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

Human group II secretory phospholipase A(2) (sPLA(2)) is an enzyme found in the α granules of platelets and at inflammatory 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)-(α)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 reflecting the formation of seven disulfide bonds. Circular dichroism studies of synthetic sPLA(2) showed α-helix, β-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 phospholipid-independent 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 structure–activity studies of anticoagulant, lipolytic, or inflammatory activities.