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Expression of C5a-like biological activity by the fifth component of human complement upon limited digestion with non-complement enzymes without release of polypeptide fragments
ABSTRACTS
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FORMATION OF DISULFIDE LINKED C9 DIME& DURING ASSEMBLY OF THE 29SMEMBRANE ATTACK COMPLEX (MC5b-912 OF HUMAN COMPLEMENT. Carl F. Ware’ and William P. Kolb. Dept. of Biochemistry, TheUniversiTy of Texas Health Science Center at San Antonio, TX 78284. The 158,000 m.w. protein previously designated as C5c, present in fully assembled complement (C) membrane attack complexes (MC5b-9) has been identified as a disulfide bonded dimer of C9. This conclusion was based on the observations that: 1) a portion of the 1251-radiolabeled precursor C9 incorporated into MC5b-9 complexes comigrated with the 158,000 m.w. protein band in SDS-polyacrylamide slab gels 2) monospecific antisera produced against native C9 and the 158,000 m.w. protein imnative C9 by double immunodiffusion and with munologically cross reacted with monomeric, monomeric C9 and the 158,000 m.w. protein by immunoreplication procedures and 3) two dimensional SDS-polyacrylamide slab gel electrophoresis, in which the second dimension was conducted under reducing conditions, revealed the 158,000 m.w. protein contained two identical subunits of 71,000 m.w. which &migrated with monomeric C9. Molar ratio estimates indicated that one mole of C5b, C9 dimer, C6. C7 and C8 with 3 to 4 moles of C9 monomer were present oer MC5b-9 comolex. Each fullv assembled; membrane bound MC5b-9 complex would therefore possess a calculated m.w. of 982,000: Quantitation of C9 dimers in a CS-dependent hemolytic assay revealed a strong correlation (r = .955) between C9 dimer formation and cell lysis. In addition, the presence of C9 dimers in the hemolytically active POS-dimeric form of the MC5b-9 complex and the absence of C9 dimers in the hemolytically inactive, 23Smonomeric form of the fluid phase SC5b-9 complex strongly suggests an important role for C9 dimer formation MC5b-9 complex structure and function. The most probable function of C9 dimers would be the formation of inter-complex disulfide crosslinks which would provide a mechanism to stabilize the assembly of MC5b-9 into aggregates of increasing size on the target membrane surface which would thus be responsible for the observed pore size heterogeneity of functional C lesions. (Sup ported by Grant CA-24447, NCI.) DUAL REQUIREMENT FOR CHEMOTACTIC FACTORS AND PROSTAGLANDINS IN PROMOTING INCREASED PULMONARY VASCULAR PERMEABILITY IN RABBITS. Robert 0. Webster’, Gary L. Larsen, and Peter M. Henson. Dept. of Pediatrics, National Jewish Hospital, Denver, CO 80206. Several investigators have suggested that intravascular complement activation plays a role in the pathogenesis of human lung diseases. In previous studies, we have demonstrated that systemic complement activation by cobra venom factor (CVF), or intravascular administration of rabbit C5a, cause sequestration of neutrophils in the pulmonary vasculature, but do not cause detectable injury to rabbit lungs. On the other hand, if animals were exposed to a transient episode of hypoxia following complement-activation, leukocyte emigration and increased pulmonary permeability were observed. Because hypoxia causes prostaglandin release, we then asked whether prostaglandins, together with chemotactic factors would cause lung injury. Rabbits were treated with 10 ug PGE2 intravenously in 20 ml saline at 1 ml per min. Midway into the infusion, 300 U CVF (in which phospholipase A2 had been inactivated) was administered as a single bolus intravenously. Arterial blood gas and respiratory function measurements showed significant changes in PaOq, dynamic compliance, and pulmonary resistance as monitored over 4 hours. No changes were noted with CVF alone or PGE2 alone. Histologic examination revealed both alveolar and interstitial neutrophil accumulation only in rabbits receiving both CVF and PGEp. Increased vascular permeability manifested by the accumulation of ‘3’l-rabbit serum albumin in lavage fluids was only noted in rabbits receiving both CVF an PGEp. These studies demonstrate further the requirements for factor(s) (in this case, prostaglandins) in addition to chemotaxin for the production of substantial leukocyte emigration and subsequent tissue damage in this model of lung injury. EXPRESSION OF C5a-LIKE BIOLOGICAL ACTIVITY BY THE FIFTH COMPONENT OF HUMAN COMPLEMENT UPON LIMITED DIGESTION WITH NON-COMPLEMENT ENZYMES WITHOUT RELEASE OF POLYPEPTIDE FRAGMENTS. Rick A. Wetse/* and William P. Kolb, Univ. of Texas Health Science Center, San Antonio, TX 78284. We have reported previously that, unlike the C5 convertase cleavage of human C5 to C5a and C5b, limited trypsin hydrolysis of C5 results in the expression of C5a-like biological activities without production of C5a and C5b fragments. The first three trypsin cleavage events generate C5 achain fragments (QI-a5) which remain disulfide bonded to the parent molecule thereby generating modified forms of C5 (C5’, C5”, C5”‘), all of which express a MW of 210,000 [Fed. Proc. 40:1016(1981)]. To correlate the site of trypsin cleavage with the expression of biological activity, C5 was incubated with 0.008 moles trypsinlmole C5 at 37°C and multiple samples were collected after various time periods. One set was made 2.5 mM in PMSF and analyzed by SDS-slab-PAGE under reducing conditions. A second set was concurrently analyzed for PMN lysosomal enzyme releasing and chemotactic activities. The stained SDS-slab gels were analyzed by quantitative densitometric scans and a plot of the percent of maximum C5al fragment produced as a function of time was superimposible with the appearance of biological activity. Therefore, the first trypsin fragmentation event which cleaves the CSachain to C5al and C5a5 (C5’) activates C5 to express biological activity. This conclusion was confirmed by preincubation of 1251-C5’with 2% SDS followed by gel filtration in the presence of 0.2% SDS. No released radiolabeled fragments were detected, and after extensive dialysis, the C5’ containing fractions expressed PMN lysosomal enzyme releasing activity which was inhibitable by anti-C5 antibodies. Our interpretation of these results is that the C5acontaining portion of the C5achain is sequestered in native C5. Trypsin activates C5 by hydrolyzing the C5achain in a manner which results in the exposure of the C5a region on the exterior of the C5’ molecule, and the externalized C5a sequence can now react with Cell surface C5a receptors without being released from the parent C5’ molecule. Our results also indicate that a-thrombin activates C5 to express biological activity by a similar mechanism. (Supported by grant CA-24447, NCI.)
1409
FORMATION OF DISULFIDE LINKED C9 DIME& DURING ASSEMBLY OF THE 29SMEMBRANE ATTACK COMPLEX (MC5b-912 OF HUMAN COMPLEMENT. Carl F. Ware’ and William P. Kolb. Dept. of Biochemistry, TheUniversiTy of Texas Health Science Center at San Antonio, TX 78284. The 158,000 m.w. protein previously designated as C5c, present in fully assembled complement (C) membrane attack complexes (MC5b-9) has been identified as a disulfide bonded dimer of C9. This conclusion was based on the observations that: 1) a portion of the 1251-radiolabeled precursor C9 incorporated into MC5b-9 complexes comigrated with the 158,000 m.w. protein band in SDS-polyacrylamide slab gels 2) monospecific antisera produced against native C9 and the 158,000 m.w. protein imnative C9 by double immunodiffusion and with munologically cross reacted with monomeric, monomeric C9 and the 158,000 m.w. protein by immunoreplication procedures and 3) two dimensional SDS-polyacrylamide slab gel electrophoresis, in which the second dimension was conducted under reducing conditions, revealed the 158,000 m.w. protein contained two identical subunits of 71,000 m.w. which &migrated with monomeric C9. Molar ratio estimates indicated that one mole of C5b, C9 dimer, C6. C7 and C8 with 3 to 4 moles of C9 monomer were present oer MC5b-9 comolex. Each fullv assembled; membrane bound MC5b-9 complex would therefore possess a calculated m.w. of 982,000: Quantitation of C9 dimers in a CS-dependent hemolytic assay revealed a strong correlation (r = .955) between C9 dimer formation and cell lysis. In addition, the presence of C9 dimers in the hemolytically active POS-dimeric form of the MC5b-9 complex and the absence of C9 dimers in the hemolytically inactive, 23Smonomeric form of the fluid phase SC5b-9 complex strongly suggests an important role for C9 dimer formation MC5b-9 complex structure and function. The most probable function of C9 dimers would be the formation of inter-complex disulfide crosslinks which would provide a mechanism to stabilize the assembly of MC5b-9 into aggregates of increasing size on the target membrane surface which would thus be responsible for the observed pore size heterogeneity of functional C lesions. (Sup ported by Grant CA-24447, NCI.) DUAL REQUIREMENT FOR CHEMOTACTIC FACTORS AND PROSTAGLANDINS IN PROMOTING INCREASED PULMONARY VASCULAR PERMEABILITY IN RABBITS. Robert 0. Webster’, Gary L. Larsen, and Peter M. Henson. Dept. of Pediatrics, National Jewish Hospital, Denver, CO 80206. Several investigators have suggested that intravascular complement activation plays a role in the pathogenesis of human lung diseases. In previous studies, we have demonstrated that systemic complement activation by cobra venom factor (CVF), or intravascular administration of rabbit C5a, cause sequestration of neutrophils in the pulmonary vasculature, but do not cause detectable injury to rabbit lungs. On the other hand, if animals were exposed to a transient episode of hypoxia following complement-activation, leukocyte emigration and increased pulmonary permeability were observed. Because hypoxia causes prostaglandin release, we then asked whether prostaglandins, together with chemotactic factors would cause lung injury. Rabbits were treated with 10 ug PGE2 intravenously in 20 ml saline at 1 ml per min. Midway into the infusion, 300 U CVF (in which phospholipase A2 had been inactivated) was administered as a single bolus intravenously. Arterial blood gas and respiratory function measurements showed significant changes in PaOq, dynamic compliance, and pulmonary resistance as monitored over 4 hours. No changes were noted with CVF alone or PGE2 alone. Histologic examination revealed both alveolar and interstitial neutrophil accumulation only in rabbits receiving both CVF and PGEp. Increased vascular permeability manifested by the accumulation of ‘3’l-rabbit serum albumin in lavage fluids was only noted in rabbits receiving both CVF an PGEp. These studies demonstrate further the requirements for factor(s) (in this case, prostaglandins) in addition to chemotaxin for the production of substantial leukocyte emigration and subsequent tissue damage in this model of lung injury. EXPRESSION OF C5a-LIKE BIOLOGICAL ACTIVITY BY THE FIFTH COMPONENT OF HUMAN COMPLEMENT UPON LIMITED DIGESTION WITH NON-COMPLEMENT ENZYMES WITHOUT RELEASE OF POLYPEPTIDE FRAGMENTS. Rick A. Wetse/* and William P. Kolb, Univ. of Texas Health Science Center, San Antonio, TX 78284. We have reported previously that, unlike the C5 convertase cleavage of human C5 to C5a and C5b, limited trypsin hydrolysis of C5 results in the expression of C5a-like biological activities without production of C5a and C5b fragments. The first three trypsin cleavage events generate C5 achain fragments (QI-a5) which remain disulfide bonded to the parent molecule thereby generating modified forms of C5 (C5’, C5”, C5”‘), all of which express a MW of 210,000 [Fed. Proc. 40:1016(1981)]. To correlate the site of trypsin cleavage with the expression of biological activity, C5 was incubated with 0.008 moles trypsinlmole C5 at 37°C and multiple samples were collected after various time periods. One set was made 2.5 mM in PMSF and analyzed by SDS-slab-PAGE under reducing conditions. A second set was concurrently analyzed for PMN lysosomal enzyme releasing and chemotactic activities. The stained SDS-slab gels were analyzed by quantitative densitometric scans and a plot of the percent of maximum C5al fragment produced as a function of time was superimposible with the appearance of biological activity. Therefore, the first trypsin fragmentation event which cleaves the CSachain to C5al and C5a5 (C5’) activates C5 to express biological activity. This conclusion was confirmed by preincubation of 1251-C5’with 2% SDS followed by gel filtration in the presence of 0.2% SDS. No released radiolabeled fragments were detected, and after extensive dialysis, the C5’ containing fractions expressed PMN lysosomal enzyme releasing activity which was inhibitable by anti-C5 antibodies. Our interpretation of these results is that the C5acontaining portion of the C5achain is sequestered in native C5. Trypsin activates C5 by hydrolyzing the C5achain in a manner which results in the exposure of the C5a region on the exterior of the C5’ molecule, and the externalized C5a sequence can now react with Cell surface C5a receptors without being released from the parent C5’ molecule. Our results also indicate that a-thrombin activates C5 to express biological activity by a similar mechanism. (Supported by grant CA-24447, NCI.)