Recombinant expression of the autocatalytic complement protease MASP-1 is crucially dependent on co-expression with its inhibitor, C1 inhibitor

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.

Graphical abstractFigure optionsDownload full-size imageDownload as PowerPoint slideHighlights► MASP-1 is an aggressive autocatalytic protease prone to auto-degradation. ► Auto-degradation occurs both intracellularly and extracellularly. ► Co-expression with MBL or ficolins does not attenuate auto-degradation. ► Co-expression with C1 inhibitor allows expression of the zymogen form of MASP-1. ► Zymogen MASP-2 can similarly be expressed by co-expression with C1 inhibitor.