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John E. Volanakis - One of the best experts on this subject based on the ideXlab platform.
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complement Factor D a novel serine protease
Protein Science, 2008Co-Authors: John E. Volanakis, Sthanam V. L. NarayanaAbstract:Factor D is unique among serine proteases in that it requires neither enzymatic cleavage for expression of proteolytic activity nor inactivation by a serpin for its control. Regulation of Factor D activity is insteaD attaineD by a novel mechanism that DepenDs on reversible conformational changes for expression anD control of catalytic activity. These conformational changes are believeD to be inDuceD by the single natural substrate, C3bB, anD to result in realignment of the catalytic triaD, the specificity pocket, anD the nonspecific substrate binDing site, all of which have atypical conformations. Mutational stuDies have DefineD structural Determinants responsible for these unique structural features of Factor D anD for the resultant low reactivity with synthetic esters.
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complement activation in Factor D Deficient mice
Proceedings of the National Academy of Sciences of the United States of America, 2001Co-Authors: Yuanyuan Xu, Gregory C Ippolito, Harry W Schroeder, Michael C Carroll, John E. VolanakisAbstract:To assess the contribution of the alternative pathway in complement activation anD host Defense anD its possible role in the regulation of systemic energy balance in vivo, Factor D-Deficient mice were generateD by gene targeting. The mutant mice have no apparent abnormality in Development anD their boDy weights are similar to those of Factor D-sufficient littermates. Complement activation coulD not be initiateD in the serum of Deficient mice by the alternative pathway activators rabbit erythrocytes anD zymosan. Surprisingly, injection of cobra venom Factor (CVF) causeD a profounD anD reproDucible reDuction in serum C3 levels, whereas, as expecteD, there was no C3 reDuction in Factor B-Deficient mice treateD similarly. StuDies of C3 anD Factor B activation in vitro by CVF DemonstrateD that in Factor D-Deficient serum the α chain of C3 was cleaveD graDually over a perioD of 60 min without Detectable cleavage of Factor B. CVF-DepenDent C3 cleavage in the Deficient serum requireD the presence of Mg2+, whereas in normal mouse serum the presence of Divalent cations was not requireD. These results suggest that in mouse proteolytic cleavage of Factor B by Factor D is not an absolute requirement for the zymogen to active enzyme conformational transition of CVF-bounD Factor B. Kinetics of opsonization of Streptococcus pneumoniae by C3 fragments was much slower in Factor D-Deficient serum, suggesting a significant contribution of the alternative pathway to antibacterial host Defense early after infection.
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structural basis of proFactor D activation from a highly flexible zymogen to a novel self inhibiteD serine protease complement Factor D
The EMBO Journal, 1999Co-Authors: H Jing, Dwight Moore, Lawrence J Delucas, Kevin Macon, John E. Volanakis, Sthanam V. L. NarayanaAbstract:The crystal structure of proFactor D, DetermineD at 2.1 A resolution with an Rfree anD an R-Factor of 25.1 anD 20.4%, respectively, Displays highly flexible or DisorDereD conformation for five regions: N-22, 71-76, 143-152, 187-193 anD 215-223. A comparison with the structure of its mature serine protease, complement Factor D, revealeD major conformational changes in the similar regions. Comparisons with the zymogen-active enzyme pairs of chymotrypsinogen, trypsinogen anD prethrombin-2 showeD a similar Distribution of the flexible regions. However, proFactor D is the most flexible of the four, anD its mature enzyme Displays inactive, self-inhibiteD active site conformation. Examination of the surface properties of the N-terminus-binDing pocket inDicates that Ile16 may play the initial positioning role for the N-terminus, anD Leu17 probably also helps in inDucing the requireD conformational changes. This process, perhaps shareD by most chymotrypsinogen-like zymogens, is followeD by a Factor D-unique step, the re-orientation of an external Arg218 to an internal position for salt-briDging with Asp189, leaDing to the generation of the self-inhibiteD Factor D.
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structures of native anD complexeD complement Factor D implications of the atypical his57 conformation anD self inhibitory loop in the regulation of specific serine protease activity
Journal of Molecular Biology, 1998Co-Authors: H Jing, J M Kilpatrick, John E. Volanakis, Yarlagadda S. Babu, David Moore, Sthanam V. L. NarayanaAbstract:Abstract Factor D is a serine protease essential for the activation of the alternative pathway of complement. The structures of native Factor D anD a complex formeD with isatoic anhyDriDe inhibitor were DetermineD at resolution of 2.3 anD 1.5 A, respectively, in an isomorphous monoclinic crystal form containing one molecule per asymmetric unit. The native structure was compareD with structures DetermineD previously in a triclinic cell containing two molecules with Different active site conformations. The current structure shows greater similarity with molecule B in the triclinic cell, suggesting that this may be the Dominant Factor D conformation in solution. The major conformational Differences with molecule A in the triclinic cell are locateD in four regions, three of which are close to the active site anD incluDe some of the resiDues shown to be critical for Factor D catalytic activity. The conformational flexibility associateD with these regions is proposeD to proviDe a structural basis for the previously proposeD substrate-inDuceD reversible conformational changes in Factor D. The high-resolution structure of the Factor D/isatoic anhyDriDe complex reveals the binDing moDe of the mechanism-baseD inhibitor. The higher specificity towarDs Factor D over trypsin anD thrombin is baseD on hyDrophobic interactions between the inhibitor benzyl ring anD the aliphatic siDe-chain of Arg218 that is salt briDgeD with Asp189 at the bottom of the primary specificity (S1) pocket. Comparison of Factor D structural variants with other serine protease structures revealeD the presence of a unique “self-inhibitory loop”. This loop (214–218) Dictates the resting-state conformation of Factor D by (1) preventing His57 from aDopting active tautomer conformation, (2) preventing the P1 to P3 resiDues of the substrate from forming anti-parallel β-sheets with the non-specific substrate binDing loop, anD (3) blocking the accessibility of Asp189 to the positive1y chargeD P1 resiDue of the substrate. The conformational switch from resting-state to active-state can only be inDuceD by the single macromolecular substrate, C3b-bounD Factor B. This self-inhibitory mechanism is highly correlateD with the unique functional properties of Factor D, which incluDe high specificity towarD Factor B, low esterolytic activity towarD synthetic substrates, anD absence of regulation by zymogen anD serpin-like or other natural inhibitors in blooD.
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Structure of Diisopropyl fluorophosphate-inhibiteD Factor D.
Acta Crystallographica Section D Biological Crystallography, 1997Co-Authors: L B Cole, J M Kilpatrick, John E. Volanakis, Sthanam V. L. Narayana, N Chu, Yarlagadda S. BabuAbstract:Factor D (D) is a serine protease, crucial for the activation of the alternative complement pathway. Only a limiteD number of general serine protease inhibitors are known to inhibit D, most of which covalently binD to the serine hyDroxyl of the catalytic triaD. The structure of the first enzyme:inhibitor covalent aDDuct of D with Diisopropyl fluorophosphate (DIP:D) to a resolution of 2.4 A is DescribeD. The inhibiteD enzyme is similar in overall structure to the native enzyme anD to trypsin, yet exhibits notable Differences in the active site. One region of the active site is conserveD between D anD trypsin with respect to amino-aciD sequence anD to conformation. Another reflects the amino-aciD substitutions anD conformational flexibility between these enzymes. The active-site histiDine resiDue is observeD in the gauche+ conformation, not the normal gauche- orientation seen in the classic catalytic triaD arrangement requireD for enzymatic activity in serine proteases. Comparisons of the active sites between native D, the DIP:D aDDuct, anD DIP-inhibiteD trypsin have proviDeD funDamental insights currently being employeD in the Design of novel small-molecule pharmaceutical agents capable of moDulating the alternative complement pathway.
Sthanam V. L. Narayana - One of the best experts on this subject based on the ideXlab platform.
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complement Factor D a novel serine protease
Protein Science, 2008Co-Authors: John E. Volanakis, Sthanam V. L. NarayanaAbstract:Factor D is unique among serine proteases in that it requires neither enzymatic cleavage for expression of proteolytic activity nor inactivation by a serpin for its control. Regulation of Factor D activity is insteaD attaineD by a novel mechanism that DepenDs on reversible conformational changes for expression anD control of catalytic activity. These conformational changes are believeD to be inDuceD by the single natural substrate, C3bB, anD to result in realignment of the catalytic triaD, the specificity pocket, anD the nonspecific substrate binDing site, all of which have atypical conformations. Mutational stuDies have DefineD structural Determinants responsible for these unique structural features of Factor D anD for the resultant low reactivity with synthetic esters.
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structural basis of proFactor D activation from a highly flexible zymogen to a novel self inhibiteD serine protease complement Factor D
The EMBO Journal, 1999Co-Authors: H Jing, Dwight Moore, Lawrence J Delucas, Kevin Macon, John E. Volanakis, Sthanam V. L. NarayanaAbstract:The crystal structure of proFactor D, DetermineD at 2.1 A resolution with an Rfree anD an R-Factor of 25.1 anD 20.4%, respectively, Displays highly flexible or DisorDereD conformation for five regions: N-22, 71-76, 143-152, 187-193 anD 215-223. A comparison with the structure of its mature serine protease, complement Factor D, revealeD major conformational changes in the similar regions. Comparisons with the zymogen-active enzyme pairs of chymotrypsinogen, trypsinogen anD prethrombin-2 showeD a similar Distribution of the flexible regions. However, proFactor D is the most flexible of the four, anD its mature enzyme Displays inactive, self-inhibiteD active site conformation. Examination of the surface properties of the N-terminus-binDing pocket inDicates that Ile16 may play the initial positioning role for the N-terminus, anD Leu17 probably also helps in inDucing the requireD conformational changes. This process, perhaps shareD by most chymotrypsinogen-like zymogens, is followeD by a Factor D-unique step, the re-orientation of an external Arg218 to an internal position for salt-briDging with Asp189, leaDing to the generation of the self-inhibiteD Factor D.
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structures of native anD complexeD complement Factor D implications of the atypical his57 conformation anD self inhibitory loop in the regulation of specific serine protease activity
Journal of Molecular Biology, 1998Co-Authors: H Jing, J M Kilpatrick, John E. Volanakis, Yarlagadda S. Babu, David Moore, Sthanam V. L. NarayanaAbstract:Abstract Factor D is a serine protease essential for the activation of the alternative pathway of complement. The structures of native Factor D anD a complex formeD with isatoic anhyDriDe inhibitor were DetermineD at resolution of 2.3 anD 1.5 A, respectively, in an isomorphous monoclinic crystal form containing one molecule per asymmetric unit. The native structure was compareD with structures DetermineD previously in a triclinic cell containing two molecules with Different active site conformations. The current structure shows greater similarity with molecule B in the triclinic cell, suggesting that this may be the Dominant Factor D conformation in solution. The major conformational Differences with molecule A in the triclinic cell are locateD in four regions, three of which are close to the active site anD incluDe some of the resiDues shown to be critical for Factor D catalytic activity. The conformational flexibility associateD with these regions is proposeD to proviDe a structural basis for the previously proposeD substrate-inDuceD reversible conformational changes in Factor D. The high-resolution structure of the Factor D/isatoic anhyDriDe complex reveals the binDing moDe of the mechanism-baseD inhibitor. The higher specificity towarDs Factor D over trypsin anD thrombin is baseD on hyDrophobic interactions between the inhibitor benzyl ring anD the aliphatic siDe-chain of Arg218 that is salt briDgeD with Asp189 at the bottom of the primary specificity (S1) pocket. Comparison of Factor D structural variants with other serine protease structures revealeD the presence of a unique “self-inhibitory loop”. This loop (214–218) Dictates the resting-state conformation of Factor D by (1) preventing His57 from aDopting active tautomer conformation, (2) preventing the P1 to P3 resiDues of the substrate from forming anti-parallel β-sheets with the non-specific substrate binDing loop, anD (3) blocking the accessibility of Asp189 to the positive1y chargeD P1 resiDue of the substrate. The conformational switch from resting-state to active-state can only be inDuceD by the single macromolecular substrate, C3b-bounD Factor B. This self-inhibitory mechanism is highly correlateD with the unique functional properties of Factor D, which incluDe high specificity towarD Factor B, low esterolytic activity towarD synthetic substrates, anD absence of regulation by zymogen anD serpin-like or other natural inhibitors in blooD.
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Structure of Diisopropyl fluorophosphate-inhibiteD Factor D.
Acta Crystallographica Section D Biological Crystallography, 1997Co-Authors: L B Cole, J M Kilpatrick, John E. Volanakis, Sthanam V. L. Narayana, N Chu, Yarlagadda S. BabuAbstract:Factor D (D) is a serine protease, crucial for the activation of the alternative complement pathway. Only a limiteD number of general serine protease inhibitors are known to inhibit D, most of which covalently binD to the serine hyDroxyl of the catalytic triaD. The structure of the first enzyme:inhibitor covalent aDDuct of D with Diisopropyl fluorophosphate (DIP:D) to a resolution of 2.4 A is DescribeD. The inhibiteD enzyme is similar in overall structure to the native enzyme anD to trypsin, yet exhibits notable Differences in the active site. One region of the active site is conserveD between D anD trypsin with respect to amino-aciD sequence anD to conformation. Another reflects the amino-aciD substitutions anD conformational flexibility between these enzymes. The active-site histiDine resiDue is observeD in the gauche+ conformation, not the normal gauche- orientation seen in the classic catalytic triaD arrangement requireD for enzymatic activity in serine proteases. Comparisons of the active sites between native D, the DIP:D aDDuct, anD DIP-inhibiteD trypsin have proviDeD funDamental insights currently being employeD in the Design of novel small-molecule pharmaceutical agents capable of moDulating the alternative complement pathway.
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crystal structure of a complement Factor D mutant expressing enhanceD catalytic activity
Journal of Biological Chemistry, 1995Co-Authors: Sthanam V. L. Narayana, John E. VolanakisAbstract:Abstract Complement Factor D is a serine protease regulateD by a novel mechanism that DepenDs on conformational changes rather than cleavage of a zymogen for expression of proteolytic activity. The conformational changes are presumeD to be inDuceD by the single natural substrate, C3bB, anD to result in reversible reorientation of the catalytic center anD of the substrate binDing site of Factor D, both of which have atypical conformations. Here we report that replacement of Ser94, Thr214, anD Ser215 of Factor D (chymotrypsinogen numbering has been useD for comparison purposes) with the corresponDing resiDues of trypsin, Tyr, Ser, anD Trp, is sufficient to inDuce substantially higher catalytic activity associateD with a typical serine protease alignment of the catalytic triaD resiDues His57, Asp102, anD Ser195. These results proviDe a partial structural explanation for the low reactivity of “resting-state” Factor D towarD synthetic substrates.
Toshio Miyata - One of the best experts on this subject based on the ideXlab platform.
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Evaluation of the Proteolytic Activity of Factor D AccumulateD as an Active Serine Protease in Patients with Chronic Renal Failure
Nephron, 1994Co-Authors: Reiko Inagi, Kenji Maeda, Toshio Miyata, Kozo InoueAbstract:Complement Factor D, a complement system serine protease, circulating in vivo as its active form, accumulates in patients with chronic renal failure. The pathophysiological role of this active protease in these patients was examineD by stuDies on activities of excess Factor D on 10 synthetic peptiDe substrates for some usual serine proteases. The most sensitive of these substrates to Factor D was Boc-Gln-Ala-Arg-MCA, which is useD as a substrate for trypsin. The proteolytic activity of Factor D (2.17 unit/mg/h) on this substrate was estimateD to be 10–5-folD that of trypsin (2.18 × 105 unit/mg/h). The activities of Factor D on other synthetic substrates were lower. Thus the proteolytic activity of Factor D is consiDereD to be very specific for its natural substrate, complement Factor B bounD with C3b, even when it is highly accumulateD in vivo. The inhibitory effects of some serine protease inhibitors useD clinically (nafamostat mesilate, sepinostat mesilate, camostat mesilate anD gabexate mesilate) on the proteolytic activity of Factor D on its natural substrate, Factor B, were also investigateD. Of these synthetic compounDs, nafamostat mesilate was the most effective inhibitor (ID50:25 µM) of the activity of Factor D on Factor B.
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Effects of excess Factor D on early- anD late-phase activation of the complement cascaDe.
Nihon Jinzo Gakkai shi, 1992Co-Authors: Hiroyuki Kobayakawa, Reiko Inagi, Toshio Miyata, Takahiro Shinzato, Kenji MaedaAbstract:The present stuDy was unDertaken to examine the effects of excess Factor D builD-up in the boDy of enD-stage renal Disease (ESRD) patients upon the activation of the alternative pathway anD the terminal pathway in the fluiD phase. First, to clarify the effect of excess Factor D on the alternative pathway, purifieD Factor D from an ESRD patient was aDDeD to normal serum anD the changes in concentrations of C3a-Des-Arg anD C5a-Des-Arg were investigateD. The results showeD that once the serum Factor D level reacheD a concentration corresponDing to 15 micrograms/ml in the serum of the ESRD patient, the C3a-Des-Arg anD C5a-Des-Arg levels haD climbeD to about 1.7-folD the concentration in normal serum. Next, in orDer to clarify the effect of excess Factor D on the terminal pathway, purifieD Factor D was aDDeD to normal serum, anD the changes in C5b6 generation were examineD. The results inDicateD that as the Factor D level increaseD in the serum, the C5b6 level rose graDually also; anD when the Factor D concentration reacheD 15 micrograms/ml, the C5b6 generation haD risen to approximately 1.5-folD the level in normal serum. The present results therefore suggest that Factor D builD-up in ESRD patients proviDes a uremic toxin that can cause abnormal activation of the whole complement cascaDe.
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fluiD phase activation of the alternative pathway of complement by excess Factor D in regularly DialyzeD patients
Nephron, 1992Co-Authors: Toshio Miyata, Akio Miyama, Reiko Inagi, Kozo Inoue, Kyogsu Hong, Yoshiyasu Iida, Taroh Kinoshita, Kenji MaedaAbstract:We examineD the effect of excess Factor D on the alternative pathway of complement (APC). First, we DemonstrateD that the proDuction of C3a is accelerateD in the fluiD-phase with the aDDition of purifieD Factor D. Analysis by soDium DoDecylsulfate polyacrylamiDe gel electrophoresis unDer reDucing conDitions showeD that the serum iC3b level was elevateD when incubateD with excess Factor D. SeconDly, we DemonstrateD, by measuring the C5a-Des-Arg level, that the generation of C5a was promoteD in the fluiD-phase with the aDDition of purifieD Factor D. We then stuDieD whether activation of APC is elevateD in the blooD of patients on maintenance hemoDialysis whose sera containeD a high concentration of Factor D. First, we DetecteD, by fluorescence activateD cell sorter analysis, greater amounts of C3D on erythrocytes from the patients (mean fluorescence intensity ± SD: 7.7 ± 1.7 arbitrary units) than those from healthy inDiviDuals (5.4 ± 0.5 arbitrary units; p
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Deterioration of Immune Complex Solubilization Activity of Serum by IncreaseD Concentration of Factor D
Nephron, 1991Co-Authors: Toshio Miyata, Akio Miyama, Reiko Inagi, Itaru Inoue, Hidechika Okada, Izumi Nakashima, Kenji MaedaAbstract:We investigateD the effect of excess complement Factor D on the immune complex solubilization activity (ICSA) of serum. First, we estimateD the concentration of Factor D, ICSA anD the hemolytic activity via the classical complement pathway (CH50) in the sera of 16 healthy inDiviDuals anD 36 patients on hemoDialysis for enD-stage renal failure. The serum concentration of Factor D in these patients (mean ± SD: 12.12 ± 2.38 μg/ml) was significantly higher (p
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Molecular anD functional iDentification anD purification of complement component Factor D from urine of patients with chronic renal failure.
Molecular Immunology, 1990Co-Authors: Toshio Miyata, A. Mlyama, Kayo Maeda, Reiko Inagi, Izumi Nakashima, Satoru Sugiyama, N YamanakaAbstract:Urine proteins of normal subject anD patients with impaireD renal function were analyzeD by two-Dimensional polyacrylamiDe gel electrophoresis. As a result, a clear spot was DetecteD specifically in urine from patients with obvious renal Dysfunction. The isoelectrical point of this unique spot was pH 7.1–7.2 anD the flow-rate (Rf) was 0.50–0.55 as that of albumin was 1.0. Partial amino aciD sequence analysis revealeD that the NH2-terminal to 22nD amino aciD sequence was iDentical with that of complement Factor D. We purifieD 22 mg of this protein (Factor D) from 5000 ml of urine from a patient on hemoDialysis by three Chromatographic steps using DEAE-SephaDex A-50 anD Sephacryl S-200. The purifieD urine Factor D gave a single banD in soDium DoDecyl sulfate polyacrylamiDe gel electrophoresis at the position of 23 kD, anD DisplayeD normal Factor D hemolytic activity. The concentrations of Factor D estimateD by hemolytic assay were 1.9 μgml of normal serum,
Reiko Inagi - One of the best experts on this subject based on the ideXlab platform.
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Evaluation of the Proteolytic Activity of Factor D AccumulateD as an Active Serine Protease in Patients with Chronic Renal Failure
Nephron, 1994Co-Authors: Reiko Inagi, Kenji Maeda, Toshio Miyata, Kozo InoueAbstract:Complement Factor D, a complement system serine protease, circulating in vivo as its active form, accumulates in patients with chronic renal failure. The pathophysiological role of this active protease in these patients was examineD by stuDies on activities of excess Factor D on 10 synthetic peptiDe substrates for some usual serine proteases. The most sensitive of these substrates to Factor D was Boc-Gln-Ala-Arg-MCA, which is useD as a substrate for trypsin. The proteolytic activity of Factor D (2.17 unit/mg/h) on this substrate was estimateD to be 10–5-folD that of trypsin (2.18 × 105 unit/mg/h). The activities of Factor D on other synthetic substrates were lower. Thus the proteolytic activity of Factor D is consiDereD to be very specific for its natural substrate, complement Factor B bounD with C3b, even when it is highly accumulateD in vivo. The inhibitory effects of some serine protease inhibitors useD clinically (nafamostat mesilate, sepinostat mesilate, camostat mesilate anD gabexate mesilate) on the proteolytic activity of Factor D on its natural substrate, Factor B, were also investigateD. Of these synthetic compounDs, nafamostat mesilate was the most effective inhibitor (ID50:25 µM) of the activity of Factor D on Factor B.
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Effects of excess Factor D on early- anD late-phase activation of the complement cascaDe.
Nihon Jinzo Gakkai shi, 1992Co-Authors: Hiroyuki Kobayakawa, Reiko Inagi, Toshio Miyata, Takahiro Shinzato, Kenji MaedaAbstract:The present stuDy was unDertaken to examine the effects of excess Factor D builD-up in the boDy of enD-stage renal Disease (ESRD) patients upon the activation of the alternative pathway anD the terminal pathway in the fluiD phase. First, to clarify the effect of excess Factor D on the alternative pathway, purifieD Factor D from an ESRD patient was aDDeD to normal serum anD the changes in concentrations of C3a-Des-Arg anD C5a-Des-Arg were investigateD. The results showeD that once the serum Factor D level reacheD a concentration corresponDing to 15 micrograms/ml in the serum of the ESRD patient, the C3a-Des-Arg anD C5a-Des-Arg levels haD climbeD to about 1.7-folD the concentration in normal serum. Next, in orDer to clarify the effect of excess Factor D on the terminal pathway, purifieD Factor D was aDDeD to normal serum, anD the changes in C5b6 generation were examineD. The results inDicateD that as the Factor D level increaseD in the serum, the C5b6 level rose graDually also; anD when the Factor D concentration reacheD 15 micrograms/ml, the C5b6 generation haD risen to approximately 1.5-folD the level in normal serum. The present results therefore suggest that Factor D builD-up in ESRD patients proviDes a uremic toxin that can cause abnormal activation of the whole complement cascaDe.
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fluiD phase activation of the alternative pathway of complement by excess Factor D in regularly DialyzeD patients
Nephron, 1992Co-Authors: Toshio Miyata, Akio Miyama, Reiko Inagi, Kozo Inoue, Kyogsu Hong, Yoshiyasu Iida, Taroh Kinoshita, Kenji MaedaAbstract:We examineD the effect of excess Factor D on the alternative pathway of complement (APC). First, we DemonstrateD that the proDuction of C3a is accelerateD in the fluiD-phase with the aDDition of purifieD Factor D. Analysis by soDium DoDecylsulfate polyacrylamiDe gel electrophoresis unDer reDucing conDitions showeD that the serum iC3b level was elevateD when incubateD with excess Factor D. SeconDly, we DemonstrateD, by measuring the C5a-Des-Arg level, that the generation of C5a was promoteD in the fluiD-phase with the aDDition of purifieD Factor D. We then stuDieD whether activation of APC is elevateD in the blooD of patients on maintenance hemoDialysis whose sera containeD a high concentration of Factor D. First, we DetecteD, by fluorescence activateD cell sorter analysis, greater amounts of C3D on erythrocytes from the patients (mean fluorescence intensity ± SD: 7.7 ± 1.7 arbitrary units) than those from healthy inDiviDuals (5.4 ± 0.5 arbitrary units; p
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Deterioration of Immune Complex Solubilization Activity of Serum by IncreaseD Concentration of Factor D
Nephron, 1991Co-Authors: Toshio Miyata, Akio Miyama, Reiko Inagi, Itaru Inoue, Hidechika Okada, Izumi Nakashima, Kenji MaedaAbstract:We investigateD the effect of excess complement Factor D on the immune complex solubilization activity (ICSA) of serum. First, we estimateD the concentration of Factor D, ICSA anD the hemolytic activity via the classical complement pathway (CH50) in the sera of 16 healthy inDiviDuals anD 36 patients on hemoDialysis for enD-stage renal failure. The serum concentration of Factor D in these patients (mean ± SD: 12.12 ± 2.38 μg/ml) was significantly higher (p
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Molecular anD functional iDentification anD purification of complement component Factor D from urine of patients with chronic renal failure.
Molecular Immunology, 1990Co-Authors: Toshio Miyata, A. Mlyama, Kayo Maeda, Reiko Inagi, Izumi Nakashima, Satoru Sugiyama, N YamanakaAbstract:Urine proteins of normal subject anD patients with impaireD renal function were analyzeD by two-Dimensional polyacrylamiDe gel electrophoresis. As a result, a clear spot was DetecteD specifically in urine from patients with obvious renal Dysfunction. The isoelectrical point of this unique spot was pH 7.1–7.2 anD the flow-rate (Rf) was 0.50–0.55 as that of albumin was 1.0. Partial amino aciD sequence analysis revealeD that the NH2-terminal to 22nD amino aciD sequence was iDentical with that of complement Factor D. We purifieD 22 mg of this protein (Factor D) from 5000 ml of urine from a patient on hemoDialysis by three Chromatographic steps using DEAE-SephaDex A-50 anD Sephacryl S-200. The purifieD urine Factor D gave a single banD in soDium DoDecyl sulfate polyacrylamiDe gel electrophoresis at the position of 23 kD, anD DisplayeD normal Factor D hemolytic activity. The concentrations of Factor D estimateD by hemolytic assay were 1.9 μgml of normal serum,
J M Kilpatrick - One of the best experts on this subject based on the ideXlab platform.
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structures of native anD complexeD complement Factor D implications of the atypical his57 conformation anD self inhibitory loop in the regulation of specific serine protease activity
Journal of Molecular Biology, 1998Co-Authors: H Jing, J M Kilpatrick, John E. Volanakis, Yarlagadda S. Babu, David Moore, Sthanam V. L. NarayanaAbstract:Abstract Factor D is a serine protease essential for the activation of the alternative pathway of complement. The structures of native Factor D anD a complex formeD with isatoic anhyDriDe inhibitor were DetermineD at resolution of 2.3 anD 1.5 A, respectively, in an isomorphous monoclinic crystal form containing one molecule per asymmetric unit. The native structure was compareD with structures DetermineD previously in a triclinic cell containing two molecules with Different active site conformations. The current structure shows greater similarity with molecule B in the triclinic cell, suggesting that this may be the Dominant Factor D conformation in solution. The major conformational Differences with molecule A in the triclinic cell are locateD in four regions, three of which are close to the active site anD incluDe some of the resiDues shown to be critical for Factor D catalytic activity. The conformational flexibility associateD with these regions is proposeD to proviDe a structural basis for the previously proposeD substrate-inDuceD reversible conformational changes in Factor D. The high-resolution structure of the Factor D/isatoic anhyDriDe complex reveals the binDing moDe of the mechanism-baseD inhibitor. The higher specificity towarDs Factor D over trypsin anD thrombin is baseD on hyDrophobic interactions between the inhibitor benzyl ring anD the aliphatic siDe-chain of Arg218 that is salt briDgeD with Asp189 at the bottom of the primary specificity (S1) pocket. Comparison of Factor D structural variants with other serine protease structures revealeD the presence of a unique “self-inhibitory loop”. This loop (214–218) Dictates the resting-state conformation of Factor D by (1) preventing His57 from aDopting active tautomer conformation, (2) preventing the P1 to P3 resiDues of the substrate from forming anti-parallel β-sheets with the non-specific substrate binDing loop, anD (3) blocking the accessibility of Asp189 to the positive1y chargeD P1 resiDue of the substrate. The conformational switch from resting-state to active-state can only be inDuceD by the single macromolecular substrate, C3b-bounD Factor B. This self-inhibitory mechanism is highly correlateD with the unique functional properties of Factor D, which incluDe high specificity towarD Factor B, low esterolytic activity towarD synthetic substrates, anD absence of regulation by zymogen anD serpin-like or other natural inhibitors in blooD.
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Structure of 3,4-Dichloroisocoumarin-inhibiteD Factor D.
Acta crystallographica. Section D Biological crystallography, 1998Co-Authors: L B Cole, J M Kilpatrick, N Chu, Y S BabuAbstract:Factor D (D) is a serine protease essential in the activation of the alternative complement pathway. Only a few of the common serine protease inhibitors inhibit D, binDing covalently to the serine hyDroxyl of the catalytic triaD. 3,4-Dichloroisocoumarin (DCI) is a mechanism-baseD inhibitor which inhibits most serine proteases anD many esterases, incluDing D. The structure of the enzyme:inhibitor covalent aDDuct of D with DCI, DCI:D, to a resolution of 1.8 A is DescribeD, which represents the first structural analysis of D with a mechanism-baseD inhibitor. The siDe chain of the ring-openeD DCI moiety of the protein aDDuct unDergoes chemical moDification in the buffereD solution, resulting in the formation of an alpha-hyDroxy aciD moiety through the nucleophilic substitution of both Cl atoms. The inhibiteD enzyme is similar in overall structure to the native enzyme, as well as to a variety of isocoumarin-inhibiteD trypsin anD porcine pancreatic elastase (PPE) structures, yet notable Differences are observeD in the active site anD binDing moDe of these small-molecule inhibitors. One region of the active site (resiDues 189-195) is relatively conserveD between Factor D, trypsin, anD elastase with respect to amino-aciD sequence anD to conformation. Another region (resiDues 214-220) reflects the amino-aciD substitutions anD conformational flexibility between these enzymes. The carbonyl O atom of the DCI moiety was founD to be orienteD away from the oxyanion hole, which greatly contributes to the stability of the DCI:D aDDuct. The comparisons of the active sites between native Factor D, DCI-inhibiteD Factor D, anD various inhibiteD trypsin anD elastase (PPE) molecules are proviDing the chemical bases Directing our Design of novel, small-molecule pharmaceutical agents capable of moDulating the alternative complement pathway.
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Structure of Diisopropyl fluorophosphate-inhibiteD Factor D.
Acta Crystallographica Section D Biological Crystallography, 1997Co-Authors: L B Cole, J M Kilpatrick, John E. Volanakis, Sthanam V. L. Narayana, N Chu, Yarlagadda S. BabuAbstract:Factor D (D) is a serine protease, crucial for the activation of the alternative complement pathway. Only a limiteD number of general serine protease inhibitors are known to inhibit D, most of which covalently binD to the serine hyDroxyl of the catalytic triaD. The structure of the first enzyme:inhibitor covalent aDDuct of D with Diisopropyl fluorophosphate (DIP:D) to a resolution of 2.4 A is DescribeD. The inhibiteD enzyme is similar in overall structure to the native enzyme anD to trypsin, yet exhibits notable Differences in the active site. One region of the active site is conserveD between D anD trypsin with respect to amino-aciD sequence anD to conformation. Another reflects the amino-aciD substitutions anD conformational flexibility between these enzymes. The active-site histiDine resiDue is observeD in the gauche+ conformation, not the normal gauche- orientation seen in the classic catalytic triaD arrangement requireD for enzymatic activity in serine proteases. Comparisons of the active sites between native D, the DIP:D aDDuct, anD DIP-inhibiteD trypsin have proviDeD funDamental insights currently being employeD in the Design of novel small-molecule pharmaceutical agents capable of moDulating the alternative complement pathway.
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structure of human Factor D a complement system protein at 2 0 a resolution
Journal of Molecular Biology, 1994Co-Authors: Sthanam V. L. Narayana, J M Kilpatrick, John E. Volanakis, David Moore, Ossama Elkabbani, Charles E Bugg, M Carson, Xi Chen, Lawrence J DelucasAbstract:Abstract Factor D, an essential enzyme for the activation of the alternative pathway of the complement system, belongs to the serine protease superfamily. The crystal structure of the enzyme was solveD by a combination of multiple isomorphous replacement anD molecular replacement methoDs. The present moDel was refineD to an R -Factor of 18·8% using 23,681 observeD reflections between 7·5 anD 2·0 A resolution, with a root-mean-square Deviation from stanDarD bonD lengths of 0·016 A. The two non-crystallographically relateD molecules in the triclinic unit cell have Distinctive active site conformations. The protein has the general structural folD of a serine protease, but there are several unique amino aciD substitutions resulting in significant alterations in the critical loops responsible for catalysis anD substrate specificity in serine proteases. Factor D is the first complement serine protease whose three-Dimensional structure has been DetermineD.
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Purification anD properties of human Factor D.
Methods in Enzymology, 1993Co-Authors: John E. Volanakis, Susan R. Barnum, J M KilpatrickAbstract:Publisher Summary This chapter Describes the simpler, higher yielD methoD for purifying Factor D from urine of patients with tubular Dysfunction anD from peritoneal Dialysis fluiD. The chapter also Discusses the recent information on the structure anD properties of Factor D. Complement Factor D is an essential component of the alternative pathway of complement activation. It is a serine proteinase with low catalytic activity against synthetic substrates 2 anD a single known natural substrate, complement Factor B. Several hemolytic assays are available for measuring Factor D activity. This assay involves the use of rabbit erythrocytes anD of a serum reagent DepleteD of Factor D (RD). The amino aciD sequence of Factor D shows a high Degree of homology to that of other serine proteinases, exhibiting 33–35% amino aciD resiDue iDentity with pancreatic bovine trypsin, bovine chymotrypsin A, porcine elastase, anD human neutrophil elastase. In aDDition to RD proceDures, a number of more quantitative assays using cellular intermeDiates anD purifieD complement components are also DescribeD in the chapter.
