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Mode of interaction of different polyanions with the first (C1, C1 ), the second (C2) and the fourth (C4) component of complement-III
Immunochemistry, 1976, Vol. 13, pp. 789-791. Pergamon Press. Printed in Great Britain
M O D E OF INTERACTION OF DIFFERENT POLYANIONS WITH THE FIRST (C1, C]-), THE SECOND (C2) AND THE FOURTH (C4) COMPONENT OF COMPLEMENT--III. INHIBITION
OF C4 AND
C2 BINDING
SITE(S) O N C l g B Y P O L Y A N I O N S *
M I C H A E L LOOS,t J O H N E. VOLANAKIS and ROBERT M. STROUD~ Institute for Medical Microbiology, Johannes Gutenberg-University, 65 Mainz, Germany; and the Department of Medicine, University of Alabama in Birmingham, Birmingham, AL 35294, U.S.A. (Received 29 January 1976) Al~raet--PA§ inhibited the fluid phase consumption of C4 and C2 by CI~. In the presence of 100 #g/ml of dextran sulfate, heparin or pentosanpolysulfoester (Sp54) approx 30-40 times more CI~ was required to consume the same amount of C2. Similar effects were obtained for C4 consumption: 100-150 times more CI~ was needed to consume the same amount of C4 in the presence of 100 #g/ml of PA. Dose response data indicate that PA inhibition of C4 and C2 consumption was highly dependent on the relative concentration of polyanion to CI~ at a given C4 or C2 concentration. In contrast to C4 and C2, PA had no effect on the esterolytic activity of CI~ against the synthetic substrate TAMe, indicating that PA do not interfere with the enzymatic site of CI~. Therefore we conclude that the PA inhibition of C4 and C2 consumption by CI~ is due to interference of these substances with the binding of C4 and C2 to Clg.
INTRODUCTION
In a previous paper (Raepple et al., 1975) we described the effect of different polyanions (PA) on the first three components of complement, C]-, C4 and C2. There was no irreversible inhibition of C4 and C2 by the polyanions tested. In contrast, most of these polyanions inhibited C]- by interacting with the binding of C]- to antigen-antibody complexes through an interaction with Clq. In additional experiments we showed that the consumption of C4 and C2 by C]- in the presence of PA was reduced (Raepple et al., 1975). From these experiments it was not clear if the effect of PA on the C4 and C2 consumption by Ci- was due to the interaction of PA with C l q in the C]- macro-molecule or due to a direct effect of PA on the esterase, Clg. To distinguish between the effect of PA on C l q and the inhibition of the esterolytic activity of C]- (Clg) against C4 and C2, the effect of different polyanions on the consumption of C4 and C2 by highly purified CI~ was studied and the results reported here show that in the presence of PA the consumption of C4 and C2 by Clg was markedly reduced; however, the hydrolysis of the small synthetic substrate TAMe
(p-Tosyl-l-arginine methylester) by Clg was not affected by polyanions. MATERIALS AND METHODS
The polyanions used in the experiments were: dextran sulfate, sodium salt, mol. wt 500,000, eat. No. 18707; polyvinyl sulfate, potassium salt, cat. No. 33426 (Serva, Feinbiochemica, Heidelberg, Germany); pentosanpolysulfoester, Sp 54IR} (Bene-Chemie, Munich-Solln, Germany); sodium heparin, aqueous (Lipo-HeparintR}, 1000 USP anits/ml (lot. 22989), Parker Laboratories, Northridge, CA, U.S.A.; the polyanions were dissolved in isotonic-Veronal buffered saline (# = 0.15, pH 7.4) to a concentration of 50 mg/ml. These stock solutions were stored at 0°C; dilutions were made in Veronal-buffered glucose saline containing gelatin (0.1%) and metals 1 mM Ca 2+ and 15 mM Mg 2+. The hydrolysis of TAMe by CI~ was tested according to Nagaki & Stroud (1969); TAMe: p-Tosyl-l-arginine methylester HCI (lot U 2516) from Mann Research Laboratories, NY. Sheep erythrocytes sensitized with rabbit antibody to boiled sheep erythocytes (EA) were prepared as described by Mayer (1961). EAC4 cells were prepared as described by Rapp and Borsos (1970). Veronal-buffered glucose saline (/~ = 0.065, pH 7.4), was prepared by mixing isotonic Veronal-buffered saline (# = 0.147, pH 7.4) and 5% glucose in water. This buffer contained MgC12, CaC12 and gelatin to a final concentration of 1 raM, 15 mM and 0.1% respectively. EDTA *The complement nomenclature follows the WHO buffer was prepared by making a 1:10 dilution of stock recommendations (Bull. Wld Hlth Org. 39, 939, 1968). 0.1 M Tris-sodium ehtylendiamine tetraacetate (pH 7.4) t Supported by the Deutsche Forschungsgemeinschaft with isotonic Veronal buffered saline, containing 0.1% gelaSFB 107/A2. To whom reprint requests should be sent, tin. Functionally pure guinea pig C2 was prepared using at: Institut ftir Medizinische Mikrobiologie, 65 Mainz, the method of Nelson et al. (1966). Functionally pure Augustusplatz, Hochhaus, Germany. human C4 (lot No. 31183) was purchased from the Cordis :~Supported by grants from the NIH and Project Corporation, Miami, FL. Human Clg was purified accord8197-01, VA Hospital, Birmingham, AL 35294, U.S.A. ing to Sakai & Stroud (1973). C-EDTA was prepared by §Abbreviations used: DS, dextran sulfate; PVS, poly- diluting guinea pig serum 1:50 with EDTA buffer. C1~ vinyl sulfate; Sp 54, pentosanpolysulfoester; liquoid, activity was measured as the ability of Clg to form polyanethol sulfonate; PA, polyanion; TAMe, p-tosyl-l- EAC4a2b from EAC4b and C2: EAC4b (1.5 x 10s arginine-methylester. cells/ml) were incubated with varying dilutions of Clg (1.32 789
790
MICHAEL LOOS, JOHN E. VOLANAKIS and ROBERT M. STROUD Table l. Effect of polyanions on the consumption of C2 or C4 by different Clg concentrations=
100",\Z~.....
\ U 60-
10
""
100
1000
-I0000
C1§ SFU/mI (1SFUofClt ~tO.O08/ug protein)
Fig. 1. Effect of polyanions on the consumption of C2 by CI~ as a function of CI~ concentration. (Incubation time: 30 min at 37°C. © O, control; × x, DS; /x A, heparin; ® ®, SP 54) mg protein/ml) and with C2 (20-50 eft. molecules/cell) for 10 min at 30°C; C-EDTA was then added to determine the number of SAC4-b--~formed, analogously to the C]titration method of Borsos & Rapp (1963). Under these conditions, one Clg site forming unit (SFU) was the amount of Clg which generated one SAC4-b-~ site. The Clg preparation used contained 1 Clg SFU/0.008 #g protein. C2 activity was tested by incubation of varying dilutions of C2 with EAC4b and purified Clg (ca. 200 CI~ SFU/ml) similarly to the CI~ procedure. C4 activity was tested according the one-step method described by Gaither et al. (1974) using serum from the C4 deficient strain of guinea pigs. RESULTS In preliminary experiments the direct effect of polyanions on the hemolytic activity of C4, C2 or Clg was examined: equal volumes of Clg (ca. 160,000 SFU/ml), C4 (ca. 2000 sites/cell) or C2 (ca. 2000 sites/ cell) were incubated with equal volumes of polyanions (100 #g/ml of DS, PVS, liquoid, Sp54 and heparin) for 10 rain at 30°C. Each mixture was then diluted in Veronal-buffered glucose saline and its hemolytic activity was determined. Under these conditions the polyanions were sufficiently diluted during the hemolytic assay and had no residual effect on Clg, C4 and C2. The effect of polyanions on the consumption of C4 or C2 by CI~ was first tested as a function of Clg concentration. Equal volumes of different Clg dilutions were incubated with a constant amount of C2 (1000 eft. molecules/cell and ml) and a constant amount of dextran sulfate (100 gg/ml), heparin (100 #g/ml) or Sp54 (100 #g/ml); in controls, buffer was substituted for PA. After incubation for 30 min at 37°C the residual C2 activity was measured by making a large dilution, so that the PA would not affect the binding of C2 to EAC4b (Looset al., 1976). The results shown in Fig. 1 indicate that dextran sulfate, heparin, as well as Sp54 inhibited the consumption of C2 by Clg. Under the experimental conditions used, 26 Clg SFU were sufficient to consume 50~o of the added C2 (control). In the presence of 100 #g of dextran sulfate, heparin or Sp54, 1050, 810 or 760 Clg SFU/ml respectively, were necessary to obtain the same amount of C2 consumption (Table 1). Similar results were obtained when consumption of C4 (1000 sites/cell) by CI~ was examined in the
100 #g/ml
C4
C2
Dextran sulfate Sp 54 Heparin Buffer
34b 27 43 0.27
1050 760 810 26
a Different concentrations of Clg were incubated with ca. 1000 C2 sites per cell or with ca. 1000 C4 sites per cell for 30 min at 37°C in the presence of different polyanions or buffer. bThe figures indicate that number of Clg SFU/ml which are necessary to consume 50~o of C2 or C4 under these experimental conditions. presence of dextran sulfate, heparin or Sp54 (100 #g/ml). From the data summarized in Table 1, it can be seen that 0.27 Clg SFU/ml are necessary to consume 50% of C4 added (control). This indicates that the consumption of C4 by CI~ is 100 times more efficient than the consumption of C2 by CI~. All polyanions tested inhibited the consumption of C4 by Clg. On a weight basis, there was almost no difference between the inhibitory effect of dextran sulfate, heparin or Sp54. The consumption of C2 by Clg as a function of polyanion concentrations was examined next. Equal volumes of CI~ (1000 SFU/ml), of C2 (1000 sites/eell) and of different concentrations of dextran sulfate or Sp 54 were incubated for 30 min at 37°C. Residual C2 activity was then measured and compared to a C2 plus buffer control. The results shown in Fig. 2 indicate that under these conditions 150/~g/ml of dextran sulfate and 190 #g/ml of Sp 54 inhibited the consumption of C2 by 50°/0. This experiment confirms the finding of Table 1 that on a weight basis, the different polyanions showed no significant differences in their efficiency to inhibit the consumption of C2 by CI~. To examine whether polyanions interfere with the enzymatic site of CI~, their effect on the hydrolysis of TAMe by C lg was examined. The hydrolysis of TAMe by CI~ was tested according to Nagaki & Stroud (1969) following the change of O.D. at 247
100.~ 8060t?
"~ 40. 20"
~b
"rio Mglmt
Fig. 2. Effect of polyanions on the consumption of C2 by CI~ as a function of PA concentration. (Incubation time: 30 min at 37°C; O O, SP 54; H , DS)
Interaction of Polyanions with C4 and C2 Binding Site(s) on CI~
controt •3
PA
2
0i'~=T
,o~i"~
2. ~
.1.
/I
~"
.
io
.
.
.
zb minute s at
~'o 30°C
io
Fig. 3. Effect of different concentrations of dextran sulfate on the hydrolysis of TAMe at 30°C. (Relative PA concentration 2 and 25/~g/SFU of Clg). nm. TAMe (1.6 mM) was incubated with 5000 SFU of Clg in 0.1 M Tris, pH 7.4 at 25°C in the presence or absence of polyanions. As shown in Fig. 3, two different concentrations of dextran sulfate (2 and 25 /~g/SFU of CI~) did not significantly affect the rate of TAMe hydrolysis by CIR. The highest concentration of dextran sulfate was 2-3 times higher than those required to completely inhibit C2 consumption, and 12-13 times higher than that required to inhibit C4 consumption by CIR. In additional similar experiments, PVS, liquoid and heparin also failed to affect the cleavage of TAMe by CI~.
791
occurring or synthetic negatively charged molecules, so called polyanions (PA). A number of PA's were described to bind directly to Clq, a subunit of the first component of complement, as shown by precipitation (Yachnin et al., 1964; Borsos et al., 1965; Agnello et al., 1969) or reduction of the C]- inhibiting effect of PA by pre-incubation with purified Clq (Raepple et al., 1975). In a comparative study it was shown that PA's differ in their binding affinity for C1 (Raepple et al., 1975). In the present study we present evidence for a second type of interaction of PA with C1, in this case, with highly purified Cl-esterase, CI~. In a first set of experiments it is shown that PA do not react irreversibly with isolated CI~. PA's have also no effect on the substrates of Clg, C4 or C2 alone, but do inhibit the consumption of C4 or C2 by C1R. This inhibitory effect depends upon the CI~ concentration as well as on the PA concentration as shown in Fig. 1. An influence of the substrate concentration is also reasonable, although it was not tested. The results of Figs. 1 and 2 suggest that the extent of inhibition depends upon the ratio of the amounts of PA and CI~ at a given substrate concentration. The polyanions tested, e.g. dextran sulfate, heparin or Sp 54 showed on a weight basis, no difference in their inhibitory effect, although the mol. wts are different (dextran sulfate 500,000; heparin 15,000; Sp 54 2000). Since the sulfur content ranged from 11% to 17%, it seems possible that the inhibitory effect of PA's depends only on the number of negatively charged groups and not on the size of the carrier. Although polyanions inhibited the consumption of C4 and C2 by CIR, the hydrolysis of TAMe, a small synthetic substrate of CI~, was not inhibited (Fig. 3). We conclude from these experiments that polyanions interfere with a presumably positively charged binding site(s) for C4 or C2 on CI~ as schematically shown in Fig. 4.
DISCUSSION
The development of compounds which inhibit or control complement component interactions and thus have possible therapeutic significance, depends upon a proper understanding of the mechanisms of their action. One group of such compounds are naturally
C"PA: dextransul fofe
I
C2
J "t"
polyvinylsulfate
I
I
cT inoctivator
polyanetholsulfono/e
DFP
pent~onpolysu I fo-
TAMe guonld]ne, amldine
ester (Sp541
hepaHn
1 -
I
Fig. 4. Mode of interference of polyanions with the consumption of C4 or C2 by CI~.
REFERENCES
Agnello V., Carr R. I., Koffler D. & Kunkel H. G. (1969) Fedn. Proc. 28, 2447. Borsos T. & Rapp H. J. (1963) J. lmmun. 91, 851. Borsos T., Rapp H. J. & Crisler C. (1965) J. Immun. 94, 662. Gaither T. A., Alling D. W. & Frank M. M. (1974) J. Immuno. 113, 574. Loos M., Volanakis J. E. & Stroud R. M. (1976) lmmunochemistry 13, 257. Mayer M. M. (1961) Experimental Immunochemistry, (Edited by Kabat E. A. & Mayer M. M. ), 2nd Edn, Thomas, Springfield, IL. Nagaki K. & Stroud R. M. (1969) J. hmnun. 102, 421. Nelson R. A., Jensen J., Gigli J. & Tamura N. (1966) Immunochemistry 3, 1l 1.
Raepple E., Hill H.-I1. & Loos M. (1976) Immunochemistry 13, 251. Rapp H. J. & Borsos T. (1970) Molecular Basis of Complement Action, Appleton-Century-Crofts, NY. Sakai K. & Stroud R. M. (1973) J. Immun. 110, 1010. Yachnin S., Rosenblum D. & Chatman D. (1964) J. lmmun. 93, 540.
M O D E OF INTERACTION OF DIFFERENT POLYANIONS WITH THE FIRST (C1, C]-), THE SECOND (C2) AND THE FOURTH (C4) COMPONENT OF COMPLEMENT--III. INHIBITION
OF C4 AND
C2 BINDING
SITE(S) O N C l g B Y P O L Y A N I O N S *
M I C H A E L LOOS,t J O H N E. VOLANAKIS and ROBERT M. STROUD~ Institute for Medical Microbiology, Johannes Gutenberg-University, 65 Mainz, Germany; and the Department of Medicine, University of Alabama in Birmingham, Birmingham, AL 35294, U.S.A. (Received 29 January 1976) Al~raet--PA§ inhibited the fluid phase consumption of C4 and C2 by CI~. In the presence of 100 #g/ml of dextran sulfate, heparin or pentosanpolysulfoester (Sp54) approx 30-40 times more CI~ was required to consume the same amount of C2. Similar effects were obtained for C4 consumption: 100-150 times more CI~ was needed to consume the same amount of C4 in the presence of 100 #g/ml of PA. Dose response data indicate that PA inhibition of C4 and C2 consumption was highly dependent on the relative concentration of polyanion to CI~ at a given C4 or C2 concentration. In contrast to C4 and C2, PA had no effect on the esterolytic activity of CI~ against the synthetic substrate TAMe, indicating that PA do not interfere with the enzymatic site of CI~. Therefore we conclude that the PA inhibition of C4 and C2 consumption by CI~ is due to interference of these substances with the binding of C4 and C2 to Clg.
INTRODUCTION
In a previous paper (Raepple et al., 1975) we described the effect of different polyanions (PA) on the first three components of complement, C]-, C4 and C2. There was no irreversible inhibition of C4 and C2 by the polyanions tested. In contrast, most of these polyanions inhibited C]- by interacting with the binding of C]- to antigen-antibody complexes through an interaction with Clq. In additional experiments we showed that the consumption of C4 and C2 by C]- in the presence of PA was reduced (Raepple et al., 1975). From these experiments it was not clear if the effect of PA on the C4 and C2 consumption by Ci- was due to the interaction of PA with C l q in the C]- macro-molecule or due to a direct effect of PA on the esterase, Clg. To distinguish between the effect of PA on C l q and the inhibition of the esterolytic activity of C]- (Clg) against C4 and C2, the effect of different polyanions on the consumption of C4 and C2 by highly purified CI~ was studied and the results reported here show that in the presence of PA the consumption of C4 and C2 by Clg was markedly reduced; however, the hydrolysis of the small synthetic substrate TAMe
(p-Tosyl-l-arginine methylester) by Clg was not affected by polyanions. MATERIALS AND METHODS
The polyanions used in the experiments were: dextran sulfate, sodium salt, mol. wt 500,000, eat. No. 18707; polyvinyl sulfate, potassium salt, cat. No. 33426 (Serva, Feinbiochemica, Heidelberg, Germany); pentosanpolysulfoester, Sp 54IR} (Bene-Chemie, Munich-Solln, Germany); sodium heparin, aqueous (Lipo-HeparintR}, 1000 USP anits/ml (lot. 22989), Parker Laboratories, Northridge, CA, U.S.A.; the polyanions were dissolved in isotonic-Veronal buffered saline (# = 0.15, pH 7.4) to a concentration of 50 mg/ml. These stock solutions were stored at 0°C; dilutions were made in Veronal-buffered glucose saline containing gelatin (0.1%) and metals 1 mM Ca 2+ and 15 mM Mg 2+. The hydrolysis of TAMe by CI~ was tested according to Nagaki & Stroud (1969); TAMe: p-Tosyl-l-arginine methylester HCI (lot U 2516) from Mann Research Laboratories, NY. Sheep erythrocytes sensitized with rabbit antibody to boiled sheep erythocytes (EA) were prepared as described by Mayer (1961). EAC4 cells were prepared as described by Rapp and Borsos (1970). Veronal-buffered glucose saline (/~ = 0.065, pH 7.4), was prepared by mixing isotonic Veronal-buffered saline (# = 0.147, pH 7.4) and 5% glucose in water. This buffer contained MgC12, CaC12 and gelatin to a final concentration of 1 raM, 15 mM and 0.1% respectively. EDTA *The complement nomenclature follows the WHO buffer was prepared by making a 1:10 dilution of stock recommendations (Bull. Wld Hlth Org. 39, 939, 1968). 0.1 M Tris-sodium ehtylendiamine tetraacetate (pH 7.4) t Supported by the Deutsche Forschungsgemeinschaft with isotonic Veronal buffered saline, containing 0.1% gelaSFB 107/A2. To whom reprint requests should be sent, tin. Functionally pure guinea pig C2 was prepared using at: Institut ftir Medizinische Mikrobiologie, 65 Mainz, the method of Nelson et al. (1966). Functionally pure Augustusplatz, Hochhaus, Germany. human C4 (lot No. 31183) was purchased from the Cordis :~Supported by grants from the NIH and Project Corporation, Miami, FL. Human Clg was purified accord8197-01, VA Hospital, Birmingham, AL 35294, U.S.A. ing to Sakai & Stroud (1973). C-EDTA was prepared by §Abbreviations used: DS, dextran sulfate; PVS, poly- diluting guinea pig serum 1:50 with EDTA buffer. C1~ vinyl sulfate; Sp 54, pentosanpolysulfoester; liquoid, activity was measured as the ability of Clg to form polyanethol sulfonate; PA, polyanion; TAMe, p-tosyl-l- EAC4a2b from EAC4b and C2: EAC4b (1.5 x 10s arginine-methylester. cells/ml) were incubated with varying dilutions of Clg (1.32 789
790
MICHAEL LOOS, JOHN E. VOLANAKIS and ROBERT M. STROUD Table l. Effect of polyanions on the consumption of C2 or C4 by different Clg concentrations=
100",\Z~.....
\ U 60-
10
""
100
1000
-I0000
C1§ SFU/mI (1SFUofClt ~tO.O08/ug protein)
Fig. 1. Effect of polyanions on the consumption of C2 by CI~ as a function of CI~ concentration. (Incubation time: 30 min at 37°C. © O, control; × x, DS; /x A, heparin; ® ®, SP 54) mg protein/ml) and with C2 (20-50 eft. molecules/cell) for 10 min at 30°C; C-EDTA was then added to determine the number of SAC4-b--~formed, analogously to the C]titration method of Borsos & Rapp (1963). Under these conditions, one Clg site forming unit (SFU) was the amount of Clg which generated one SAC4-b-~ site. The Clg preparation used contained 1 Clg SFU/0.008 #g protein. C2 activity was tested by incubation of varying dilutions of C2 with EAC4b and purified Clg (ca. 200 CI~ SFU/ml) similarly to the CI~ procedure. C4 activity was tested according the one-step method described by Gaither et al. (1974) using serum from the C4 deficient strain of guinea pigs. RESULTS In preliminary experiments the direct effect of polyanions on the hemolytic activity of C4, C2 or Clg was examined: equal volumes of Clg (ca. 160,000 SFU/ml), C4 (ca. 2000 sites/cell) or C2 (ca. 2000 sites/ cell) were incubated with equal volumes of polyanions (100 #g/ml of DS, PVS, liquoid, Sp54 and heparin) for 10 rain at 30°C. Each mixture was then diluted in Veronal-buffered glucose saline and its hemolytic activity was determined. Under these conditions the polyanions were sufficiently diluted during the hemolytic assay and had no residual effect on Clg, C4 and C2. The effect of polyanions on the consumption of C4 or C2 by CI~ was first tested as a function of Clg concentration. Equal volumes of different Clg dilutions were incubated with a constant amount of C2 (1000 eft. molecules/cell and ml) and a constant amount of dextran sulfate (100 gg/ml), heparin (100 #g/ml) or Sp54 (100 #g/ml); in controls, buffer was substituted for PA. After incubation for 30 min at 37°C the residual C2 activity was measured by making a large dilution, so that the PA would not affect the binding of C2 to EAC4b (Looset al., 1976). The results shown in Fig. 1 indicate that dextran sulfate, heparin, as well as Sp54 inhibited the consumption of C2 by Clg. Under the experimental conditions used, 26 Clg SFU were sufficient to consume 50~o of the added C2 (control). In the presence of 100 #g of dextran sulfate, heparin or Sp54, 1050, 810 or 760 Clg SFU/ml respectively, were necessary to obtain the same amount of C2 consumption (Table 1). Similar results were obtained when consumption of C4 (1000 sites/cell) by CI~ was examined in the
100 #g/ml
C4
C2
Dextran sulfate Sp 54 Heparin Buffer
34b 27 43 0.27
1050 760 810 26
a Different concentrations of Clg were incubated with ca. 1000 C2 sites per cell or with ca. 1000 C4 sites per cell for 30 min at 37°C in the presence of different polyanions or buffer. bThe figures indicate that number of Clg SFU/ml which are necessary to consume 50~o of C2 or C4 under these experimental conditions. presence of dextran sulfate, heparin or Sp54 (100 #g/ml). From the data summarized in Table 1, it can be seen that 0.27 Clg SFU/ml are necessary to consume 50% of C4 added (control). This indicates that the consumption of C4 by CI~ is 100 times more efficient than the consumption of C2 by CI~. All polyanions tested inhibited the consumption of C4 by Clg. On a weight basis, there was almost no difference between the inhibitory effect of dextran sulfate, heparin or Sp54. The consumption of C2 by Clg as a function of polyanion concentrations was examined next. Equal volumes of CI~ (1000 SFU/ml), of C2 (1000 sites/eell) and of different concentrations of dextran sulfate or Sp 54 were incubated for 30 min at 37°C. Residual C2 activity was then measured and compared to a C2 plus buffer control. The results shown in Fig. 2 indicate that under these conditions 150/~g/ml of dextran sulfate and 190 #g/ml of Sp 54 inhibited the consumption of C2 by 50°/0. This experiment confirms the finding of Table 1 that on a weight basis, the different polyanions showed no significant differences in their efficiency to inhibit the consumption of C2 by CI~. To examine whether polyanions interfere with the enzymatic site of CI~, their effect on the hydrolysis of TAMe by C lg was examined. The hydrolysis of TAMe by CI~ was tested according to Nagaki & Stroud (1969) following the change of O.D. at 247
100.~ 8060t?
"~ 40. 20"
~b
"rio Mglmt
Fig. 2. Effect of polyanions on the consumption of C2 by CI~ as a function of PA concentration. (Incubation time: 30 min at 37°C; O O, SP 54; H , DS)
Interaction of Polyanions with C4 and C2 Binding Site(s) on CI~
controt •3
PA
2
0i'~=T
,o~i"~
2. ~
.1.
/I
~"
.
io
.
.
.
zb minute s at
~'o 30°C
io
Fig. 3. Effect of different concentrations of dextran sulfate on the hydrolysis of TAMe at 30°C. (Relative PA concentration 2 and 25/~g/SFU of Clg). nm. TAMe (1.6 mM) was incubated with 5000 SFU of Clg in 0.1 M Tris, pH 7.4 at 25°C in the presence or absence of polyanions. As shown in Fig. 3, two different concentrations of dextran sulfate (2 and 25 /~g/SFU of CI~) did not significantly affect the rate of TAMe hydrolysis by CIR. The highest concentration of dextran sulfate was 2-3 times higher than those required to completely inhibit C2 consumption, and 12-13 times higher than that required to inhibit C4 consumption by CIR. In additional similar experiments, PVS, liquoid and heparin also failed to affect the cleavage of TAMe by CI~.
791
occurring or synthetic negatively charged molecules, so called polyanions (PA). A number of PA's were described to bind directly to Clq, a subunit of the first component of complement, as shown by precipitation (Yachnin et al., 1964; Borsos et al., 1965; Agnello et al., 1969) or reduction of the C]- inhibiting effect of PA by pre-incubation with purified Clq (Raepple et al., 1975). In a comparative study it was shown that PA's differ in their binding affinity for C1 (Raepple et al., 1975). In the present study we present evidence for a second type of interaction of PA with C1, in this case, with highly purified Cl-esterase, CI~. In a first set of experiments it is shown that PA do not react irreversibly with isolated CI~. PA's have also no effect on the substrates of Clg, C4 or C2 alone, but do inhibit the consumption of C4 or C2 by C1R. This inhibitory effect depends upon the CI~ concentration as well as on the PA concentration as shown in Fig. 1. An influence of the substrate concentration is also reasonable, although it was not tested. The results of Figs. 1 and 2 suggest that the extent of inhibition depends upon the ratio of the amounts of PA and CI~ at a given substrate concentration. The polyanions tested, e.g. dextran sulfate, heparin or Sp 54 showed on a weight basis, no difference in their inhibitory effect, although the mol. wts are different (dextran sulfate 500,000; heparin 15,000; Sp 54 2000). Since the sulfur content ranged from 11% to 17%, it seems possible that the inhibitory effect of PA's depends only on the number of negatively charged groups and not on the size of the carrier. Although polyanions inhibited the consumption of C4 and C2 by CIR, the hydrolysis of TAMe, a small synthetic substrate of CI~, was not inhibited (Fig. 3). We conclude from these experiments that polyanions interfere with a presumably positively charged binding site(s) for C4 or C2 on CI~ as schematically shown in Fig. 4.
DISCUSSION
The development of compounds which inhibit or control complement component interactions and thus have possible therapeutic significance, depends upon a proper understanding of the mechanisms of their action. One group of such compounds are naturally
C"PA: dextransul fofe
I
C2
J "t"
polyvinylsulfate
I
I
cT inoctivator
polyanetholsulfono/e
DFP
pent~onpolysu I fo-
TAMe guonld]ne, amldine
ester (Sp541
hepaHn
1 -
I
Fig. 4. Mode of interference of polyanions with the consumption of C4 or C2 by CI~.
REFERENCES
Agnello V., Carr R. I., Koffler D. & Kunkel H. G. (1969) Fedn. Proc. 28, 2447. Borsos T. & Rapp H. J. (1963) J. lmmun. 91, 851. Borsos T., Rapp H. J. & Crisler C. (1965) J. Immun. 94, 662. Gaither T. A., Alling D. W. & Frank M. M. (1974) J. Immuno. 113, 574. Loos M., Volanakis J. E. & Stroud R. M. (1976) lmmunochemistry 13, 257. Mayer M. M. (1961) Experimental Immunochemistry, (Edited by Kabat E. A. & Mayer M. M. ), 2nd Edn, Thomas, Springfield, IL. Nagaki K. & Stroud R. M. (1969) J. hmnun. 102, 421. Nelson R. A., Jensen J., Gigli J. & Tamura N. (1966) Immunochemistry 3, 1l 1.
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