Functional Characteristics of a Phospholipase A2Inhibitor from Notechis ater Serum

Abstract

A phospholipase A 2 inhibitor has been purified from the serum of Notechis ater using DEAE-Sephacel chromatography. The inhibitor was found to be composed of two protein subunits (␣ and ␤) that form the intact complex of approximately 110 kDa. The ␣-chain is a 30-kDa glycoprotein and the ␤-chain a nonglycosylated, 25-kDa protein. N-terminal sequence analysis reveals a high level of homology to other snake phospholipase A 2 inhibitors. The inhibitor was shown to be extremely pH and temperature stable. The inhibitor was tested against a wide variety of phospholipase A 2 enzymes and inhibited the enzymatic activity of all phospholipase A 2 enzymes tested, binding with micromole to nanomole affinity. Furthermore, the inhibitor was compared with the Eli-Lilly compound LY311727 and found to have a higher affinity for human secretory nonpancreatic phospholipase A 2 than this chemical inhibitor. The role of the carbohydrate moiety was investigated and found not to affect the in vitro function of the inhibitor.For many years research on PLA 2 1 enzymes has centred on snake venom PLA 2 s, largely because of the fact that PLA 2 enzymes are extremely abundant in snake venoms and are easily purified. PLA 2 enzymes have also been identified and purified from bovine, porcine, and human pancreas (1-3) and in human synovial fluid aspirates from rheumatoid and osteoarthritis patients (4, 5). PLA 2 enzymes have been grouped according to structural and sequence characteristics. Pancreatic PLA 2 enzymes belong to group I, whereas the human secretory nonpancreatic PLA 2 is a group II enzyme. PLA 2 enzymes from snake venom belong to group I or II (6 -8).Recently PLA 2 enzymes have been implicated as playing a role in a range of diseases including rheumatoid and osteoarthritis, asthma, acute pancreatitis, psoriasis, multiple organ failure, septic shock, and adult respiratory distress syndrome (9 -11). Several review articles (9,10,(12)(13)(14) have examined the physiological and potential disease role of hsPLA 2 -II. Of major importance are the reaction by-products of PLA 2 activity, a free fatty acid and a lysophospholipid. The fatty acid is often arachidonic acid, one of the main constituents of the cell membrane in several tissues (15)(16)(17). Arachidonic acid is the precursor for inflammatory mediators such as thromboxanes, leukotrienes, and prostaglandins (18,19). Lysophospholipids are the precursor of platelet activating factor (20).The suggestion that PLA 2 enzymes play a role in diseases process has intensified research on PLA 2 inhibitors. Many investigators have purified and characterized PLIs from numerous sources including plant, fungi, and bacteria (21-28). A large research effort has gone into the design of synthetic compounds, such as the Eli-Lilly compound LY311727, which specifically inhibits hsPLA 2 -II (29). Additionally, a number of PLIs have been purified from the serum of elapid and crotalid snakes. These PLIs have all been shown to be homologous or heterologous complexes that interact with PLA 2 enzymes to inhib...

Purification and inhibitory profile of phospholipase A2 inhibitors from Australian elapid sera

“…All other snake PLIs also have at least one chain glycosylated, suggesting the carbohydrate might have a functional role in the inhibitory activity of the PLIs. However, work performed in our laboratory indicates that the carbohydrate moiety has little or no role in the inhibition of enzymic activity [47]. This finding has recently been confirmed for another elapid PLI [25].…”

Purification and characterization of a glycoprotein inhibitor of toxic phospholipase from Withania somnifera

“…The a-chains are 20-to 30-kDa glycoprotein subunits and the b-chains are nonglycosylated 20-to 25-kDa protein subunits. The carbohydrate moiety is found not to effect the in vitro function of the inhibitor [50,51]. The WSG is also an acidic glycoprotein similar to the achain of the PLIs but in contrast it is composed of a single protein.…”

Functional site of endogenous phospholipase A2 inhibitor from python serum

“…It has the highest sequence identity (57–61%) to the mature PLIs from the sera of Crotalidae snakes , Agkistrodon blomhoffii siniticus [14], Crotalus durissus terrificus [11,13], and Trimeresurus flavoviridis ( Protobothrops flavoviridis ) [9,15], with sequence identities of 61, 60 and 57%, respectively. PIP also has a significant (57%) homology to the sequences of mature PLIs of a nonvenomous snake Elaphe quadrivirgata [21], and also to those of the PLIs from the sera of Australian Elapidaes, Notechis ater , Notechis scutatus , and Oxyuranus scutellatus [19], with sequence identities in the vicinity of 56%.…”

“…A number of PLIs have been purified and characterized from a variety of sources, including plant, fungi, and bacteria [6–8]. PLIs that interact with PLA 2 s and inhibit their enzymatic activity have been identified in the sera of venomous snakes belonging to Elapidae and Crotalidae families [9–20]. The discovery of specific sPLA 2 inhibitors has also been reported in the blood serum of nonvenomous snakes [21,22].…”