Identification, affinity characterisation and biological interactions of lectin‐like peptide–carbohydrate complexes derived from human TNF‐α using high‐resolution mass spectrometry

Abstract

A cyclic disulfide heptadecapeptide (TIP17ox; 2) derived from the lectin-like 17-amino acid domain of human tumor necrosis factor-alpha [TNF-alpha (100-116)] was synthesised and demonstrated to bind specifically to N,N-diacetylchitobiose, a disaccharide present in many glycan structures of glycoproteins. Although the TIP domain forms a loop structure in the native TNF-alpha protein, we show in this study by high-resolution ESI-FTICR mass spectrometry that a homologous linear heptadecapeptide (TIP17rd; 1) binds with comparable affinity to chitobiose, suggesting that cyclisation is not essential for carbohydrate binding. ESI-FTICR-MS was used as an efficient tool for the direct molecular characterisation of TIP peptide-carbohydrate complexes. The specific binding of the TNF-TIP domain to chitobiose and other carbohydrate motifs in glycoproteins may explain the high proteolytic stability of these peptides in biological fluids. A considerably higher proteolytic stability in human plasma was found by mass spectrometric analysis for the cyclic TIP peptide 2, compared to the linear peptide 1. Furthermore, affinity-proteomics studies using immobilised cyclic TIP peptide 2 provided the identification of specific interacting glycoproteins in plasma.

A Computational Study of the Oligosaccharide Binding Sites in the Lectin-Like Domain of Tumor Necrosis Factor and the TNF-derived TIP Peptide

“…After energy minimization and MD equilibration with YASARA, the folding of the TIP_A peptide resembled most the crystal structure of native TNF, demonstrating its structural stability and was therefore selected as the only model for the further study. Even though TNF represents the trimer of the lectin-like domains (or TIP peptides), no trimer formation of TIP peptides was reported [23], leading to the conclusion that single TIP peptide molecules are able to mediate the lectin-like activity of TNF [4, 19]. Our docking results obtained with the TNF trimer have identified a unique binding site for chitobiose and trimannose ligands.…”

“…A reported mass spectroscopic study of the interaction between the TIP peptide and oligosaccharides has shown that cellobiose binds with a 20-fold lower affinity to the TIP peptide than does chitobiose [23]. This disagreement between the experimental and our computational methods could be due to the fact that 100 ns of MD simulation was not long enough to observe dissociation of cellobiose.…”

“…Since no experimentally determined 3D structure of the TIP peptide is available, we have made use of a model structure of the TIP peptide, based on the crystal structure of TNF [22]. Molecular docking and molecular dynamics (MD) simulations, which allow the identification of specific binding sites and key elements involved in the interactions, revealed that cellobiose is theoretically unable to form stable complexes with TNF and the TIP peptide, as was indeed confirmed by experimental results [3, 4, 19, 23]. In general, the results of this study can thus improve our understanding of the lectin-like interactions between a cytokine domain or its peptide mimic and specific oligosaccharides.…”

Mass spectrometry of peptides and proteins from human blood

“…A lectin-like 17-amino acid domain of human tumor necrosis factor-alpha was demonstrated to bind specifically to N,N-diacetylchitobiose, a disaccharide present in many glycan structures of glycoproteins (Marquardt et al, 2007). A specific serum alpha-N-acetylgalactosaminidase activity and its target's glycosylation levels were observed to vary significantly between stages of melanoma ).…”

The Lectin-like Domain of TNF Increases ENaC Open Probability through a Novel Site at the Interface between the Second Transmembrane and C-terminal Domains of the α-Subunit

“…This suggests that glycosylated residues in the extracellular loop of ENaC-␣ are crucial for activation of the channel by TNF. However, a mutant TIP peptide in which three residues (one Thr and two Glu) crucial for ENaC activation were changed to Ala still efficiently associates with oligosaccharides to which the lectin-like domain of TNF binds, such as N,NЈ-diacetylchitobiose (39), which argues against the hypothesis that the glycosylation sites in the extracellular loop are sufficient for channel activation. Moreover, the TIP peptide associates with an as yet unidentified site proximal to the C-terminal domain of recombinant, and thus non-glycosylated, ENaC-␣ (29).…”