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Activity Stability, Binding to MBL, and Enzymatic Polymorphisms in Mannan-Binding Lectin (MBL)-Associated Serine Protease 2 Affect Stability, Binding to MBL, and Enzymatic Activity This information is current as of September 26, 2021. Steffen Thiel, Martin Kolev, Søren Degn, Rudi Steffensen, Annette G. Hansen, Marieta Ruseva and Jens C. Jensenius J Immunol 2009; 182:2939-2947; ; doi: 10.4049/jimmunol.0802053 http://www.jimmunol.org/content/182/5/2939 Downloaded from References This article cites 31 articles, 14 of which you can access for free at: http://www.jimmunol.org/content/182/5/2939.full#ref-list-1 http://www.jimmunol.org/ Why The JI? Submit online. • 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 by guest on September 26, 2021 *average 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 © 2009 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology Polymorphisms in Mannan-Binding Lectin (MBL)-Associated Serine Protease 2 Affect Stability, Binding to MBL, and Enzymatic Activity1 Steffen Thiel,2* Martin Kolev,* Søren Degn,* Rudi Steffensen,† Annette G. Hansen,* Marieta Ruseva,* and Jens C. Jensenius* Mannan-binding lectin-associated serine protease 2 (MASP-2) is an enzyme of the innate immune system. MASP-2 forms com- plexes with the pattern recognition molecules mannan-binding lectin (MBL), H-ficolin, L-ficolin, or M-ficolin, and is activated when one of these proteins recognizes microorganisms and subsequently cleaves complement factors C4 and C2, thus initiating the activation of the complement system. Missense polymorphisms of MASP-2 exist in different ethnic populations. To further char- acterize the nature of these, we have produced and characterized rMASP-2s representing the following naturally occurring Downloaded from polymorphisms: R99Q, D120G, P126L, H155R, 156_159dupCHNH (CHNHdup), V377A, and R439H. Only very low levels of CHNHdup were secreted from the cells, whereas quantities similar to wild-type MASP-2 were found intracellularly, indicating that this mutation results in a misfolded protein. We found that D120G and CHNHdup could not associate with MBL, whereas R99Q, P126L, H155R, V377A, R439H, and wild-type MASP-2 bound equally well to MBL. Accordingly, when D120G and CHNHdup were mixed with MBL, no activation of complement factor C4 was observed, whereas R99Q, P126L, and V377A cleaved C4 with an activity comparable to wild-type MASP-2 and H155R slightly better. In contrast, the R439H variant was http://www.jimmunol.org/ deficient in this process despite its normal binding to MBL. This variant was also not able to autoactivate in the presence of MBL and mannan. We find the R439H variant is common in Sub-Saharan Africans with a gene frequency of 10%. Our results indicate that individuals with different types of MASP-2 defects may be identified through genotyping. The Journal of Immunology, 2009, 182: 2939–2947. nderstanding the workings of the complement system is pathway are all found in complexes with the same set of proen- important for unraveling the innate immune defense and zymes, the MBL-associated serine proteases (MASPs): MASP-1, its interactions with the adaptive immune response (1, MASP-2, and MASP-3, as well as MBL-associated protein of 19 U by guest on September 26, 2021 2). The complement system includes more than 30 soluble and kDa, MAp19 (3–5). MASP-1, MASP-2, and MASP-3 all present membrane-bound proteins. A number of these proteins are serine domain structures identical with those of the C1 proteases, C1r proteases found as zymogens in the circulation. To activate these and C1s, and all five proenzymes are activated through the proenzymes, three pathways have evolved over time, as follows: 1) cleavage of the polypeptide chain between the A segment and the classical pathway is activated when the C1 complex recognizes the B segment, with the latter presenting the protease domain patterns of Fc regions from Ig molecules; 2) the lectin pathway is (exemplified by the structure of MASP-2 in Fig. 1). MAp19 3 activated when mannan-binding lectin (MBL) or one of the three presents the two N-terminal domains (of six domains) of ficolins (H-ficolin, L-ficolin, or M-ficolin) recognizes a pattern of MASP-2, and in addition, four unique amino acids, and is de- carbohydrates or acetylated molecules; and 3) the alternative path- void of enzymatic activity (6). When MBL or ficolin in complex way is activated when the balance between inhibition and activa- with MASP recognizes a ligand, the MASPs are activated. The tion of C3 activation is shifted and is also important for amplifying physiological relevant substrates for activated MASP-1 and the first two pathways. The recognition molecules of the lectin MASP-3 are still under debate, although MASP-1 has been shown to cleave C2 and to possess some thrombin-like activity (7, 8). The activity of MASP-2, in contrast, is clearer. Activated *Department of Medical Microbiology and Immunology, University of Aarhus, Aar- MASP-2 very efficiently cleaves the complement factors C4 and hus, Denmark; and †Regional Centre for Blood Transfusion and Clinical Immunol- ogy, Aalborg Hospital, Aalborg, Denmark C2 to the fragments C4b and C4a, and C2b and C2a, respec- Received for publication June 25, 2008. Accepted for publication December 17, 2008. tively, and C4b and C2b join to form a C3 convertase (9, 10). MASP-2 is thus a central molecule in the activation of the com- The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance plement system as it translates the recognition of microorgan- with 18 U.S.C. Section 1734 solely to indicate this fact. isms (or altered self) by four pattern recognition molecules, i.e., 1 This work was partly supported by the Danish Research Council and Novo Nordic MBL, H-ficolin, L-ficolin, and M-ficolin, into initiation of the Foundation. enzymatic cascades of the complement system. 2 Address correspondence and reprint requests to Dr. Steffen Thiel, Department of Medical Microbiology and Immunology, University of Aarhus, Aarhus, Denmark. Uncovering mutations resulting in selective deficiencies in the E-mail address: [email protected] immune system may not only further our understanding of basic 3 Abbreviations used in this paper: MBL, mannan-binding lectin; EGF, epidermal mechanisms, but may also have significant clinical implications. growth factor; MASP, MBL-associated serine protease; wt, wild type; SNP, single We and others have defined a number of nonsynonymous poly- nucleotide polymorphism. morphisms in the gene encoding MASP-2. We have previously Copyright © 2009 by The American Association of Immunologists, Inc. 0022-1767/09/$2.00 identified and characterized the distribution of naturally occurring www.jimmunol.org/cgi/doi/10.4049/jimmunol.0802053 2940 ACTIVITIES OF MASP-2 VARIANTS Table I. The primers used for site-directed mutagenesis Mutants Primer R99Qa Forward: 5Ј-GCACAGACACGGAGCAGGCCCCTGGCAAGGAC-3Ј Reverse: 5Ј-GTCCTTGCCAGGGGCCTGCTCCGTGTCTGTGC-3Ј P126L Forward: 5Ј-GACTACTCCAACGAGAAGCTGTTCACGGGGTTCGAG-3Ј Reverse: 5Ј-CTCGAACCCCGTGAACAGCTTCTCGTTGGAGTAGTC-3Ј H155R Forward: 5Ј-CCCACCTGCGACCACCGCTGCCACAACCACCTG-3Ј Reverse: 5Ј-CAGGTGGTTGTGGCAGCGGTGGTCGCAGGTGGG-3Ј CHNHdup Forward: 5Ј-GAAACCGCCCAGGTGGTTGTGGCAGTGGTTGTGGCAG-3Ј Reverse: 5Ј-CTGCCACAACCACTGCCACAACCACCTGGGCGGTTTC-3Ј V377A Forward: 5Ј-CTACCCAGTGGCCGAGCGGAGTACATCACAGGTC-3Ј Reverse: 5Ј-GACCTGTGATGTACTCCGCTCGGCCACTGGGTAG-3Ј R439H Forward: 5Ј-CTACCCAGTGGCCGAGCGGAGTACATCACAGGTC-3Ј Reverse: 5Ј-GACCTGTGATGTACTCCGCTCGGCCACTGGGTAG-3Ј a Variant of rMASP-2. missense polymorphisms of MASP-2 in different ethnic popula- change R444Q (Fig. 1). This is a change at the P1 position of the cleavage tions, i.e., Caucasians, Hong Kong Chinese, Sub-Saharan Africans, between the A chain and the B chain. This mutant cannot be cleaved and Downloaded from South-African Indians, and Inuits, and have observed associations is thus not activated. with circulating levels of MASP-2 (11). In the present study, we Production of rMASP-2 characterize the functional effects of the resulting naturally occur- Plasmids containing the MASP-2 cDNA inserts were used for the trans- ring variants of the MASP-2 protein through the production of the fection of HEK293 cells (Freestyle 293-F cells; Invitrogen). Briefly, plas- recombinant forms of the different MASP-2 allotypes. Most have mids (1 ␮g/ml) were mixed with lipofectamine 2000 (Invitrogen) and Opti- no effect on the function of the MBL pathway of complement MEM (Invitrogen), according to the manufacturer’s instructions, and used 6 http://www.jimmunol.org/ activation, but one blocks, not the synthesis, but the secretion of for transfection of early passage 293-F cells (30 ml of 10 cells/ml). Cells were cultured for 4 days in Freestyle expression medium (Invitrogen), and the variant, another binds to MBL as potently as the wild type (wt), supernatants
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