Total chemical synthesis of enzymatically active human type II secretory phospholipase A ₂

Abstract

Human group II secretory phospholipase A 2 (sPLA 2 ) is an enzyme found in the ␣ granules of platelets and at inf lammatory sites. Although its physiological function is unclear, sPLA 2 can inhibit blood coagulation reactions independent of its lipolytic action. To study the molecular basis of PLA 2 activities, we developed a total chemical synthesis of sPLA 2 by chemical ligation of large unprotected peptides. The synthetic segments PLA 2 -(1-58)-␣ COSCH 2 COOH and PLA 2 -(59-124) were prepared by stepwise solid-phase peptide synthesis and ligated to yield a peptide bond between Gly 58 and Cys 59 . The 124-residue polypeptide product (mass: 13,920 ؎ 2 Da) was folded to yield one major product (mass: 13,905 ؎ 1 Da), the loss of 15 ؎ 3 Da ref lecting the formation of seven disulfide bonds. Circular dichroism studies of synthetic sPLA 2 showed ␣-helix, ␤-structure, and random coil contents consistent with those found in the crystal structure of sPLA 2 . Synthetic sPLA 2 had k cat and K m values identical to those of recombinant sPLA 2 for hydrolysis of 1,2-bis(heptanoylthio)-phosphatidylcholine. Synthetic sPLA 2 , like recombinant sPLA 2 , inhibited thrombin generation from prothrombinase complex (factors Xa, V, II, Ca 2؉ , and phospholipids). In the absence of phospholipids, both synthetic and recombinant sPLA 2 inhibited by 70% prothrombin activation by factors Xa, Va, and Ca 2؉ . Thus, synthetic sPLA 2 is a phospholipidindependent anticoagulant like recombinant or natural sPLA 2 . This study demonstrates that chemical synthesis of sPLA 2 yields a fully active native-like enzyme and offers a straightforward tool to provide sPLA 2 analogs for structureactivity studies of anticoagulant, lipolytic, or inf lammatory activities.

Synthesis of Native Proteins by Chemical Ligation

“…It does not matter how many additional internal Cys residues are present in either segment (62,64). No protecting groups are necessary for any of the side-chain functional groups normally found in proteins, and quantitative yields of the ligation product are obtained.…”

Total chemical synthesis of polypeptides and proteins: chemistry of ligation techniques and beyond

“…The synthesis was performed by ligating PLA 2 -(1e58) (9 mg/ml, 0.4 mmol) with PLA 2 -(59e124) (9 mg/ml, 0.4 mmol). 44 The reaction was complete in about 22 h, as monitored by ES-MS and HPLC. ES-MS analysis of the crude sample confirmed the presence of the 124-amino acid protein chain (13,919.5AE1.6 Da; calculated value 13,918 Da).…”

Total synthesis by modern chemical ligation methods and high resolution (1.1 Å) X‐ray structure of ribonuclease A

“…2,3 Since that time, solid phase peptide synthesis has been integral in the total chemical synthesis of a number of other enzyme molecules including HIV-1 protease (HIV-1 PR) 4 ; a tethered dimer of HIV-1 PR 5 ; 4-oxalocrotonate tautomerase (4-OT ) 6 ; human type II secretory phospholipase A2 (PLA2) 7 ; barnase 8 ; and the electron transport protein cytochrome b562. 9 Unlike Gutte and Merrifield's original groundbreaking stepwise synthesis of RNase A, 2,3 all of these enzyme syntheses (except for the synthesis of the 62 residue 4-OT 6 ) have relied on some sort of peptide ligation chemistry in addition to solid phase peptide synthesis.…”