title: Mass spectrometric identification of the trypsin cleavage pathway in lysyl-proline containing oligotuftsin peptides.
abstract: Trypsin cleaves specifically peptide bonds at the C-terminal side of lysine and arginine residues, except for -Arg-Pro- and -Lys-Pro- bonds which are normally resistant to proteolysis. Here we report evidence for a -Lys-Pro- tryptic cleavage in modified oligotuftsin derivatives, Ac-[TKPKG]4-NH2) (1), using high-resolution mass spectrometry and HPLC as primary methods for analysis of proteolytic reactions. The proteolytic susceptibility of -Lys-Pro- bonds was strongly dependent on flanking residues, and the flexibility of the peptide backbone might be a prerequisite for this unusual cleavage. While -Lys-Gly- bonds in 1 were rapidly cleaved, the modification of these Lys residues by the attachment of a ss-amyloid(4-10) epitope to yield -Lys(X)-Gly derivatives prevented cleavage of this bond, and provided trypsin cleavage of -Lys-Pro- bonds, the pathway of this degradation being independent on the type of Lys-N(epsilon)-side chains (acetyl group, amino acid, peptide). Substitution of the Lys residues by Ala at the P'2 positions decreased the tryptic cleavage, while replacement of the bulky side chain of Thr at the P2 positions strongly increased the cleavage of -Lys-Pro- bonds. Circular dichroism (CD) data of the modified oligotuftsin derivatives are in accord with enhanced flexibility of the peptide backbone, as a prerequisite for increased susceptibility to cleavage of -Lys-Pro- bonds. These results obtained of oligotuftsin derivatives might have implications for the proteolytic degradation of target peptides that require specific conformational preconditions.
authors: Department of Chemistry, Laboratory of Analytical Chemistry and Biopolymer Structure Analysis, University of Konstanz, 78457 Konstanz, Germany.
authors: Marilena
authors: M
authors: Manea
authors: Gábor
authors: G
authors: Mezo
authors: Ferenc
authors: F
authors: Hudecz
authors: Michael
authors: M
authors: Przybylski
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D016229:Amyloid beta-Peptides / Q000737:chemistry; D002851:Chromatography, High Pressure Liquid; D002942:Circular Dichroism; D004151:Dipeptides / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry; D011487:Protein Conformation; D017550:Spectroscopy, Fourier Transform Infrared / Q000379:methods*; D014357:Trypsin / Q000378:metabolism*; D014405:Tuftsin / Q000032:analysis / Q000737:chemistry / Q000378:metabolism*
chemical_list: D016229:Amyloid beta-Peptides; D004151:Dipeptides; D010446:Peptide Fragments; C039356:lysylproline; D014357:Trypsin; D014405:Tuftsin