Action of phospholipase C (Bacillus cereus) on isolated myelin sheath preparations

The action of phospholipase C (Bacillus cereus) on the phospholipids of myelin sheath preparations has been investigated. With freshly isolated bovine brain myelin about 40% of the total phospholipid could be hydrolyzed by this enzyme. With bovine spinal cord myelin the phospholipid seemed more resistant to attack whereas the opposite was the case with myelin from guinea-pig brain or rat brain. With fresh bovine brain myelin, phosphatidylcholine and the ethanolamine-containing phospholipids were the main targets for the enzyme with lesser extents of hydrolysis occurring with phosphatidylserine and sphingomyelin. The effect of exposing bovine brain myelin to structural perturbants prior to enzyme digestion indicated that trypsin pretreatment had no significant effect, whereas marked enhancement of the extent of phospholipid hydrolysis occurred following lyophilization + rehydration, or pretreatment of myelin with HCl, Triton TX-100/ammonium acetate or deoxycholate. The effect of myelin pretreatment on the degradation of the individual phospholipid classes was also studied.     

The reaction of myelin phospholipids with phospholipase C and D

Phospholipase C and D hydrolyze the membrane-bound phospholipids of isolated, untreated myelin. When the membrane is treated with detergents or solvents which disrupt the membrane structure, the activity of the enzymes against the membrane-bound lipids increases. Myelin in the central nervous system is derived from the cell membrane of the oligodendroglial cell. Because the phospholipids in erythrocyte cell membranes are strikingly resistent to phospholipase C and D hydrolysis the question is raised of whether myelin in situ, as opposed to isolated myelin, is susceptible to phospholipase hydrolysis.     

The role of phospholipases from inflammatory macrophages in demyelination

Activated macrophages harvested from rat peritoneum were shown to contain phospholipase A₁, A₂ and lysophospholipase activities which were defined on a series of radiolabelled phospholipid substrates. During in vitro culture of these elicited macrophage populations, phospholipase enzymes were secreted into the culture medium. Radiolabelled myelin, prepared from young rats after intracerebral injection of¹⁴C acetate, was used as a substrate to analyze the susceptibility of central nervous system (CNS) myelin to attack by cell-associated and secreted macrophage enzymes. Homogenates of peritoneal macrophages degraded the myelin lipids at acid pH; phosphatidyl choline (PC) and ethanolamine phosphatide (EP) were both degraded with liberation of free fatty acid and small amounts of lysolipids. The ethanolamine lipids were most vulnerable; up to 20% of this fraction was degraded in six hours. Selected batches of macrophage culture supernatant similarly degraded the myelin EP at acid pH. These results suggest that phospholipase enzymes, released from activated macrophages in close proximity to the myelin sheath, may participate in primary demyelination in inflammatory CNS lesions.     

Phosphatidylinositol distribution and translocation in sonicated vesicles. A study with exchange protein and phospholipase C

The distribution of phosphatidylinositol and phosphatidylcholine in sonicated phospholipid vesicles (phosphatidylcholine: diphosphatidylglycerol: phosphatidylinositol, 90:5:5 mol%) has been determined by the use of exchange protein from beef heart and phosphatidylinositol-specific phospholipase C from Staphylococcus aureus. Approximately 70% of the phosphatidylinositol in the sonicated vesicles was accessible to the exchange protein and 70–75% was accessible to the phospholipase C. A similar proportion (65%) of the phosphatidylcholine was accessible to the exchange protein suggesting that phosphatidylinositol was not preferentially located in either surface of the phospholipid bilayer. The rate of translocation of both phospholipids was very slow but the rate for phosphatidylcholine ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 4–7}\text{days}$) appeared to be greater than that for phosphatidylinositol ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 8–60}\text{days}$). Production of asymmetric vesicles by removing phosphatidylinositol from the outer surface with either exchange protein or phospholipase C did not induce rapid phospholipid translocation.     

In vitro synthesis of phosphatidylinositol and phosphatidylcholine by phospholipase D

Phosphatidylinositol (PI) was prepared from egg lecithin by a one-step transphosphatidylation reaction catalysed by phospholipase D in the presence of myo-inositol. Similarly phosphatidylcholine (PC) has been synthesized by the same technique from egg phosphatidylethanolamine using phospholipase D and choline chloride.The yield of PI was ca 25 % and that of PC ca 28 %. The transphosphatidylase function of phospholipase D offers a useful route for the synthesis of different classes of phospholipids.     

An expedient synthesis of fluorescent labeled ceramide-1-phosphate analogues

A synthesis for fluorescent analogs of ceramide-1-phosphate bearing 9-anthrylvinyl or 4,4-difluoro-3a,4a-diaza-s-indacene-8-yl (Me₄-BODIPY) fluorophore at co-position of fatty acid residue was carried out. The key stage of the synthesis is hydrolysis of corresponding sphingomyelins catalyzed by phospholipase D from Streptomyces chromofuscus; the enzymatic yield has been raised to 50–70% by appliance of organic solvent in the incubation medium.     

Asymmetrical orientation of phospholipids and their interactions with marker enzymes in pig heart mitochondrial inner membrane

The transverse distribution of phospholipids and their interactions with marker enzymes were investigated in pig heart mitoplasts and inverted vesicles, using phospholipase A₂ from N. naja venom and chemical labeling with TNBS and FDNB. Morphological integrity was checked by freeze-fracturing. Fifty percent of phosphatidylcholine was hydrolyzed in mitoplasts as well as in inverted vesicles, suggesting an even distribution of this phospholipid on the two halves of the inner membrane; however, the fatty acid distribution did not appear the same in the two membrane fractions. Cardiolipin is exclusively hydrolyzed in inverted vesicles proving its location on the inner face of the inner membrane. The results obtained from phospholipase hydrolysis and TNBS labeling suggest that three different pools of phosphatidylethanolamine occur in the membrane: a first pool—about 50–60% of the total membrane phosphatidylethanolamine–is quickly accessible from the two sides of the membrane, a second pool—about 20–30% is slowly available, and finally 20–30% are buried within the membrane and inaccessible to the phospholipase and the probe. The cytochrome c oxidase activity increased in mitoplasts with the phospholipase attack suggesting a better accessibility of added cytochrome c after the attack. The rotenone-sensitive NADH-cytochrome c reductase was activated in mitoplasts but completely inactivated in inverted vesicles by the attack; the addition of cardiolipin liposomes restored the latter activity. The soluble matricial malate dehydrogenase was released, but the particulate form of this enzyme, strongly associated to the membrane, was detached only after attack of inverted vesicles.     

On the mechanism of activation of protein kinase FA (an activating factor of ATP.Mg-dependent protein phosphatase) in brain myelin

Protein kinase F_A (an activating factor of ATP·Mg-dependent protein phosphatase) has been characterized to exist in two forms in the purified brain myelin. One form of kinase F_A is spontaneously active and trypsin-labile, whereas the other form of kinase F_A is inactive and trypsin-resistant, suggesting a different membrane topography with active F_A exposed on the outer face of the myelin membrane and inactivu F_Q buried within the myelin membrane. When myelin was solubilized in 1% Triton X-100, all kinase F_A became active and trypsin-labile. Phospholipid reconstitution studies further indicated that when kinase F_A was reconstituted in acidic phospholipids, such as phosphatidylinositol and phosphatidylserine, the enzyme activity was inhibited in a dose-dependent manner, suggesting that kinase F_A interacts with acidic phospholipids which inhibit its activity. Furthermore, when myelin was incubated with exogenous phospholipase C, the inactive/trypsin-resistant F_A could be converted to the active/trypsin-labile F_A in a time- and dose-dependent manner. Taken together, it is concluded that membrane phospholipids play an important role in modulating the activity of kinase F_A in the brain myelin. It is suggested that phospholipase C may mediate the activation-sequestration of inactive/trypsin-resistant kinase F_A in the brain myelin through the phospholipase C-katalyzed degradation of acidic membrane phospholipids. The activation-sequestration of protein Kinase F_A may represent one mode of control modulating the activity of kinase F_A in the central nervous system myelin.     

On the mechanism of activation of protein kinase FA (an activating factor of ATP·Mg-dependent protein phosphatase) in brain myelin

Protein kinase F_A (an activating factor of ATP·Mg-dependent protein phosphatase) has been characterized to exist in two forms in the purified brain myelin. One form of kinase F_A is spontaneously active and trypsin-labile, whereas the other form of kinase F_A is inactive and trypsin-resistant, suggesting a different membrane topography with active F_A exposed on the outer face of the myelin membrane and inactivu F_Q buried within the myelin membrane. When myelin was solubilized in 1% Triton X-100, all kinase F_A became active and trypsin-labile. Phospholipid reconstitution studies further indicated that when kinase F_A was reconstituted in acidic phospholipids, such as phosphatidylinositol and phosphatidylserine, the enzyme activity was inhibited in a dose-dependent manner, suggesting that kinase F_A interacts with acidic phospholipids which inhibit its activity. Furthermore, when myelin was incubated with exogenous phospholipase C, the inactive/trypsin-resistant F_A could be converted to the active/trypsin-labile F_A in a time- and dose-dependent manner. Taken together, it is concluded that membrane phospholipids play an important role in modulating the activity of kinase F_A in the brain myelin. It is suggested that phospholipase C may mediate the activation-sequestration of inactive/trypsin-resistant kinase F_A in the brain myelin through the phospholipase C-katalyzed degradation of acidic membrane phospholipids. The activation-sequestration of protein Kinase F_A may represent one mode of control modulating the activity of kinase F_A in the central nervous system myelin.     

Role of the carbohydrate moiety of phospholipase B from Penicillium notatum in enzyme activity

Two types of phospholipase B from Penicillium notatum—the native enzyme and enzyme modified by endogenous protease (T. Okumura, S. Kimura, and K. Saito (1980) Biochim. Biophys. Acta, 617, 264–273)—were treated with endoglycosidase H (endo-β-N-acetylglucosaminidase H, Streptomyces griseus) to investigate the orientational change of the sugar chains associated with the lower activity of the modified enzyme. On measurement of release of sugar chains, by periodic acid-Schiff staining of endoglycosidase H-treated phospholipase B on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by direct sugar analysis of the isolated endoglycosidase H-treated phospholipase B, distinct curves were obtained for release of sugar chains from the native and modified enzymes with ultimately loss of about 30 and 55%, respectively, of the carbohydrate. Removal of sugar chains from the two enzymes resulted in similar increases in phospholipase B activity (phosphatidylcholine hydrolysis) and their phospholipase A₁ and A₂ activities in the presence of Triton X-100, but no change of lysophospholipase activity (lysophosphatidylcholine hydrolysis). The three former activities of the native and modified enzymes increased to almost 170 and 350%, respectively, of their initial values. However, little increase in phospholipase B activity was observed when the activity was assayed in the absence of Triton X-100, and none when it was assayed in the presence of sodium taurocholate. These findings suggest that the carbohydrate moiety of phospholipase B greatly influence the phospholipase B activity, especially in the presence of Triton X-100, and that the low phospholipase B activity of the modified enzyme is due to excess exposure of sugar chains on the surface of the molecule as a result of protease attack.     

Use of phospholipase A to compare phospholipid organization in synaptic membranes,myelin, and liposomes

Summary The pattern of fatty acid release from rat synaptic membranes in the presence of phospholipase A₂ (Vipera russelli) was compared to that from liposomes comprised of phospholipids. Phospholipase A₂ more readily attacked myelin and synaptic membranes than liposomes prepared from total phospholipids derived from myelin. Although hydrolysis of liposomal phospholipids occurred in the absence of added calcium, the presence of 2mm CaCl₂ or 2% bovine serum albumin significantly enhanced the phospholipase attack of liposomes, but not synaptic membranes or myelin. Phospholipase exhibited a marked preference for phospholipids containing docosahexaenoic acid (226) in the synaptic membranes, while with liposomes the pattern of released fatty acid reflected the fatty acid composition in the two-position of the phospholipids. Although either calcium or albumin markedly increased the phospholipase hydrolysis of liposomes, neither affected the hydrolysis of synaptic membranes or the pattern of fatty acid release from liposomes. It was concluded that the nonlipid constituents, particularly the proteins, of biomembranes were responsible for the organization of the phospholipids and accounted for the observed differences between liposomes and synaptic membranes with respect to enzymic accessibility.     

The PMP22 Gene and Its Related Diseases

Peripheral myelin protein-22 (PMP22) is primarily expressed in the compact myelin of the peripheral nervous system. Levels of PMP22 have to be tightly regulated since alterations of PMP22 levels by mutations of the PMP22 gene are responsible for >50 % of all patients with inherited peripheral neuropathies, including Charcot–Marie–Tooth type-1A (CMT1A) with trisomy of PMP22, hereditary neuropathy with liability to pressure palsies (HNPP) with heterozygous deletion of PMP22, and CMT1E with point mutations of PMP22. While overexpression and point-mutations of the PMP22 gene may produce gain-of-function phenotypes, deletion of PMP22 results in a loss-of-function phenotype that reveals the normal physiological functions of the PMP22 protein. In this article, we will review the basic genetics, biochemistry and molecular structure of PMP22, followed by discussion of the current understanding of pathogenic mechanisms involving in the inherited neuropathies with mutations in PMP22 gene.     

Characteristics of magnesium-dependent, Ca2+ -stimulated endogenous protein kinase-catalyzed phosphorylation of basic proteins in myelin isolated from rat brain stem white matter

Purified myelin fraction isolated from rat brain white matter contained Mg²⁺-dependent protein kinase capable of phosphorylation of myelin basic proteins. The Mg²⁺-supported kinase was markedly stimulated (two- to fivefold) by micromolar concentrations of free Ca²⁺ with and without Triton X-100 in the assay, the degree of stimulation being greater with the detergent present. Cyclic AMP, on the other hand, failed to show any effect on phosphorylation of myelin in the absence of Triton X-100 and in the presence of Triton caused only 25–30% stimulation. The phosphorylation reaction was temperature dependent and exhibited a pH optimum at pH 6.5. Apparent affinity toward MgATP^2? was found to be about 70 μm and Ca²⁺ had no effect on this parameter. Dependence on MgCl₂ of myelin phosphorylation indicated the presence of high- and low-affinity sites toward Mg²⁺; Ca²⁺ appeared to influence the low-affinity site. Maximal level of phosphorylation was attained by 10–15 min at 30 °C and it declined at longer incubation times due to phosphatase activity present in the preparation. Stimulatory effect of Ca²⁺ on phosphorylation was not due to inhibition of phosphatase activity. Dephosphorylation experiments showed that neither cyclic AMP nor Ca²⁺ influenced the myelin phosphatase activity. Autoradiographic analysis revealed that phosphorylation of myelin basic proteins accounted for nearly 90% of total myelin phosphorylation. This was supported by the observation that the HCl extract of myelin contained 85% of total activity and comigrated with purified myelin basic proteins. Basal and Ca²⁺-stimulated phosphorylation of basic proteins were due to phosphorylation of serines mainly, although threonine was phosphorylated to a minor extent. Within myelin, Ca²⁺ and cyclic AMP kinases are differentially bound. It appears that the myelin kinase (studied in vitro) is primarily influenced by Ca²⁺ rather than cyclic AMP. Inhibitors (Type I and Type II) of cyclic nucleotide-stimulated protein kinases had no effect on the Ca²⁺-stimulated phosphorylation although basal and cyclic AMP-stimulated phosphorylation was inhibited, indicating that the Ca²⁺ kinase is a separate and distinct enzyme from the cyclic AMP-stimulated and basal kinase(s). Also, leupeptin, a protease inhibitor, did not influence basal, cyclic AMP-stimulated, or Ca²⁺-stimulated myelin phosphorylation, indicating that under the conditions used protease(s) did not alter the myelin kinase activity. The potential significance of phosphorylation of myelin basic proteins and the stimulatory action of Ca²⁺ on this reaction are discussed.     

Producción de fosfolipasa y proteinasa en cepas de Malassezia pachydermatis aisladas de perros con otitis y sin otitis

BackgroundMalassezia pachydermatis is part of the skin microbiota of dogs and cats. M. pachydermatis has been associated with external otitis and seborrhoeic dermatitis, reported more often in dogs than in cats. When the physical, chemical or immunological mechanisms of the skin are altered, M. pachydermatis could act as a pathogen. Thus, several virulence factors, such as the ability to produce esterase, lipase, lipoxygenase, protease, chondroitin sulphatase, and hyaluronidase, have been studied.AimsIn the present study, we aim to identify the phospholipase activity measured at pH 6.3, and the proteinase activity measured at pH 6.3 and pH 6.8 (pH from ears of dogs with external otitis) of M. pachydermatis strains isolated from dogs with and without external otitis.MethodsThe phospholipase activity was measured using a semi-quantitative method with egg yolk, and the proteinase activity with a semi-quantitative method using bovine serum albumin agar. The study was performed on 96 isolates of M. pachydermatis, 43 isolated from dogs without clinical symptoms of otitis, and 52 isolated from dogs with otitis.ResultsIn our study, 75.8% of the isolates showed phospholipase activity at pH 6.3, and 81 and 97.9% of them showed proteinase activity measured at pH 6.3 and 6.8, respectively. A higher phospholipase activity was detected in strains isolated from dogs with otitis. The proteinase activity was increased at a pH of 6.8 (97.9%) in comparison to a pH of 6.3 (81%).ConclusionsOur results suggest that the phospholipase activity may play an important role in the invasion of host tissues in chronic canine otitis cases. The proteinase activity results obtained in this study suggest that a reduction in the pH of the treatment may improve its efficacy in the resolution of M. pachydermatis otitis.     

Tissue eosinophil numbers and phospholipase B activity in mice infected with Trichinella spiralis

Wilkes S. D. and Goven A. J. 1984. Tissue eosinophil numbers and phospholipase B activity in mice infected with Trichinella spiralis. International Journal for Parasitology14. 479–482. Tissue eosinophils were counted and phospholipase B activity was assayed in the intestines of mice infected with 200 Trichinella spiralis larvae. The numbers of intestinal eosinophils and phospholipase B activity increased, peaked and returned to normal levels during the same time period. The findings support the hypothesis that a parasite-induced tissue eosinophilia is the source of elevated phospholipase B activity present in parasitized tissues.     

Decreased iodination of the red cell surface following phospholipase C treatment

Human red blood cells were treated with phospholipase C from Clostridium welchii. Lipase concentrations which produced <1% hemolysis and 10–15% hydrolysis of the membrane phospholipids reduced markedly ($\text{>80\%}$) the accessibility of mambrane proteins to the external surface as measured by lactoperoxidase-catalyzed iodianation.     

Lipid abnormalities in succinate semialdehyde dehydrogenase (Aldh5a1−/−) deficient mouse brain provide additional evidence for myelin alterations

Earlier work from our laboratory provided evidence for myelin abnormalities (decreased quantities of proteins associated with myelin compaction, decreased sheath thickness) in cortex and hippocampus of Aldh5a1^−/− mice, which have a complete ablation of the succinate semialdehyde dehydrogenase protein [E.A. Donarum, D.A. Stephan, K. Larkin, E.J. Murphy, M. Gupta, H. Senephansiri, R.C. Switzer, P.L. Pearl, O.C. Snead, C. Jakobs, K.M. Gibson, Expression profiling reveals multiple myelin alterations in murine succinate semialdehyde dehydrogenase deficiency, J. Inher. Metab. Dis. 29 (2006) 143–156]. In the current report, we have extended these findings via comprehensive analysis of brain phospholipid fractions, including quantitation of fatty acids in individual phospholipid subclasses and estimation of hexose-ceramide in Aldh5a1^−/− brain. In comparison to wild-type littermates (Aldh5a1^+/+), we detected a 20% reduction in the ethanolamine glycerophospholipid content of Aldh5a1^−/−mice, while other brain phospholipids (choline glycerophospholipid, phosphatidylserine and phosphatidylinositol) were within normal limits. Analysis of individual fatty acids in each of these fractions revealed consistent alterations in n-3 fatty acids, primarily increased 22:6n-3 levels (docosahexaenoic acid; DHA). In the phosphatidyl serine fraction there were marked increases in the proportions of polyunsaturated fatty acids with corresponding decreases of monounsaturated fatty acids. Interestingly, the levels of hexose-ceramide (glucosyl- and galactosylceramide, principal myelin cerebrosides) were decreased in Aldh5a1^−/− brain tissue (one-tailed t test, p = 0.0449). The current results suggest that lipid and myelin abnormalities in this animal may contribute to the pathophysiology.     

Phosphatidylinositol as the endogenous activator of the (Na+ + K+)-ATPase in microsomes of rabbit kidney

Incubation of rabbit kidney microsomes with pig pancreatic phospholipase A₂ produced residual membrane preparations with very low $\text{(Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}\text{)-ATPase}$ activity. The activity could be restored by recombination with lipid vesicles of negatively-charged glycerophospholipids. Vesicles of pure phosphatidylcholine and phosphatidylethanolamine were virtually inactive in this respect, but could reactivate in the presence of cholate.Incubation of the microsomes with a combination of phospholipase C (Bacillus cereus) and sphingomyelinase C (Staphylococcus aureus) resulted in 90–95% release of the phospholipids. The residual membrane contained only phosphatidylinositol and still showed 50–100% of the $\text{(Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}\text{)-ATPase}$ activity.     

Tuning PAK Activity to Rescue Abnormal Myelin Permeability in HNPP

Schwann cells in the peripheral nervous systems extend their membranes to wrap axons concentrically and form the insulating sheath, called myelin. The spaces between layers of myelin are sealed by myelin junctions. This tight insulation enables rapid conduction of electric impulses (action potentials) through axons. Demyelination (stripping off the insulating sheath) has been widely regarded as one of the most important mechanisms altering the action potential propagation in many neurological diseases. However, the effective nerve conduction is also thought to require a proper myelin seal through myelin junctions such as tight junctions and adherens junctions. In the present study, we have demonstrated the disruption of myelin junctions in a mouse model (Pmp22+/-) of hereditary neuropathy with liability to pressure palsies (HNPP) with heterozygous deletion of Pmp22 gene. We observed a robust increase of F-actin in Pmp22+/- nerve regions where myelin junctions were disrupted, leading to increased myelin permeability. These abnormalities were present long before segmental demyelination at the late phase of Pmp22+/- mice. Moreover, the increase of F-actin levels correlated with an enhanced activity of p21-activated kinase (PAK1), a molecule known to regulate actin polymerization. Pharmacological inhibition of PAK normalized levels of F-actin, and completely prevented the progression of the myelin junction disruption and nerve conduction failure in Pmp22+/- mice. Our findings explain how abnormal myelin permeability is caused in HNPP, leading to impaired action potential propagation in the absence of demyelination. We call it “functional demyelination”, a novel mechanism upstream to the actual stripping of myelin that is relevant to many demyelinating diseases. This observation also provides a potential therapeutic approach for HNPP.