A Keratin Peptide Inhibits Mannose-Binding Lectin

Abstract

Complement plays a significant role in mediating endothelial injury following oxidative stress. We have previously demonstrated that the lectin complement pathway (LCP), which is initiated by deposition of the mannose-binding lectin (MBL), is largely responsible for activating complement on endothelial cells following periods of oxidative stress. Identifying functional inhibitors that block MBL binding will be useful in characterizing the role of the LCP in disease models. The human cytokeratin peptide SFGSGFGGGY has been identified as a molecular mimic of N-acetyl-d-glucosamine (GlcNAc), a known ligand of MBL. Thus, we hypothesized that this peptide would specifically bind to MBL and functionally inhibit the LCP on endothelial cells following oxidative stress. Using a BIAcore 3000 optical biosensor, competition experiments were performed to demonstrate that the peptide SFGSGFGGGY inhibits binding of purified recombinant human MBL to GlcNAc in a concentration-dependent manner. Solution affinity data generated by BIAcore indicate this peptide binds to MBL with an affinity (KD) of 5 × 10−5 mol/L. Pretreatment of human serum (30%) with the GlcNAc-mimicking peptide (10–50 μg/ml) significantly attenuated MBL and C3 deposition on human endothelial cells subjected to oxidative stress in a dose-dependent manner, as demonstrated by cell surface ELISA and confocal microscopy. Additionally, this decapeptide sequence attenuated complement-dependent VCAM-1 expression following oxidative stress. These data indicate that a short peptide sequence that mimics GlcNAc can specifically bind to MBL and functionally inhibit the proinflammatory action of the LCP on oxidatively stressed endothelial cells.

Selective Inhibition of the Lectin Pathway of Complement with Phage Display Selected Peptides against Mannose-Binding Lectin-Associated Serine Protease (MASP)-1 and -2: Significant Contribution of MASP-1 to Lectin Pathway Activation

“…The 100-fold difference in the IC 50 values convincingly demonstrates that MASP-1 has a significant contribution to the activation of MASP-2. Although MASP-2 alone can autoactivate and initiate complement activation (9,39), the presence of MASP-1 appears to facilitate this process.…”

“…First, we measured the inhibition of activated MASP-2 by adding purified C4 to the immobilized, mannan-activated MBL-MASP complexes. In this assay format, the trend of the IC 50 values mirror those of the K I data: the MASP-2-selective SFMI-2 is 10-fold more effective in this assay than SFMI-1, which compared with SFMI-2, inhibits MASP-2 six times weaker (Fig. 4A, 4B).…”

“…Specific inhibitors are extremely useful tools for basic research and therapeutic purposes. Previously, there were attempts to develop pathway-selective inhibitors by preventing the binding of the recognition molecules (C1q and MBL) to their targets (50,51). Each activation pathway is associated with unique proteases, which could be appropriate targets for such inhibitors.…”

The Role of Nanometer-Scaled Ligand Patterns in Polyvalent Binding by Large Mannan-Binding Lectin Oligomers

“…They have been used to measure the binding between MBL and ligands (18,24,25), as well the complex formation between MBL and the MBL-associated serine proteases (MASPs) (26). The analysis of the impact of higherorder oligomerization of MBL 3 failed to reveal any functional difference between the oligomers, with only an ∼2-fold difference in binding kinetics and equilibrium constants reported for human 33MBL 3 and 43MBL 3 (18).…”

“…Establishing an SPR spectroscopy-based assay for quantifying the binding between MBL oligomers and ligands SPR spectroscopy has been used for studies of the interaction between MBL and ligands (18,24). Based on the observation made earlier that mcBSA supports stronger MBL binding than does Nacetyl glucosamine-conjugated BSA, we prepared SPR chip surfaces with mcBSA.…”