Structure of recombinant human rheumatoid arthritic synovial fluid phospholipase A2 at 2.2 Å resolution

Abstract

Phospholipases A2 (PLA2s) may be grouped into distinct families of proteins that catalyse the hydrolysis of the 2-acyl bond of phospholipids and perform a variety of biological functions. The best characterized are the small (relative molecular mass approximately 14,000) calcium-dependent, secretory enzymes of diverse origin, such as pancreatic and venom PLA2s. The structures and functions of several PLA2s are known. Recently, high-resolution crystal structures of complexes of secretory PLA2s with phosphonate phospholipid analogues have provided information about the detailed stereochemistry of transition-state binding, confirming the proposed catalytic mechanism of esterolysis. By contrast, studies on mammalian nonpancreatic secretory PLA2s (s-PLA2s) have only recently begun; s-PLA2s are scarce in normal cells and tissues but large amounts are found in association with local and systemic inflammatory processes and tissue injury in animals and man. Such s-PLAs have been purified from rabbit and rat inflammatory exudate, from synovial fluid from patients with rheumatoid arthritis and from human platelets. Cloning and sequencing shows that the primary structure of the human s-PLA2 has about 37% homology with that of bovine pancreatic PLA2 and 44% homology with that of Crotalus atrox PLA2. The human s-PLA2 is an unusually basic protein, yet contains most of the highly conserved amino-acid residues and sequences characteristic of the PLA2s sequenced so far. Here we report the refined, three-dimensional crystal structure at 2.2 A resolution of recombinant human rheumatoid arthritic synovial fluid PLA2. This may aid the development of potent and specific inhibitors of this enzyme using structure-based design.

Generation of lyso-phospholipids from surfactant in acute lung injury is mediated by type-II phospholipase A2 and inhibited by a direct surfactant protein A-phospholipase A2 protein interaction.

“…Taken together, these data indicate that, in contrast to annexins (50), SP-A does not alter sPLA2-II activity by sequestering phospholipid substrates but, as shown by BIAcore analysis, through a direct protein-protein interaction. The high selectivity of SP-A interaction with sPLA2s-II may indicate that the catalytic site of the enzyme is not directly involved, since amino acid residues of the catalytic domain of sPLA2s are highly conserved among all secretory PLA2s types (51). However, the CRD of SP-A shares sequence homology with venom sPLA2 inhibitors purified from the plasma of the Habu snake T. flavoridis sPLA2-II (18).…”

Microsomal Prostaglandin E Synthase Is Regulated by Proinflammatory Cytokines and Glucocorticoids in Primary Rheumatoid Synovial Cells

“…It has become apparent over the last several years that there are distinct pathways by which PGs are formed. Constitutively produced PGs mediating homeostatic functions and PGs produced immediately following cellular activation are synthesized via enzymes expressed under basal conditions, including cytosolic phospholipase A 2 (PLA 2 ) 3 and cyclooxygenase (COX)-1 (2). In contrast, induction of high level PG production in a time-and tissue-specific manner occurs via a set of synthetic enzymes whose expression is tightly regulated by pathologic and physiologic stimuli.…”

“…In contrast, induction of high level PG production in a time-and tissue-specific manner occurs via a set of synthetic enzymes whose expression is tightly regulated by pathologic and physiologic stimuli. In inflammatory arthritis, there is a marked increase in soluble PLA 2 in the joint fluid providing transcellular arachidonic acid for eicosanoid biosynthesis (3,4). Cytosolic PLA 2 levels are also increased by treatment of cultured synovial cells with IL-1 (5, 6).…”

Native Peptide Inhibition

“…It is also found in fluids derived from patients with inflammatory conditions (7,8) and is induced in several cell types in response to inflammatory stimuli (5). Despite differences in their primary sequences (ϳ30% homology only), crystal structures of PLA 2 from bovine pancreas (type I) and human synovial fluid (type II) are almost superimposable and, not surprisingly, the active sites from both types of enzymes are virtually identical (9). Asp-99 and His-48 form an essential catalytic dyad in the fashion of serine proteases (10).…”