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PMID:12538697

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Citation

Ambrus, G, Gál, P, Kojima, M, Szilágyi, K, Balczer, J, Antal, J, Gráf, L, Laich, A, Moffatt, BE, Schwaeble, W, Sim, RB and Závodszky, P (2003) Natural substrates and inhibitors of mannan-binding lectin-associated serine protease-1 and -2: a study on recombinant catalytic fragments. J. Immunol. 170:1374-82

Abstract

Mannan-binding lectin-associated serine protease (SP) (MASP)-1 and MASP-2 are modular SP and form complexes with mannan-binding lectin, the recognition molecule of the lectin pathway of the complement system. To characterize the enzymatic properties of these proteases we expressed their catalytic region, the C-terminal three domains, in Escherichia coli. Both enzymes autoactivated and cleaved synthetic oligopeptide substrates. In a competing oligopeptide substrate library assay, MASP-1 showed extreme Arg selectivity, whereas MASP-2 exhibited a less restricted, trypsin-like specificity. The enzymatic assays with complement components showed that cleavage of intact C3 by MASP-1 and MASP-2 was detectable, but was only approximately 0.1% of the previously reported efficiency of C3bBb, the alternative pathway C3-convertase. Both enzymes cleaved C3i 10- to 20-fold faster, but still at only approximately 1% of the efficiency of MASP-2 cleavage of C2. We believe that C3 is not the natural substrate of either enzyme. MASP-2 cleaved C2 and C4 at high rates. To determine the role of the individual domains in the catalytic region of MASP-2, the second complement control protein module together with the SP module and the SP module were also expressed and characterized. We demonstrated that the SP domain alone can autoactivate and cleave C2 as efficiently as the entire catalytic region, while the second complement control protein module is necessary for efficient C4 cleavage. This behavior strongly resembles C1s. Each MASP-1 and MASP-2 fragment reacted with C1-inhibitor, which completely blocked the enzymatic action of the enzymes. Nevertheless, relative rates of reaction with alpha-2-macroglobulin and C1-inhibitor suggest that alpha-2-macroglobulin may be a significant physiological inhibitor of MASP-1.

Links

PubMed

Keywords

Binding, Competitive/immunology; Catalytic Domain/immunology; Complement C1 Inactivator Proteins/metabolism; Complement C3/metabolism; Humans; Hydrolysis; Mannose-Binding Lectin/antagonists & inhibitors; Mannose-Binding Lectin/metabolism; Mannose-Binding Protein-Associated Serine Proteases; Oligopeptides/metabolism; Peptide Fragments/biosynthesis; Peptide Fragments/genetics; Peptide Fragments/isolation & purification; Peptide Fragments/metabolism; Recombinant Proteins/biosynthesis; Recombinant Proteins/genetics; Recombinant Proteins/isolation & purification; Recombinant Proteins/metabolism; Serine Endopeptidases/genetics; Serine Endopeptidases/metabolism; Serine Proteinase Inhibitors/metabolism; Substrate Specificity/immunology; alpha-Macroglobulins/metabolism

Significance

Annotations

Gene product Qualifier GO Term Evidence Code with/from Aspect Extension Notes Status

HUMAN:MASP2

enables

GO:0008233: peptidase activity

ECO:0000314: direct assay evidence used in manual assertion

F

Seeded From UniProt

complete

HUMAN:A2MG

involved_in

GO:0001869: negative regulation of complement activation, lectin pathway

ECO:0000314: direct assay evidence used in manual assertion

P

Seeded From UniProt

complete

HUMAN:A2MG

enables

GO:0004867: serine-type endopeptidase inhibitor activity

ECO:0000314: direct assay evidence used in manual assertion

F

Seeded From UniProt

complete

HUMAN:MASP1

enables

GO:0008233: peptidase activity

ECO:0000314: direct assay evidence used in manual assertion

F

Seeded From UniProt

complete


See also

References

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