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367.Full.Pdf Human Complement Factor I Does Not Require Cofactors for Cleavage of Synthetic Substrates This information is current as Stefanos A. Tsiftsoglou and Robert B. Sim of October 2, 2021. J Immunol 2004; 173:367-375; ; doi: 10.4049/jimmunol.173.1.367 http://www.jimmunol.org/content/173/1/367 Downloaded from References This article cites 41 articles, 17 of which you can access for free at: http://www.jimmunol.org/content/173/1/367.full#ref-list-1 Why The JI? Submit online. http://www.jimmunol.org/ • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication *average by guest on October 2, 2021 Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2004 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology Human Complement Factor I Does Not Require Cofactors for Cleavage of Synthetic Substrates1 Stefanos A. Tsiftsoglou2 and Robert B. Sim Complement factor I (fI) plays a major role in the regulation of the complement system. It circulates in an active form and has very restricted specificity, cleaving only C3b or C4b in the presence of a cofactor such as factor H (fH), complement receptor type 1, membrane cofactor protein, or C4-binding protein. Using peptide-7-amino-4-methylcoumarin derivatives, we investigated the substrate specificity of fI. There is no previous report of synthetic substrate cleavage by fI, but five substrates were found in this study. A survey of 15 substrates and a range of inhibitors showed that fI has specificity similar to that of thrombin, but with much lower catalytic activity than that of thrombin. fI amidolytic activity has a pH optimum of 8.25, typical of serine proteases and is insensitive to ionic strength. This is in contrast to its proteolytic activity within the fI-C3b-fH reaction, in which the pH optimum for C3b cleavage is <5.5 and the reaction rate is highly dependent on ionic strength. The rate of cleavage of tripeptide 7-amino- Downloaded from 4-methylcoumarins by fI is unaffected by the presence of fH or C3(NH3). The amidolytic activity is inhibited by the synthetic thrombin inhibitor Z-D-Phe-Pro-methoxypropylboroglycinepinanediol ester, consistent with previous reports, and by benzene- sulfonyl fluorides such as Pefabloc SC. Suramin inhibits fI directly at concentration of 1 mM. Within a range of metal ions tested, ,only Cr2؉ and Fe3؉ were found to inhibit both the proteolytic and amidolytic activity of fI. The Journal of Immunology, 2004 173: 367–375. http://www.jimmunol.org/ he complement system is a major recognition and effector human fI (accession numbers: cDNA, Y00318; genomic, X78594) mechanism of innate immunity. Seven key serine pro- is localized on chromosome 4q25. T teases, factor D (fD),3 MBL-associated serine protease fI has very restricted specificity limited to cleavage of arginyl (MASP)-2, C1s, C1r, factor B (fB), C2, and factor I (fI), play bonds in its natural protein substrates C3b and C4b. Cofactor pro- crucial roles in the generation of complement activities in the teins such as factor H (fH), complement receptor type 1, mem- phases of amplification and regulation of the cascade reactions (1, brane cofactor protein, or C4-binding protein are required for 2). Two additional homologues of MASP-2, namely, MASP-1 and cleavage. During natural substrate cleavage, fI forms a ternary MASP-3, have been identified, but their roles in complement ac- complex with the substrate and the cofactor (5). Certain aspects of tivation have yet to be determined. They all carry domains homol- the fI-cofactor-substrate complex remain unclear, such as whether by guest on October 2, 2021 ogous to the trypsin family and, with the exception of fD, addi- binding of the cofactor to both fI and the substrate is required for tional protein modules that influence the orientation and substrate orientation or is necessary for inducing appropriate con- localization of protein substrates and mediate complex formation formations in either the substrate or enzyme (1). through protein-protein interactions. fI is synthesized as a single polypeptide chain, which is glyco- fI plays an essential role in the modulation of the complement sylated and processed before secretion. No circulating zymogen cascade by the regulation of the C3 convertase of the classical and form has been identified. The mature protein consists of a N-ter- alternative activation pathways (3, 4). It is also essential for con- minal H chain with 317 aa residues and a C-terminal L chain with version of C3b into iC3b, a major opsonin. The gene encoding 244 residues (6, 7) that are covalently linked via a disulfide bond between residues Cys309 and Cys435 (S. A. Tsiftsoglou and A. C. Willis, unpublished data). Each chain contains three occupied N- Medical Research Council Immunochemistry Unit, Department of Biochemistry, Uni- linked glycosylation sites contributing 20–25% (w/w) of the ap- versity of Oxford, South Parks Road, Oxford, United Kingdom parent protein molecular mass (8, 9). Analysis of the primary Received for publication March 15, 2004. Accepted for publication April 21, 2004. structure of fI reveals a unique linear arrangement of domains; a The costs of publication of this article were defrayed in part by the payment of page N-terminal fI membrane attack complex domain, an scavenger re- charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. ceptor cysteine-rich domain, and two class A low density lipopro- 1 This work was supported by the Medical Research Council (U.K.) and by a fees tein receptor domains in the noncatalytic H chain (6, 7, 10). The scholarship from the Department of Biochemistry, University of Oxford (to S.A.T.). C-terminal consists entirely of a trypsin-like L chain containing the 2 Address correspondence and reprint requests to Dr. Stefanos A. Tsiftsoglou, residues that define the His-Asp-Ser catalytic triad. In addition, Medical Research Council Immunochemistry Unit, Department of Biochemistry, residues are present that define the specificity pocket Asp189 and University of Oxford, South Parks Road, Oxford OX1 3QU, U.K. E-mail address: 214 215 216 [email protected] the extended substrate binding sites Ser , Trp , and Gly . 3 Abbreviations used in this paper: fD, factor D; MASP, MBL-associated serine Among complement proteases, the serine protease domain of protease; fB, factor B; fI, factor I; fH, factor H; BoroMPG, Z-D-Phe-Pro- human fI is most similar to fD (28% amino acid sequence identity), methoxypropylboroglycinepinanediol ester; DFP, diisopropylfluorophosphate; but among all human serine proteases, it exhibits the closest sim- suramin, 8,8Ј-{carbonylbis[imino-3,1-phenylenecarbonylimino(4-methyl-3,1- phenylene)carbonylimino]}bis-1,3,5-naphthalenetrisulfonic acid; buffer A, 10 ilarity to human tissue plasminogen activator (41% sequence iden- mM potassium phosphate, 0.5 mM EDTA, 0.1% Tween 20, pH 6.2; buffer B, 25 tity), and human plasma kallikrein (37% sequence identity). fD mM Bicine, 0.5 mM EDTA, pH 8.25; AMC, 7-amino-4-methylcoumarin; ⑀ACA, ⑀-aminocaproic acid; SBTI, soybean trypsin inhibitor; LBTI, lima bean trypsin and fI both cleave their natural substrates in the presence of co- inhibitor type IIL. factors, and their rates of inhibition by substituted isocoumarins Copyright © 2004 by The American Association of Immunologists, Inc. 0022-1767/04/$02.00 368 SUBSTRATES AND INHIBITORS OF HUMAN COMPLEMENT fI are very low (11). There is structural evidence to suggest reversible substrate-induced conformational change within fD that may be required for optimal alignment of the catalytic triad (12, 13). This could account for the very low esterolytic activity (14) of isolated fD. A C3b-induced realignment of the catalytic site of fI has been proposed by Ekdahl et al. (15), on the basis of reactivity of fI with the serine protease inhibitor diisopropylfluorophosphate (DFP). To facilitate measurement of fI enzyme activity, we examined its amidolytic activity against 15 fluorogenic substrates. Kam et al. (11) examined 50 peptide thiobenzyl esters but found that none was cleaved by fI. Before this report, there has been no data de- scribing synthetic substrates for fI. 7-Amino-4-methylcoumarin (AMC) derivatives provide greater sensitivity in substrate assays than their thiobenzyl ester counterparts and have been used in stud- ies involving highly selective and low activity serine proteases, such as blood coagulation factors IXa (16) and VIIa (17) and the complement proteases fD, C2, and fB (14). Here, we provide the first report of the cleavage of synthetic substrates by fI in the absence of cofactors. Native fI cleaves pep- Downloaded from tides with a thrombin-like specificity, targeting Arg at the P1 po- sition, but with a lower catalytic activity than that of thrombin. The FIGURE 1. SDS-PAGE analysis of the protein preparations used in this work. fI, fH, and C3(NH ) were analyzed by SDS-10% PAGE under re- presence of cofactor has no significant influence on the enzymatic 3 duced and nonreduced conditions with Coomassie blue staining. Under turnover of these synthetic substrates, indicating that the cofactor reduced conditions, the heavily glycosylated (20–25% w/w) fI appears as does not alter the primary active site conformation of fI.
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