Magdalena Tchorbadjieva

Purification of C-phycocyanin from Spirulina (Arthrospira) fusiformis

C-phycocyanin was purified from Spirulina (Arthrospira) fusiformis by a multi-step treatment of the crude extract with rivanol in a ratio 10:1 (v/v), followed by 40% saturation with ammonium sulfate. After removal of rivanol by gel-filtration on Sephadex G-25, the pigment solution was saturated to 70% with ammonium sulfate. After the last step of purification, C-phycocyanin had an emission and absorption maxima at 620 and 650 nm, respectively and absorbance ratio A(620)/A(280) of 4.3, which are specific for the pure biliprotein. Its homogeneity was demonstrated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, yielding two bands of molecular masses 19500 and 21500 kDa, corresponding to alpha and beta subunits of the pigment, respectively. The yield of C-phycocyanin was approximately 46% from its content in the crude extract.

Mutational Analyses of the Recombinant Globular Regions of Human C1q A, B, and C Chains Suggest an Essential Role for Arginine and Histidine Residues in the C1q-IgG Interaction

The first step in the activation of the classical complement pathway by immune complexes involves the binding of the globular domain (gC1q) of C1q to the Fc regions of aggregated IgG or IgM. Each gC1q domain is a heterotrimer of the C-terminal halves of one A (ghA), one B (ghB), and one C (ghC) chain. Our recent studies have suggested a modular organization of gC1q, consistent with the view that ghA, ghB, and ghC are functionally autonomous modules and have distinct and differential ligand-binding properties. Although C1q binding sites on IgG have been previously identified, the complementary interacting sites on the gC1q domain have not been precisely defined. The availability of the recombinant constructs expressing ghA, ghB, and ghC has allowed us, for the first time, to engineer single-residue substitution mutations and identify residues on the gC1q domain, which are involved in the interaction between C1q and IgG. Because C1q is a charge pattern recognition molecule, we have sequentially targeted arginine and histidine residues in each chain. Consistent with previous chemical modification studies and the recent crystal structure of gC1q, our results support a central role for arginine and histidine residues, especially Arg114 and Arg129 of the ghB module, in the C1q-IgG interaction.

New insight into the autoimmunogenicity of the complement protein C1q

C1q along with its physiological role in maintenance of homeostasis and normal function of the immune system is involved in pathological conditions associated with repetitive generation of anti-C1q autoantibodies. The time and events that cause their first appearance are still unknown. We addressed this issue by analyzing the immunogenicity of C1q in two target groups-one of non-diseased humans and the other of lupus nephritis (LN) patients whose autoimmune disorder is associated with high titers of anti-C1q autoantibodies. The non-diseased humans were represented by pregnant women because the sex hormones are thought to be involved in triggering autoimmune pathologies by their ability to tip the balance of female adaptive immune response to production of antibodies. We screened, using ELISA, 31 sera from healthy pregnant women for the presence of IgM and IgG classes of autoantibodies, recognizing epitopes within the native C1q molecule, its collagen-like region (CLR) and globular head fragment (gC1q). The latter was represented by recombinant analogs of the three globular fragments of A, B and C chains, comprising C1q-ghA, ghB and ghC. We did not find IgM antibodies for all test-antigens which suggest that the natural IgM antibodies are not involved in triggering autoimmunity to C1q. Still more, we did not detect anti-CLR antibodies which have been proved pathogenic in already manifested LN. We completed the analysis with comparative epitope mapping of gC1q and we found similar immunogenic behavior in both target groups-ghA and ghC contained the immunodominant epitopes. This implies that the initial immune response to C1q might occur when the molecule has interacted with its ligands via ghB as part of gC1q. The presence of anti-gC1q in both healthy and diseased humans also implies that these antibodies, unlike anti-CLR, may have a contribution to an onset of autoimmunity.

TWO-DIMENSIONAL PROTEIN PATTERN ANALYSIS OF EXTRACELLULAR PROTEINS SECRETED BY EMBRYOGENIC AND NON-EMBRYOGENIC SUSPENSION CULTURES OF DACTYLIS GLOMERATA L.

ABSTRACT Proteins from the medium of orchardgrass (Dactylis glomerata L.) embryogenic suspension culture during defined stages of somatic embryogenesis were compared with those of non-embryogenic suspension culture during unorganized cell proliferation. After separation on two-dimensional polyacrylamide gel electrophoresis, we were able to classify 40 proteins in each of 6 groups: a group common to both embryogenic and non-embryogenic suspension cultures, a group specific only for embryogenic, respectively non-embryogenic suspension cultures and 3 groups of microcluster-, proembryogenic masses- and somatic embryo-specific proteins. One of the groups is particularly interesting as it corresponds to polypeptides that are related to the earliest stages of somatic embryo-genesis-the transition of microclusters into proembryogenic masses when no morphological changes are visible yet. Using this experimental approach, we identified the major extracellular proteins involved in somatic embryo development and we discuss on the possibility for their use as early markers for somatic embryogenesis.

Biochemical analysis of the epitope specificities of anti-C1q autoantibodies accompanying human lupus nephritis reveals them as a dynamic population in the course of the disease

We analyzed the epitope specificities of the polyclonal anti-C1q antibodies, present in human LN sera, searching to deduce the structural characteristics of C1q associated with its transition to an autoantigen. We screened 78 serum samples from LN patients distributed in three clinical groups - non-active, moderately active and severely active. We found three classes of C1q autoepitopes: (a) neo-epitopes, exposed upon immobilization due to conformational changes; (b) epitopes formed by sequences that are brought together by the conformation of the whole molecule; (c) cryptic epitopes that become exposed only after fragmentation of C1q. The latter suggest that the immunogen involved in the initiation of anti-C1q autoantibodies might be an extrinsic molecule that shares some degree of structural similarity to C1q. None of the tested epitope specificities was associated with active LN. We found a prevalence of anti-gC1q antibodies among the non-active LN patients suggesting that they might be the fraction of the polyclonal anti-C1q, preceding the initiation of autoimmunity to C1q, or alternatively, preceding LN flare.

In vivo spectroscopy and NMR metabolite fingerprinting approaches to connect the dynamics of photosynthetic and metabolic phenotypes in resurrection plant Haberlea rhodopensis during desiccation and recovery

The resurrection plant Haberlea rhodopensis was used to study dynamics of drought response of photosynthetic machinery parallel with changes in primary metabolism. A relation between leaf water content and photosynthetic performance was established, enabling us to perform a non-destructive evaluation of the plant water status during stress. Spectroscopic analysis of photosynthesis indicated that, at variance with linear electron flow (LEF) involving photosystem (PS) I and II, cyclic electron flow around PSI remains active till almost full dry state at the expense of the LEF, due to the changed protein organization of photosynthetic apparatus. We suggest that, this activity could have a photoprotective role and prevent a complete drop in adenosine triphosphate (ATP), in the absence of LEF, to fuel specific energy-dependent processes necessary for the survival of the plant, during the late states of desiccation. The NMR fingerprint shows the significant metabolic changes in several pathways. Due to the declining of LEF accompanied by biosynthetic reactions during desiccation, a reduction of the ATP pool during drought was observed, which was fully and quickly recovered after plants rehydration. We found a decline of valine accompanied by lipid degradation during stress, likely to provide alternative carbon sources for sucrose accumulation at late stages of desiccation. This accumulation, as well as the increased levels of glycerophosphodiesters during drought stress could provide osmoprotection to the cells.

Advances in Proteomics of Somatic Embryogenesis

Plants are sessile organisms and, as such, have evolved a remarkable developmental plasticity allowing them to cope with the adverse effects of numerous biotic and abiotic factors from the environment. One of the most intriguing examples of this plasticity is somatic embryogenesis during which somatic cells dedifferentiate into cells that are capable to form embryos. The latter are morphologically similar to zygotic embryos and can regenerate whole plants. The transition of somatic cells into embryogenic ones is the most intriguing and the part of somatic embryogenesis least understood. To better understand the mechanisms of somatic embryogenesis, comparative proteomic approaches have been used, and in recent years, hundreds of proteins have been identified in embryogenic and nonembryogenic cultures, in somatic and zygotic embryos, and during distinct stages of somatic embryogenesis in both angiosperms and gymnosperms.

Developmental and Organ-Specific Expression of Transferrin in Drosophila Melanogaster

ABSTRACT Polyclonal antibodies raised against recombinant D. melanogaster transferrin were used to study both developmental and organ-specific expression of D. melanogaster transferrin. It was shown that transferrin is present at all developmental stages except late embryos and early larvae. The organ distribution of transferrin reveals that it is abundant in the hemolymph and from all organs examined was detected only in the ovaries. The maternal origin of the transferrin in early embryos, the activation of its synthesis and its accumulation in the oocytes are discussed.

Differentially Secreted Proteins of Antarctic and Mesophilic Strains of Synechocystis Salina and Chlorella Vulgaris after UV-B and Temperature Stress Treatment

ABSTRACT Exponential-phase cell cultures from Antarctic and mesophilic strains of the cyanobacterium Synechocystis salina and the unicellular green alga Chlorella vulgaris were subjected to UV-B and temperature stress and investigated for alterations in the spectrum of secreted proteins. In addition to constitutively secreted proteins, strain-specific stress proteins were detected on silver-stained gels. Extracellular protein patterns within a given strain, as well as between strains after exposure to each UV-B and temperature stress were notably different. UV-B exposure induced only moderate changes of the secreted proteins in both Antarctic and mesophilic Synechocystis salina strains. However, a 21 kDa protein band present in Antarctic Synechocystis salina only was identified using LC-MS/MS analysis, to contain two proteins—a peroxiredoxin and Fe-superoxide dismutase (Fe-SOD). Their presence might modulate the level of reactive oxygen species generated in membranes during UV-B stress and, thus, contribute to the higher survival rate of Antarctic Synechocystis salina. In addition, proteolytic activity in the extracellular protein samples of both species was detected on 1D zymography in a polyacrylamide gel containing gelatin as substrate. Antarctic Synechocystis salina secreted cysteine-, serine-, and metalloproteases, while mesophilic Synechocystis salina secreted mainly cysteine proteases. Subtle changes of the protease activity bands in Antarctic Synechocystis salina and Chlorella vulgaris were observed after UV-B and temperature stress. In contrast, mesophilic Synechocystis salina responded with a considerable increase in protease activity and number of activity bands after 30 min and 60 min exposure to UV-B, as well as when grown at 35°C and 40°C. Peptidases are key players in structuring and adapting cells under various stressful conditions. The varying pattern of secreted proteases among the Antarctic and mesophilic strains of Synechocystis salina and Chlorella vulgaris suggests different mechanisms for coping with UV-B and temperature stress. Characterization of extracellular proteins discovered in the present study will provide further understanding of the rich defense arsenal and mechanisms of survival of cyanobacteria and microalgae.

Generation of Recombinant Antibodies against Orchardgrass Acidic nsLTP-Like Proteins

Embryogenic and non-embryogenic suspension cultures of orchardgrass (Dactylis glomerata L.) secreted into the culture medium a set of proteins, among which low molecular mass (11/12 kDa) proteins were found. However, only the 11/12 kDa proteins from the embryogenic suspension cultures reacted specifically with an antiserum raised against the carrot EP2 non-specific lipid transfer protein (nsLTP). Two-dimensional (2-D) electrophoretic analysis revealed that the extracellular nsLTP-like proteins from the embryogenic lines were acidic proteins, with pI values ranging between 4.3 and 6.4, and the 11/12 kDa proteins of the nonembryogenic lines were basic ones (pI 8-9.3). This is only the second case to report on the accumulation of extracellular acidic nsLTP-like proteins in the culture medium during somatic embryogenesis. A naı¨ve phage display Griffin1. library was used to select single-chain phage antibodies, which specifically bind to acidic nsLTP-like proteins. Nine phage clones were selected after four rounds of biopanning of the target proteins blotted on a nitrocellulose membrane. Three soluble monoclonal single-chain phage antibodies, expressed in the non-suppressor E. coli strain HB2151, were purified by metal affinity chromatography and found to be highly specific for the acidic nsLTP-like proteins from the embryogenic suspension cultures. The application of the selected monoclonal antibodies for localization and elucidation of the role of the acidic nsLTP-like proteins in vivo is discussed.