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Unique structural features of human factor D and its zymogen form
37 E F F E C T O F H I G H D O S E A D M I N I S T R A T I O H OF METHYLPREDHISOLOHE ON COMPLEMENT SYSTEM IN PATIENTS WITH CONNECTIVE TISSUE DIEEASES S.Nakanishi, S.Takemura, M.Deguchi, N.Ichio, K.Ashihara,R.Nakahara, T.Doi, N.Seto, Y.Kasamatsu, M.Okamoto,K.Yanagida, W.Fukuda, H.Onodera, M.Ueda, S.Sugino and M.Kondo Department of Internal Medicine, Kyoto Prefectural University of Medicine, Kyoto 602, Japan High dose of methylprednisolone(MP),"pulse therapy", is effective in various diseases with activation of the complement system, but the precise mechanism is obscure. We studied the changes of complement profiles in 4 patients with connective tissue diseases after "pulse therapy". Samples were collected before and after 1000mg MP drip infusion. CH50 was measured by Mayer's method and C1 activity by hemolytic assay. The amounts of complement fragments, C4d, Bb, iC3b, SC5b-9 and CIs-CIINH complex were tested by ELISA. ClINH activity was analyzed by inhibiting effect of Cls on C4 consumption. CH50 and C1 activity increased at 6hr after MP administration in all cases. The complement fragments, C4d and Bb were decreased after 3hrs. SC5b-9 and iC3b decreased at 3hrs followed by increase at 6hrs. CIs-CIINH complex decreased after 6hrs in contrast to CIINH activity. These results suggested that MP inhibit complement activation in vivo by the inhibition or inactivation of Cls generated by immune complexes.
EXPRESSIONS ANO CHARACTERIZATIONS OF RECOMBINANT MACIFS (CD59) OF THREE DIFFERENT LENGTHS Yasuko NakanoI ), Yuji Sugita2), Takashi "robeI ), Nam-Ho Miura1) and Motowo TomitaI ) 1)Dept. Physiological Chemistry, School of Pharmaceutical Sciences, Showa Univ. and 2)Molecular Biology Dept., Central Research Lab., Yamanouchi Pharmaceutical Co. Ltd. MACIF (CD59) is a 18 kDa GPl-anohoredglycoproteinconsistlng of 77 amino i:Ids. The amino acld sequence predlotedfrom its eDNA nucleotlde sequmtce cousists of 128 amino aclds and congainsa signal ~ at carboxy tennious he,des amino terminus. We constructed three expression plasmides whose inserts coded Met plus 77 amino aclds, rMACIF77, Met plus 86 amino acids which had a I~lroohlUc Weeh of carboxy-tenninal signal pelXide, rMACIF86, alld Met plus 103 amino acids had a full length of ced)oxy-tenninal signal peptide, rMACIF103. These recombinant proteins were produced in E.coli in inclusion bodies and bed to be denatured and rofolded to ol0tainthe aotive forms. Ntar af~lyour~aUon Usinga ~ a n ~ U U ~ t F ~3F~pr, a,-o~e,sm=uaJ ma~sus and ~t~Uon aotiviCyana~/~s of the three rMACIFs were ioerloffned alndcornl~lred wtlh each others. Amino-terminal sequences and disulMe bridges of rMACIFs were same as that of natureMACIR rMAClF7"/aed rMACIF86 were soluble whlle rMACIF103 tended to aggregate and became iusolubte v ~ dete~genL because of U h y d r ~ c w b o x y ~ i n a l sequsnce, rMACIF77 and 88 hed weak inhlbiUon ac~dlles whlch were comparableto solubio MACIF in urine and could not be incofl)~ated Into memlxaa~. On the other hand, rMACIF103 could be incoq~-ated into m o n t a n e ~ h ~ sequonoe.
UNIQUE HUMAN FORM
STRUCTURAL FEATURES OF FACTOR D AND ITS ZYMOGEN
S.V.L. Narayana, D. Moore, L.J. DeLucas, Kilpatrick, Y. Yamauchi, K.J. Macon, and Volanakis. University of Alabama at Birmingham, USA.
J.M. J.E.
The structure of factor D has been determined and refined to 2.0~ resolution by using x-ray diffraction data from two crystal forms (PI and P21). Factor D retains the general structural fold of s e r i n e proteases with more than 40% of the residues included in two antiparallel 13-sheet structures. However, the orientation of the side chains of His-57 and Asp-102 (chymotrypsinogen numbering) of the catalytic triad differs considerably from that of other scrinc proteases. Furthermore, the two noncrystallographically related molecules in the unit cell, A and B, display distinctive active center conformations. In molecule A, Asp-102 is pointed away from His-57 and is fully accessible to the solvent. In molecule B, the imidazole ring of His-57 is in the energetically-favored trans conformation and the space it occupies in other serine proteases is filled by the side chain of Ser-215. In either case the catalytic triad, Asp-102/His-57/Ser-195, seems to be out of alignment and non-functional. These findings support the hypothesis that the active conformation of the catalytic center of factor D is induced by the substrate, C3bB. To further characterize factor D, we have recently obtained crystals of a r e c o m b i n a n t zymogen form of the enzyme. The determination of the structure of this proD is in progress. Structural differences from the structure of native factor D will be discussed.
RECEPTOR-INDEPENDENT ACTIVATION OF GTP-BINDING, REGULATORY PROTEINS BY C5b-9 Florin I. Niculescu, Horea G. Rus, and Moon L. Shin, University Maryland, School of Medicine, Baltimore, MD 21201 Sublytic terminal complement complexes (TCC) induce hydrolysis of major membrane phospholipids, produce signal messengers like Ca2+, cAMP, DAG, ceramide, and increase PKC activity in metabolically active nucleated cells. Since DAG increase by TCC in human JY cell lines was inhibited by pertussis toxin (PTX), PTX-sensitive G proteins that may be involved in TCC-induced cellular activities was investigated by assessing G protein activation in JY25 and JY5, a mutant deficient in GPI-anchor proteins derived from wild type JY25. TCC assembly induced time-dependent increase in GTP¥S binding, 4-fold in JY25 and 8-fold in JY5 at 15 min. Similar increase in GTP hydrolysis was also induced by TCC in both membranes. G protein activation was noted first with C5b-7 assembly, which increased further with C5b-8 and C5b-9 formation. PTX, but not cholera toxin (CTX), induced ADP-ribosylation of 41 kDa Gc~ subunits in intact membrane, which was reduced to 20% in the presence of inserted TCC. Immunoprecipitation of TCC-treated membrane lysates with antibodies to C5b-9 neoantigen, C7, or C8 revealed the presence of PTX-inducible, ADP-ribosylating Go~i of 41kDa. The G-proteisns complexed with TCC were functionally active, as shown by GTPyS binding, increased with time. Enhanced TCC elimination from the cell surface induced by signals generated through TCC, mimicked by mastoparan, was inhibited by PTX, but not with CTX. These findings suggest that the biological activities induced by TCC are, in part, mediated through activation of PTX-sensitive G-proteins which interact directly with inserted TCC peptides.
EXPRESSIONS ANO CHARACTERIZATIONS OF RECOMBINANT MACIFS (CD59) OF THREE DIFFERENT LENGTHS Yasuko NakanoI ), Yuji Sugita2), Takashi "robeI ), Nam-Ho Miura1) and Motowo TomitaI ) 1)Dept. Physiological Chemistry, School of Pharmaceutical Sciences, Showa Univ. and 2)Molecular Biology Dept., Central Research Lab., Yamanouchi Pharmaceutical Co. Ltd. MACIF (CD59) is a 18 kDa GPl-anohoredglycoproteinconsistlng of 77 amino i:Ids. The amino acld sequence predlotedfrom its eDNA nucleotlde sequmtce cousists of 128 amino aclds and congainsa signal ~ at carboxy tennious he,des amino terminus. We constructed three expression plasmides whose inserts coded Met plus 77 amino aclds, rMACIF77, Met plus 86 amino acids which had a I~lroohlUc Weeh of carboxy-tenninal signal pelXide, rMACIF86, alld Met plus 103 amino acids had a full length of ced)oxy-tenninal signal peptide, rMACIF103. These recombinant proteins were produced in E.coli in inclusion bodies and bed to be denatured and rofolded to ol0tainthe aotive forms. Ntar af~lyour~aUon Usinga ~ a n ~ U U ~ t F ~3F~pr, a,-o~e,sm=uaJ ma~sus and ~t~Uon aotiviCyana~/~s of the three rMACIFs were ioerloffned alndcornl~lred wtlh each others. Amino-terminal sequences and disulMe bridges of rMACIFs were same as that of natureMACIR rMAClF7"/aed rMACIF86 were soluble whlle rMACIF103 tended to aggregate and became iusolubte v ~ dete~genL because of U h y d r ~ c w b o x y ~ i n a l sequsnce, rMACIF77 and 88 hed weak inhlbiUon ac~dlles whlch were comparableto solubio MACIF in urine and could not be incofl)~ated Into memlxaa~. On the other hand, rMACIF103 could be incoq~-ated into m o n t a n e ~ h ~ sequonoe.
UNIQUE HUMAN FORM
STRUCTURAL FEATURES OF FACTOR D AND ITS ZYMOGEN
S.V.L. Narayana, D. Moore, L.J. DeLucas, Kilpatrick, Y. Yamauchi, K.J. Macon, and Volanakis. University of Alabama at Birmingham, USA.
J.M. J.E.
The structure of factor D has been determined and refined to 2.0~ resolution by using x-ray diffraction data from two crystal forms (PI and P21). Factor D retains the general structural fold of s e r i n e proteases with more than 40% of the residues included in two antiparallel 13-sheet structures. However, the orientation of the side chains of His-57 and Asp-102 (chymotrypsinogen numbering) of the catalytic triad differs considerably from that of other scrinc proteases. Furthermore, the two noncrystallographically related molecules in the unit cell, A and B, display distinctive active center conformations. In molecule A, Asp-102 is pointed away from His-57 and is fully accessible to the solvent. In molecule B, the imidazole ring of His-57 is in the energetically-favored trans conformation and the space it occupies in other serine proteases is filled by the side chain of Ser-215. In either case the catalytic triad, Asp-102/His-57/Ser-195, seems to be out of alignment and non-functional. These findings support the hypothesis that the active conformation of the catalytic center of factor D is induced by the substrate, C3bB. To further characterize factor D, we have recently obtained crystals of a r e c o m b i n a n t zymogen form of the enzyme. The determination of the structure of this proD is in progress. Structural differences from the structure of native factor D will be discussed.
RECEPTOR-INDEPENDENT ACTIVATION OF GTP-BINDING, REGULATORY PROTEINS BY C5b-9 Florin I. Niculescu, Horea G. Rus, and Moon L. Shin, University Maryland, School of Medicine, Baltimore, MD 21201 Sublytic terminal complement complexes (TCC) induce hydrolysis of major membrane phospholipids, produce signal messengers like Ca2+, cAMP, DAG, ceramide, and increase PKC activity in metabolically active nucleated cells. Since DAG increase by TCC in human JY cell lines was inhibited by pertussis toxin (PTX), PTX-sensitive G proteins that may be involved in TCC-induced cellular activities was investigated by assessing G protein activation in JY25 and JY5, a mutant deficient in GPI-anchor proteins derived from wild type JY25. TCC assembly induced time-dependent increase in GTP¥S binding, 4-fold in JY25 and 8-fold in JY5 at 15 min. Similar increase in GTP hydrolysis was also induced by TCC in both membranes. G protein activation was noted first with C5b-7 assembly, which increased further with C5b-8 and C5b-9 formation. PTX, but not cholera toxin (CTX), induced ADP-ribosylation of 41 kDa Gc~ subunits in intact membrane, which was reduced to 20% in the presence of inserted TCC. Immunoprecipitation of TCC-treated membrane lysates with antibodies to C5b-9 neoantigen, C7, or C8 revealed the presence of PTX-inducible, ADP-ribosylating Go~i of 41kDa. The G-proteisns complexed with TCC were functionally active, as shown by GTPyS binding, increased with time. Enhanced TCC elimination from the cell surface induced by signals generated through TCC, mimicked by mastoparan, was inhibited by PTX, but not with CTX. These findings suggest that the biological activities induced by TCC are, in part, mediated through activation of PTX-sensitive G-proteins which interact directly with inserted TCC peptides.