A Role for Complement in Phagocytosis of Myelin

The mechanisms for phagocytosis of myelin in cell-mediated demyelinating diseases have not been clarified. We have previously shown with cultured phagocytic cells that myelin opsonized with antiserum to myelin constituents is phagocytized in much higher amounts than untreated myelin, indicating that Fc receptors may be involved in the demyelinating process. Using various treatments of antisera, such as heating to destroy complement, and purification of IgG, we show here that complement is a necessary factor for maximal myelin phagocytosis by cultured macrophages. If myelin is sonicated to decrease its particle size, however, complement is not an active factor. Cultured microglia, on the other hand, required complement for maximal phagocytosis of both unsonicated and sonicated myelin. Addition of serum complement greatly increased phagocytosis of untreated CNS and PNS myelin, both unsonicated and sonicated, by macrophages and microglia. From these results it appears that the most important effect of complement is to fragment the myelin, making it more easily phagocytized. Prefragmentation of myelin by sonication can substitute for complement. Complement receptors may, in addition, be important for maximal myelin phagocytosis by microglia.This work was done at the VA Medical Center in fulfillment of the research requirement at the University of Amsterdam     

Cooperative D1 Degradation in the Photosystem II Repair Mediated by Chloroplastic Proteases in Arabidopsis

Light energy constantly damages photosynthetic apparatuses, ultimately causing impaired growth. Particularly, the sessile nature of higher plants has allowed chloroplasts to develop unique mechanisms to alleviate the irreversible inactivation of photosynthesis. Photosystem II (PSII) is known as a primary target of photodamage. Photosynthetic organisms have evolved the so-called PSII repair cycle, in which a reaction center protein, D1, is degraded rapidly in a specific manner. Two proteases that perform processive or endopeptidic degradation, FtsH and Deg, respectively, participate in this cycle. To examine the cooperative D1 degradation by these proteases, we engaged Arabidopsis (Arabidopsis thaliana) mutants lacking FtsH2 (yellow variegated2 [var2]) and Deg5/Deg8 (deg5 deg8) in detecting D1 cleaved fragments. We detected several D1 fragments only under the var2 background, using amino-terminal or carboxyl-terminal specific antibodies of D1. The appearance of these D1 fragments was inhibited by a serine protease inhibitor and by deg5 deg8 mutations. Given the localization of Deg5/Deg8 on the luminal side of thylakoid membranes, we inferred that Deg5/Deg8 cleaves D1 at its luminal loop connecting the transmembrane helices C and D and that the cleaved products of D1 are the substrate for FtsH. These D1 fragments detected in var2 were associated with the PSII monomer, dimer, and partial disassembly complex but not with PSII supercomplexes. It is particularly interesting that another processive protease, Clp, was up-regulated and appeared to be recruited from stroma to the thylakoid membrane in var2, suggesting compensation for FtsH deficiency. Together, our data demonstrate in vivo cooperative degradation of D1, in which Deg cleavage assists FtsH processive degradation under photoinhibitory conditions.     

Degradation of the Major Storage Protein of Phaseolus vulgaris during Germination : Role of Endogenous Proteases and Protease Inhibitors

Cotyledons from Phaseolus vulgaris L. (var. Improved Tendergreen) were tested for their activity on α-N-benzoyl-dl-arginine-p-nitroanilide (BAPNA) and azocasein during a germination periood of 10 days. Both activities increased throughout germination when activity was expressed on the basis of dry weight or protein. That these two activities were most likely due to the action of different enzymes was indicated by the fact that (a) optimal pH for the hydrolysis of BAPNA and azocasein was 8.2 and 5.5, respectively, and (b) the digestion of azocasein was considerably enhanced by mercaptoethanol and partially inhibited by thiol protease inhibitors, N-ethylmaleimide, and E-64, whereas these same regents caused little change in activity toward BAPNA. The three subunits of the major storage protein, G1, disappeared during germination and were accompanied by the accumulation of lower molecular weight products. The breakdown of G1 by extracts of the germinated beans could be demonstrated in vitro at pH 5 to 6. This activity was enhanced by mercaptoethanol and completely abolished by N-ethylmalemide, leupeptin, and E-64. It is concluded that a thiol protease with an acid pH optimum is primarily responsible for the disappearance of the major storage protein during germination. Although an inhibitor of the plant thiol protease, papain, is present in the mature bean and decreases during germination, its role in the control of the breakdown of the storage protein remains to be elucidated.     

Isolation of Membrane Attack Complex of Complement from Myelin Membranes Treated with Serum Complement

Abstract: The interaction between complement and myelin membranes and its possible role in myelin damage and in the disposal of damaged myelin in vivo is of interest because activation of complement generates both opsonin(s) and membrane attack complex of complement. In our studies on the role of complement in demyelin-ation, we have shown that isolated myelin activates serum complement in the absence of myelin-specific antibody and that membrane attack complex of complement is the required factor in antibody-mediated demyelination of mouse cerebellar expiant cultures. In the present study, we examined whether activation of serum complement by myelin is associated with the formation of membrane attack complex of complement in myelin membranes. Extracts of myelin-associated proteins following incubation of myelin with fresh serum were studied by ultracentrifugation on a sucrose density gradient for detection of C5b-9 neoantigen. The subunit structure of C5b-9 was determined by sodium dodecyl sulfate-poly-acrylamide gel electrophoresis, electroblotting, and immunostaining. Results indicate that the macromolecular complex consisting of late-acting complement components, C5-C9, was assembled in the target myelin membranes.     

Demyelinating Antibodies to Myelin Oligodendrocyte Glycoprotein and Galactocerebroside Induce Degradation of Myelin Basic Protein in Isolated Human Myelin

Abstract: Although the specificity of multiple sclerosis (MS) brain immunoglobulins (lgs) remains unknown, the incubation of these lgs with human myelin can lead to myelin basic protein (MBP) degradation mediated by neutral proteases. In this study, we demonstrate that monoclonal antibodies (mAbs) specific to myelin components such as the CNS-specific myelin oligodendrocyte glycoprotein (MOG) and galactocerebroside (GalC) are found to induce a significant loss of MBP mediated by neutral proteases in myelin. By contrast, antibodies to periaxonal and structural components of myelin, such as MBP and myelin-associated glycoprotein, are ineffective in inducing such MBP degradation. Among the 11 different anti-MOG mAbs directed to externally located epitopes of MOG, only two were found to induce a significant degradation of MBP, suggesting that antibody-induced MBP degradation is not only antigen specific but also epitope specific. Based on the inhibition of MBP degradation in the presence of EGTA and the analysis of the degradation products obtained following incubation of myelin with mAbs to GalC and MOG (8-18C5), the neutral protease involved in this antibody-induced degradation of MBP could be calcium-activated neutral protease. Taken together, these results suggest that antibodies to GalC and MOG can play a major role in destabilizing myelin through MBP breakdown mediated by neutral proteases and thus have an important role to play in the pathogenesis of MS.     

Complement Regulatory Molecules on Human Myelin and Glial Cells: Differential Expression Affects the Deposition of Activated Complement Proteins

Abstract: The expression of decay-accelerating factor CD55, membrane cofactor protein CD46, and CD59 was studied on Schwann cells cultured from human sural nerve and myelin membranes prepared from human cauda equina and spinal cord. These proteins are regulatory membrane molecules of the complement system. CD55 and CD46 are inhibitors of C3 and C5 convertases and CD59 inhibits C8 and C9 incorporation into C5b-9 complex and C9-C9 polymerization. The presence of these proteins was assessed by using antibodies to each of the proteins by fluorescent microscopy, fluorescence-activated cell sorter analysis, and also sodium dodecyl sulfate-polyacrylamide gel electrophoresis and western blot analysis. Schwann cells in culture expressed CD55, CD46, and CD59. It is interesting that only CD59 was detected on myelin from both central and peripheral nerve tissue. The ability of these proteins to limit C3 peptide deposition and C9 polymerization in myelin was studied by western blot analysis. C3b deposition was readily detected on antibody-sensitized myelin incubated with normal human serum used as a source of complement but not with EDTA-treated or heat-inactivated serum. C3b deposition was not affected by anti-CD55 antibody. On the other hand, poly-C9 formation in myelin, which was maximum when 50% normal human serum was used, was increased four- to fivefold when myelin was preincubated with anti-CD59. Our data suggest that complement activation on myelin is down-regulated at the step of the assembly of terminal complement complexes, including C5b-9, due to the presence of CD59.     

Activation of Complement by Myelin: Identification of C1-Binding Proteins of Human Myelin from Central Nervous Tissue

Myelin isolated from central nervous tissue activates the classic pathway of complement by directly activating C1. Activation of C1 can proceed to form membrane attack complex, C5b-9, in the myelin. Such an interaction between myelin and complement may be important in diseases involving myelin damage, in view of the role of complement in membrane attack and inflammation. To identify the C1-activating protein, myelin was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot. The blots were incubated with C1 or with whole serum complement, followed by immunostaining for C1 or C3, respectively. A duplicate strip was stained with amido black or anti-myelin antibody to visualize the myelin proteins. The results showed that two major protein bands were capable of activating C1. An approximately 56-58-kilodalton band comigrated with the W2 protein and an approximately 45-47-kilodalton band migrated along with, but slightly behind, the W1 Wolfgram doublet.     

Regulation of Exocellular Proteases in Neurospora crassa: Role of Neurospora Proteases in Induction

Cells of Neurospora crassa strain 74A, grown on sucrose for 12 h and transferred to a medium containing protein as sole carbon source, would not produce exocellular protease in significant amounts. When a filtrate from a culture induced to make protease by normal growth on a medium containing protein as principal carbon source was added to an exponential-phase culture in protein medium, exocellular protease was made in amounts similar to those made during normal induction. The material in the culture filtrate that participated in the induction process was identified as protease by its heat lability, molecular weight, and the dependence of induction rate on units of proteolytic activity added to the exponential-phase culture. Induction of the formation of exocellular protease by exponential-phase cells appears to require a protein substrate, added proteolytic activity, and protein synthesis. The protease produced by induced exponential-phase cells was as efficient in promoting induction as normally induced enzyme, whereas constitutive intracellular enzyme was only 50% as efficient. The bacterial protease thermolysin was able to induce exocellular protease at 90.7% of the rate observed with added N. crassa exocellular protease.     

Multiple Sclerosis Brain Immunoglobulins Stimulate Myelin Basic Protein Degradation in Human Myelin: A New Cause of Demyelination

Membrane-bound proteolysis may be implicated in the pathogenesis of demyelinating disorders including multiple sclerosis (MS). We previously found that the extent of myelin basic protein (MBP) degradation by the calcium-activated neutral protease did not differ for isolated human control myelin or MS myelin. Hence we suggested that, if involved in demyelination, the myelin neutral protease must be activated in vivo by an increased availability of free calcium. The postulate was therefore tested that immunoglobulin (Ig) binding to myelin results in activation of the myelin neutral protease, possibly through release of free calcium from calcium-binding sites of myelin. Isolated myelin from the brains of controls and patients with MS were incubated with purified Igs eluted from the brains of patients with MS or controls and degradation of MBP was assessed by quantitative electroimmunoblotting. Such degradation was significantly greater in myelin incubated in the presence of MS Igs than in myelin incubated without added Igs or in the presence of control Igs. Furthermore, the degree of MBP degradation in myelin incubated with control Igs was similar to that observed in myelin incubated without added Igs. Accordingly, it is suggested that Ig in MS brain potentiates myelin breakdown. Moreover activation of membrane-bound proteolysis by Ig binding to myelin appears to represent a hitherto undescribed pathway for demyelination in MS.     

Major Role for Cysteine Proteases during the Early Phase of Acanthamoeba castellanii Encystment

Acanthamoeba castellanii is a facultative pathogen that has a two-stage life cycle comprising the vegetatively growing trophozoite stage and the dormant cyst stage. Cysts are formed when the cell encounters unfavorable conditions, such as environmental stress or food deprivation. Due to their rigid double-layered wall, Acanthamoeba cysts are highly resistant to antiamoebic drugs. This is problematic as cysts can survive initially successful chemotherapeutic treatment and cause relapse of the disease. We studied the Acanthamoeba encystment process by using two-dimensional gel electrophoresis (2DE) and found that most changes in the protein content occur early in the process. Truncated actin isoforms were found to abound in the encysting cell, and the levels of translation elongation factor 2 (EF2) were sharply decreased, indicating that the rate of protein synthesis must be low at this stage. In the advanced stage of encystment, however, EF2 levels and the trophozoite proteome were partly restored. The protease inhibitors PMSF (phenylmethylsulfonyl fluoride) and E64d [(2S,3S)-trans-epoxysuccinyl-l-leucylamido-3-methylbutane ethyl ester] inhibited the onset of encystment, whereas the protein synthesis inhibitor cycloheximide was ineffective. Changes in the protein profile, similar to those of encysting cells, could be observed with trophozoite homogenates incubated at room temperature for several hours. Interestingly, these changes could be inhibited significantly by cysteine protease inhibitors but not by inhibitors against other proteases. Taken together, we conclude that the encystment process in A. castellanii is of a bipartite nature consisting of an initial phase of autolysis and protein degradation and an advanced stage of restoration accompanied by the expression of encystment-specific genes.The bacteriovorous Acanthamoeba spp. occur ubiquitously in the environment (27) and have a two-stage life cycle consisting of the replicating and feeding trophozoite stage and the dormant, double-walled, cyst stage (16). Cysts are formed in order to survive in an inhospitable environment and are able to persist in a wide variety of habitats (4, 17). Indeed, the ubiquity of Acanthamoeba is made possible by the extreme resistance of the cyst against desiccation, temperature changes, chemicals, radiation, and prolonged starvation. Also, various antiamoebic agents, such as benzalkonium chloride and propamidine isethionate, have no effect on cysts (9, 13, 29). Since acanthamoebae are facultative pathogens that can cause Acanthamoeba keratitis (AK) and granulomatous amoebic encephalitis (GAE), encystment is also of medical relevance (16). An often occurring complication in the treatment of AK is the presence of viable cysts that remain in the corneal stroma after initial successful therapy, as these can eventually excyst again and lead to recurrent infections (23).According to Weisman (31), the encystment process comprises three phases: induction, wall synthesis, and dormancy. During the induction phase, trophozoites begin to lose their amoeboid appearance and become round. The first wall that is formed gives rise to the exocyst; this wall is 0.3 to 0.5 μm thick and consists mostly of acid-insoluble proteins. The endocyst is formed after the appearance of a well-defined layer whose major component is cellulose (31). Cell wall synthesis is usually accompanied by a decrease in cytoplasmic mass of approximately 80% through a gradual dehydration of the amoeba, thereby causing retraction of the protoplast from the cell wall (2). Rather early, autolysosomes appear and remain in the cytoplasm throughout the whole encystment process. In light of these dramatic changes in the cell''s physiology, it is surprising that the encysting cell can stop and revert the process until 15 h after induction (30). Afterwards, however, cells become committed to the completion of the encystment process.At the molecular level, a number of factors involved in the encystment process have been characterized thus far. For example, cyst-specific protein 21 (Csp21) is a cyst wall protein found in group II acanthamoebae and was reported to be synthesized approximately 12 h after induction (6). The expression of the respective gene is repressed under normal growth conditions via one or more repressor elements between the TATA box and nucleotide (nt) +63 (3). Furthermore, encystment requires serine protease activity (5, 20) and autophagy proteins (22), all of which are suggested to be involved in autolytic processes, and glycogen phosphorylase, which is necessary for the breakdown of glycogen (14). The glucose-1-phosphate that is thereby liberated is subsequently used for the buildup of cellulose in the cyst wall.In the search for additional factors, there have been several successful attempts in the past years to screen encysting Acanthamoeba castellanii for genes specifically expressed during encystment at the mRNA level (19, 21) as well as at the protein level (1, 24). However, there is still a lack of information on the extent of cellular reorganization during the encystment process at the protein level. In this study, we therefore aimed to monitor the encystment process in PAT06, a new clinical isolate of A. castellanii (10), by using two-dimensional gel electrophoresis (2DE) and to analyze the developmental and molecular processes at the proteomic level.