Chemical synthesis and structural characterization of the RGD‐protein decorsin: A potent inhibitor of platelet aggregation

Abstract

Decorsin is a 39-residue RGD-protein crosslinked by three disulfide bridges isolated from the leech Mucrobdellu decoru belonging to the family of GPIIb-IIIa antagonists and acting as a potent inhibitor of platelet aggregation. Here we report the solid-phase synthesis of decorsin using the Fmoc strategy. The crude polypeptide was purified by reverse-phase HPLC in its reduced form and allowed to refold in the presence of glutathione. The homogeneity of the synthetic oxidized decorsin was established by reverse-phase HPLC and capillary zone electrophoresis. The results of amino acid analysis after acid hydrolysis of the synthetic protein, NH2-terminal sequencing and mass determination (4,377 Da) by electrospray mass spectrometry were in full agreement with this theory. The correct pairing of the three disulfide bridges in synthetic decorsin was determined by a combined approach of both peptide mapping using proteolytic enzymes and analysis of the disulfide chirality by CD spectroscopy in the near-UV region. Synthetic decorsin inhibited human platelet aggregation with an ICso of -0.1 p M , a figure quite similar to that determined utilizing decorsin from natural source. In particular, the synthetic protein was 2,000-fold more potent than a model RGD-peptide (e.g., Arg-Gly-Asp-Ser) in inhibiting platelet aggregation. Thermal denaturation experiments of synthetic decorsin, monitored by CD spectroscopy, revealed its high thermal stability (T, -74 "C). The features of the oxidative refolding process of reduced decorsin, as well as the thermal stability of the oxidized species, were compared with those previously determined for the NH2-terminal core domain fragment 1-41 or 1-43 from hirudin. This fragment shows similarity in size, pairing of the three disulfides and three-dimensional structure with those of decorsin, even if very low sequence similarity. It is suggested that the less efficient oxidative folding and the enhanced thermal stability of decorsin in respect to those of hirudin core domain likely can be ascribed to the presence of the six Pro residues in the decorsin chain, whereas none is present in the hirudin domain. The results of this study indicate that decorsin can be obtained by solid-phase methodology in purity and quantities suitable for structural and functional studies and thus open the way to prepare by chemical methods novel decorsin derivatives containing unusual amino acids or even non-peptidic moieties.

Cysteine racemization during the Fmoc solid phase peptide synthesis of the Nav1.7‐selective peptide – protoxin II

“…After obtaining a racemization‐minimized crude linear protoxin II, crude peptide was then air oxidized to form disulfide bridges. When disulfides are in the Cys1–Cys4, Cys2–Cys5 and Cys3–Cys6 bridging pattern, air oxidation is a robust method to form these disulfide bridges with high yield. Steiner and Bulaj reported a summary of oxidative folding conditions in which it was observed that a pH of 6–8 was optimal and the ratio between reduced and oxidized glutathione does not significantly affect the efficiency.…”

“…Cysteine residues are known to racemize during coupling in Fmoc solid phase peptide synthesis (Fmoc‐SPPS) , and this problematic issue is even more complex when synthesizing toxins from venomous animals because of the complex arrangement of multiple disulfide bonds. There are numerous reports on the Fmoc‐SPPS of naturally occurring inhibitory cystine knot peptides such as protoxin II , but interestingly, none of these previous publications mention the extent (or lack thereof) of cysteine racemization and thus create possible ambiguity regarding the chiral purity of the reported peptides. Given that each cysteine may racemize (5–50%) during the synthetic process, the desired crude linear toxin may only comprise a small fraction of the crude product, making it difficult to isolate and subsequently purify.…”

Chirality of the Disulfide in the Prion Proteins

“…The stabilizing role of disulfide bridges in proteins is well illustrated in the structural studies of decorsin, a 39-residue protein isolated from the blood-sucking leech, Macobdella decora, which has the powerful ability to prevent blood clot formation . Decorsin has been intensively studied as a potential therapeutic agent or a model for other inhibitors of platelet aggregation.…”