title: Recombinant expression of the autocatalytic complement protease MASP-1 is crucially dependent on co-expression with its inhibitor, C1 inhibitor.
abstract: MASP-1 is a protease of the lectin pathway of complement. It is homologous with MASP-2, previously thought both necessary and sufficient for lectin pathway activation. Recently MASP-1 has taken centre stage with the observation that it is crucial to the activation of MASP-2 and thus central to complement activation. Numerous additional functions have been suggested for MASP-1 and its importance is obvious. Yet, thorough analyses of proteolytic activities and physiological roles in the human scenario have been hampered by difficulties in purifying or producing full-length human MASP-1. We present the successful expression of full-length recombinant human MASP-1 entirely in the zymogen form in a mammalian expression system. We found that the catalytic activity of MASP-1 suppresses its expression through rapid auto-activation and auto-degradation. This auto-degradation was not inhibited by the addition of inhibitors to the culture medium, and it was subsequently found to occur intracellularly. Numerous mutations aimed at attenuating auto-activation or preventing auto-degradation failed to rescue expression, as did also attempts at stabilizing the protease by co-expression with MBL or ficolins or expression in hepatocyte cell lines, representing the natural site of synthesis. The active protease was finally produced through co-expression with the serine protease inhibitor C1 inhibitor. We demonstrate that the expressed protease is capable of binding MBL and auto-activating, and is catalytically active. We have generalized the concept to the expression also of MASP-2 entirely in its zymogen form and with improved yields. We suggest a general advantage of expressing aggressive, autocatalytic proteases with their cognate inhibitors.
authors: Department of Biomedicine, Aarhus University, DK-8000 Aarhus C, Denmark. sdegn@microbiology.au.dk
authors: Søren E
authors: SE
authors: Degn
authors: Steffen
authors: S
authors: Thiel
authors: Jens C
authors: JC
authors: Jensenius
mesh_terms: D000818:Animals; D002460:Cell Line; D003001:Cloning, Molecular / Q000379:methods; D003174:Complement C1 Inactivator Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D004792:Enzyme Precursors / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D015870:Gene Expression; D005822:Genetic Vectors / Q000235:genetics; D056945:Hep G2 Cells; D006801:Humans; D037102:Lectins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D037601:Mannose-Binding Lectin / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D050606:Mannose-Binding Protein-Associated Serine Proteases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D000097604:Ficolins
chemical_list: D003174:Complement C1 Inactivator Proteins; D004792:Enzyme Precursors; D037102:Lectins; D037601:Mannose-Binding Lectin; D050606:Mannose-Binding Protein-Associated Serine Proteases; D011994:Recombinant Proteins; D000097604:Ficolins; C084067:MASP1 protein, human; C105296:MASP2 protein, human