Phospholipase A2 from human synovial fluid: purification and structural homology to the placental enzyme

Phospholipase A2 (PLA2) has been purified to homogeneity from synovial fluid of arthritis patients. The 3-step purification procedure included: a) dialysis against 5mM NH4-acetate, pH 5.5, in which PLA2 precipitated with euglobulins, followed by extraction with 0.4 M NaCl/0.05 M NH4-acetate, pH 5, b) chromatography on CM-cellulose, c) preparative gel electrophoresis in the presence of 0.1% Na-dodecyl sulfate and electroelution of the band containing the enzyme. Automated sequence analysis has indicated that the protein is pure, with the following NH2-terminal sequence: Asn-Leu-Val-Asn-Phe-His-Arg-Met-Ile-Lys-Leu-Thr-Thr-. A computer search revealed that all proteins with greater than 75% analogies in NH2-terminal sequences were PLA2's from various snake venoms. When PLA2 was purified from human placental membranes and analyzed, it was found to contain an identical sequence of 13 residues from the NH2-terminus. This and other characteristics suggest that the two human enzymes are closely related, if not identical.     

Purification of a soluble phospholipase A2 from synovial fluid in rheumatoid arthritis

A soluble phospholipase A2 (PLA2) was purified 4,500-fold from human rheumatoid synovial fluid. Preparative sodium dodecyl sulfate polyacrylamide gel electrophoresis yielded two bands of PLA2 activity of molecular weights 15,000 and 17,000 and pl 4.2-5.0. Purified PLA2 had absolute 2-acyl specificity, and hydrolyzed phosphatidylcholine with optimal activity at pH 7.5-8.0 and phosphatidylethanolamine with optimal activity at pH 7.0. Human synovial fluid PLA2 did not cross-react with anti-human pancreatic PLA2, as tested by radioimmunoassay.     

Detection of three distinct phospholipases A2 in cultured mast cells.

Phospholipase A2 activity in lysates of mast cells such as rat mastocytoma RBL-2H3 cells and mouse bone marrow-derived IL-3-dependent mast cells (BMMC) was measured using phosphatidylcholine (PC), phosphatidylethanolamine (PE), or phosphatidylserine (PS) as a substrate. Both types of cells exhibited phospholipase A2 activity with a similar pH profile; the optimum pH observed with PS as a substrate was 5.5-7.4, whereas that with PE or PC was 8.0-9.0. PE and PC bearing an arachidonate at the sn-2 position were cleaved more efficiently by PE, PC-hydrolyzing phospholipase A2 than phospholipids with a linoleate. A monoclonal antibody raised against rabbit platelet 85-kDa cytosolic phospholipase A2 absorbed the PE, PC-hydrolyzing activity. PS-hydrolyzing activity was purified from RBL-2H3 cells and BMMC by sequential heparin-Sepharose, butyl-Toyo-pearl, and reverse-phase HPLC. On reverse-phase HPLC, the PS-hydrolyzing activity of RBL cells was separated into two peaks, A and B. The peak B activity was inhibited by the anti-rat 14-kDa group II phospholipase A2 antibody, while the peak A activity was not. The partially purified peak A activity hydrolyzed PS about 10-fold more efficiently than PE at optimum pH of 5.5-7.4. No appreciable hydrolysis was observed with PC or phosphatidylinositol (PI). Thus, mast cells may express at least three distinct phospholipases A2; 14-kDa group II phospholipase A2, 85-kDa cytosolic arachidonate preferential phospholipase A2, and a novel phospholipase A2 that shows high substrate specificity for PS.     

Limitations of phosphatidylcholine/deoxycholate mixtures for the analysis of phospholipase A2 inhibition and activation: illustration with annexins.

The effects of human recombinant lipocortin I (annexin I) and bovine lung calpactin I (annexin II) on porcine pancreatic phospholipase A2 (PLA2) activity in phosphatidylcholine (PC)/deoxycholate (DOC) mixtures were investigated. Annexin-associated decreases in PLA2 activity were observed under some conditions, for example, at high DOC/PC molar ratios; however, activation was observed under other conditions. NaCl, which lowers the non-critical micellar concentration (NCMC) of deoxycholate, caused significant decreases in control PLA2 activity in the absence of annexins, and greater decreases in PLA2 activity in annexin-containing samples, resulting in an apparent increase in inhibition. The PC/DOC substrate mixtures themselves appeared unstable. Despite a large excess of detergent, precipitates were, at times, observed upon incubation of some PC/DOC mixtures at 37 degrees C. Such behavior is of interest in view of the numerous reports of PLA2 inhibition by annexins and annexin-derived peptides in the PC/DOC system. The influence of the annexins on activity in this system is consistent with effects on the phase behavior of the PC/DOC mixture and/or competition with the enzyme for available Ca2+. These results caution against use of the PC/DOC system for analysis of potential PLA2 inhibitors unless the phase behavior of the system is more fully delineated.     

Enzymatic activity and distribution of phospholipase A2 in human cartilage

Extracellular phospholipase A2 (PLA2) with proinflammatory activity has recently been discovered in synovial fluids in inflammatory arthritides. In the search for the sources of synovial fluid PLA2, human synovium and articular cartilage were found to contain large quantities of the enzyme. In rheumatoid arthritis (RA), PLA2 activity in synovium, superficial and deep layers of articular cartilage was 20 +/- 14 (SEM), 168 +/- 62 and 533 +/- 176 nmol/min/mg protein respectively. Corresponding values in osteoarthritis (OA) were 49 +/- 11, 569 +/- 109 and 1709 +/- 243 nmol/min/mg protein, all significantly higher (p less than .01) than in RA. Nasal septal cartilage contained much less PLA2, 19 +/- 5.6. PLA2 in human articular and nasal cartilage has sn-2 specificity, a neutral pH optimum and absolute calcium dependence. High PLA2 concentration in articular cartilage may imply that, at least in part, cartilage is the source of PLA2 in the joint space. Since RA cartilage and synovium have less PLA2 activity than the corresponding OA tissues, additional sources of PLA2 in RA synovial fluids are implicated.     

Human extracellular recombinant phospholipase A2 induces an inflammatory response in rabbit joints

Phospholipase A2 (PLA2) enzymes hydrolyze membrane phospholipids liberating fatty acid and lysophospholipid. This event is thought to be the rate-limiting step in the generation of the lipid proinflammatory mediators, the eicosanoids and possibly platelet-activating factor. For this reason, extracellular forms of PLA2 have been postulated to be a component of the inflammatory cascade in certain biologic settings. In the synovial fluid of patients with inflammatory arthritides, substantial amounts of PLA2 activity have been found, and subsequently, one enzyme was purified, cloned, and expressed. Here we show that the pure recombinant enzyme free of any proinflammatory contaminants elicits a dramatic inflammatory, arthritogenic response when injected into the joint space of healthy rabbits. Within 24 h, extensive leukocyte infiltration and hyperplasia of the synovial lining cells were observed, and prostaglandin production in the joint space increased. In comparison, pancreatic PLA2(2) had little activity in this system, whereas a very inflammatory cobra venom enzyme was intermediate in its effects. In view of the presence of the enzyme in inflamed joints, the fact that its synthesis and secretion can be induced by proinflammatory cytokines and its proinflammatory biologic activity, we suggest that synovial PLA2 plays an exacerbating role in acute episodes in chronic inflammatory conditions such as rheumatoid arthritis.     

Kallikrein in synovial fluid with rheumatoid arthritis

The levels of kallikrein and collagenase in synovial fluid from rheumatoid arthritis (RA) patients were examined and the role of kallikrein in procollagenase activation is discussed. Both prekallikrein and active kallikrein in synovial fluid from patients with RA were significantly elevated when compared to synovial fluid from patients with osteoarthritis (OA). In RA synovial fluid, the ratio of the active form to total kallikrein was also higher than that in OA synovial fluid. Both active collagenase and the alpha 2-macroglobulin (alpha 2M)-collagenase complex in RA synovial fluid were higher than in OA synovial fluid. A partial correlation (r = 0.58) between active kallikrein and total collagenase (active and alpha 2M-collagenase complex) was observed in RA synovial fluid. These observations indicate that both kallikrein and collagenase are associated with the destruction of cartilage, but the role of kallikrein in procollagenase activation was not fully clarified.     

Purification and characterization of extracellular phospholipase A2 from human synovial fluid in rheumatoid arthritis

Extracellular phospholipase A2 was purified about 1.7 X 10(5) fold to near homogeneity from human synovial fluid of rheumatoid arthritis by sequential use of column chromatographies on heparin-Sepharose, butyl-Toyopearl, and reversed-phase HPLC. The final preparation showed a single band on SDS-polyacrylamide gel electrophoresis, and its molecular mass was estimated to be approximately 13,700 daltons. The purified enzyme had a pH optimum of 9.0 and required Ca2+ for maximum activity. It hydrolyzed phosphatidyl-ethanolamine more effectively than phosphatidylserine and phosphatidylcholine. These properties were similar to those of an extracellular phospholipase A2 detected in the peritoneal cavity of caseinate-treated rats.     

Recombinant human secretory phospholipase A2: purification and characterization of the enzyme for active site studies.

A secreted form of phospholipase A2 (PLA2) is thought to play an important role in inflammatory diseases. To characterize this enzyme the cDNA encoding a low molecular weight PLA2 was cloned from a human placental cDNA library. The cDNA encoding the human PLA2 was subcloned into an expression vector and subsequently transfected into Chinese hamster ovary (CHO) cells. A stable CHO cell clone, secreting ca 1 mg/L of recombinant PLA2 into the medium, was scaled up in culture to 180 L. The recombinant enzyme was purified from the cell supernatant to apparent homogeneity by a novel procedure combining adsorption to poly(vinylidene difluoride) membranes, ion exchange chromatography and size exclusion chromatography. The final recovery of PLA2 activity was 58%. A direct comparison between the purified recombinant human PLA2 and PLA2 purified from human synovial fluid, including molecular weight, antigenicity, ionic dependence, substrate specificity and sensitivity to known PLA2 inhibitors, indicated that the two enzymes exhibit identical biochemical properties. These results show that the recombinant PLA2 can be efficiently expressed and purified in sufficient quantities to characterize the enzyme active site, to aid in the rational development of PLA2 inhibitors as potential anti-inflammatory drugs, and to investigate further the role of PLA2 in inflammatory disease.     

Preparative ion-exchange high-performance liquid chromatography of bacterial ribosomal proteins

We have developed analytical and preparative ion-exchange HPLC methods for the separation of bacterial ribosomal proteins. Proteins separated by the TSK SP-5-PW column were identified with reverse-phase HPLC and gel electrophoresis. The 21 proteins of the small ribosomal subunit were resolved into 18 peaks, and the 32 large ribosomal subunit proteins produced 25 distinct peaks. All peaks containing more than one protein were resolved using reverse-phase HPLC. Peak volumes were typically a few milliliters. Separation times were 90 min for analytical and 5 h for preparative columns. Preparative-scale sample loads ranged from 100 to 400 mg. Overall recovery efficiency for 30S and 50S subunit proteins was approximately 100%. 30S ribosomal subunit proteins purified by this method were shown to be fully capable of participating in vitro reassembly to form intact, active ribosomal subunits.     

An albumin-associated PLA2-like activity inactivates surfactant phosphatidylcholine secreted from fetal type II pneumocytes

Type II pneumocytes are responsible for the synthesis and secretion of pulmonary surfactant, which reduces surface tension in lung alveoli, thus decreasing their tendency to collapse during expiration. For this effect to be sustained, the integrity of the surface-active components of surfactant must be maintained. This study has shown that, when cultured type II pneumocytes are exposed to lipoprotein-free serum (LFS), the level of lyso-phosphatidylcholine (lyso-PC) in the secreted surfactant phospholipids is markedly elevated with a concomitant decline in the level of phosphatidylcholine (PC). This effect is the result of hydrolysis of surfactant PC by a phospholipase A(2) (PLA(2))-like activity present within serum. Anion-exchange chromatography, gel filtration chromatography and preparative electrophoresis of human LFS have shown that this PLA(2)-like activity coelutes with albumin and is biochemically distinct from the secretory form of PLA(2). Furthermore, specific inhibitors of PLA(2) such as p-bromophenacyl bromide, aristolochic acid, and palmitoyl trifluoromethyl ketone do not inhibit this activity of serum. Commercially purified human serum albumin fraction V and recombinant human serum albumin (rHSA) are almost as effective as LFS in enhancing the level of lyso-PC in the media. The latter finding implies that rHSA directly generates lyso-PC from secreted PC and suggests that this PLA(2)-like activity may be an intrinsic attribute of albumin.     

Regulation of synovial cell growth: basic fibroblast growth factor synergizes with interleukin 1 beta stimulating phospholipase A2 enzyme activity, phospholipase A2 activating protein production and release of prostaglandin E2 by rheumatoid arthritis synovial cells in culture.

Cytokines have been implicated in the regulation of eicosanoid synthesis and synovial cell proliferation. To further define these mechanisms, we have compared the effects of basic fibroblast growth factor and platelet-derived growth factor on cell growth, prostaglandin E2 (PGE2) production and phospholipase A2 enzyme activity in long-term cultures of synovial cells from rheumatoid arthritis (RA) patients capable of proliferating in serum-free medium. Compared with serum-free medium alone, RA synovial cell growth was significantly enhanced by adding either basic fibroblast growth factor (bFGF) or platelet-derived growth factor (PDGF) to the culture medium. Growing RA synovial cells for 14 days in serum-free medium plus bFGF caused them to spontaneously release significant amounts of PGE2, an effect not seen if cells were grown in serum-free medium alone, or serum-free medium plus PDGF. Enhanced release of PGE2 occurred when arachidonic acid was added to bFGF but not PDGF-treated RA synovial cells, suggesting that bFGF increased cyclooxygenase enzyme activity in these cells. Moreover, phospholipase A2 (PLA2) enzyme activity was found to be significantly greater in RA synovial cells grown for 14 days in serum-free medium containing bFGF alone, or bFGF plus interleukin 1 beta (IL-1 beta) compared with cells grown in either serum-free medium alone, or serum-free medium plus PDGF. Similarly, bFGF plus IL-1 beta-stimulated release of PLA2 activating protein, a novel mammalian phospholipase stimulator found in high concentrations in RA synovial fluid.(ABSTRACT TRUNCATED AT 250 WORDS)     

A simple procedure for the preparation and purification of the oligosaccharide components of the glycosylphosphatidylinositol anchor of membrane proteins

The oligosaccharide components of the glycosylphosphatidylinositol anchors of Trypanosoma brucei variant surface glycoproteins have been prepared and purified by treatment with hydrolytic enzymes and solvent extraction procedures followed by HPLC purification using a specific oligosaccharide binding matrix (Glyco-Pak N, by Waters). Three oligosaccharide peaks (peaks I, II and III) were resolved by a single isocratic HPLC step (70% acetonitrile in water). The material from these peaks was hydrolyzed in acid and analyzed by GC/MS. GC/MS analysis of the material obtained from each peak demonstrated the presence of inositol, glucosamine, and mannose in a 1:1:3 ratio. A variable number of galactose residues were detected in each peak. The galactose:inositol ratios of the purified components were 1:1, 2:1, and 3:1 for peaks I, II and III, respectively, suggesting that the separation obtained depends primarily on the number of sugar residues present in each fraction.     

Isoelectric focusing of glutathione S-transferases from rat liver and kidney

The glutathione S-transferases that were purified to homogeneity from liver cytosol have overlapping but distinct substrate specificities and different isoelectric points. This report explores the possibility of using preparative electrofocusing to compare the composition of the transferases in liver and kidney cytosol. Hepatic cytosol from adult male Sprague–Dawley rats was resolved by isoelectric focusing on Sephadex columns into five peaks of transferase activity, each with characteristic substrate specificity. The first four peaks of transferase activity (in order of decreasing basicity) are identified as transferases AA, B, A and C respectively, on the basis of substrate specificity, but the fifth peak (pI6.6) does not correspond to a previously described transferase. Isoelectric focusing of renal cytosol resolves only three major peaks of transferase activity, each with narrow substrate specificity. In the kidney, peak 1 (pI9.0) has most of the activity toward 1-chloro-2,4-dinitrobenzene, peak 2 (pI8.5) toward p-nitrobenzyl chloride, and peak 3 (pI7.0) toward trans-4-phenylbut-3-en-2-one. Renal transferase peak 1 (pI9.0) appears to correspond to transferase B on the basis of pI, substrate specificity and antigenicity. Kidney transferase peaks 2 (pI8.5) and 3 (pI7.0) do not correspond to previously described glutathione S-transferases, although kidney transferase peak 3 is similar to the transferase peak 5 from focused hepatic cytosol. Transferases A and C were not found in kidney cytosol, and transferase AA was detected in only one out of six replicates. Thus it is important to recognize the contribution of individual transferases to total transferase activity in that each transferase may be regulated independently.     

Application of a novel protein biochip technology for detection and identification of rheumatoid arthritis biomarkers in synovial fluid

We compared protein profiles of the synovial fluid of patients with rheumatoid arthritis and osteoarthritis by using surface-enhanced laser desorption/ionization mass spectrometry technology. With this approach, we identified a protein expressed specifically in the synovial fluid of the patients with rheumatoid arthritis. During the investigation, we found several reproducible and discriminatory biomarker candidates for distinction between rheumatoid arthritis and osteoarthritis. Among these candidates, a 10 850 Da protein peak was the clearest example of a single signal found specifically in the rheumatoid arthritis samples. This candidate was purified using a size-exclusion spin column followed by gel electrophoresis and subsequently identified by peptide mapping and post-source decay (PSD) analysis. The results clearly indicate that the protein is myeloid-related protein 8, which was verified by the enzyme immunoassay. It is known that the myeloid-related protein 8 level in serum and synovial fluid is related to disease activity in juvenile rheumatoid arthritis. The results suggest that the ProteinChip platform is useful to detect and identify protein biomarkers expressed specifically in diseases or in some stage of diseases.     

Calmodulin resolution of multiple peaks of activity by preparative electrofocusing.

When the supernatant fractions from rat brain homogenates were subjected to preparative electrofocusing in a bed of Sephadex G75, several peaks of calmodulin were resolved. A minor peak representing free calmodulin migrated with a pI of 3.8 --4.4. Other peaks of calmodulin activity were observed with isoelectric points at pH 4.8, 5.2, 6.0 and 6.8. The peak of calmodulin activity at 5.2 co-migrated with phosphodiesterase activity which was stimulated 1.8-fold by calcium. A second peak of phosphodiesterase activity detected at pH 8.0 was stimulated 1.2-fold by calcium and occurred in an area where no calmodulin activity could be detected. If isoelectric focusing was done in the presence of 8 M urea only one peak of calmodulin activity was observed with a pI of 4.0--4.4. It is suggested that the multiple peaks of calmodulin resolved by electrofocusing represent calmodulin associated with various proteins which are subject to modulation by calmodulin and calcium.     

Prorenin-renin axis in synovial fluid in patients with rheumatoid arthritis and osteoarthritis.

This study was undertaken 1) to determine whether or not renin is present in synovial fluid in patients with rheumatoid arthritis and osteoarthritis, and, if present, 2) to investigate whether it is synthesized in synovial fluid, or it is only transported from the circulation into the synovial cavity. The active renin concentration (indirect) was measured with angiotensin I radioimmunoassay kits. Inactive renin was converted into active renin with Sepharose-bound trypsin. Both active and inactive forms of renin were found in synovial fluid. They were significantly higher in patients with rheumatoid arthritis (n = 9) than in those with osteoarthritis (n = 16). In plasma, the concentration of inactive renin was significantly higher (P less than 0.001) in the former. Albumin, transferrin, alpha 2-macroglobulin, ceruloplasmin and immunoglobulins G and M were also found in synovial fluid. In each disease, a plot of the log ratio of synovial fluid to the serum concentration against the log molecular weight of each protein gave an approximately straight line curve, suggesting that these proteins are derived from the circulation and are transported into the synovial cavity. In contrast, the ratio of synovial fluid to plasma concentrations of active renin was significantly higher than that predicted on the basis of the above-mentioned interrelationships in both diseases, whereas the ratio of inactive renin was significantly lower. These findings suggest that 1) inactive and active renin are filtered into the synovial fluid from the circulation, and that 2) inactive renin is converted into the active form in the fluid.     

Transient expression of CYP1A1 in rat epithelial cells cultured in suspension

The biochemical properties of an in vivo hormonally regulated low Km cAMP phosphodiesterase (PDE) activity associated with a liver Golgi-endosomal (GE) fraction have been characterized. DEAE-Sephacel chromatography of a GE fraction solubilized by a lysosomal extract resulted in the sequential elution of three peaks of activity (numbered I, II, and III), while ion-exchange HPLC resolved five peaks of activity (numbered 1, 2, 3, 4, and 5). Based on the sensitivity of the eluted activity to cGMP and selected phosphodiesterase inhibitors, two phosphodiesterase isoforms were resolved: a cGMP-stimulated and EHNA-inhibited PDE2, eluted in DEAE-Sephacel peak I and HPLC peak 2 and a cGMP-, a cilostamide-, and ICI 118233-inhibited PDE3, eluted in DEAE-Sephacel peak III and HPLC peaks 3, 4, and 5. GE fractions isolated after acute treatments with insulin, tetraiodoglucagon, and growth hormone displayed an increase in phosphodiesterase activity relative to saline-injected controls, as did GE fractions from genetically obese and hyperinsulinemic rats relative to lean littermates. In all experimental rats, an increase in PDE3 activity associated with DEAE-Sephacel peak III and HPLC peaks 4 and 5 was observed relative to control animals. Furthermore, in genetically obese Zucker rats, an increase in the sensitivity of PDE activity to cilostamide and in the amount of PDE activity immunoprecipitated by an antibody to adipose tissue PDE3 was observed relative to lean littermates. These results extend earlier studies on isolated hepatocytes and show that liver PDE3 is the main if not sole PDE isoform activated by insulin, glucagon, and growth hormone in vivo.     

Measurement of sulfidopeptide leukotrienes and their metabolism in human synovial fluid of patients with rheumatoid arthritis

Leukotriene (LT)C4 in the synovial fluid of patients with osteoarthritis deformans (OA) and rheumatoid arthritis (RA) was measured by radioimmunoassay (RIA) after extraction with Sep-Pak C18 cartridge. The amounts of immunoreactive LTC4 (i-LTC4) in samples from patients with OA and RA were not significantly different, being 0.198 +/- 0.018 pmol/ml (n = 11) and 0.179 +/- 0.016 pmol/ml (n = 12), respectively. After separation by high performance liquid chromatography (HPLC) and measurement by RIA, the levels of other sulfidopeptide LTs, such as LTD4 and LTE4, in synovial fluid from patients with RA were found to be significantly higher than those in fluid from patients with OA. The leukocyte number in synovial fluids did not correlate with the i-LTC4 level. The metabolic activities of these synovial fluids were determined by incubating them with 3H-LTC4 and then separating sulfidopeptide LTs by HPLC. The conversion of LTC4 to LTD4 in synovial fluids of patients with OA and RA were similar, but the dipeptidase activity converting LTD4 to LTE4 was higher in fluid from patients with RA. It is suggested that a high level of LTE4 may contribute to exudation of synovial fluid, since LTE4 increases vascular permeability.