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The x-ray crystal structure of mannose-binding lectin-associated serine proteinase-3 reveals the structural basis for enzyme inactivity associated with the Carnevale, Mingarelli, Malpuech, and Michels (3MC) syndrome.

ABSTRACT: The mannose-binding lectin associated-protease-3 (MASP-3) is a member of the lectin pathway of the complement system, a key component of human innate and active immunity. Mutations in MASP-3 have recently been found to be associated with Carnevale, Mingarelli, Malpuech, and Michels (3MC) syndrome, a severe developmental disorder manifested by cleft palate, intellectual disability, and skeletal abnormalities. However, the molecular basis for MASP-3 function remains to be understood. Here we characterize the substrate specificity of MASP-3 by screening against a combinatorial peptide substrate library. Through this approach, we successfully identified a peptide substrate that was 20-fold more efficiently cleaved than any other identified to date. Furthermore, we demonstrated that mutant forms of the enzyme associated with 3MC syndrome were completely inactive against this substrate. To address the structural basis for this defect, we determined the 2.6-? structure of the zymogen form of the G666E mutant of MASP-3. These data reveal that the mutation disrupts the active site and perturbs the position of the catalytic serine residue. Together, these insights into the function of MASP-3 reveal how a mutation in this enzyme causes it to be inactive and thus contribute to the 3MC syndrome.

SUBMITTER: Yongqing T

PROVIDER: S-EPMC3829330 | biostudies-literature | 2013 Aug

REPOSITORIES: biostudies-literature

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The x-ray crystal structure of mannose-binding lectin-associated serine proteinase-3 reveals the structural basis for enzyme inactivity associated with the Carnevale, Mingarelli, Malpuech, and Michels (3MC) syndrome.

Yongqing Tang T Wilmann Pascal G PG Reeve Shane B SB Coetzer Theresa H TH Smith A Ian AI Whisstock James C JC Pike Robert N RN Wijeyewickrema Lakshmi C LC

The Journal of biological chemistry 20130621 31

The mannose-binding lectin associated-protease-3 (MASP-3) is a member of the lectin pathway of the complement system, a key component of human innate and active immunity. Mutations in MASP-3 have recently been found to be associated with Carnevale, Mingarelli, Malpuech, and Michels (3MC) syndrome, a severe developmental disorder manifested by cleft palate, intellectual disability, and skeletal abnormalities. However, the molecular basis for MASP-3 function remains to be understood. Here we chara ...[more]

PMID: 23792966

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Project description:Serine proteases (SPs) are typically synthesized as precursors, termed proenzymes or zymogens, and the fully active form is produced via limited proteolysis by another protease or by autoactivation. The lectin pathway of the complement system is initiated by mannose-binding lectin (MBL)-associated SPs (MASP)-1, and MASP-2, which are known to be present as proenzymes in blood. The third SP of the lectin pathway, MASP-3, was recently shown to be the major activator, and the exclusive "resting blood" activator of profactor D, producing factor D, the initiator protease of the alternative pathway. Because only activated MASP-3 is capable of carrying out this cleavage, it was presumed that a significant fraction of MASP-3 must be present in the active form in resting blood. Here, we aimed to detect active MASP-3 in the blood by a more direct technique and to quantitate the active to zymogen ratio. First, MASPs were partially purified (enriched) from human plasma samples by affinity chromatography using immobilized MBL in the presence of inhibitors. Using this MASP pool, only the zymogen form of MASP-1 was detected by Western blot, whereas over 70% MASP-3 was in an activated form in the same samples. Furthermore, the active to zymogen ratio of MASP-3 showed little individual variation. It is enigmatic how MASP-3, which is not able to autoactivate, is present mostly as an active enzyme, whereas MASP-1, which has a potent autoactivation capability, is predominantly proenzymic in resting blood. In an attempt to explain this phenomenon, we modeled the basal level fluid-phase activation of lectin pathway proteases and their subsequent inactivation by C1 inhibitor and antithrombin using available and newly determined kinetic constants. The model can explain extensive MASP-3 activation only if we assume efficient intracomplex activation of MASP-3 by zymogen MASP-1. On the other hand, the model is in good agreement with the fact that MASP-1 and -2 are predominantly proenzymic and some of them is present in the form of inactive serpin-protease complexes. As an alternative hypothesis, MASP-3 activation by proprotein convertases is also discussed.

Dataset Information

The x-ray crystal structure of mannose-binding lectin-associated serine proteinase-3 reveals the structural basis for enzyme inactivity associated with the Carnevale, Mingarelli, Malpuech, and Michels (3MC) syndrome.

Publications

The x-ray crystal structure of mannose-binding lectin-associated serine proteinase-3 reveals the structural basis for enzyme inactivity associated with the Carnevale, Mingarelli, Malpuech, and Michels (3MC) syndrome.

Similar Datasets

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