Chimeric Proteins Containing MAP-1 and Functional Domains of C4b-Binding Protein Reveal Strong Complement Inhibitory Capacities

Abstract

The complement system is a tightly regulated network of proteins involved in defense against pathogens, inflammatory processes, and coordination of the innate and adaptive immune responses. Dysregulation of the complement cascade is associated with many inflammatory disorders. Thus, inhibition of the complement system has emerged as an option for treatment of a range of different inflammatory diseases. MAP-1 is a pattern recognition molecule (PRM)-associated inhibitor of the lectin pathway of the complement system, whereas C4b-binding protein (C4BP) regulates both the classical and lectin pathways. In this study we generated chimeric proteins consisting of MAP-1 and the first five domains of human C4BP (C4BP1−5) in order to develop a targeted inhibitor acting at different levels of the complement cascade. Two different constructs were designed and expressed in CHO cells where MAP-1 was fused with C4BP1−5 in either the C- or N-terminus. The functionality of the chimeric proteins was assessed using different in vitro complement activation assays. Both chimeric proteins displayed the characteristic Ca2+-dependent dimerization and binding to PRMs of native MAP-1, as well as the co-factor activity of native C4BP. In ELISA-based complement activation assays they could effectively inhibit the lectin and classical pathways. Notably, MAP-1:C4BP1−5 was five times more effective than rMAP-1 and rC4BP1−5 applied at the same time, emphasizing the advantage of a single inhibitor containing both functional domains. The MAP-1/C4BP chimeras exert unique complement inhibitory properties and represent a novel therapeutic approach targeting both upstream and central complement activation.

Combining MAP‐1:CD35 or MAP‐1:CD55 fusion proteins with pattern‐recognition molecules as novel targeted modulators of the complement cascade

“…It has been reported that MAP‐1 forms calcium‐dependent homodimers (13) and that this characteristic can be employed to dimerize MAP‐1–containing engineered complement inhibitors (25, 26). To ensure that the addition of CD35 and CD55 functional domains in the C terminus of MAP‐1 did not hinder its ability to dimerize, we subjected MAP‐1:CD35 1–3 and MAP‐1:CD55 1–4 to SEC under physiologic concentrations of calcium and in the presence of the calcium chelator EDTA.…”

“…We tested the in vitro inhibitory activity of the chimeric proteins in the LP using an established ELISA‐based setup (26). We incubated the chimeric proteins on an MBL‐mannan‐coated plate before adding MBL‐defect serum as complement source.…”

“…MAP‐1–containing proteins were purified by affinity chromatography using the mAb anti–MAP‐1, clone 20C4 (12), as previously described (25, 26). Briefly, PBS was used as a binding buffer, and a solution of 0.25% (v/v) citric acid (pH 2.5) was used as elution buffer.…”

“…MAP‐1:CD35 1–3 and MAP‐1:CD55 1–4 cofactor activity for the FI‐mediated proteolytic degradation of C4b and C3b was determined as previously described by Hertz et al . (26) with minor adaptations. Briefly, 200 nM C3b or C4b was incubated alone or in combination with 100 nM FI and 400 nM factor H (FH), C4BP (all from Complement Technology, Tyler, TX, USA), rMAP‐1 (in house), full‐length rCD35 (5748‐CD050; R&D Systems), full‐length rCD55 (ab168708; Abcam, Cambridge, United Kingdom), MAP‐1:CD35 1–3 , or MAP‐1:CD55 1–4 for 2 h at 37°.…”

“…MAP-1:CD35 123 and MAP-1:CD55 124 dimerize and bind to the LP PRMs in a calcium-dependent manner Previous studies have shown that MAP-1 (13) and MAP-1-containing chimeric proteins (25,26) form homodimers in the presence of physiologic concentrations of calcium via the interaction of CUB 1-EGF domains (37). We confirmed that the fusion proteins retain the ability of MAP-1 to homodimerize in a calcium-dependent fashion (Fig.…”