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Cleavage products of C4b produced by enzymes in human serum
lnmmnochemistry, 1975, Vol. 12, pp. 935 939. Pergamon Press. Printed in Great Britain.
CLEAVAGE PRODUCTS OF C4b PRODUCED BY ENZYMES IN HUMAN SERUM* SATOSHI SHIRAISHI and ROBERT M. S T R O U D t Division of Clinical Immunology and Rheumatology, Department of Medicine, University of Alabama in Birmingham, University Station, Birmingham, AL 35294, U.S.A. (First received 10 March 1975; in revised fi)rm 2 April 1975)
Abstract--When purified human C4 is reacted with purified Cls, two fragments called C4a and C4b are produced. On subsequent incubation with normal human serum or C4 deficient serum, C4b undergoes a further cleavage as indicated by a cathodal shift of its ¢/ arc on immunoelectrophoresis and generation of a new arc with 2 mobility. The results show that after CTs cleaves C4 to C4a and C4b, C4b is subsequently cleaved to C4c and C4d by other serum enzymes. The mol. wt of C4c (fl mobility) and C4d (2 mobility) as estimated by Sephadex G-150 gel filtration are approximately 140,000 and 50,000, respectively. When EAC4 cells are incubated in serum the hemolytic activity of C4 is destroyed. After whole serum is fractionated on G-150, the fraction which has the most destructive effect on EAC4 does not generate C4d from C4b until fractions from higher mol. wt regions are added. The partial purification of these enzymes indicated that fractions rich in the C3b inactivator bring about destruction of cell bound C4b but another enzyme is necessary for generation of the C4d fragment from C4b. Furthermore, C3b inactivator deficient serum will not generate the C4d fragment until partially purified C3b inactivator is restored. INTRODUCTION The first component of complement (CI) is an assembly of subcomponents designated Clq, C l r and Cls (Lepow et al., 1963). When C1 is activated (C]-), C l s is converted to its active form (Cls). The fourth component of complement (C4) is one of the natural substrates of C l s and is known to be cleaved by C]-s with a loss of hemolytic activity if there are no suitable sensitized cell receptors (EA) close at hand. After cleavage a large fragment C4b and a small fragment, C4a are produced. These two fragments remain associated at neutral p H but dissociate after acidification. This dissociation is not reversible (Patrick et al., 1970, and Budzko et al., 1970). On immunoelectrophoresis (IEP) C4, after cleavage by C l s exhibits an increased net negative charge and gives rise to an elongated precipitin line suggesting molecular heterogeneity (Mtiller-Eberhard and Lepow, 1965). However, other split product(s) have not been demonstrated on IEP. A n t i g e ~ a n t i b o d y crossed electrophoresis has been applied to study the structural polymorphism of C4 (Rosenfeld et al., 1969). These authors described partial conversion of C4 during agarose electrophoresis of serum. They also demonstrated a sing_le conversion product produced by the action of Cl on purified C4 and the lack of hemolytic inactivation of C4 when C1 was removed from serum by euglobulin precipitation before electrophoresis. Conversion products of C4 on crossed immunoelectrophoresis in serum and in aged EDTA-plasma were also reported by Sjoholm
* This work was supported in part by grants from the N.I.H. and credit is given to project 8197-01, VA Hospital, Birmingham, AL t To whom all reprint requests should be sent.
and Laurell, (1973). They found a conversion product in the fl region when Cls, trypsin, chyrnotrypsin, plasmin or thrombin was added to partially purified C4. CTs, trypsin and chymotrypsin produced cleavage products in the fl and e regions in the presence of EDTA. They suspected that some of the products were complexes with other serum proteins, however, our preliminary experiments suggested the possibility of further fragmentation by other serum enzyme(s) acting on the C l s cleaved CA (Perrin et al., 1974; Shiraishi and Stroud, 1974). These experiments extend this information and document the cleavage of C4b. The smaller and larger products are referred to as C4d and C4c, respectively. Some characteristics of the enzyme(s) which cleave C4 in the fluid phase and cell bound C4 are examined. MATERIALS AND METHODS
Normal human serum and plasma
Blood from healthy adults was drawn into tubes without anticoagulant or into tubes containing 0.004 M EDTA and immediately centrifuged and kept at - 7 0 C . Outdated blood was from the Blood Bank at the University of Alabama in Birmingham, AL. C4 deficient human serum was a gift from Dr. Gerald Hauptmann, Strasbourg, France. C2 deficient serum was drawn from a previously described C2 deficient patient (Klemperer et al., 1966). C3b inactivator deficient serum (T.J.) was a gift from Dr. Richard Johnston (University of Alabama in Birmingham, AL). Cls was purified according to Sakai and Stroud (1973). The C]-s was homogeneous on immunoelectrophoresis against antiwhole human serum (Institute for Microbiology, Osaka University, Osaka, Japan) and on analytical acrylamide gel electrophoresis. Cellulose chromatography
Fibrous DEAE cellulose (Whatman DE23) used for the purification of C4 was purchased from H. Reeve Angel, 935
936
SATOSHI SH1RAISHI and ROBERT M. STROUD
Inc., Clifton, NJ and prepared according to their instructions. DEAE cellulose was equilibrated with 0.005 M phosphate buffer, 0-002 M EDTA (pH 7%, RSC = 0.0b~l.
Preparation of affinity resin Sepharose 6B was purchased l?om Pharmacia Fine Chemicals, Inc., Piscataway, NJ. Human lgG covtdently linked to Sepharose 6B under basic conditions with cyanogen bromide was prepared according to Patrick et al. (1973). A crude (74 fraction precipitated by Na2SO4 from psuedoglobulin of human serum and subsequently eluted from DEAE cellulose was used for further purification on a column of Sepharose 6B coupled with lgG. Gel .filtratio~ Sephadex G- 150 was boiled lot l0 min, washed and then suspended in 0.15M NaC1 for packing. The packed column was equilibrated with buffer for 3 days before use. Marker molecules were cytochrome C (Sigma Chemical Co.): ovalbumin (Sigma Chemical Co): bovine serum albumin (Armour Pharmaceutical Co.): human lgG (Mann Research Laboratories, Inc., NYI: catalase (Sigma Chemical Co.) assuming the mol, wt to be 12,400, 45,000, 67,000, 160,000 and 244,000, respectively.
lmmunodiffi*sion Radial lmmunodiffusion (RID) was carried out according to a modification of Mancini et .1. (1965) with I% agarose containing an optimal amount of antibody to human C4. lmmunoelectrophoresis was carried out with a modification of the method of Scheidegger with 1% agarose in Veronal acetate buffer containing 0.01 M EDTA (pH ,~.6, RSC = 0.06).
13l~l]~'r The relative salt concentration (RSC) of a buffer relers to the molarity of a solution of NaCI giving the same electrical resistance as the buffer at 0 (7. Isotonic Veronal-buffered saline IVBS) with optimal amounts of Ca ~ , Mg 2~ and gelatin (GVB 2~) was prepared according to Mayer (1961L Veronal-buffered dextrose saline containing Ca 2". Mg 2 " and gelatin (DGVB 2 " ) was prepared according to Nelson el a/. (19661.
tfemolytic titration C4 hemolytic titrations were carried out in microtiter plates or in tubes as described by Nelson et al. (1966) using EAC1 cells, functionally purified C2 and C-EDTA. C4 activity could also bc detected using C4 deficient guinea pig scrum according to Gaithcr et al. (1974). The C4 deficient guinea pigs were a gift from Dr. Michael Frank, NIH, Bethesda, MD. EAC4 cells were prepared according to the method of Borsos and Rapp (19671. RESULTS
c a purification and antibody By using Na2SO,~, two columns and acrylamide gel eleetrophoresis, C4 could be purified for immunization as shown schematically in Fig. 1. Outdated normal human serum was subjected to two precipitations to remove the greater part of CI and C5 as described by Vroon et al. (1970). The supernatant of the second precipitate after adjusting the pH to 6-8 was mixed with Na2SOa at a final concentration of 14'% and the resulting precipitates were discarded. Crude C4 was obtained after further precipitation with Na2SO4 at a final concentration of 18~'{; and was applied to a D E A E cellulose column at pH 7.5 and RSC of 0'08. C4 was eluted with a linear salt gradient up to a
limiting RSC of 0"3. Thc C4 rich fractions, concentrated by ultrafiltration, were applied to a column of Sepharose 6B coupled with human lgG ;_it pH 75 and a RSC of 0.02 according to Patrick et al. (19731. Elution was carried out stepwise. After twice repeating acrylamide gel electrophorcsis, the get slices and eluates containing hemolytically active C4 were pulverized and emulsified with equal volumes of Freund*s complete adjuvant. Usually one or two segments from a single second gel run were injected subcutaneously into the back and gluteal region of a goat. Three weeks later the goat was boosted b,, intracutaneous injection of a similar amount of C4 in tile same adjuvant. One week after boosting antibodies were lound in a significant titer. Since the antisera was monospecific against fresh normal human serum, absorption was unnecessary (Perrin eta/., 1974~.
C4 ./i'agments produced b.l' (7]-.~ aml otlz~'r wr,m ~dI1Z VD1CS
The immunoelectrophoretic pattern of (7'4 was studied after incubation with CIs or aggregated human gamma globulin (agg. H G G ) and fresh human serum. As shown in Fig. 2, a new fragment exhibiting mobility is generated in a fresh normal serum pool (NHS) or in C2 deficient serum alter incubation with CTs or agg. H G G but is absent m NHS incubated with buffer and in C4 deficient serum. After generation of the fragment with ~ mobility, the precipitation arc in the fl region showed a slight but reproducibly increased cathodal migration.
MolecHlar u'eight eslimatioJl (!f the ~. fi'a~tmt'*It A column of Sephadcx G-150 was equilibrated with 0-1 M Tris buffer, containing 0.2M NaCl, 0.002M EDTA, 002",0 NAN3, pH 7.6. The run was perlormed in an upward direction at a flow rate of 25 ml/hr. Six millilitres of NHS was incubated with 105 pg of C l s for 1 hr at 3 7 C and applied to the column and 5 ml fractions were collected. The C4 protein of each fl-action was detected by radial immunodiffusion. From the elution volume, the tool, wt of the y. fragment was calculated to be approximately 50,000 by using the method of Andrews (1964}. The fragment in the fi precipitin arc on IEP was calculated to have a mol. wt of approximately 140,0{R).
The stepwise nature o/ C4 clearoge I. Prool of fio'ther clearaqe bv ,wrum enzyme,s clem~ing the (~(s treated (7'4 (C4b). ('4 deficient human serum which did not show any lines with anti-C4 antibodies and has no delectable hemolytic C4 was used as a reagent containing the enzyme(s) in the second step. About 120/~g/100/~1 of partially purified 674 was incubated with 6 F~g (in 10#1) of purified C l s for 1 hr at 37'~C, then 50/xl of 6'4 deficient serum was added to the above mixture followed by incubation for another hr at 3 7 C . After the second incubation, all samples were analyzed on IEP as shown in Fig. 3. The electrophoretic mobility of C4 treated with Cls, C4b, is faster than native C4. Using C4 deficient serum as a source of the C4b cleaving enzyme(s) a new fragment exhibiting ,~ mobility is generated and the precipitin arc in the fi region shifts to a position cathodal to native C4. We have desigmated these arcs.
937
Generation of C4d
Purification of C4 Normal Human Serum
/~Dilution RSC .04 7.5 Sup.
Sup.
/~O]oN%SO, Sup. //J~
% Nazs04
Ppt.
I DEAE
+-
I
Sepharose 6B coupled With IgG
I
Gel Electrophoresis (2 x) immunize with 2nd. gel segment Fig. I. Flow chart of C4 purification. A, 1st electrophoresis run; B, 2nd run. Arrow indicates hemolyti-
cally active C4. representing cleavage products of C4b as C4d and C4c respectively. 2. Existence of two cooperating enzymes which are capable of generating the orfragment. In order to determine the nature of these enzyme(s), fresh normal human serum was applied on Sephadex G-150. As shown in Fig. 4, three protein peaks were detected by O.D. measurement at 280 nm and six pools designated as shown in Fig. 4, P-l, -II, -III, -IV, -V and -VI were concentrated to about the original serum volume by utilizing an Amicon concentration device with a UM 10 filter. None of the pools generated an ~ fragment on IEP, but only the combination P-I and P-IV produced an c~ fragment when incubated with C4b generated from C4 and Cls. We have not observed a change in mobility after incubation with peak I.
serum or serum fractions from Sephadex G-150 were diluted with 0.01 M EDTA GVB and incubated with equal volumes of EAC4 cells (1-5 × 108/ml) for 2 hr at 37°C. The cells were washed twice with ice cold EDTA GVB and GVB 2+ and were then re-suspended to the original volume and incubated at 30°C with C4 deficient guinea pig serum diluted 1/100 in order to measure the residual SAC4. The reaction was stopped by the addition of ice cold EDTA GVB when the control tube showed approximately 75% lysis. As shown in Fig. 5, it is clear that there is a loss of SAC4 sites after incubation with serum. Furthermore, this inhibitory effect is only found in fraction P-IV (Fig. 4) and the addition of any otl~er pool did not produce a significant increase in the inhibitory effects.
Inhibitory effects on EAC4 cells by serum and serum fractions To determine the effects on EAC4 cells by serum, it was necessary to absorb the Forssman antigen according to the method of Mayer (1961). Absorbed
DISCUSSION The data presented in this paper indicate further cleavage of C4 by other enzyme(s) acting after the action of Cls. When purified C4 is treated with purified Cls, two fragments called C4a and C4b are pro-
938
SATOSHI SHIRAISHI and ROBERT M, STROt!D Gel Filtrotion
G-I50
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o~2ooo
I~ 1
I000
i
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____
150
P-~
I
200
250 300 Volume {roll
..J_
350
400
Fig. 4. Chromatography of fresh normal human scrum on Sephadex (;-[51~. See text. Fig. 2. lmmuntNlectrophoretic analysis of various sera incubated with C Is and agg. HGG: a, fresh normal human serum (NHS) + C]-s: b, NHS + buffer: c. NHS + agg. HGG; d, C4 deficient serum (('4d)+ buffer: e. C4d + Cls; f. C4d + agg. HGG; g, C2 deficient serum (C2d) + C]-s; h, C2d + buffer. Cis (15 /~g:10 /d), agg. HGG. (200,ug/10Hl) and 10/tl of buffet were incubated witfi 100 HI of serum at 37 C for 60 min before the 1EP. Anode is at the right. Anti-C4 in trough. duced. O n subsequent incubation with C4 deficient serum as a source of these enzymes, C4b which migrates anodally to native C4 on IEP undergoes further cleavage as indicated by a shift of its fl arc to a position cathodal to C4b and native C4 and by the generation of a new fragment exhibiting :~ mobility. These findings suggest that after C l s cleaves C4 to C4a and C4b, C4b is subsequently cleaved to C4c and C4d by other enzymes. The result possibly coincides with the results of Sjoholm and Laurell (1973), who found C4 conversion products in the [1 and :~ region on crossed immunoelectrophoresis when C]s, trypsin and chymotrypsin were added to E D T A -
serum. ]'he tool. wt of ('4c and C4d as estimated by Sephadex G-150 gel tiltration were approximately 140,000 and 50,000, respecti,,el 5. The sum of the tool. wt of the two fragments, 190,000 is in close agreement with the mol. wt of ('4 as found by. Schreiber and Miiller-Eberhard (1974l. Our mol. wt estimates and the mobility on IEP show that ('4d is not identical to the known fragments C4a and C4b. The following facts lend support to the concept that C4b undergoes further fragmentation: 1. C4d is absent in fresh serum. fresh EDTA-plasma and fresh EDTA-serum but present in aged materials: 2. C4d is generated after incubation with C l s and NHS, EDTA-plasma or E D T A serum but not generated after activation of the alternative pathway by insulin (Perrin et al., 1974): 3. C4d is not detected in C4 deficient serum either in its native form nor after incubation with C l s or agg. H G G : and 4. C4d is not the 42 complex (C4d is generated in C2 deficient serumi. It is of interest to compare this proposed hagmentation scheme tbr C'4 with current knowledge of the fragmentation and structure of C3. C3 is cleaved by the action of C3 convertasc 1C42), factor B or trypsin into a small fragment, C3a and a large fragment C3b. Then C3b inactivator or K A F cleaves C3b into C3c and C3d. Activated C3b has two biologically Loss of SAC4 After Incubotion With Serum I00
.-r "E
50
8 Q_
Fig. 3. Immunophoretic analysis of the cleavage of ('4b by serum enzymes present in C4d serum. Anti-C4 in trough, a, C4 + Cls: b, C4 + C]-s + C4 deficient serum: c, C4 deficient serum: d, ('4 solo.
i i
i
i
32 16
8
4
hi Serum
Dilution
Fig. 5. l,oss of SAC4 alter incubation vdth scrum
Generation of C4d
Fig. 6. Immunoelectrophoretic analysis of C4 cleavage by C3b inactivator deficient serum with or without added C3b inactivator 1st incubation a. C4 + C]-s
2nd incubation
+C3b inactivator deficient serum b. C4 + C ] - s +C3b inactivator rich fraction c. Same as (a) Same as (a) d. Same as (b) Same as (b) e. Normal human serum pool
939
shows that two enzymes or serum fractions are necessary to generate C4d from C4b. Quantitative data on these enzymes will be published separately, but it should be noticed that the mobility of the C4b arc is more anodal when incubated with the C3b inactivator deficient serum. This suggests that a serum fraction reacts with C4b to make an intermediate cleavage product or complex, but cannot produce the C4d fragment without the C3b inactivator. Since high concentrations of C4d are found in the synovial fluid of patients with rheumatoid arthritis and in hereditary angioneurotic edema (HANE) sera, detection and quantitation of this fragment will be clinically useful in these diseases and perhaps in other diseases which activate the classical pathway. We have described recently the generation of this fragment by incubation of serum with agg. HGG. which "clearly relates its significance to immune complex diseases (Perrin et al., 1974).
3rd incubation +C3b inactivator Rich fraction +C3b inactivator Deficient serum + Buffer + Buffer
The double C4 arc is due to excess antibody
important binding sites; a labile binding site which allows the attachment of C3b to cell surfaces and a stable binding site which enables C3b to react with the C3b receptor on the surface of various cells (Miiller-Eberhard et al., 1966; Nishioka and Linscott, 1963). Recently a similarity between the two polypeptide chains of C3 and two of the three polypeptide chains of C4 was presented (Schreiber and MfillerEberhard, 1974). Furthermore, a receptor for C3b on human erythrocytes, B-lymphocytes, and polymorphonuclear leukocytes has been reported (Cooper, 1969; Ross and Polley, 1974; Sobel and Bokisch, 1974) and it was shown that C4b was cleaved by the C3b inactivator (Bokisch and Sobel, 1974; Cooper, 1975), rendering cell bound C4b unreactive in immune adherence or in rosette formation with erythrocytes or lymphocytes. Our data show that EAC4 cells treated with serum fractions lose their hemolytic activity suggesting the presence of a C4b inactivator(s). This observation thus would be analogous to the activity of the C3b inactivator on cell bound C3b. The purification of this inactivator(s) is underway. We have evidence that partially pure fractions rich in the C3b inactivator bring about destruction of cell bound C4b hemolytic sites but do not produce C4d fragments from C4b fragments. In addition the C3b inactivator deficient serum cannot generate C4d fragments until the C3b inactivator is restored (Fig. 6). These results support the results of Fig. 4 which
REFERENCES
Andrews P. (1964) Biochem. J. 91, 222. Bokisch V. A. and Sobel A. T. (1974) J. exp. Med. 140, 1336. Borsos T. and Rapp H. J. (1967) J. lmmun, 99, 263. Budzko D. B. and Miiller-Eberhard H. J. (1970) lmmunochemistry 7, 227. Cooper N. R. (1969) Science 165, 396. Cooper N. R, (1975) J. exp. Med. 141, 890. Gaither T. A., Ailing D. W. and Frank M. M. (1974) J. lmmun. 113, 574. Klemperer M. R., Woodworth H. C., Rosen F. S. and Austen K. F. (1966) J. clin. Invest. 45, 880. Lepow I. H., Naff G. B., Todd E. W., Pensky J. and Hinz C. F. (1963) J. exp. Med. 117, 983. Mancini G., Carbonara O. and Heremens J. F. (1965) lmmunochemistry 2, 235. Mayer M. M. (1961) in Experimental lmmunochemistry, 2rid Edn. Thomas, Springfield, IL. Miiller-Eberhard H. J. and Lepow I. H. (1965) J. exp. Med. 121, 819. Miiller-Eberhard H. J., Dalmasso A. P. and Calcott M. A. (1966) J. exp. Med. 123, 33. Nelson R. A. Jr., Jensen J., Gigli I. and Tamura N. (1966) lmmunochemistry 3, 111.
Nishioka K. and Linscott W. D. (1963) J. exp. Med. 118, 767. Patrick R. A., Taubman S. B. and Lepow I. H. (1970) lmmunochemistry 7, 217.
Patrick R. A., Mernitz J. and Bing D. H. (1973) J. hnmun. I11,296. Perrin L. H., Shiraishi S., Stroud R. M. and Lambert P. H. (1975) J. Immun. 115, 32. Rosenfeld S. J., Ruddy S. and Austen K. F. (1969) J. clin. Invest. 48, 2283. Ross G. D. and Polley M. J. (1974) Fedn Proc. 33, 759. Sakai K. and Stroud R. M. (1973) J. Immun. 110, 1010. Schreiber R. D. and Mi~ller-Eberhard H. J. (1974) J. exp. Med. 140, 1324. Shiraishi S. and Stroud R. M. (1974) Submitted to American Rheumatism Association, Southeastern Regional Meeting, 13-14 Dec, 1974. SjOholm A. G. and Laurell A.-B. (1973) Clin. exp. lmmunol. 14, 515.
CLEAVAGE PRODUCTS OF C4b PRODUCED BY ENZYMES IN HUMAN SERUM* SATOSHI SHIRAISHI and ROBERT M. S T R O U D t Division of Clinical Immunology and Rheumatology, Department of Medicine, University of Alabama in Birmingham, University Station, Birmingham, AL 35294, U.S.A. (First received 10 March 1975; in revised fi)rm 2 April 1975)
Abstract--When purified human C4 is reacted with purified Cls, two fragments called C4a and C4b are produced. On subsequent incubation with normal human serum or C4 deficient serum, C4b undergoes a further cleavage as indicated by a cathodal shift of its ¢/ arc on immunoelectrophoresis and generation of a new arc with 2 mobility. The results show that after CTs cleaves C4 to C4a and C4b, C4b is subsequently cleaved to C4c and C4d by other serum enzymes. The mol. wt of C4c (fl mobility) and C4d (2 mobility) as estimated by Sephadex G-150 gel filtration are approximately 140,000 and 50,000, respectively. When EAC4 cells are incubated in serum the hemolytic activity of C4 is destroyed. After whole serum is fractionated on G-150, the fraction which has the most destructive effect on EAC4 does not generate C4d from C4b until fractions from higher mol. wt regions are added. The partial purification of these enzymes indicated that fractions rich in the C3b inactivator bring about destruction of cell bound C4b but another enzyme is necessary for generation of the C4d fragment from C4b. Furthermore, C3b inactivator deficient serum will not generate the C4d fragment until partially purified C3b inactivator is restored. INTRODUCTION The first component of complement (CI) is an assembly of subcomponents designated Clq, C l r and Cls (Lepow et al., 1963). When C1 is activated (C]-), C l s is converted to its active form (Cls). The fourth component of complement (C4) is one of the natural substrates of C l s and is known to be cleaved by C]-s with a loss of hemolytic activity if there are no suitable sensitized cell receptors (EA) close at hand. After cleavage a large fragment C4b and a small fragment, C4a are produced. These two fragments remain associated at neutral p H but dissociate after acidification. This dissociation is not reversible (Patrick et al., 1970, and Budzko et al., 1970). On immunoelectrophoresis (IEP) C4, after cleavage by C l s exhibits an increased net negative charge and gives rise to an elongated precipitin line suggesting molecular heterogeneity (Mtiller-Eberhard and Lepow, 1965). However, other split product(s) have not been demonstrated on IEP. A n t i g e ~ a n t i b o d y crossed electrophoresis has been applied to study the structural polymorphism of C4 (Rosenfeld et al., 1969). These authors described partial conversion of C4 during agarose electrophoresis of serum. They also demonstrated a sing_le conversion product produced by the action of Cl on purified C4 and the lack of hemolytic inactivation of C4 when C1 was removed from serum by euglobulin precipitation before electrophoresis. Conversion products of C4 on crossed immunoelectrophoresis in serum and in aged EDTA-plasma were also reported by Sjoholm
* This work was supported in part by grants from the N.I.H. and credit is given to project 8197-01, VA Hospital, Birmingham, AL t To whom all reprint requests should be sent.
and Laurell, (1973). They found a conversion product in the fl region when Cls, trypsin, chyrnotrypsin, plasmin or thrombin was added to partially purified C4. CTs, trypsin and chymotrypsin produced cleavage products in the fl and e regions in the presence of EDTA. They suspected that some of the products were complexes with other serum proteins, however, our preliminary experiments suggested the possibility of further fragmentation by other serum enzyme(s) acting on the C l s cleaved CA (Perrin et al., 1974; Shiraishi and Stroud, 1974). These experiments extend this information and document the cleavage of C4b. The smaller and larger products are referred to as C4d and C4c, respectively. Some characteristics of the enzyme(s) which cleave C4 in the fluid phase and cell bound C4 are examined. MATERIALS AND METHODS
Normal human serum and plasma
Blood from healthy adults was drawn into tubes without anticoagulant or into tubes containing 0.004 M EDTA and immediately centrifuged and kept at - 7 0 C . Outdated blood was from the Blood Bank at the University of Alabama in Birmingham, AL. C4 deficient human serum was a gift from Dr. Gerald Hauptmann, Strasbourg, France. C2 deficient serum was drawn from a previously described C2 deficient patient (Klemperer et al., 1966). C3b inactivator deficient serum (T.J.) was a gift from Dr. Richard Johnston (University of Alabama in Birmingham, AL). Cls was purified according to Sakai and Stroud (1973). The C]-s was homogeneous on immunoelectrophoresis against antiwhole human serum (Institute for Microbiology, Osaka University, Osaka, Japan) and on analytical acrylamide gel electrophoresis. Cellulose chromatography
Fibrous DEAE cellulose (Whatman DE23) used for the purification of C4 was purchased from H. Reeve Angel, 935
936
SATOSHI SH1RAISHI and ROBERT M. STROUD
Inc., Clifton, NJ and prepared according to their instructions. DEAE cellulose was equilibrated with 0.005 M phosphate buffer, 0-002 M EDTA (pH 7%, RSC = 0.0b~l.
Preparation of affinity resin Sepharose 6B was purchased l?om Pharmacia Fine Chemicals, Inc., Piscataway, NJ. Human lgG covtdently linked to Sepharose 6B under basic conditions with cyanogen bromide was prepared according to Patrick et al. (1973). A crude (74 fraction precipitated by Na2SO4 from psuedoglobulin of human serum and subsequently eluted from DEAE cellulose was used for further purification on a column of Sepharose 6B coupled with lgG. Gel .filtratio~ Sephadex G- 150 was boiled lot l0 min, washed and then suspended in 0.15M NaC1 for packing. The packed column was equilibrated with buffer for 3 days before use. Marker molecules were cytochrome C (Sigma Chemical Co.): ovalbumin (Sigma Chemical Co): bovine serum albumin (Armour Pharmaceutical Co.): human lgG (Mann Research Laboratories, Inc., NYI: catalase (Sigma Chemical Co.) assuming the mol, wt to be 12,400, 45,000, 67,000, 160,000 and 244,000, respectively.
lmmunodiffi*sion Radial lmmunodiffusion (RID) was carried out according to a modification of Mancini et .1. (1965) with I% agarose containing an optimal amount of antibody to human C4. lmmunoelectrophoresis was carried out with a modification of the method of Scheidegger with 1% agarose in Veronal acetate buffer containing 0.01 M EDTA (pH ,~.6, RSC = 0.06).
13l~l]~'r The relative salt concentration (RSC) of a buffer relers to the molarity of a solution of NaCI giving the same electrical resistance as the buffer at 0 (7. Isotonic Veronal-buffered saline IVBS) with optimal amounts of Ca ~ , Mg 2~ and gelatin (GVB 2~) was prepared according to Mayer (1961L Veronal-buffered dextrose saline containing Ca 2". Mg 2 " and gelatin (DGVB 2 " ) was prepared according to Nelson el a/. (19661.
tfemolytic titration C4 hemolytic titrations were carried out in microtiter plates or in tubes as described by Nelson et al. (1966) using EAC1 cells, functionally purified C2 and C-EDTA. C4 activity could also bc detected using C4 deficient guinea pig scrum according to Gaithcr et al. (1974). The C4 deficient guinea pigs were a gift from Dr. Michael Frank, NIH, Bethesda, MD. EAC4 cells were prepared according to the method of Borsos and Rapp (19671. RESULTS
c a purification and antibody By using Na2SO,~, two columns and acrylamide gel eleetrophoresis, C4 could be purified for immunization as shown schematically in Fig. 1. Outdated normal human serum was subjected to two precipitations to remove the greater part of CI and C5 as described by Vroon et al. (1970). The supernatant of the second precipitate after adjusting the pH to 6-8 was mixed with Na2SOa at a final concentration of 14'% and the resulting precipitates were discarded. Crude C4 was obtained after further precipitation with Na2SO4 at a final concentration of 18~'{; and was applied to a D E A E cellulose column at pH 7.5 and RSC of 0'08. C4 was eluted with a linear salt gradient up to a
limiting RSC of 0"3. Thc C4 rich fractions, concentrated by ultrafiltration, were applied to a column of Sepharose 6B coupled with human lgG ;_it pH 75 and a RSC of 0.02 according to Patrick et al. (19731. Elution was carried out stepwise. After twice repeating acrylamide gel electrophorcsis, the get slices and eluates containing hemolytically active C4 were pulverized and emulsified with equal volumes of Freund*s complete adjuvant. Usually one or two segments from a single second gel run were injected subcutaneously into the back and gluteal region of a goat. Three weeks later the goat was boosted b,, intracutaneous injection of a similar amount of C4 in tile same adjuvant. One week after boosting antibodies were lound in a significant titer. Since the antisera was monospecific against fresh normal human serum, absorption was unnecessary (Perrin eta/., 1974~.
C4 ./i'agments produced b.l' (7]-.~ aml otlz~'r wr,m ~dI1Z VD1CS
The immunoelectrophoretic pattern of (7'4 was studied after incubation with CIs or aggregated human gamma globulin (agg. H G G ) and fresh human serum. As shown in Fig. 2, a new fragment exhibiting mobility is generated in a fresh normal serum pool (NHS) or in C2 deficient serum alter incubation with CTs or agg. H G G but is absent m NHS incubated with buffer and in C4 deficient serum. After generation of the fragment with ~ mobility, the precipitation arc in the fl region showed a slight but reproducibly increased cathodal migration.
MolecHlar u'eight eslimatioJl (!f the ~. fi'a~tmt'*It A column of Sephadcx G-150 was equilibrated with 0-1 M Tris buffer, containing 0.2M NaCl, 0.002M EDTA, 002",0 NAN3, pH 7.6. The run was perlormed in an upward direction at a flow rate of 25 ml/hr. Six millilitres of NHS was incubated with 105 pg of C l s for 1 hr at 3 7 C and applied to the column and 5 ml fractions were collected. The C4 protein of each fl-action was detected by radial immunodiffusion. From the elution volume, the tool, wt of the y. fragment was calculated to be approximately 50,000 by using the method of Andrews (1964}. The fragment in the fi precipitin arc on IEP was calculated to have a mol. wt of approximately 140,0{R).
The stepwise nature o/ C4 clearoge I. Prool of fio'ther clearaqe bv ,wrum enzyme,s clem~ing the (~(s treated (7'4 (C4b). ('4 deficient human serum which did not show any lines with anti-C4 antibodies and has no delectable hemolytic C4 was used as a reagent containing the enzyme(s) in the second step. About 120/~g/100/~1 of partially purified 674 was incubated with 6 F~g (in 10#1) of purified C l s for 1 hr at 37'~C, then 50/xl of 6'4 deficient serum was added to the above mixture followed by incubation for another hr at 3 7 C . After the second incubation, all samples were analyzed on IEP as shown in Fig. 3. The electrophoretic mobility of C4 treated with Cls, C4b, is faster than native C4. Using C4 deficient serum as a source of the C4b cleaving enzyme(s) a new fragment exhibiting ,~ mobility is generated and the precipitin arc in the fi region shifts to a position cathodal to native C4. We have desigmated these arcs.
937
Generation of C4d
Purification of C4 Normal Human Serum
/~Dilution RSC .04 7.5 Sup.
Sup.
/~O]oN%SO, Sup. //J~
% Nazs04
Ppt.
I DEAE
+-
I
Sepharose 6B coupled With IgG
I
Gel Electrophoresis (2 x) immunize with 2nd. gel segment Fig. I. Flow chart of C4 purification. A, 1st electrophoresis run; B, 2nd run. Arrow indicates hemolyti-
cally active C4. representing cleavage products of C4b as C4d and C4c respectively. 2. Existence of two cooperating enzymes which are capable of generating the orfragment. In order to determine the nature of these enzyme(s), fresh normal human serum was applied on Sephadex G-150. As shown in Fig. 4, three protein peaks were detected by O.D. measurement at 280 nm and six pools designated as shown in Fig. 4, P-l, -II, -III, -IV, -V and -VI were concentrated to about the original serum volume by utilizing an Amicon concentration device with a UM 10 filter. None of the pools generated an ~ fragment on IEP, but only the combination P-I and P-IV produced an c~ fragment when incubated with C4b generated from C4 and Cls. We have not observed a change in mobility after incubation with peak I.
serum or serum fractions from Sephadex G-150 were diluted with 0.01 M EDTA GVB and incubated with equal volumes of EAC4 cells (1-5 × 108/ml) for 2 hr at 37°C. The cells were washed twice with ice cold EDTA GVB and GVB 2+ and were then re-suspended to the original volume and incubated at 30°C with C4 deficient guinea pig serum diluted 1/100 in order to measure the residual SAC4. The reaction was stopped by the addition of ice cold EDTA GVB when the control tube showed approximately 75% lysis. As shown in Fig. 5, it is clear that there is a loss of SAC4 sites after incubation with serum. Furthermore, this inhibitory effect is only found in fraction P-IV (Fig. 4) and the addition of any otl~er pool did not produce a significant increase in the inhibitory effects.
Inhibitory effects on EAC4 cells by serum and serum fractions To determine the effects on EAC4 cells by serum, it was necessary to absorb the Forssman antigen according to the method of Mayer (1961). Absorbed
DISCUSSION The data presented in this paper indicate further cleavage of C4 by other enzyme(s) acting after the action of Cls. When purified C4 is treated with purified Cls, two fragments called C4a and C4b are pro-
938
SATOSHI SHIRAISHI and ROBERT M, STROt!D Gel Filtrotion
G-I50
3.000
o~2ooo
I~ 1
I000
i
P"
____
150
P-~
I
200
250 300 Volume {roll
..J_
350
400
Fig. 4. Chromatography of fresh normal human scrum on Sephadex (;-[51~. See text. Fig. 2. lmmuntNlectrophoretic analysis of various sera incubated with C Is and agg. HGG: a, fresh normal human serum (NHS) + C]-s: b, NHS + buffer: c. NHS + agg. HGG; d, C4 deficient serum (('4d)+ buffer: e. C4d + Cls; f. C4d + agg. HGG; g, C2 deficient serum (C2d) + C]-s; h, C2d + buffer. Cis (15 /~g:10 /d), agg. HGG. (200,ug/10Hl) and 10/tl of buffet were incubated witfi 100 HI of serum at 37 C for 60 min before the 1EP. Anode is at the right. Anti-C4 in trough. duced. O n subsequent incubation with C4 deficient serum as a source of these enzymes, C4b which migrates anodally to native C4 on IEP undergoes further cleavage as indicated by a shift of its fl arc to a position cathodal to C4b and native C4 and by the generation of a new fragment exhibiting :~ mobility. These findings suggest that after C l s cleaves C4 to C4a and C4b, C4b is subsequently cleaved to C4c and C4d by other enzymes. The result possibly coincides with the results of Sjoholm and Laurell (1973), who found C4 conversion products in the [1 and :~ region on crossed immunoelectrophoresis when C]s, trypsin and chymotrypsin were added to E D T A -
serum. ]'he tool. wt of ('4c and C4d as estimated by Sephadex G-150 gel tiltration were approximately 140,000 and 50,000, respecti,,el 5. The sum of the tool. wt of the two fragments, 190,000 is in close agreement with the mol. wt of ('4 as found by. Schreiber and Miiller-Eberhard (1974l. Our mol. wt estimates and the mobility on IEP show that ('4d is not identical to the known fragments C4a and C4b. The following facts lend support to the concept that C4b undergoes further fragmentation: 1. C4d is absent in fresh serum. fresh EDTA-plasma and fresh EDTA-serum but present in aged materials: 2. C4d is generated after incubation with C l s and NHS, EDTA-plasma or E D T A serum but not generated after activation of the alternative pathway by insulin (Perrin et al., 1974): 3. C4d is not detected in C4 deficient serum either in its native form nor after incubation with C l s or agg. H G G : and 4. C4d is not the 42 complex (C4d is generated in C2 deficient serumi. It is of interest to compare this proposed hagmentation scheme tbr C'4 with current knowledge of the fragmentation and structure of C3. C3 is cleaved by the action of C3 convertasc 1C42), factor B or trypsin into a small fragment, C3a and a large fragment C3b. Then C3b inactivator or K A F cleaves C3b into C3c and C3d. Activated C3b has two biologically Loss of SAC4 After Incubotion With Serum I00
.-r "E
50
8 Q_
Fig. 3. Immunophoretic analysis of the cleavage of ('4b by serum enzymes present in C4d serum. Anti-C4 in trough, a, C4 + Cls: b, C4 + C]-s + C4 deficient serum: c, C4 deficient serum: d, ('4 solo.
i i
i
i
32 16
8
4
hi Serum
Dilution
Fig. 5. l,oss of SAC4 alter incubation vdth scrum
Generation of C4d
Fig. 6. Immunoelectrophoretic analysis of C4 cleavage by C3b inactivator deficient serum with or without added C3b inactivator 1st incubation a. C4 + C]-s
2nd incubation
+C3b inactivator deficient serum b. C4 + C ] - s +C3b inactivator rich fraction c. Same as (a) Same as (a) d. Same as (b) Same as (b) e. Normal human serum pool
939
shows that two enzymes or serum fractions are necessary to generate C4d from C4b. Quantitative data on these enzymes will be published separately, but it should be noticed that the mobility of the C4b arc is more anodal when incubated with the C3b inactivator deficient serum. This suggests that a serum fraction reacts with C4b to make an intermediate cleavage product or complex, but cannot produce the C4d fragment without the C3b inactivator. Since high concentrations of C4d are found in the synovial fluid of patients with rheumatoid arthritis and in hereditary angioneurotic edema (HANE) sera, detection and quantitation of this fragment will be clinically useful in these diseases and perhaps in other diseases which activate the classical pathway. We have described recently the generation of this fragment by incubation of serum with agg. HGG. which "clearly relates its significance to immune complex diseases (Perrin et al., 1974).
3rd incubation +C3b inactivator Rich fraction +C3b inactivator Deficient serum + Buffer + Buffer
The double C4 arc is due to excess antibody
important binding sites; a labile binding site which allows the attachment of C3b to cell surfaces and a stable binding site which enables C3b to react with the C3b receptor on the surface of various cells (Miiller-Eberhard et al., 1966; Nishioka and Linscott, 1963). Recently a similarity between the two polypeptide chains of C3 and two of the three polypeptide chains of C4 was presented (Schreiber and MfillerEberhard, 1974). Furthermore, a receptor for C3b on human erythrocytes, B-lymphocytes, and polymorphonuclear leukocytes has been reported (Cooper, 1969; Ross and Polley, 1974; Sobel and Bokisch, 1974) and it was shown that C4b was cleaved by the C3b inactivator (Bokisch and Sobel, 1974; Cooper, 1975), rendering cell bound C4b unreactive in immune adherence or in rosette formation with erythrocytes or lymphocytes. Our data show that EAC4 cells treated with serum fractions lose their hemolytic activity suggesting the presence of a C4b inactivator(s). This observation thus would be analogous to the activity of the C3b inactivator on cell bound C3b. The purification of this inactivator(s) is underway. We have evidence that partially pure fractions rich in the C3b inactivator bring about destruction of cell bound C4b hemolytic sites but do not produce C4d fragments from C4b fragments. In addition the C3b inactivator deficient serum cannot generate C4d fragments until the C3b inactivator is restored (Fig. 6). These results support the results of Fig. 4 which
REFERENCES
Andrews P. (1964) Biochem. J. 91, 222. Bokisch V. A. and Sobel A. T. (1974) J. exp. Med. 140, 1336. Borsos T. and Rapp H. J. (1967) J. lmmun, 99, 263. Budzko D. B. and Miiller-Eberhard H. J. (1970) lmmunochemistry 7, 227. Cooper N. R. (1969) Science 165, 396. Cooper N. R, (1975) J. exp. Med. 141, 890. Gaither T. A., Ailing D. W. and Frank M. M. (1974) J. lmmun. 113, 574. Klemperer M. R., Woodworth H. C., Rosen F. S. and Austen K. F. (1966) J. clin. Invest. 45, 880. Lepow I. H., Naff G. B., Todd E. W., Pensky J. and Hinz C. F. (1963) J. exp. Med. 117, 983. Mancini G., Carbonara O. and Heremens J. F. (1965) lmmunochemistry 2, 235. Mayer M. M. (1961) in Experimental lmmunochemistry, 2rid Edn. Thomas, Springfield, IL. Miiller-Eberhard H. J. and Lepow I. H. (1965) J. exp. Med. 121, 819. Miiller-Eberhard H. J., Dalmasso A. P. and Calcott M. A. (1966) J. exp. Med. 123, 33. Nelson R. A. Jr., Jensen J., Gigli I. and Tamura N. (1966) lmmunochemistry 3, 111.
Nishioka K. and Linscott W. D. (1963) J. exp. Med. 118, 767. Patrick R. A., Taubman S. B. and Lepow I. H. (1970) lmmunochemistry 7, 217.
Patrick R. A., Mernitz J. and Bing D. H. (1973) J. hnmun. I11,296. Perrin L. H., Shiraishi S., Stroud R. M. and Lambert P. H. (1975) J. Immun. 115, 32. Rosenfeld S. J., Ruddy S. and Austen K. F. (1969) J. clin. Invest. 48, 2283. Ross G. D. and Polley M. J. (1974) Fedn Proc. 33, 759. Sakai K. and Stroud R. M. (1973) J. Immun. 110, 1010. Schreiber R. D. and Mi~ller-Eberhard H. J. (1974) J. exp. Med. 140, 1324. Shiraishi S. and Stroud R. M. (1974) Submitted to American Rheumatism Association, Southeastern Regional Meeting, 13-14 Dec, 1974. SjOholm A. G. and Laurell A.-B. (1973) Clin. exp. lmmunol. 14, 515.