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Zymogen forms of complement factor D
63 THE C7 M/N POLYMORPHISM IS D E T E R M I N E D BY A NEUTRAL AMINO ACID SUBSTITUTION OUTSIDE THE EPITOPE OF THE ALLOSPECIFIC MONOCLONAL ANTIBODY WU 4-15 R. Wiirzner, B.A. Fernie, M.J. Hobart, A. Orren, P.J. Lachmann Molecular Immunopathology Unit, MRC Centre, Cambridge, UK Homozygous and heterozygous individuals of the C7 M/N protein polymorphism are identified by a combination of two ELISA assays, one of which is based on the allospecific monoclonal antibody (mab) WU 4-15. In order to determine the molecular basis of this polymorphism, C7 cDNA subclones (DiScipio & Hugli 1989) or polymemse chain reaction (PCR) amplified C7 coding sequences were expressed as fusion proteins with betagalactosidase using the pUEX expression system (Bressan & Stanley 1987). Recombinant colonies were screened for reaction with the allospecific mab WU 4-15 and the region of C7 containing the epitope was mapped to the boundary region of the two Short Consensus Repeats (SCRs) of C7 (residues 600-613). However, two observations were made which suggested that the allotypic behaviour of WU 4-15 might not depend on the composition of residues 600-613. First, isoelectric focusing followed by electroblotting and immunoprobing could not distinguish C7 M from C7 N samples as the latter also reacted with the allospecific mab, suggesting that the epitope is present in C7 N samples but not accessible in the native form for conformational reasons; and second, a possible molecular basis for such a conformation-altering substitution was detected at about 40 amino acid residues aminoterminal from the epitope: the published 565-Pro codon (DiScipio et al. 1988) was found to be replaced by a 565-Thr codon. Such an exchange is electrically neutral, as postulated, and results in a loss of a Sau961 restriction site and a creation of an RsaI site. PCR amplification of genomic DNA of homo- and heterozygous individuals and analysis of Rsal and Sau961 digests of the amplified products showed a perfect correlation of RsaI + / Sau96 1 - with C7 M and RsaI - / Sau96 + with C7 N. Thus, a threonine at amino acid residue 565 allows access of the mab to its epitope, but its substitution with proline completely inhibits access in the native molecule. This interesting finding will not only shed more light on the three-dimensional structure of C7 but also of SCRs in general as both the polymorphic site and the epitope reside within the two SCRs of C7.
THE DYSMORPHIC BUT FUNCTIONALLY ACTIVE C6 OF SUBTOTAL C6 DEFICIENT SUBJECTS IS CARBOXYTERMINALLY TRUNCATED R. Wiirzner, D. Mewar, P.J. Lachmann Molecular Immunopathology Unit, MRC Centre, Cambridge, UK The C6 of 8 subtotal C6 deficient subjects was previously found to be unequivocally present (1-2% of the normal mean), able to incorporate into the terminal complement complex (TCC), haemolytically active but dysmorphic as it was 14% smaller than normal C6. In ELISA assays one anti-C6 specific monoclonal antibody (mab) did, and another did not, bind to this dysmorphic C6. Using the pUEX expression system (Bressan & Stanley 1987) and C6 cDNA subclones (DiScipio & Hugli 1989) the epitopes of the mabs were mapped to the third Thrombospondin Repeat (TSP) or to the second Factor I Module (FIM) of C6, respectively, the latter indicating that the dysmorphic C6 is carboxyterminally truncated. Since the absence of as much as one-seventh of C6 still permits functional activity, the missing FIM 2 of C6 appears to be of minor importance. In contrast, that region was thought to represent a part of the C5b-binding region (DiScipio 1991), whereas another study mapped this binding site to the two short consensus repeats (SCRs) of C6 (Nakano et al. 1991). A third study indicated that a fragment containing the SCRs and the FIMs of C6 bound more weakly to C5b than whole C6 (Haefliger et al. 1989) indicating an involvement of aminoterminal neighbouring domains to facilitate binding to C5b. The mab anti-C6 which binds to the third TSR of C6 (WU 6-4) was previously shown to be capable of markedly inhibiting TCC formation. Using a reactive lysis assay, TCC formation could not be inhibited when this mab was introduced after C5b6 formation and in an ELISA no binding of WU 6-4 to its epitope was detected when C5b6 served as antigen. Both assays suggest that WU 6-4 inhibits C5b6 formation, favouring the adjacent (in the primary structure) SCRs rather than the FIMs as C5bbinding site. The molecular basis of the subtotal C6 deficiency most likely resides within intron 15 of C6, preventing a splicing out, and furthermore might be identical to the defect resulting in an apparently identical dysmorphic C6 (same isoelectric focusing pattern) of combined C6/C7 deficient subjects (see abstract by Hobart, Femie, W~zner, Morgan, Lachmann).
THE MAJORITY OF HUMAN C7 SYNTHESISED I N V I V O IS O F E X T R A H E P A T I C ORIGIN R. Wttrzner, H. Morgan:~, V. Joysey:~, P.J. Lachmann Molecular Immunopathology Unit, MRC Centre, and :~Tissue Typing Laboratory Addenbrookes Hospital, Cambridge, UK C7 M/N typing, the determination of the allotypes of the C7 M/N protein polymorphism, was carried out on samples from donors and recipients of liver transplantations in order to find out if the liver is the predominant site of 'in vivo' synthesis of complement protein C7. That determination which is based on the reactivity of an allotypic monoclonal antibody in an ELISA assay offered two great advantages: first, since C7*N is much more frequent than C7"2, the second most common allele of the C7 IEF polymorphism, informative pairings were expected in Caucasians, and second, since C7 M/N typing is based on ratios, the contribution of the transplanted liver towards the allotype was quantifiable. Out of 35 recipients 13 received a liver from a donor with a C7 M/N phenotype different from their own and the determination of the C7 concentrations and ratios of 3 to 6 post transplantation (p.t.) samples revealed that there was only a contribution of the transplanted liver towards the C7 M/N phenotype at 2-4 weeks p.t.; that influence decreased with time and was between 0-20% after 4-26 weeks p.t., thus excluding the liver as the predominant site of in vivo C7 synthesis. The transient contribution of the donor phenotype could be attributable to cells of the monocyte / macrophage lineage, Kupffer cells in particular. C7 is thus the only terminal complement component not predominantly synthesised by hepatocytes in contrast to C6 (Hobart et al. 1976), C8 (Alper et al. 1980) and probably C9, as the latter is an acute phase protein (Adinolfi and Lehner 1988). This finding is also consistent with observations that high C7 levels are maintained in liver failure. Although sufficient C7 is usually present in serum, a modulation of complement activity by local C7 synthesis by monocytes or macrophages cannot be excluded as the initiating C5b6 molecule of terminal complement attack is quite stable with C7 often being the limiting factor for terminal complement complex generation (Lachmann and Thompson 1970).
ZYMOGEN FACTOR
FORMS
OF
COMPLEMENT
D
Y. Y a m a u c h i , J.W. S t e v e n s , K.J. M a c o n , a n d Volanakis. U n i v e r s i t y o f A l a b a m a at B i r m i n g h a m , USA.
J.E.
W e u s e d a b a c u l o v i r u s s y s t e m to e x p r e s s the h u m a n factor D e D N A , hg31 (R.T. White et al., J. Biol. C h e m . 267:9210, 1992) in Sf9 cells. The recombinant p r o t e i n (r-proD) w a s purified by c h r o m a t o g r a p h y on Bit-Rex 70 a n d MonoS and was hemolytically inactive. On S D S - P A G E , r-proD had a slightly lower mobility t h a n a u t h e n t i c f a c t o r D. Amino acid s e q u e n c i n g indicated that it c o n s i s t e d of two f o r m s o f proD with r e s p e c t i v e a c t i v a t i o n p e p t i d e s , A A P P R G R and A P P R G R . Catalytic a m o u n t s o f trypsin converted r-proD to active factor D, h a v i n g S D S - P A G E mobility and specific hemolytic activity similar to t h o s e o f the native enzyme. A b o u t 90% o f t r y p s i n - a c t i v a t e d rproD had the s a m e N H 2 - t e r m i n u s as a u t h e n t i c factor D while t h e r e m a i n d e r had b e e n c l e a v e d at A r g 5 . T r y p s i n - a c t i v a t a b l e p r o D was also purified f r o m the u r i n e o f a patient with F a n c o n i ' s s y n d r o m e . This n a t i v e proD (n-proD) r e p r e s e n t s less than 1% of the total a n t i g e n i c D in the urine and h a s the dipeptide G R as a c t i v a t i o n p e p t i d e . Activation of n-proD r e q u i r e s h i g h e r c o n c e n t r a t i o n s o f t r y p s i n t h a n rproD and only 1/3 o f the resulting factor D h a s the s a m e specific h e m o l y t i c activity and N H 2 - t e r m i n u s as native D. T h e r e m a i n d e r o f the protein is cleaved at Arg5. Apparently, n-proD is produced by cleavage at Arg(-3) by an enzyme with trypsin-like s p e c i f i c i t y w h i c h m a y be i d e n t i c a l to t h e p r o D processing enzyme that cleaves at A r g ( - 1 ) but d i f f e r e n t from the leader p e p t i d a s e that p r o d u c e s rproD.
THE DYSMORPHIC BUT FUNCTIONALLY ACTIVE C6 OF SUBTOTAL C6 DEFICIENT SUBJECTS IS CARBOXYTERMINALLY TRUNCATED R. Wiirzner, D. Mewar, P.J. Lachmann Molecular Immunopathology Unit, MRC Centre, Cambridge, UK The C6 of 8 subtotal C6 deficient subjects was previously found to be unequivocally present (1-2% of the normal mean), able to incorporate into the terminal complement complex (TCC), haemolytically active but dysmorphic as it was 14% smaller than normal C6. In ELISA assays one anti-C6 specific monoclonal antibody (mab) did, and another did not, bind to this dysmorphic C6. Using the pUEX expression system (Bressan & Stanley 1987) and C6 cDNA subclones (DiScipio & Hugli 1989) the epitopes of the mabs were mapped to the third Thrombospondin Repeat (TSP) or to the second Factor I Module (FIM) of C6, respectively, the latter indicating that the dysmorphic C6 is carboxyterminally truncated. Since the absence of as much as one-seventh of C6 still permits functional activity, the missing FIM 2 of C6 appears to be of minor importance. In contrast, that region was thought to represent a part of the C5b-binding region (DiScipio 1991), whereas another study mapped this binding site to the two short consensus repeats (SCRs) of C6 (Nakano et al. 1991). A third study indicated that a fragment containing the SCRs and the FIMs of C6 bound more weakly to C5b than whole C6 (Haefliger et al. 1989) indicating an involvement of aminoterminal neighbouring domains to facilitate binding to C5b. The mab anti-C6 which binds to the third TSR of C6 (WU 6-4) was previously shown to be capable of markedly inhibiting TCC formation. Using a reactive lysis assay, TCC formation could not be inhibited when this mab was introduced after C5b6 formation and in an ELISA no binding of WU 6-4 to its epitope was detected when C5b6 served as antigen. Both assays suggest that WU 6-4 inhibits C5b6 formation, favouring the adjacent (in the primary structure) SCRs rather than the FIMs as C5bbinding site. The molecular basis of the subtotal C6 deficiency most likely resides within intron 15 of C6, preventing a splicing out, and furthermore might be identical to the defect resulting in an apparently identical dysmorphic C6 (same isoelectric focusing pattern) of combined C6/C7 deficient subjects (see abstract by Hobart, Femie, W~zner, Morgan, Lachmann).
THE MAJORITY OF HUMAN C7 SYNTHESISED I N V I V O IS O F E X T R A H E P A T I C ORIGIN R. Wttrzner, H. Morgan:~, V. Joysey:~, P.J. Lachmann Molecular Immunopathology Unit, MRC Centre, and :~Tissue Typing Laboratory Addenbrookes Hospital, Cambridge, UK C7 M/N typing, the determination of the allotypes of the C7 M/N protein polymorphism, was carried out on samples from donors and recipients of liver transplantations in order to find out if the liver is the predominant site of 'in vivo' synthesis of complement protein C7. That determination which is based on the reactivity of an allotypic monoclonal antibody in an ELISA assay offered two great advantages: first, since C7*N is much more frequent than C7"2, the second most common allele of the C7 IEF polymorphism, informative pairings were expected in Caucasians, and second, since C7 M/N typing is based on ratios, the contribution of the transplanted liver towards the allotype was quantifiable. Out of 35 recipients 13 received a liver from a donor with a C7 M/N phenotype different from their own and the determination of the C7 concentrations and ratios of 3 to 6 post transplantation (p.t.) samples revealed that there was only a contribution of the transplanted liver towards the C7 M/N phenotype at 2-4 weeks p.t.; that influence decreased with time and was between 0-20% after 4-26 weeks p.t., thus excluding the liver as the predominant site of in vivo C7 synthesis. The transient contribution of the donor phenotype could be attributable to cells of the monocyte / macrophage lineage, Kupffer cells in particular. C7 is thus the only terminal complement component not predominantly synthesised by hepatocytes in contrast to C6 (Hobart et al. 1976), C8 (Alper et al. 1980) and probably C9, as the latter is an acute phase protein (Adinolfi and Lehner 1988). This finding is also consistent with observations that high C7 levels are maintained in liver failure. Although sufficient C7 is usually present in serum, a modulation of complement activity by local C7 synthesis by monocytes or macrophages cannot be excluded as the initiating C5b6 molecule of terminal complement attack is quite stable with C7 often being the limiting factor for terminal complement complex generation (Lachmann and Thompson 1970).
ZYMOGEN FACTOR
FORMS
OF
COMPLEMENT
D
Y. Y a m a u c h i , J.W. S t e v e n s , K.J. M a c o n , a n d Volanakis. U n i v e r s i t y o f A l a b a m a at B i r m i n g h a m , USA.
J.E.
W e u s e d a b a c u l o v i r u s s y s t e m to e x p r e s s the h u m a n factor D e D N A , hg31 (R.T. White et al., J. Biol. C h e m . 267:9210, 1992) in Sf9 cells. The recombinant p r o t e i n (r-proD) w a s purified by c h r o m a t o g r a p h y on Bit-Rex 70 a n d MonoS and was hemolytically inactive. On S D S - P A G E , r-proD had a slightly lower mobility t h a n a u t h e n t i c f a c t o r D. Amino acid s e q u e n c i n g indicated that it c o n s i s t e d of two f o r m s o f proD with r e s p e c t i v e a c t i v a t i o n p e p t i d e s , A A P P R G R and A P P R G R . Catalytic a m o u n t s o f trypsin converted r-proD to active factor D, h a v i n g S D S - P A G E mobility and specific hemolytic activity similar to t h o s e o f the native enzyme. A b o u t 90% o f t r y p s i n - a c t i v a t e d rproD had the s a m e N H 2 - t e r m i n u s as a u t h e n t i c factor D while t h e r e m a i n d e r had b e e n c l e a v e d at A r g 5 . T r y p s i n - a c t i v a t a b l e p r o D was also purified f r o m the u r i n e o f a patient with F a n c o n i ' s s y n d r o m e . This n a t i v e proD (n-proD) r e p r e s e n t s less than 1% of the total a n t i g e n i c D in the urine and h a s the dipeptide G R as a c t i v a t i o n p e p t i d e . Activation of n-proD r e q u i r e s h i g h e r c o n c e n t r a t i o n s o f t r y p s i n t h a n rproD and only 1/3 o f the resulting factor D h a s the s a m e specific h e m o l y t i c activity and N H 2 - t e r m i n u s as native D. T h e r e m a i n d e r o f the protein is cleaved at Arg5. Apparently, n-proD is produced by cleavage at Arg(-3) by an enzyme with trypsin-like s p e c i f i c i t y w h i c h m a y be i d e n t i c a l to t h e p r o D processing enzyme that cleaves at A r g ( - 1 ) but d i f f e r e n t from the leader p e p t i d a s e that p r o d u c e s rproD.