Pathway Function MASP-1
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The Pro-Factor D Cleaving Activity of MASP-1/-3 Is Not Required for Alternative Pathway Function This information is current as Søren E. Degn, Jens C. Jensenius and Steffen Thiel of October 3, 2021. J Immunol 2014; 192:5447-5448; ; doi: 10.4049/jimmunol.1400777 http://www.jimmunol.org/content/192/12/5447 Downloaded from References This article cites 8 articles, 7 of which you can access for free at: http://www.jimmunol.org/content/192/12/5447.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 3, 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 © 2014 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Th eJournal of Letters to the Editor Immunology The Pro-Factor D Cleaving Activity Furthermore, our observation (1) of marked alternative path- of MASP-1/-3 Is Not Required for way activity in the MASP-1/-3 deficient human suggested that any such backup pathways were highly functional, calling Alternative Pathway Function into question the purported essential role of MASP-1/-3. Recently, fresh observations have shed new light on the basis e recently reported in The Journal of Immunology of this controversy, prompting us to readdress the issue in this (1) the presence of a functional alternative path- letter. In their recent article, Ruseva and colleagues (7) dem- W way in a human patient with Malpuech–Michels– onstrated that MASP-1 and MASP-3 are not essential for Mingarelli–Carnevale (3MC) syndrome caused by a genetic activation of the complement system via the alternative path- defect in the MASP1 gene. This defect abolished the pro- way in vivo [(7); murine complement]. However, they noted duction of all three splice products of this gene: mannan- that this occurred in the absence of detectable mature fD [(7); binding lectin (MBL)–associated serine proteases (MASP)-1 murine complement]. This reconciles the previous seemingly and MASP-3, and MBL-associated protein of 44 kDa disparate findings, as it confirms both the observation of (MAp44) (human complement). Takahashi et al. (4) on the role of MASP-1/-3 in cleaving Downloaded from Our paper generated debate because the findings directly profD and our observation of a functioning alternative path- conflicted with results obtained in a mouse model of complete way in the absence of MASP-1/-3. Two possible scenarios MASP1 deficiency (2, 3). In 2010 Takahashi and colleagues present themselves: either undetectable but catalytically func- (4) reported in the Journal of Experimental Medicine that tional levels of mature fD are generated during alternative MASP-1 was the protease responsible for cleaving profactor D pathway activation via the bypass pathways suggested by (profD) and thus essential for the presence of mature, active Takahashi and colleagues; or, in contrast to what was previ- http://www.jimmunol.org/ fD in the blood [(4); murine complement]. Thus, in a mouse ously reported [(8); human complement], profD has sig- model of genetic deficiency of all three Masp1 gene products, nificant activity toward the fBC3(H2O) substrate. Further Takahashi et al. (4) reported complete deficiency of the al- studies are needed to answer this question. ternative pathway. They determined this to be caused by a Meanwhile, the discrepancy between results of Ruseva et al. complete absence of mature, active fD in the Masp1 knockout [(7); murine complement] and Takahashi et al. [(4); murine serum, and demonstrated that recombinant murine MASP-1 complement] regarding alternative pathway activity in the could cleave profD, rescuing alternative pathway activation in Masp1 knockout mouse serum prompted us to reexamine this vitro. The group followed up on these findings (5) reporting activity. We report in this letter that the difference arises from by guest on October 3, 2021 that MASP-3 could also cleave profD, and in addition, at low the use of different buffer systems. At physiological ionic efficiency, factor B to Bb [(5); murine complement]. The strength, alternative pathway activity is observed, albeit at a suggestion of an essential role of MASP-1/-3 in alternative lower level than in wild type mice, thus confirming the recent pathwayfunctionwascorroboratedbyastudyusingthe results of Ruseva et al. [(7); murine complement] (Fig. 1A, 1B). murine anti-collagen Ab-induced model of inflammatory Conversely, the high ionic strength buffer used by Takahashi arthritis, which is known to be sensitive to alterations in the et al. [(4); murine complement] suppresses the weaker alter- alternative pathway [(6); murine complement]. native pathway activity of the Masp1 knockout mouse to Surprisingly, our results (1), based on the 3MC syndrome background levels (Fig. 1C). This suggests that either the patient and in vitro studies with recombinant human MASP-1, interaction of backup enzymes with profD or the profD in- indicated that in humans neither MASP-1 nor MASP-3 are teraction with fBC3(H2O) substrate is sensitive to high ionic required for alternative pathway function [(1); human com- strength. plement]. It was suggested that the activity we observed was Regarding the discrepancy between the role of MASP-1/-3 due to backup pathways, with thrombin, kallikrein, and plas- in vivo in the anti-collagen Ab-induced arthritis model of Banda min being able to cleave profD (3). Because of natural variation et al. [(6); murine complement] and in the fH deficient model in alternative pathway hemolytic activity and the availability of of Ruseva et al. [(7); murine complement], we also attempt to only a single deficient human, we could not determine whether offer some insight. The former model is dependent on the al- MASP-1/-3 deficiency caused a quantitative deficiency, but ternative pathway for initiation and propagation of injury, and simply noted the existence of significant alternative pathway due to the weaker alternative pathway activation in the Masp1 activity, indicating that mature fD was indeed present in the knockout, significant protection is observed. The latter model absence of MASP-1/-3 (2). is based on the absence of a crucial regulator of the alternative The issue remained that the complete absence of alternative pathway: factor H. In this scenario, the lower level alternative pathway activity in the MASP-1/-3 deficient mouse was not pathway activity in Masp1 knockouts is sufficient to initiate reconcilable with the existence of the proposed backup pathways. formation of C3 convertase, which in the absence of factor H forms an unregulated positive amplification loop. Hence, the two scenarios are mechanistically fundamentally different, ex- Copyright Ó 2014 by The American Association of Immunologists, Inc. 0022-1767/14/$16.00 plaining the different outcomes. 5448 LETTERS TO THE EDITOR Downloaded from FIGURE 1. Influence of buffer composition on the activity of the alternative pathway of complement activation in mice in the presence and the absence of the Masp1 gene products: MASP-1, MASP-3, and MAp44. (A) Rabbit erythrocyte lysis as a function of a 1.5-fold dilution series in gelatin-containing Veronal buffer (GVB) with EGTA and MgCl2 (GVB/EGTA/Mg: 5 mM barbital, 145 mM NaCl, 10 mM EGTA, 5 mM MgCl2, pH 7.4, with 0.1% [w/v] gelatin) of either wild-type C57BL/6 serum or Masp1 knockout serum. Note the narrow dynamic range in such assays. Mean and SD of duplicate http://www.jimmunol.org/ measurements. Experiment repeated with two different dilution series. GVB/EGTA/Mg 5 buffer only (i.e., background signal). H2O 5 maximal signal following hypotonic lysis. (B) C3 deposition in microtiter wells coated with the alternative pathway activator zymosan, as a function of a 2-fold dilution series of C57BL/6 serum in various buffers. Mean and SD of duplicates, experiment repeated twice. VBS/Ca, Veronal-buffered saline (5 mM barbital, 145 mM NaCl, pH 7.4) with 5 mM CaCl2 5 all complement pathways are active. VBS/EGTA/Mg, VBS with 10 mM EGTA and 5 mM MgCl2 5 only the alternative pathway is active. Boric acid/EGTA/Mg, boric acid-buffered saline (200 mM boric acid, 140 mM NaCl, pH 8.0) with 30 mM EGTA and 35 mM MgCl2 5 alternative pathway in presence of high ionic strength (as in Ref. 4). VBS/EDTA, VBS with 10 mM EDTA 5 no pathways active (i.e., background signal). (C)Asin(B), but for Masp1 knockout serum. Note the influence of high ionic strength on alternative pathway activity, especially in the Masp1 knockout mouse serum. by guest on October 3, 2021 Søren E. Degn, Jens C. Jensenius, and Steffen Thiel collagen antibody-induced model of inflammatory arthritis. J. Immunol. 185: 5598–5606. 7. Ruseva, M. M., M. Takahashi, T. Fujita, and M. C. Pickering. 2014. C3 dysregulation Department of Biomedicine, Aarhus University, DK-8000 Aarhus C, Denmark due to factor H deficiency is mannan-binding lectin-associated serine proteases Clin. Exp. Immunol. Address correspondence and reprint requests to Dr. Søren E. Degn, Department (MASP)-1 and MASP-3 independent in vivo. 176: 84–92. 8. Yamauchi, Y., J. W. Stevens, K. J. Macon, and J. E. Volanakis. 1994. Recombinant of Biomedicine, Aarhus University, Bartholin Building, Wilhelm Meyers Alle´ 4, J.