MASP-1 and MASP-2 do not activate pro-factor D in resting human blood, whereas MASP-3 is a potential activator: Kinetic analysis involving specific MASP-1 and MASP-2 inhibitors

Gábor Oroszlán, Elod Kortvely, Dávid Szakács, Andrea Kocsis, Sascha Dammeier, Anne Zeck, Marius Ueffing, Péter Závodszky, Gábor Pál, Péter Gál, József Dobo

Research output: Contribution to journalArticle

23 Citations (Scopus)

Abstract

It had been thought that complement factor D (FD) is activated at the site of synthesis, and only FD lacking a propeptide is present in blood. The serum of mannose-binding lectin-associated serine protease (MASP)-1/3(2/2) mice contains pro-FD and has markedly reduced alternative pathway activity. It was suggested that MASP-1 and MASP-3 directly activate pro-FD; however, other experiments contradicted this view. We decided to clarify the involvement of MASPs in pro-FD activation in normal, as opposed to deficient, human plasma and serum. Human pro-FD containing an APPRGR propeptide was produced in insect cells. We measured its activation kinetics using purified active MASP-1, MASP-2, MASP-3, as well as thrombin.We found all these enzymes to be efficient activators, whereas MASP proenzymes lacked such activity. Pro-FD cleavage in serum or plasma was quantified by a novel assay using fluorescently labeled pro-FD. Labeled pro-FD was processed with t1/2s of ∼3 and 5 h in serum and plasma, respectively, showing that proteolytic activity capable of activating pro-FD exists in blood even in the absence of active coagulation enzymes. Our previously developed selective MASP-1 and MASP-2 inhibitors did not reduce pro-FD activation at reasonable concentration. In contrast, at very high concentration, the MASP-2 inhibitor, which is also a poor MASP-3 inhibitor, slowed down the activation. When recombinant MASPs were added to plasma, only MASP-3 could reduce the half-life of pro-FD. Combining our quantitative data, MASP-1 and MASP-2 can be ruled out as direct pro-FD activators in resting blood; however, active MASP-3 is a very likely physiological activator.

Original languageEnglish
Pages (from-to)857-865
Number of pages9
JournalJournal of Immunology
Volume196
Issue number2
DOIs
Publication statusPublished - Jan 15 2016

ASJC Scopus subject areas

  • Immunology and Allergy
  • Immunology

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