Pilely K, Rosbjerg A, Genster N, Gal P, Pal G, Stahi GL, Mollnes TE, Espevik T, Garred P

Cholesterol crystals activate the lectin complement pathway via ficolin-2 and MBL - implications for the progression of atherosclerosis

JOURNAL OF IMMUNOLOGY 196:(1) p. 1. 1 p.

Pilely K, Rosbjerg A, Genster N, Gal P, Pal G, Halvorsen B, Holm S, Aukrust P, Bakke SS, Sporsheim B, Nervik I, Niyonzima N, Bartels ED, Stahl GL, Mollnes TE, Espevik T, Garred P

Cholesterol Crystals Activate the Lectin Complement Pathway via Ficolin-2 and Mannose-Binding Lectin: Implications for the Progression of Atherosclerosis.

JOURNAL OF IMMUNOLOGY 196:(12) pp. 5064-5074. (2016)

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

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.

JOURNAL OF IMMUNOLOGY 196:(2) pp. 857-865.

Kiss B, Kalmár L, Nyitray L, Pál G

Structural determinants governing S100A4-induced isoform-selective disassembly of non-muscle myosin II filaments.

FEBS JOURNAL 283: pp. 2164-2180.

Dobó József, Szakács Dávid, Oroszlán Gábor, Kortvely Elod, Kiss Bence, Boros Eszter, Szász Róbert, Závodszky Péter, Gál Péter, Pál Gábor

MASP-3 is the exclusive pro-factor D activator in resting blood: the lectin and the alternative complement pathways are fundamentally linked

SCIENTIFIC REPORTS 2016:(6) Paper 31877. 12 p.

MASP-3 was discovered 15 years ago as the third mannan-binding lectin (MBL)-associated serine protease of the complement lectin pathway. Lacking any verified substrate its role remained ambiguous. MASP-3 was shown to compete with a key lectin pathway enzyme MASP-2 for MBL binding, and was therefore considered to be a negative complement regulator. Later, knock-out mice experiments suggested that MASP-1 and/or MASP-3 play important roles in complement pro-factor D (pro-FD) maturation. However, studies on a MASP-1/MASP-3-deficient human patient produced contradicting results. In normal resting blood unperturbed by ongoing coagulation or complement activation, factor D is present predominantly in its active form, suggesting that resting blood contains at least one pro-FD activating proteinase that is not a direct initiator of coagulation or complement activation. We have recently showed that all three MASPs can activate pro-FD in vitro. In resting blood, however, using our previously evolved MASP-1 and MASP-2 inhibitors we proved that neither MASP-1 nor MASP-2 activates pro-FD. Other plasma proteinases, particularly MASP-3, remained candidates for that function. For this study we evolved a specific MASP-3 inhibitor and unambiguously proved that activated MASP-3 is the exclusive pro-FD activator in resting blood, which demonstrates a fundamental link between the lectin and alternative pathways.

The complement system1,2 is an integral part of the innate immune response, and it is responsible for the elimination of invading microorganisms and altered self-structures. It contributes to the immune homeostasis and through its classical pathway (CP), which is primarily triggered by antigen-antibody complexes, it is also linked to the adaptive immune system. Complexes of the lectin pathway (LP) are structurally similar to C13, the first component of the CP, and both pathways lead to formation of the same C3 convertase, C4b2a. The alternative pathway (AP), which can be activated on its own, serves as an amplification loop for all three pathways4. C3 convertases cleave C3, generating C3b, which then binds factor B (FB). The pro-convertase, C3bB, is cleaved by factor D (FD)5 yielding the AP C3 convertase, C3bBb, which in turn generates even more C3b molecules. C3b carries a newly exposed thioester bond and can covalently attach to the activating surface, where it serves as an opsonin. Further activation of the complement cascade generates a membrane attack complex resulting in the lysis of certain pathogens.

The LP is composed of a heterogeneous mixture of complexes, each containing one pattern recognition molecule (PRM) and one or two dimers of associated serine proteases and other related non-enzymatic proteins6,7. At least five different LP PRMs have been described: mannose-binding lectin (MBL), three ficolins, and collectin-LK3,8. The LP is triggered when the PRMs bind to surfaces displaying carbohydrate arrays or certain acetylated compounds. Two MBL-associated serine proteases, MASP-1 and MASP-2, elicit the enzymatic signal upon LP activation. Both are essential: MASP-1 autoactivates first, then it activates MASP-29,10,11,12, and while both enzymes cleave C2, only MASP-2 cleaves C4. The roles of the third associated protease, MASP-3, and the other associated proteins, MAp19 (aka sMAP, MAP-2) and MAp44 (aka MAP-1), have been unclear, but initially all three were considered as negative LP regulators13,14,15. Mutations of the MASP1 gene that specifically affect MASP-3 function16,17 cause developmental abnormalities (called the 3MC syndrome) suggesting that MASP-3 has an important physiological role, which might even be unrelated to the complement system.

For a long time, complement FD was presumed to be intracellularly activated at the site of its synthesis18. This assumption was based on observations that only active FD was purified from normal blood19,20, and predominantly active FD was detected in mammalian cell cultures21,22. However, in MASP1 knock-out mice lacking both MASP-1 and MASP-3 no AP activity was observed, and proenzyme FD (pro-FD) was detected in the serum23. Both MASP-123 and later MASP-3 were implicated as pro-FD activators, and even proenzyme MASP-3 was considered to play this role24. In marked contrast, AP activity was detected in the serum of a MASP-1/3 deficient 3MC patient12, questioning the involvement of any of these enzymes in pro-FD activation in humans. The complexity of the problem increased when the sera of MASP-1 and −3 deficient 3MC patients were shown to contain only (or predominantly) pro-FD25. The mechanism of AP activity in these patients has remained unresolved26.

To clarify the above controversy we initiated a comprehensive study, and in a recent paper presented our first findings as follows: i) normal resting human blood has a pro-FD activating capacity; ii) none of the MASP proenzymes can activate pro-FD, but iii) all three activated MASPs are able to activate pro-FD in vitro; and iv) pro-FD activating capacity of resting human blood is resistant to selective MASP-1 and MASP-2 inhibition. Based on these we concluded that out of the three MASPs, only MASP-3 is a plausible pro-FD activator in normal resting human blood27. Nevertheless, lacking a specific MASP-3 inhibitor we could not directly prove this potential MASP-3 function or exclude the possibility that many different pro-FD activating proteases act simultaneously in resting human blood.

To quantitatively determine the contribution of MASP-3 to pro-FD activation in resting blood, we developed a specific, high-affinity MASP-3 inhibitor via directed protein evolution using phage display. With our monospecific inhibitor we unequivocally identified an essential physiological role of MASP-3 in normal human resting blood.

Dobó József, Pál Gábor, Cervenak László, Gál Péter

The emerging roles of mannose-binding lectin-associated serine proteases (MASPs) in the lectin pathway of complement and beyond


Lin Y, Kardos J, Imai M, Ikenoue T, Kinoshita M, Sugiki T, Ishimori K, Goto Y, Lee YH.

Amorphous aggregation of cytochrome c with inherently low amyloidogenicity is characterized by the metastability of supersaturation and the phase diagram.

Langmuir. 2016 Jan 29.

Despite extensive studies on the folding and function of cytochrome c, the mechanisms underlying its aggregation remain largely unknown. We herein examined the aggregation behavior of the physiologically relevant two types of cytochrome c, metal-bound cytochrome c, and its fragment with high amyloidogenicity as predicted in alcohol/water mixtures. Although the aggregation propensity of holo cytochrome c was low due to high solubility, markedly unfolded apo cytochrome c, lacking the heme prosthetic group, strongly promoted the propensity for amorphous aggregation with increases in hydrophobicity. Silver-bound apo cytochrome c increased the capacity of fibrillar aggregation (i.e., protofibrils or immature fibrils) due to subtle structural changes of apo cytochrome c by strong binding of silver. However, mature amyloid fibrils were not detected for any of the cytochrome c variants or its fragment, even with extensive ultrasonication, which is a powerful amyloid inducer. These results revealed the intrinsically low amyloidogenicity of cytochrome c, which is beneficial for its homeostasis and function by facilitating the folding and minimizing irreversible amyloid formation. We propose that intrinsically low amyloidogenicity of cytochrome c is attributed to the low metastability of supersaturation. The phase diagram constructed based on solubility and aggregate type is useful for a comprehensive understanding of protein aggregation. Furthermore, amorphous aggregation, which is also viewed as a generic property of proteins, and amyloid fibrillation can be distinguished from each other by the metastability of supersaturation.