The Pro- 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 September 25, 2021. J Immunol 2014; 192:5447-5448; ; doi: 10.4049/jimmunol.1400777 http://www.jimmunol.org/content/192/12/5447 Downloaded from

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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 via the alternative path- defect in the MASP1 . 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 (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 Downloaded from () (human complement). Takahashi et al. (4) on the role of MASP-1/-3 in cleaving 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 pathway activation via the bypass pathways suggested by

MASP-1 was the protease responsible for cleaving profactor D http://www.jimmunol.org/ (profD) and thus essential for the presence of mature, active Takahashi and colleagues; or, in contrast to what was previ- 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 September 25, 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 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 , , 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: . 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 September 25, 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. Immunol. DK-8000 Aarhus C, Denmark. E-mail address: [email protected] and native zymogen forms of human complement factor D. 152: 3645–3653. Abbreviations used in this article: MAp44, MBL-associated protein of 44 kDa; MASP, mannan-binding lectin–associated ; MBL, mannan-binding lectin; 3MC, www.jimmunol.org/cgi/doi/10.4049/jimmunol.1400777 Malpuech–Michels–Mingarelli–Carnevale; profD, profactor D. Comment on “The Pro-Factor D References 1. Degn, S. E., L. Jensen, A. G. Hansen, D. Duman, M. Tekin, J. C. Jensenius, and Cleaving Activity of MASP-1/-3 Is S. Thiel. 2012. Mannan-binding lectin-associated serine protease (MASP)-1 is Not Required for Alternative crucial for activation in human serum, whereas neither MASP-1 nor MASP-3 is required for alternative pathway function. J. Immunol. 189: 3957– Pathway Function” 3969. 2. Degn, S. E., J. C. Jensenius, and S. Thiel. 2013. Response to comment on “Mannan-binding lectin-associated serine protease (MASP)-1 is crucial for lectin pathway activation in human serum, whereas neither MASP-1 nor MASP-3 is e would like to express our deepest gratitude to required for alternative pathway function.” J. Immunol. 190: 2477–2478. the editor for giving us the opportunity to discuss 3. Takahashi, M., H. Sekine, Y. Endo, and T. Fujita. 2013. Comment on “Mannan- binding lectin-associated serine protease (MASP)-1 is crucial for lectin pathway W Degn and colleagues’ argument. We previously activation in human serum, whereas neither MASP-1 nor MASP-3 is required claimed (1) in the Journal of Experimental Medicine that for alternative pathway function.” J. Immunol. 190: 2477. 4. Takahashi, M., Y. Ishida, D. Iwaki, K. Kanno, T. Suzuki, Y. Endo, Y. Homma, and mannan-binding lectin (MBL)–associated serine protease T. Fujita. 2010. Essential role of mannose-binding lectin-associated serine protease-1 (MASP)-1 has an essential role in converting profactor D in activation of the complement factor D. J. Exp. Med. 207: 29–37. (profD) into its active form, because the vast majority of fD 5. Iwaki, D., K. Kanno, M. Takahashi, Y. Endo, M. Matsushita, and T. Fujita. 2011. The role of mannose-binding lectin-associated serine protease-3 in activation of in the blood of MASP-1/-3 knockout mice was identified as the alternative complement pathway. J. Immunol. 187: 3751–3758. profD by Western blot. We also observed an absence of al- 6. Banda, N. K., M. Takahashi, B. Levitt, M. Glogowska, J. Nicholas, K. Takahashi, G. L. Stahl, T. Fujita, W. P. Arend, and V. M. Holers. 2010. Essential role of ternative pathway activity in the knockout sera by zymosan complement mannose-binding lectin-associated serine proteases-1/3 in the murine assay. However, Degn et al. (2) reported contradictory results The Journal of Immunology 5449 obtained using samples derived from human patients with Malpuech–Michels–Mingarelli–Carnevale (3MC) syndrome with a genetic deficiency in the MASP1 gene. Therefore, it was crucial to determine whether profD exists in the sera of 3MC patients. Degn and colleagues’ group kindly provided us with their samples derived from 3MC patients, as well as from healthy controls, in a blinded fashion. We com- FIGURE 1. ProfD was found in human MASP-1/-3–deficient sera. pared the m.w. of the fD by Western blot of each of the four Degn’s group kindly provided us with their samples derived from 3MC samples. As shown in Fig. 1, two out of the four samples patients and healthy controls in a blinded fashion (lanes 2–5). The sample lane 1 showed a slightly larger m.w. of fD compared with the others. in is a freshly isolated healthy control. We performed Western blotting by using anti-fD Ab to compare the m.w. of fD. From the After we sent our results to Degn et al., they informed us that results, we noticed that the m.w. of fD from the samples in lanes 3and the two samples that showed a larger m.w. of fD were indeed 5 is slightly different from the others. After we sent this figure to Degn’s derived from the 3MC patients. Thus, we successfully dis- group, they informed us that the samples in lanes 3 and 5 were derived tinguished the difference in m.w. of fD between normal and from the 3MC patients, whereas the samples in lanes 2 and 4 were from 3MC patients with 100% accuracy. Coinciding with our pre- healthy controls. vious results (1) using MASP-1/-3 knockout mice, 3MC pa- tients with a genetic deficiency in the MASP1 gene have profD In conclusion, MASP-1 and MASP-3 are crucial enzymes for in their sera. This fact strongly advocates that MASP-1 and alternative pathway activation through the activation of profD Downloaded from MASP-3 are the main enzymes for converting profD into into its active form, and this is similar to murine results. The active form in both species. conclusion proposed by Degn et al., “the pro-factor D cleaving It has been reported (3, 4) that thrombin, kallikrein, and activity of MASP-1/-3 is not required for alternative pathway have the ability to cleave profD, suggesting another function,” is liable to cause a profound misunderstanding in possibility, which is that they could act as backup enzymes, that neither MASP-1/-3 nor active fD has a role for the alter- producing an undetectable amount of active fD in both native pathway function. Therefore, it is reasonable to propose http://www.jimmunol.org/ MASP-1/-3 knockout mice and 3MC patients. In a recent that the pro-factor D cleaving activity of MASP-1/-3 is required paper, Ruseva and colleagues (5) demonstrated in vivo acti- for the full alternative pathway function. vation of the alternative pathway in sera of MASP-1/-3 and factor H doubleknockout mice. The data suggested that Minoru Takahashi, Hideharu Sekine, and MASP-1 and MASP-3 are not essential for the activation of Teizo Fujita the alternative pathway (5). Their results (5) indicated that uncontrolled activation of the alternative pathway occurs in Department of Immunology, Fukushima Medical University School of Medicine, Fukushima 960-1295, Japan

the absence of factor H. In this condition, an undetectable by guest on September 25, 2021 Address correspondence and reprint requests to Dr. Minoru Takahashi, Department of amount of active fD in Masp-1/-3 knockout mice might be Immunology, Fukushima Medical University School of Medicine, 1 Hikarigaoka, sufficient to trigger activation of the alternative pathway. Fukushima 960-1295, Japan. E-mail address: [email protected] Degnandcolleaguesobservedthattheionicstrengthin Abbreviations used in this article: MASP, mannan-binding lectin–associated serine rabbit erythrocyte lysis assay affects the outcome of the results protease; MBL, mannan-binding lectin; 3MC, Malpuech–Michels–Mingarelli–Carne- vale; prof D, profactor D. (figure 1B, 1C in their letter). Formerly, Dijk and colleagues (6) investigated the optimal reaction conditions for hemolytic assay of alternative pathway activity in mouse serum. We fol- References lowed their method because it avoids all nonspecific erythrocyte 1. Takahashi, M., Y. Ishida, D. Iwaki, K. Kanno, T. Suzuki, Y. Endo, Y. Homma, and T. Fujita. 2010. Essential role of mannose-binding lectin-associated serine lysis other than by fD. Degn et al. also showed that samples that protease-1 in activation of the complement factor D. J. Exp. Med. 207: 29–37. have high concentrations of serum from MASP-1/-3–deficient 2. Degn, S. E., L. Jensen, A. G. Hansen, D. Duman, M. Tekin, J. C. Jensenius, and S. Thiel. 2012. Mannan-binding lectin-associated serine protease (MASP)-1 is mice still have the ability to activate alternative pathway (figure crucial for lectin pathway activation in human serum, whereas neither MASP-1 1A in their letter). However, it should be noted that MASP-1/- nor MASP-3 is required for alternative pathway function. J. Immunol. 189: 3957– 3969. 3–deficient serum had a lower ability to lyse rabbit erythrocytes 3. Takahashi, M., H. Sekine, Y. Endo, and T. Fujita. 2013. Comment on “Mannan- compared with MASP-1/-3–sufficient serum under a low se- binding lectin-associated serine protease (MASP)-1 is crucial for lectin pathway activation in human serum, whereas neither MASP-1 nor MASP-3 is required for rum concentration, suggesting that the total quantity of active alternative pathway function”. J. Immunol. 190: 2477. fD that is required for full alternative pathway function is not 4. Yamauchi, Y., J. W. Stevens, K. J. Macon, and J. E. Volanakis. 1994. Recombinant and equivalent between MASP-1/-3–deficient and MASP-1/-3– native zymogen forms of human complement factor D. J. Immunol. 152: 3645–3653. 5. Ruseva, M. M., M. Takahashi, T. Fujita, and M. C. Pickering. 2014. C3 dysregulation sufficient serum. MASP-1–deficient human serum also had lower due to factor H deficiency is mannan-binding lectin-associated serine proteases 1 2 1 1 hemolytic ability compared with MASP1 / and MASP1 / (MASP)-1 and MASP-3 independent in vivo. Clin. Exp. Immunol. 176: 84–92. 6. Van Dijk, H., P. M. Rademaker, J. P. Klerx, and J. M. Willers. 1985. Study of the human serum under low serum concentrations (figure 2A in optimal reaction conditions for assay of the mouse alternative complement Ref. 2). However, these important findings were not discussed pathway. J. Immunol. Methods 85: 233–243. in the reports from Degn and colleagues. www.jimmunol.org/cgi/doi/10.4049/jimmunol.1400766