Serum concentration and interaction properties of MBL/ficolin associated protein-1

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The activity of the complement system is tightly controlled by many fluid-phase and tissue-bound regulators. Mannose-binding lectin (MBL)/ficolin/collectin-11–associated protein-1 (MAP-1) is a recently discovered plasma protein that acts as an upstream inhibitor of the lectin complement pathway (LCP). It has previously been shown that MAP-1 can compete with the MBL/ficolin/collectin-11–associated serine proteases (MASPs) in binding to MBL and the ficolins. However, this mechanism may only partly explain the inhibitory complement effect of MAP-1. We hypothesized that MAP-1 is also involved in heterocomplex formation with the MASPs thereby breaking the stoichiometry of the activation complexes of the LCP, which could represent an alternative mechanism of MAP-1–mediated complement inhibition. We assessed the heterocomplex formation with ELISA, size-exclusion chromatography, and immunoblotting using both recombinant proteins and serum/plasma. We found that rMAP-1 can engage in heterocomplexes with rMASP-1 and rMASP-3 in a calcium-dependent manner. Moreover, we discovered that rMASP-1 and rMASP-3 also form heterocomplexes under these conditions. Complexes containing both MAP-1 and MASP-1 or -3 were detected in normal human serum and plasma, and depletion of the LCP recognition molecules from ficolin-3–deficient human serum showed that free circulating heterocomplexes also exist in the blood, although the major part appears to be associated with the LCP recognition molecules. Altogether, these findings suggest that MASPs can associate in various combinations and bring new perspectives to the complexity of lectin pathway–driven complement activation.

Section: In Vivo Map-1 Heterocomplexes In Depleted Ficolin-3-deficiensupporting

confidence: 74%

supporting

confidence: 77%

Heterocomplex Formation between MBL/Ficolin/CL-11–Associated Serine Protease-1 and -3 and MBL/Ficolin/CL-11–Associated Protein-1

Rosbjerg

Munthe-Fog

Garred

et al. 2014

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“…3). Binding of MASP-1/-3/MAP-1 was detected using specific mouse mAb 8B3, recognizing the common H chain on all three molecules (40). A significant binding (p , 0.05) of native MASP-1/-3/ MAP-1 to CC was observed (Fig.…”

Section: Masp Binding and Ficolin-2-mediated C4 Depositionmentioning

confidence: 87%

“…The following Abs were used: FITC-conjugated polyclonal rabbit anti-rat Ab (F1763, Sigma-Aldrich), FITC-conjugated polyclonal goat anti-rabbit Ab (F1262, Sigma-Aldrich), FITC-conjugated polyclonal goat anti-mouse Ab (F0479, Dako), mouse IgG1k isotype control (BD Biosciences), mouse IgG2a isotype control (BD Biosciences), rabbit IgG isotype control (Invitrogen), rat IgG1k isotype control (BD Biosciences), mouse anti-human MBL mAb 131-10 (IgG1k) (BioPorto Diagnostics), mouse anti-human MBL 131-01 (IgG1k) (BioPorto Diagnostics), MBL inhibitory mAb mouse anti-human MBL 3F8 (IgG1) (38), MBL binding mAb mouse anti-human MBL 1C10 (IgG2) (39), rat antihuman MASP-2 mAb 8B5 (HM2190, Hycult Biotech), mouse anti-human complement component C5b-9 mAb (IgG2a) (011-01, Antibody Shop), polyclonal rabbit anti-human C4c Ab (Q0369, Dako), polyclonal rabbit antihuman C1q (A0136, Dako), rabbit anti-human IgM (0425, Dako), and rabbit anti-human IgG (0423, Dako). In-house-produced mAbs included: mouse anti-human ficolin-1 FCN106 (IgG1k) (15), mouse anti-human ficolin-2 FCN219 (IgG2a) (16), ficolin-2 inhibitory Ab mouse antihuman ficolin-2 FCN212 (IgG1k) (unpublished), mouse anti-human ficolin-3 FCN334 (IgG1k) (17), and mouse anti-human MASP-1/-3/ MAP-1 8B3 (IgG1k) (40,41).…”

Section: Reagentsmentioning

confidence: 99%

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

Pilely

Rosbjerg

Genster

et al. 2016

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Cholesterol crystals (CC) play an essential role in the formation of atherosclerotic plaques. CC activate the classical and the alternative complement pathways, but the role of the lectin pathway is unknown. We hypothesized that the pattern recognition molecules (PRMs) from the lectin pathway bind CC and function as an upstream innate inflammatory signal in the pathophysiology of atherosclerosis. We investigated the binding of the PRMs mannose-binding lectin (MBL), ficolin-1, ficolin-2, and ficolin-3, the associated serine proteases, and complement activation products to CC in vitro using recombinant proteins, specific inhibitors, as well as deficient and normal sera. Additionally, we examined the deposition of ficolin-2 and MBL in human carotid plaques by immunohistochemistry and fluorescence microscopy. The results showed that the lectin pathway was activated on CC by binding of ficolin-2 and MBL in vitro, resulting in activation and deposition of complement activation products. MBL bound to CC in a calcium-dependent manner whereas ficolin-2 binding was calcium-independent. No binding was observed for ficolin-1 or ficolin-3. MBL and ficolin-2 were present in human carotid plaques, and binding of MBL to CC was confirmed in vivo by immunohistochemistry, showing localization of MBL around CC clefts. Moreover, we demonstrated that IgM, but not IgG, bound to CC in vitro and that C1q binding was facilitated by IgM. In conclusion, our study demonstrates that PRMs from the lectin pathway recognize CC and provides evidence for an important role for this pathway in the inflammatory response induced by CC in the pathophysiology of atherosclerosis.

“…Recombinant human proteins MAP-1, MASP-1, MASP-2, MASP-3, MBL, and ficolin-3 were produced, as described previously (19)(20)(21), using the CHO DG44 expression system and serum-free medium (CHO-CD1; Lonza). Purified human plasma-derived C1-INH was purchased from CompTech (catalog no.…”

Section: Recombinant Proteins and Purified C1-inhmentioning

confidence: 99%

The Levels of the Lectin Pathway Serine Protease MASP-1 and Its Complex Formation with C1 Inhibitor Are Linked to the Severity of Hereditary Angioedema

Hansen

Csuka

Munthe-Fog

et al. 2015

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C1 inhibitor (C1-INH) is known to form complexes with the lectin complement pathway serine proteases MASP-1 and MASP-2. Deficiency of C1-INH is associated with hereditary angioedema (HAE), an autosomal inherited disease characterized by swelling attacks caused by elevated levels of bradykinin. MASP-1 was shown to cleave high m.w. kininogen into bradykinin; therefore, we hypothesized that MASP-1 levels and the quantity of MASP-1/C1-INH complexes might be associated with different paraclinical and clinical outcomes of HAE. We measured MASP-1 serum concentrations and endogenous MASP-1/C1-INH complex levels in 128 HAE patients and 100 controls. Relatively high levels of pre-existing MASP-1/C1-INH complexes were observed in normal serum, and we found that both the serum levels of MASP-1 and the complex formation between MASP-1 and C1-INH were significantly reduced in HAE patients compared with matched controls (p < 0.0001). The level of MASP-1 and MASP-1/C1-INH complexes in HE patients correlated with the level of C1-INH (p = 0.0009 and p = 0.0047, respectively), the level of C4 (p = 0.0084 and p < 0.0001, respectively), and the number of attacks in the year of blood sampling (p = 0.0075 and p = 0.0058, respectively). In conclusion, we show that MASP-1/C1-INH complexes circulate in normal human blood. The levels of MASP-1 and MASP-1/C1-INH complexes are reduced in HAE patients compared with controls. Both MASP-1 and MASP-1/C1-INH complexes are related to the degree of complement C4 consumption, as well as the severity of disease. These results suggest that MASP-1 may exert a previously unrecognized role in the pathophysiology of HAE.