-C1 complexes than when the proteases are free in solution. 7'3~'35 A number of studies 7'34''~'~':~9'46have established that interaction of Cl-Inh with antibody-antigen-C1 leads, under physiological conditions, to dissociation of the C1 complex. Cl-Inh reacts rapidly with the Cls in antibody-antigen-C1 complexes, and more slowly with the Clr. 35 Binding of Cl-Inh to the Clr leads to immediate dissociation of Clr, Cls, and Cl-Inh from the antibody-antigen-C1 complexes? 5 The proteins released are in the form of a Clrl-Clsl-Cl-Inh2 complexfl 9 which has MW ~e B. J. Johnson, J. Pharm. Sci. 66, 1367 (1977). ~:~ I. H. Lepow, G. B. Naff, and J. Pensky, Ciba Found. Syrup. [N.S.] 57, 74 (1965). H K. Takahashi, S. N a g a s a w a , and J. K o y a m a , Biochim. Biophys. Acta 61 I, 196 (1980). ~ M. Kondo, I. Gigli, and K. F. A u s t e n , Immunology 22, 305 (1972). "~ A.-B. Laurell, U. Johnson, U. M~rtensson, and A. G. Sj6holm, Acta Pathol. Microbiol. Stand., Sect. C 86, 299 (1978).
54
COMPLEMENT
[6]
330,000-382,000? 9 Specific assays for the Clri-Clsl-Cl-Inh2 complex in human serum have been developed by Laurell and colleagues 47 and these may be used as an indicator of C1 activation in patient plasma. A simple screening assay for Cl-Inh activity has been developed/7 based on the alteration of antigenic properties of C-lr when it is incorporated into the C l r l - C l s l - C l - I n h complex. Dissociation of the bound C1 complex by reaction with C-l-Inh leaves most of the C lq still associated with the immune complex or other surface responsible for C1 activation. Clq "exposed" in this way may then interact with lymphoblastoid cells possessing Clq receptors. 4r A.-B. Laurell, U. M~trtensson, and A. G. Sj6holm, Acta Pathol. Microbiol. S t a n d . , Sect. C 87, 79 (1979).
[6] T h e S e c o n d C o m p o n e n t of H u m a n C o m p l e m e n t By MICHAEL A. KERR
C2 is one of the least abundant of the complement components in human plasma, being present at a concentration of 15-20 mg/liter. ~''-' This and the extreme susceptibility of C2 to proteolytic digestion have in the past hampered its purification and molecular characterization. Functional studies using guinea pig and human preparations have, however, clearly identified the role of C2 in the classical pathway C3 and C5 convertases, and this has been reviewed extensively? -~ The classical pathway C3 convertase is assembled from C2 and C4 on cleavage of these proteins by the Cls subcomponent of the C1 complex. C4 is cleaved by Cls with the release of a small peptide, C4a, to form C4b (apparent MW 198,000)6:C2 is cleaved to C2a (apparent MW 74,000) and C2b (apparent MW 34,000).7's For the generation of C3 convertase activity, C4 cleavage must precede C2 cleavage. The enzyme is not generated by the addition of C4 to a previously incubated mixture of C2 and Cls, but 1 M. A. Kerr and R. R. Porter, Biochem. J. 171, 99 (1979). 2 M. J. Polley and H. J. Mfiller-Eberhard, J. Exp. Med. 128, 533 (1968). :s R. R. Porter and K. B. M. Reid, Adv. Protein Chem. 33, 1 (1979). 4 H. J. Miiller-Eberhard, in "Molecular Basis of Biological Degradative Processes" (R. D. Berlin, H. Hermann, I. H. Lepow, and J. M. Tanzer, eds.), p. 65. Academic Press, New York, 1978. 5 j. E. Fothergill and W. H. K. Anderson, Curr. Top. Cell. Regul. 13, 259 (1979). R. A. Patrick, S. B. Taubman, and I. H. Lepow, l m m u n o c h e m i s t r y 7, 217 (1979). 7 M. A. Kerr, Biochem. J. 183, 615 (1979). s S. Nagasawa and R. M. Stroud, Proc. Natl. Acad. Sci. U.S.A. 74, 2998 (1977).
METHODSIN ENZYMOLOGY,VOL. 80
Copyright © 1981 by Academic Press, Inc. All rights of reproductionin any form reserved. ISBN 0-12-181980-9
HUMAN C1 INHIBITOR
[5] P r e p a r a t i o n
and Properties
of Human
43
C1 Inhibitor
By R. B~ SlM and A. REBOCL Introduction C1 inhibitor (Cl-Inh) is a serum glycoprotein responsible for regulating the activities of the c o m p l e m e n t system serine proteases C l r and Cls. The presence of an inhibitor of C l s in h u m a n plasma was initially observed by Ratnoff and L e p o w / and substantial purification of the inhibitor was first reported by Pensky e t al." C l - I n h is in m a n y respects a typical plasma protease inhibitor, and appears to act in a similar way to, for example antithrombin III, a2-antiplasmin, and c~l-antitrypsin. C l - I n h is, however, the only p l a s m a - p r o t e a s e inhibitor that interacts with C l r and C l s under physiological conditions? Methods for the purification and assay of C l - I n h have been described previously in this series? Using information derived from earlier metho d s / a simplified, higher-yield purification method has since been developed,'~ and further information is now available on the reaction of C l - I n h with C l r and C l s . Assay Determination of activity of C l - I n h in serum or plasma is of clinical importance for diagnosis o f hereditary angioedema, a relatively frequent genetic defect characterized by total or partial absence of C l - I n h activity. (For discussion, see Ref. 4.) C l - I n h is most conveniently assayed by determining the extent to which it inhibits the hydrolysis of amino acid esters by a standard preparation of Cls. A n u m b e r of different amino acid esters are routinely used for this purpose, including N-acetyl-L-tyrosine ethyl ester (ATEE), 0 N ~acetyl-L-lysine methyl ester ( A L M E ) , 4 N'~-tosyl-L-arginine methyl ester (TAME), '~ and N~-carbobenzoxy-L-lysine p-nitrophenol ester ( Z L N E ) . 7 o. D. Ratnoff and I. H. Lepow, J. Exp. Med. 106, 327 (1957). J. Pensky, L. R. Levy, and I. H. Lepow, J. Biol. Chem. 236, 1674 (1961). :~ R. B. Sim, A. Reboul, G. J. Arlaud, C. L. Villiers, and M. G. Colomb, FEBS Lett. 97, lll (1979). 4 p. C. Harpel, this series Vol. 45, p. 751. :' A. Reboul, G. J. Arlaud, R. B. Sim, and M. G. Colomb, FEBS Lett. 79, 45 (1977). '; F. P. Schena, C. Manno, R. D'Agostino, G. Bruno, F. Cramarossa, and L. Bonomo, J. Clin. Chem. Clin. Biochem. 18, 17 (1980). 7 R. B. Sim, G. J. Arlaud, and M. G. Colomb, Biochim. Biophys. Acta 612, 433 (1980).
METHODS IN ENZYMOLOGY, VOL. 80
Copyright © 1981 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-181980-9
44
COMPLEMENT
[5]
All four methods cited provide similar sensitivity in that approximately 1 /xg of Cl-Inh can be detected, but spectrophotometric assays using TAME and ZLNE are more rapid and require fewer reagents. An assay using ZLNE is described in detail here. A standard preparation of Cls is required for assaying Cl-Inh. For simple comparative assays, a neutral euglobulin precipitate from human serum, prepared as described by Linscott, ~ may be used as a crude Cls preparation. This material should be incubated for 1 hr at 37°C and pH 7.5 after preparation to ensure complete activation of Cls. To obtain higher sensitivity, however, it is necessary to use highly purified and fully activated Cls. This is most easily obtained by using precipitation and DEAE-cellulose purification steps as described by Arlaud and colleagues. 9 For the purpose described here, a final affinity-chromatography step described by these authors, which removes traces of contaminant Clr, may be omitted. The concentration of active Cls in a crude or highly purified preparation can be estimated by direct assay with ZLNE. "~
Reagents Cls. The stock Cls preparation is dialyzed against 10 mM Tris-HC1100 mM NaCI-1 mM EDTA, pH 8.0, and adjusted to a final concentration of 75-125/xg of active Cls/ml. Substrate. ZLNE (Sigma Chemical Co.) is dissolved to a final concentration of 3 mg/ml in a mixture of 9 parts by volume acetonitrile and 1 part by volume water. The stock substrate solution should be made up each day and kept on ice. Assay buffer. Hydrolysis of ZLNE must be monitored at pH 6.0, as the substrate is unstable near neutral pH. The assay is done in 100 mM sodium phosphate-100 mM NaC1, 15 mM EDTA, pH 6.0. Cl-Inh sample. The Cl-Inh samples to be tested should be at pH 7.0-8.0. The assay is suitable for titrating Cl-Inh in plasma or serum, as well as at various stages of purification. Plasma and serum samples should be diluted 5- to 10-fold with 10 mM TrisHCI-100 mM NaCI-1 mM EDTA, pH 8.0.
Procedure The Cls preparation (50 ~tl) and Cl-Inh (up to 500 ~1, 0-20 ~zg) are mixed and incubated for 15 min at 37°C. If the Cl-Inh samples are very dilute (<2/~g/ml), incubation should be performed for 30 min at 37°C. For W. D. Linscott, Immunochemistry 5, 311 (1968). ~ G. J. Arlaud, A. Reboul, C. M. Meyer, and M. G. Colomb, Biochim. Biophys. Acta 485, 215 (1977). ~0 R. B. Sim, this volume [41.
[5]
HUMAN CT INHIBITOR
45
each sample of Cl-Inh to be tested, a range of 3-5 different volumes should be incubated with Cls. An appropriate control, containing 50 ~1 10 mM Tris-HCl-100 mM NaCI-1 mM EDTA, pH 8.0, instead of Cls, is set up for each volume of Cl-Inh used. The controls are treated throughout in the same way as test samples. At the end of the incubation period, each Cls + Cl-Inh mixture and each control is diluted to a final volume of 3.2 ml with the pH 6.0 assay buffer. Each 3.2-ml mixture is then transferred to a spectrophotometer cuvette and 50 tzl of the stock substrate solution added. The contents of the cuvette are mixed by inversion, and the increase in absorbance at 340 nm due to p-nitrophenol release is then monitored for 2-5 min. Substrate hydrolysis may be determined at any temperature between 20°C and 37°C. Greater sensitivity and speed are obtained at higher temperatures. Increase in absorbance is linear up to an absorbance value of 0.35. The rate of change of absorbance per minute (-.XA340/min) is calculated for each sample. The -XA340/min value for each Cls + Cl-Inh mixture is corrected by subtracting from it the -XA340/min value of the corresponding control. This correction takes account of spontaneous substrate hydrolysis and the possible presence of esterases in the Cl-Inh sample. ~ For each set of assays, the activity of the uninhibited Cls must be determined by measuring the .-XA340/min for a mixture of 50 gl Cls, 3.15 ml of pH 6.0 assay buffer, and 50/xl of substrate. This value is corrected for spontaneous hydrolysis of ZLNE by subtracting from it the AA340/min developed in a mixture of 3.2 ml of pH 6.0 assay buffer and 50/sl substrate. Corrected -XAa40/min values for each Cls + Cl-Inh mixture are expressed as a percentage of the corrected -XA340/min for Cls alone, and finally the concentration of Cl-Inh in the sample is calculated from a graph of the type shown in Fig. 1. Cl-Inh concentration may be calculated in absolute terms provided that highly purified Cls of known concentration is used. Alternatively, the concentration of both Cls and Cl-Inh may be expressed as activity units or arbitrary units, as in Ref. 4. Cl-Inh may also be titrated by any of several methods in which inhibition of Cls or C1 hemolytic activity is determined. Such methods may be up to 100-fold more sensitive than the esterase assays described previously, but they require relatively complex reagents. An assay of this type has been described by Gigli and colleagues. ~2
~ Use of a split-beam spectrophotometer for these assays permits direct reading of test samples against controls, thus halving the number of individual assays to be performed. With experience in the assay method, a large number of similar Cl-Inh samples (e.g., plasmas) can be compared using only a single Cl-lnh dilution. ~ 1. Gigli, S. Ruddy, and K. F. Austen, d. Immltpto[. 10(I, 1154 (1968).
46
COMPLEMENT
[5]
100¢
C rg
#
_> ,~
~
-<3_
(~
100
200
300
v o l u m e diluted plasma (pl)
FIG. 1. Titration of Cl-Inh. Cls (5 tzg in 50 •1) was incubated as described in the assay procedure with varying volumes of two diluted human plasmas, A and B. Plasma dilution factor was 1 volume plasma: 9 volumes buffer. The titer of Cl-lnh is calculated from the volume of diluted plasma required to give 50% inhibition ($). For plasma A, this volume is 150 ~1, and for B, 96 #1. The Cl-lnh concentration may be expressed in arbitrary units, where for example 1 unit the quantity of Cl-Inh required to cause 50% inhibition of the Cls present. Undiluted plasma A therefore contains [(1000/150) × plasma dilution] = 66.7 units/ml, and undiluted plasma B, 104.2 units/ml. Alternatively, from the known molecular weights of Cls and Cl-Inh, and the 1 : 1 stoichiometry of inhibition, it can be calculated that inhibition of half the Cls present requires 2.94-3.16 /xg of Cl-Inh. Undiluted plasma A contains this quantity of Cl-lnh in 15 ~tl, and therefore contains 196-211 tzg Cl-Inh/ml. Similarly, undiluted plasma B contains 306-329 #g/ml.
Purification Cl-Inh can be purified from human plasma in three successive steps: (1) polyethylene glycol precipitation; (2) DEAE-cellulose chromatography; (3) concanavalin A-Sepharose chromatography? Since Cl-Inh participates in inhibition of several proteases and can be degraded by others (e.g., plasmin4), it is essential to avoid activation of plasma proteases. To minimize protease activation and activity, coagulation contact-activation inhibitors and protease inhibitors are used during the preparation. The plasma used for purification of Cl-Inh should not have been in contact with glass, and contact with unsiliconized glass should be avoided during steps 1 and 2 of the preparation. Plasma should be kept frozen (-20°C) until it is required. Storage at 4°C for more than 3-4 hr should be avoided to limit cold-promoted activation of Factor VII.~3 Fresh plasma, containing acid citrate-dextrose (ACD) or phosphate-citrate-dextrose (PCD) anticoagulants is ideal for the preparation, but outdated ACD- or PCD-plasma is an adequate starting material, provided it has been stored at 20°Cwith no glass contact. All purification procedures are carried out at 2-4°C unless stated otherwise. ~:~E. A. van Royen, S. Lohman, M. Voss, and K. W. Pondman, J. Lab. Clin. Med. 92, 152 (1978).
[5]
HUMAN C]- INHIBITOR
47
Protease Inhibitors Diisopropyl phosphoroflurodate (DFP; Sigma or Aldrich Chemical Co.) is made up as a 2.5 M stock solution by adding 1 g DFP to 1.2 ml anhydrous isopropanol." Polybrene. Polybrene (Aldrich Chemical Co.) is made up as a 24 mg/ml solution in 0.4 M trisodium EDTA, pH 7.5.
Procedure Human plasma (250 ml) is thawed at 37°C and centrifuged for 30 min at 10,000g to remove debris. The plasma is then made 4 mM with DFP by addition of 400/zl of the DFP stock solution. The stock polybrene solution (13.2 ml) is then added to give final polybrene and EDTA concentrations of 1.2 mg/ml and 20 mM, respectively. The plasma is then stirred gently for 30 min at 25°C, and a further 400 ~l of stock DFP solution is added. Step 1. Polyethylene Glycol Precipitation. The plasma is now cooled to 4°C, and made 6% w/v polyethylene glycol (PEG) by addition of 36.4 ml of a 50% w/v solution of PEG 6000 (Sigma). The PEG solution is added slowly over a period of 20-25 min, with constant stirring. The plasma is stirred for a further 30 rain at 4°C, then centrifuged (30 min at 10,000 g). The heavy yellow precipitate is discarded. A further 400/zl of stock DFP solution is added to the supernatant, and the supernatant is dialyzed to equilibrium against 5 liters of 20 mM sodium phosphate-50 mM NaCI-5 mM EDTA, pH 7.0. The precipitation step results in removal of 35-40% of the total plasma protein with 75-85% recovery of Cl-Inh activity, representing about 1.3fold purification. Step 2. DEAE-Celhdose Chromatography. The dialyzed supernatant is centrifuged (30 min, 10,000g). A small gellike precipitate usually forms at this stage, and is discarded. The supernatant is loaded onto a column (20 × 5 cm diameter) of DEAE-cellulose (DE-32 or DE-52, Whatman), equilibrated in the dialysis buffer. Flow rate should be 80-100 ml/hr. Protein that does not bind to the column is discarded and the column is washed with the starting buffer until the absorbance at 280 nm of the eluate is <0.05. Cl-Inh is then eluted in a linear gradient of NaCI formed by mixing 700 ml of the starting buffer with 700 ml of 20 mM sodium phosphate-300 mM NaCI-5 mM EDTA, pH 7.0. A typical elution profile ~ Since DFP is volatile and highly toxic, some laboratories may prefer to use the less toxic inhibitor phenyl methyl sulphonyl fluoride (PMSF; Sigma Chemical Co.). This material is prepared as a stock 110 m M solution in ethanol and added to aqueous solutions to a final concentration of about 1 raM. It is less effective than DFP as a general serine-protease inhibitor.
48
COMPLEMENT
[5]
2c
100 r
I <~
7o4
I
L)
~6
i
so S
18 O2 I
~
C12 0
10 20 v o l u m e e l u t e d (liters)
30
FIG. 2. DEAE-cellulose chromatography. Conditions are as described in the text. - - , A280;.... , Cl-Inh activity, expressed as % inhibition of Cls in a standard assay by 100 ~tl of eluant fraction; • •, relative salt concentration (RSC) (a solution with RSC = 0.1 M has the same conductivity as 0.1 M NaC1). An arrow marks the start of gradient elution, and a bar indicates the fractions pooled for the next purification step. is shown in Fig. 2. This step results in elimination of >99% of the total protein, with 50-55% recovery of C l - I n h activity, representing 120- to 170-fold purification. Step 3. Concanavalin A-Sepharose Chromatography: The pooled material from D E A E - c e l l u l o s e chromatography is again made 4 mM with D F P and dialyzed against 5 liters of 20 mM Tris-HCl-100 mM NaCI, pH 8.0, and then applied to a column (8 x 5 cm diameter) of concanavalin A - S e p h a r o s e (Pharmacia), equilibrated in the same buffer. Flow rate should be about 20 ml/hr. The column is washed with the starting buffer until the absorbance at 280 nm of the eluate is <0.02. C l - I n h is then eluted as a sharp peak (Fig. 3) by washing the column with 450 ml of the same buffer made 0.5% w/v with a-methylmannoside (Sigma). This purification step results in elimination of 30-50% of the remaining protein, with over 90% recovery of C l - I n h activity, and therefore represents a 1.3- to 1.8fold purification. The pool of C l - I n h is then dialyzed against 2 liters of 20 mM TrisH C I - 1 0 0 mM N a C I - 1 mM EDTA, pH 8.0, to remove c~-methylmannoside, and finally concentrated to about 1 mg/ml by ultrafiltration on a PM-10 membrane (Amicon). The purified protein is stable on storage at - 2 0 ° C for more than 1 year. The yield of C l - I n h by this method is 19-36 mg of C l - I n h (average over 14 preparations = 24 mg) from the 250 ml of starting material. The yield of activity is in the range 33-42%. The concentration of C l - I n h in
[5]
HUMAN C1 INHIBITOR
49
Cl-lnh I
<(
\ 0
~00
800
1200
v o l u m e eluted (ml
FIG. 3. Concanavalin A - S e p h a r o s e chromatography. Conditions are as described in the text. - - , A2s0. An arrow marks the start of elution with ~-methylmannoside, and a bar indicates the fractions pooled.
individual plasmas shows a relatively wide variation, and other investigators have suggested mean plasma concentrations of 235,1~ 185,1" and about 130 mg/liter.17 We have not systematically titrated Cl-Inh in plasma from single donors, but single radial immunodiffusion and functional assays performed on pooled plasmas (8-15 donors) used for preparing Cl-Inh consistently show an average Cl-Inh concentration greater than 200 mg/liter. Examination of Cl-Inh purified by the above method on sodium dodecyl sulfate (SDS)-polyacrylamide gels reveals a single major component of 100,000 apparent molecular weight, with traces (generally <5%) of two other components of apparent MW 60,000 and 30,000. These are likely to be degradation products of Cl-Inh. '~ Prolonged (over 1 month) storage of the isolated protein at 4°C in the absence of protease inhibitors leads to an increase in the quantity of the two minor components present. The final preparations are therefore likely to contain trace contamination with protease(s). Occasional contamination with transferrin and albumin is observed. Contaminating albumin can be removed by repeating step 3 of the preparation. Cl-Inh isolated by cther methods 4"~ exhibited two major bands on Coomassie blue-stained SDS-polyacrylamide gels. One species had an apparent MW 9,000-10,000 lower than Cl-Inh, and appeared to corre~:' N. Heimburger, Cold Spring Harbor Con f~ Cell Proliferation 2, 367 (1975). "; F. S. Rosen, C. A. Alper, J. Pensky, M. R. Klemperer, and V. H. Donaldson, J. Clin. Invest. 50, 2143 (1971). ~7 R. J. Ziccardi and N. R. Cooper, Clin. lmmunol. Immunopathol. 15, 465 (1980). ~ D. P. Man and J. O. Minta, Fed. Proe., Fed. A m . Soc. Exp. Biol. 37, 591 (abstr. 1991) (1978).
50
COMPLEMENT
[5]
spond to a major fragment produced by plasmin or trypsin digestion of Cl-Inh. ~9This degradation product is also observed in our preparations if protease inhibitors are not used during purification. C1--Inh in Other Species Cl-Inh has been isolated from rabbit serum in low yield,z° and has been shown to be very similar in molecular weight, temperature, and pH stability to the human protein. Rabbit Cl-Inh inhibits rabbit Clr, Cls, and plasmin, and also human Cls. "° Guinea pig Cl-Inh has also been purified, and although similar to human Cl-Inh in activity, was reported to have a much higher sedimentation coefficient (6.1 S). '~ Inhibitory activity against human Cls has been detected in the sera of numerous mammals, birds, and fish. 2'''3 The sera of several primates, but not of lower mammals, contain protein that is antigenically related to human Cl-Inh. 2'~ Partial purifications of Cl-Inh from other mammalian species have been summarized by Gigli and Austen. "4 Structural Properties Isolated human Cl-Inh is a single polypeptide chain monomer of MW 100,000-105,000 as estimated by equilibrium centrifugation and by SDSpolyacrylamide gel electrophoresis. 4'5'23 It has a sedimentation coefficient of 3.T5-4.0 S? The isoelectric point is 2.7-2.8. 2~ The amino acid and carbohydrate compositions of the protein have been determined by several groups and have been presented earlier in this series? The amino acid composition is unremarkable, except for an unusually low content of glycine and sulfur-containing amino acids. The tyrosine and tryptophan content is low, consistent with the low extinction coefficient of the protein (a~% Z X l c r n 280 nm = 4.525). Cl-Inh is, however, very heavily glycosylated, containing 69 tool hexose, 47 mol hexosamine, and 51 mol sialic acid per mole of protein. 2~ Consistent with the high carbohydrate content, the partial specific volume is low (0.667 ml/g2~). No sequence data are available for Cl-Inh. ' ' P. C. Harpel and N. R. Cooper, J. Clin. Invest. 55, 593 (1975). ~" E. Ishizaki, Y. Mori, and J. K o y a m a , J. Biochem. (Tokyo) 82, 1155 (1977). ~ M. Loos, W. Opferkuch, and R. Ringelmann, Z. Med. Microbiol. lmmunol. 156, 194 (1971). 22 L. R. L e v y and I. H. Lepow, Proc. Soc. Exp. Biol. Med. !01, 608 (1959). ~:~ V. H. Donaldson and J. Pensky, J. hnrnunol. 104, 1388 (1970). '-,4 I. Gigli and K. F. Austen, Annu. Rev. Microbiol. 25, 309 (1971). '-'~ H. Haupt, N. Heimburger, T. Kranz, and H. G. Schwick, Eur. J. Biochem. ! 7, 254 (1970).
[5]
HUMAN C1 INHIBITOR
51
Activity and Stability Isolated C l - I n h loses activity on exposure to pH values outside the range 6.0-9.5. 2 The activity is totally destroyed by heating at 60°C for 30 rain, but is stable to temperatures below 48°C. ~ Inhibitory activity is unaffected by neuraminidase treatment ~s but has been reported to be destroyed by exposure to 1 mM 2-mercaptoethanol. ~ C l - I n h has been shown to inhibit, in vitro, plasma kallikrein, ='"'~7plasrain, ~r activated Hageman Factor ( F X I I J s and its fragments, 29 and activated Factor XI (FXI~02s as well as C l r 27 and Cls. ~ It has no significant inhibitory activity against trypsin or chymotrypsin. ~~:' It is degraded by trypsin and by excess plasmin. 1'~ Inhibition of plasmin by C l - I n h does not appear to be of physiological significance since, in whole plasma, a2antiplasmin and a2-macroglobulin are more effective plasmin inhibitors, a° C l - I n h may, however, be a major physiological inhibitor of plasma kallikrein, :~':1'-' of FXII~ fragments, ~a'29":~':~3 and FXI,, a2 It is the only plasma inhibitor of C l r and Cls. a Inhibition of Clr, Cls, and plasmin by C l - I n h correlates with the formation of tightly bound complexes consisting of one molecule of inhibitor per molecule of protease, r'~:~'a4 These protease-(protease inhibitor) complexes are not dissociated by strong denaturants such as guanidine hydrochloride, urea, and SDS, or by low pH, and can be observed on S D S - p o l y a c r y l a m i d e gels after staining with Coomassie blue. a'a'r'l:''a4 The complexes are also resistant to reduction and alkylation. Patterns obtained on S D S - p o l y a c r y l a m i d e gel electrophoresis of reduced and alkylated complexes of C l - I n h with Clr, Cls, or plasmin show that the inhibitor binds to the protease through the light " B " chains, which contain the active site. a'~9 Prior inactivation of C l r or C l s with D F P prevents subsequent binding of C l - l n h 3~'aa and vice versa. :~4Thus it is likely that binding of C l - I n h to these proteins occurs via the catalytic active site. Similar formation of denaturation-resistant equimolar complexes has been described for other proteases with plasma protease inhibitors, for e,; I. Gigli, J. W. Mason, R. W. Colman, and K. F. Austen, J. Immured. 104, 574 (1970). 27 O. D. Ratnoff, J. Pensky, D. Ogston, and G. B. Naff, J. Exp. Med. 129, 315 (1969). ~ C. D. Forbes, J. Pensky, and O. D. Ratnoff, J. Lab. Clin. Med. 76, 809 (19711). z~'A. D. Schreiber, A. P. Kaplan, and K. F. Austen, J. Clin. Invest. 52, 1402 11973). a, p. C. Harpel, J. E.vp. Med. 146, 1033 (1977). :~ H. Fritz, E. Fink, and E. Truscheit, Fed. Proc.. Feel. A m . Soc. Exp. Biol. 38, 2753 (1979). :~2H. Saito, G. H. Goldsmith, M. Moroi, and N. Aoki, Proc. Natl. Acad. Sci. U.S.A. 76, 2013 (1979). a:~p. S. Damus, M. Hicks, and R. D. Rosenberg, Nature (London) 246, 355 11973). :~ G. J. Arlaud, A. Reboul, R. B. Sire, and M. G. C o l o m b , B i o c h i m . Bit, phys. Acta 576, 151 (1979). aa R. B. Sim, G. J. Arlaud, and M. G. Colomb, Biochem. J. 179, 449 (1979).
52
COMPLEMENT
[5]
example, Factor X~,-antithrombin III, 3'; t h r o m b i n - a n t i t h r o m b i n III, :~" trypsin-oq-antitrypsin, 37 trypsin-antithrombin III, 37 and plasmin-azantiplasmin. 3° Jesty 3" and Mahoney and colleagues 37 have recently shown that complexes of this type dissociate slowly on exposure to p H >9. Preliminary evidence (R. B. Sim, unpublished results; A. W. Dodds, unpublished results) indicates that this is also the case for p l a s m i n - C l - I n h and C l s - C l - I n h complexes. It is likely that the protease and inhibitor in such c o m p l e x e s are covalently associated, via formation of a tetrahedral intermediate? 7 Interaction with C l r and C l s The kinetics of interaction of C l - I n h with isolated C l r and C l s have been studied. 7 Both proteases have a similar affinity for C l - I n h (functional dissociation constant = 10 7 M).7 At 37°C, in the presence of EDTA, C l r reacts 4- to 5-fold slower with C-l-Inh (second-order rate constant kl = 2.8 × 103 M -~ sec ') than does C l s (kl - 1.2 × 104 M 1 sec ,).7 In buffers containing Ca 2+ ions, the rate of reaction of C l r is reduced 2-fold, but the reactivity of C l s toward C l - I n h is unaffected. 7 The activation energy for interaction of C l r and C l - I n h (44.3 kcal/mol) is much higher than that for the C l s - C l - I n h interaction (11.7 kcal/mol), and thus at temperatures below 37°C the disparity in the reaction rates with C l r and C l s b e c o m e s much greater. 7 Isolated C l r , which exists normally as a dimer, '° forms large complexes with C l - I n h , which are likely to have the composition Clr2-Cl-Inh2.:~ Under certain conditions, however, the C l r dimer appears to dissociate on interaction with C l - I n h , and C l r , - C l - I n h i complexes are formed. 3s Interaction of C l r that is present in a n t i b o d y - a n t i g e n - C 1 complexes, with C l - I n h , also results in dissociation of the C l r dimer. 39 Inhibition of isolated C l r and C l s by C l - I n h shows a p H o p t i m u m of 7.5-8.2 (R. B. Sim, unpublished results). The reaction of C l s with C l - I n h is insensitive to alteration in ionic strength, but the corresponding reaction with C l r proceeds optimally at an ionic strength corresponding to 110-300 m M NaCl. ~ Inhibition of C1 by C l - I n h is greatly enhanced in the presence of low concentrations of heparin. 7'4°'4~ This enhancement is due principally to an :~'~J. Jesty, J. Biol. Chem. 254, 1044 (1979). :~rW. C. Mahoney, K. Kurachi, and M. A. Hermodson, Eur. J. Biochem. 105, 545 (1980). ..~sS. Chesne, G. J. Arlaud, C. L. Villiers, and M. G. Colomb, Abstr.. Int. Congr. hnmunol.. 4th, 1980 Abstract 15.1.05 (1980). :~' R. J. Ziccardi and N. R. Cooper, J. lmmunol. 123, 788 (1979). '" R. Rent, R. Myrrmann, B. A. Fiedel, and H. Gewurz, Clin. Exp. lmmunol. 23, 264 (1976). H K. Nagaki and S. lnai, Int. Arch. Allergy Appl. lmmunol. 511, 172 (1976).
[5]
HUMAN C 1 INHIBITOR
53
acceleration of the rate of reaction of Cl-Inh with Cls. 7'4~ Heparin also increases the rate of Cl-Inh inactivation of Clr 7 and plasmin (R. B. Sim and P. E. Gasson, unpublished results), but the enhancement is much smaller than that seen with Cls. As discussed in Ref. 7, this enhancement is very similar to the effect of heparin on antithrombin III. The physiological significance of this effect is uncertain, as heparin has many inhibitory actions in the complement system?" As discussed above, there is evidence that Cl-Inh interacts directly with the active sites of the proteases that it inhibits. It is therefore to be expected that Cl-Inh inhibits in parallel all of the esterolytic and proteolytic activities of each enzyme. Parallel inhibition by Cl-Inh of Clsdependent cleavage of C2, C4, and amino acid esters has been reported? 3 However other investigators have suggested differences in the sensitivities to Cl-Inh of the C2-cleaving, C4-cleaving, and various esterase activities of Cls. 44'45 These discrepancies may be partly attributable to the diversity and complexity of the assay systems used, or to the use of insufficiently pure reagents. Nevertheless, this point has not been investigated systematically and requires further study. Interaction with C1 In the course of complement activation, Clr and Cls do not exist as free proteases in solution, but rather as Ca2+-dependent macromolecular Clr2-Cls2 complexes bound to Clq, which is in turn bound to the activating surface (e.g., antibody-antigen complexes). Cls bound within antibody-antigen-C1 reacts with C-l-Inh at the same rate as does isolated Cls, 7:~'~but the reactivity of Clr toward Cl-Inh is enhanced up to 4-fold when Clr is bound within C1. 7'35 Thus the reactivities of Clr and Cls toward Cl-Inh are much closer when Clr and Cls are within antibodyantigen-C1 complexes than when the proteases are free in solution. 7'3~'35 A number of studies 7'34''~'~':~9'46have established that interaction of Cl-Inh with antibody-antigen-C1 leads, under physiological conditions, to dissociation of the C1 complex. Cl-Inh reacts rapidly with the Cls in antibody-antigen-C1 complexes, and more slowly with the Clr. 35 Binding of Cl-Inh to the Clr leads to immediate dissociation of Clr, Cls, and Cl-Inh from the antibody-antigen-C1 complexes? 5 The proteins released are in the form of a Clrl-Clsl-Cl-Inh2 complexfl 9 which has MW ~e B. J. Johnson, J. Pharm. Sci. 66, 1367 (1977). ~:~ I. H. Lepow, G. B. Naff, and J. Pensky, Ciba Found. Syrup. [N.S.] 57, 74 (1965). H K. Takahashi, S. N a g a s a w a , and J. K o y a m a , Biochim. Biophys. Acta 61 I, 196 (1980). ~ M. Kondo, I. Gigli, and K. F. A u s t e n , Immunology 22, 305 (1972). "~ A.-B. Laurell, U. Johnson, U. M~rtensson, and A. G. Sj6holm, Acta Pathol. Microbiol. Stand., Sect. C 86, 299 (1978).
54
COMPLEMENT
[6]
330,000-382,000? 9 Specific assays for the Clri-Clsl-Cl-Inh2 complex in human serum have been developed by Laurell and colleagues 47 and these may be used as an indicator of C1 activation in patient plasma. A simple screening assay for Cl-Inh activity has been developed/7 based on the alteration of antigenic properties of C-lr when it is incorporated into the C l r l - C l s l - C l - I n h complex. Dissociation of the bound C1 complex by reaction with C-l-Inh leaves most of the C lq still associated with the immune complex or other surface responsible for C1 activation. Clq "exposed" in this way may then interact with lymphoblastoid cells possessing Clq receptors. 4r A.-B. Laurell, U. M~trtensson, and A. G. Sj6holm, Acta Pathol. Microbiol. S t a n d . , Sect. C 87, 79 (1979).
[6] T h e S e c o n d C o m p o n e n t of H u m a n C o m p l e m e n t By MICHAEL A. KERR
C2 is one of the least abundant of the complement components in human plasma, being present at a concentration of 15-20 mg/liter. ~''-' This and the extreme susceptibility of C2 to proteolytic digestion have in the past hampered its purification and molecular characterization. Functional studies using guinea pig and human preparations have, however, clearly identified the role of C2 in the classical pathway C3 and C5 convertases, and this has been reviewed extensively? -~ The classical pathway C3 convertase is assembled from C2 and C4 on cleavage of these proteins by the Cls subcomponent of the C1 complex. C4 is cleaved by Cls with the release of a small peptide, C4a, to form C4b (apparent MW 198,000)6:C2 is cleaved to C2a (apparent MW 74,000) and C2b (apparent MW 34,000).7's For the generation of C3 convertase activity, C4 cleavage must precede C2 cleavage. The enzyme is not generated by the addition of C4 to a previously incubated mixture of C2 and Cls, but 1 M. A. Kerr and R. R. Porter, Biochem. J. 171, 99 (1979). 2 M. J. Polley and H. J. Mfiller-Eberhard, J. Exp. Med. 128, 533 (1968). :s R. R. Porter and K. B. M. Reid, Adv. Protein Chem. 33, 1 (1979). 4 H. J. Miiller-Eberhard, in "Molecular Basis of Biological Degradative Processes" (R. D. Berlin, H. Hermann, I. H. Lepow, and J. M. Tanzer, eds.), p. 65. Academic Press, New York, 1978. 5 j. E. Fothergill and W. H. K. Anderson, Curr. Top. Cell. Regul. 13, 259 (1979). R. A. Patrick, S. B. Taubman, and I. H. Lepow, l m m u n o c h e m i s t r y 7, 217 (1979). 7 M. A. Kerr, Biochem. J. 183, 615 (1979). s S. Nagasawa and R. M. Stroud, Proc. Natl. Acad. Sci. U.S.A. 74, 2998 (1977).
METHODSIN ENZYMOLOGY,VOL. 80
Copyright © 1981 by Academic Press, Inc. All rights of reproductionin any form reserved. ISBN 0-12-181980-9