Investigation of peptide splicing using two‐peptide‐chain analogs of trypsin inhibitor SFTI‐1

Abstract

This study examines peptide splicing catalyzed by serine proteinases. A series of two-peptide-chain analogs of trypsin inhibitor SFTI-1 were designed and synthesized via the solid-phase method. All consisted of two peptide chains (also called N-and C-terminal fragments) joined together by one disulfide bridge. The analogs were incubated with bovine b-trypsin or bovine a-chymotrypsin. Analysis of MS data analysis showed that, after enzyme-catalyzed degradation of the single peptide bond between the Lys and Ser residues located at the C-terminus of the C-terminal peptide chain, a new peptide bond was formed. This bond brought together the separated peptide chains, and, as a result, monocyclic SFTI-1 was recovered. This proteolytic route of peptide rearrangement appears to be similar to peptide splicing catalyzed by proteasomes. However, the proteasome is much more complex than 'classical' serine proteinases.

Investigation of Serine‐Proteinase‐Catalyzed Peptide Splicing in Analogues of Sunflower Trypsin Inhibitor 1 (SFTI‐1)

“…[15] However,i ncubation of 2 with equimolar trypsin here yieldede fficient hydrolysis. HPLC analysis demonstrated complete absence of the parent peptide after 1.5 h, accompanied by absorbance increases uggestingh ydrolysis at the Lys-Anb amide bond (whereAnb is 5-amino-2-nitrobenzoica cid).…”

“…Here we report the crystal structure solution of the spliced variant in complex with the splicinge nzyme. Unexpectedly,u nder the conditions used for crystallization (equimolar concentrations of enzyme and STFI-1 analogue) splicing was observedn ot only in the previously reported analogues, [15] but also in analogues that were not spliced at catalytic enzymec oncentrations. The latter were furthere valuated in the presenceo fe quimolar amounts of enzyme, and splicing was confirmed by mass spectrometry.T herefore, under the conditions evaluated, splicing was observed regardless of the precursor containing ac leavable peptideb ondi nt he carboxyl group donating peptide.…”

“…Althought his should result in specificity www.chembiochem.org towardsc hymotrypsin instead of trypsin, our previous analysis with catalytic amounts of enzyme [15] demonstrated that 4 is neither as ubstrate for nor an inhibitoro fe ither of these enzymes. However,i ncubation with an equimolar amounto fc hymotrypsin resulted in the efficient release of the chromogenic moiety and religation of the inhibitor ( Figure 3C).…”

“…[15] However, upon incubation with equimolar enzyme, several new signals were observedi nt he MS spectra ( Figure 3A). After 1.5 hapeak appeared with m/z 1532.9 (II), which corresponds to the monocylic SFTI-1.…”

“…[15] In the presenceo facatalytic amount of enzyme (1-2mol %), P 1 -P 1 ' resynthesis was observed only in precursors containing ac leavable peptide bond in the N-terminal peptide (Table 1). This suggested that trypsin-catalyzed peptides plicing in this type of analogues or conditions takes place by direct transpeptidation.…”