Structural Insight into the Activation Mechanism of Human Pancreatic Prophospholipase A2

Abstract

Pancreatic phospholipase A2 (phospholipase A2 group 1B, G1B) belongs to the superfamily of secreted phospholipase A2 (PLA2) enzymes. G1B has been proposed to be a potential target for diseases such as hypertension, obesity, and diabetes. Human pancreatic prophospholipase A2 (pro-hG1B) is activated by cleavage of the first seven-residue propeptide (phospholipase A2 propeptide, PROP). However, questions still remain on the mode of action for pro-hG1B. In this work, we expressed prohG1B in Pichia pastoris and determined the crystal structure at 1.55-Å resolution. The x-ray structure demonstrates that prohG1B forms a trimer. In addition, PROP occupies the catalytic cavity and can be self-cleaved at 37°C. A new membrane-bound surface and activation mechanism are proposed based on the trimeric model of pro-hG1B. We also propose a new autoproteolytic mechanism for pro-hG1B by the reaction triad Asp49-Arg0-Ser(-2) that is similar to the serine protease catalytic triad.Phospholipase A2 (PLA2, 5 EC 3.1.1.4) hydrolyzes glycerophospholipids at the sn-2 acyl bond to produce free fatty acids. The PLA2 family currently comprises five categories: the secreted PLA2s, the cytosolic PLA2s, the Ca 2ϩ -independent PLA2s, the platelet-activating factor acetyl hydrolases, and the lysosomal PLA2s. To date, based on the catalytic mechanism as well as functional and structural information, 15 different groups of PLA2 have been reported and named (1).G1B is a member of the secreted PLA2 enzymes. This lipolytic enzyme releases glycerophospholipids and arachidonic acid that serve as the precursors of signal molecules that mediate a multitude of biological functions, such as inflammation. The G1B gene has been reported to be linked to hypertension in three sample populations (2). The concentration of G1B protein in serum is a potential marker for pancreatic acinar cell carcinoma (3). Knock-out mice experiments showed that G1B knockdown can prevent diet-induced obesity and obesity-related insulin resistance (4). After being fed with glucose-rich meals, knock-out mice showed lower postprandial glycemia than wild-type mice (5). A recent report also pointed to the linkage between hG1B and ophthalmic diseases (6). Therefore, hG1B is considered to be a potential target for treatment of obesity, diabetes (4, 5), or ophthalmic diseases (6).Pro-hG1B is a digestive zymogen secreted from pancreatic acinar cells in its inactive form (7). It is activated by trypsin in the duodenum. The activation of pro-hG1B by cleavage of the PROP, a heptapeptide of the sequence DSGISPR, is linked to diseases like pancreatitis (8) and acute lung injury (9). Circulating hG1B, mostly in the form of pro-hG1B, indicates pancreatic injury in acute pancreatitis (10). PROP can be used in assays to characterize the severity of acute pancreatitis (11). The C-terminal pentapeptide of PROP (GISPR) is essential for the inhibition of enzyme activity (12). Besides trypsin, pro-hG1B can also be activated by thrombin (13) and 29-kDa trypsin-like endogenous type 1-proPLA2 activator (11)...

Phospholipase A2 Enzymes: Physical Structure, Biological Function, Disease Implication, Chemical Inhibition, and Therapeutic Intervention

“…The authors suggested that human GIB sPLA 2 may use a different activation mechanism in which GIB sPLA 2 switches from monomeric to trimeric form when it associates with the membrane using the highly positive charged trimer back side. 40 The crystal structure of the trimeric form has also been shown in studies on cobra venom 41 and Naja naja GIA sPLA 2 s. 42 Anion-assisted dimer crystal structures were reported for other pancreatic sPLA 2 s. 43 While the existence of dimeric and trimeric forms may be a functionally important feature of these sPLA 2 s, the higher oligomeric states of sPLA 2 structures are found under high concentration crystallization conditions, so they may not be representative of the real physiological state. Whether the trimeric or dimmeric form is important for sPLA 2 catalysis or not requires further investigation.…”

Group X Secreted Phospholipase A2 Proenzyme Is Matured by a Furin-like Proprotein Convertase and Releases Arachidonic Acid inside of Human HEK293 Cells

“…We carried out multitemplate modeling based on the crystal structures of hGX sPLA 2 (Protein Data Bank code 1LE7 (residue nos. 43-165) (32)), PropGIB (Protein Data Bank code 1HN4 (9)), and ProhGIB sPLA 2 (Protein Data Bank code 3ELO, (33)). The overall structure and the side chain positions are conserved in the model when compared with the experimental structure of hGX.…”

Structural analysis of a group III Glu62-phospholipase A2 from the scorpion, Mesobuthus tamulus: Targeting and reversible inhibition by native peptides

“…These enzymes are classified into different groups based on their primary sequence structures and the positions of their disulfide bonds [1]. While the structures of group I and group II PLA 2 s have been well characterized from different sources including humans [2][3][4][5], there exists only one representative crystal structure known so far in the group III which is that of the bee venom PLA 2 [6]. The structure of this group III PLA 2 is completely different from those of group I Abbreviations: PLA2, phospholipase A2; ExPASy, Expert Protein Analysis System; DS, Discovery Studio; ABNR, adopted basis set Newton-Raphson; CHARMM, Chemistry at HARvard Molecular Mechanics; PROCHECK, protein structure check; r.m.s.d., root mean square deviation; ps, pico seconds.…”