title: Identification, affinity characterisation and biological interactions of lectin-like peptide-carbohydrate complexes derived from human TNF-alpha 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.
authors.affiliation: Department of Chemistry, Laboratory of Analytical Chemistry and Biopolymer Structure Analysis, University of Konstanz, 78457 Konstanz, Germany.
authors.forename: Andreas
authors.initials: A
authors.lastname: Marquardt
authors.forename: Bogdan
authors.initials: B
authors.lastname: Bernevic
authors.forename: Michael
authors.initials: M
authors.lastname: Przybylski
mesh_terms: D000595:Amino Acid Sequence; D001667:Binding, Competitive; D002236:Carbohydrate Conformation; D002241:Carbohydrates / Q000138:chemical synthesis / Q000737:chemistry* / Q000494:pharmacology; D004187:Disaccharides / Q000737:chemistry*; D004220:Disulfides / Q000737:chemistry; D006801:Humans; D037102:Lectins / Q000138:chemical synthesis / Q000737:chemistry* / Q000494:pharmacology; D008969:Molecular Sequence Data; D010456:Peptides, Cyclic / Q000138:chemical synthesis / Q000737:chemistry* / Q000494:pharmacology; D011487:Protein Conformation; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D017550:Spectroscopy, Fourier Transform Infrared / Q000379:methods; D014409:Tumor Necrosis Factor-alpha / Q000737:chemistry*
chemical_list: D002241:Carbohydrates; D004187:Disaccharides; D004220:Disulfides; D037102:Lectins; D010456:Peptides, Cyclic; D014409:Tumor Necrosis Factor-alpha; C010913:N,N-diacetylchitobiose