Ganglioside GD2-specific trifunctional surrogate antibody Surek demonstrates therapeutic activity in a mouse melanoma model.

BACKGROUND: Trifunctional bispecific antibodies (trAb) are a special class of bispecific molecules recruiting and activating T cells and accessory immune cells simultaneously at the targeted tumor. The new trAb Ektomab that targets the melanoma-associated ganglioside antigen GD2 and the signaling molecule human CD3 (hCD3) on T cells demonstrated potent T-cell activation and tumor cell destruction in vitro. However, the relatively low affinity for the GD2 antigen raised the question of its therapeutic capability. To further evaluate its efficacy in vivo it was necessary to establish a mouse model. METHODS: We generated the surrogate trAb Surek, which possesses the identical anti-GD2 binding arm as Ektomab, but targets mouse CD3 (mCD3) instead of hCD3, and evaluated its chemical and functional quality as a therapeutic antibody homologue. The therapeutic and immunizing potential of Surek was investigated using B78-D14, a B16 melanoma transfected with GD2 and GD3 synthases and showing strong GD2 surface expression. The induction of tumor-associated and autoreactive antibodies was evaluated. RESULTS: Despite its low affinity of approximately 10(7) M(-1) for GD2, Surek exerted efficient tumor cell destruction in vitro at an EC(50) of 70 ng/ml [0.47 nM]. Furthermore, Surek showed strong therapeutic efficacy in a dose-dependent manner and is superior to the parental GD2 mono-specific antibody, while the use of a control trAb with irrelevant target specificity had no effect. The therapeutic activity of Surek was strictly dependent on CD4(+) and CD8(+) T cells, and cured mice developed a long-term memory response against a second challenge even with GD2-negative B16 melanoma cells. Moreover, tumor protection was associated with humoral immune responses dominated by IgG2a and IgG3 tumor-reactive antibodies indicating a Th1-biased immune response. Autoreactive antibodies against the GD2 target antigen were not induced. CONCLUSION: Our data suggest that Surek revealed strong tumor elimination and anti-tumor immunization capabilities. The results warrant further clinical development of the human therapeutic equivalent antibody Ektomab.

Expression-based identification of genetic determinants of the bacterial symbiosis 'Chlorochromatium aggregatum'.

The phototrophic consortium 'Chlorochromatium aggregatum' is a highly structured association of green sulfur bacterial epibionts surrounding a central, motile bacterium and is the most specific symbiosis currently known between two phylogenetically distinct bacterial species. Genes and gene products potentially involved in the symbiotic interaction were identified on the genomic, transcriptomic and proteomic level. As compared with the 11 available genomes of free-living relatives, only 186 open reading frames were found to be unique to the epibiont genome. 2-D differential gel electrophoresis (2-D DIGE) of the soluble proteomes recovered 1612 protein spots of which 54 were detected exclusively in consortia but not in pure epibiont cultures. Using mass spectrometry analyses, the 13 most intense of the 54 spots could be attributed to the epibiont. Analyses of the membrane proteins of consortia, of consortia treated with cross-linkers and of pure cultures indicated that a branched chain amino acid ABC-transporter binding protein is only expressed in the symbiotic state of the epibiont. Furthermore, analyses of chlorosomes revealed that an uncharacterized 11 kDa epibiont protein is only expressed during symbiosis. This protein may be involved in the intracellular sorting of chlorosomes. Application of a novel prokaryotic cDNA suppression subtractive hybridization technique led to identification of 14 differentially regulated genes, and comparison of the transcriptomes of symbiotic and free-living epibionts indicated that 328 genes were differentially transcribed. The three approaches were mostly complementary and thereby yielded a first inventory of 352 genes that are likely to be involved in the bacterial interaction in 'C. aggregatum'. Notably, most of the regulated genes encoded components of central metabolic pathways whereas only very few (7.5%) of the unique 'symbiosis genes' turned out to be regulated under the experimental conditions tested. This pronounced regulation of central metabolic pathways may serve to fine-tune the symbiotic interaction in 'C. aggregatum' in response to environmental conditions.

Mass spectrometric characterization of membrane integral low molecular weight proteins from photosystem II in barley etioplasts.

In Photosystem II (PSII), a high number of plastid encoded and membrane integral low molecular weight proteins smaller than 10 kDa, the proteins PsbE, F, H, I, J, K, L, M, N, Tc, Z and the nuclear encoded PsbW, X, Y1, Y2 proteins have been described. Here we show that all low molecular weight proteins of PSII already accumulate in the etioplast membrane fraction in darkness, whereas PsaI and PsaJ of photosystem I (PSI) represent the only low molecular weight proteins that do not accumulate in darkness. We found by BN-PAGE separation of membrane protein complexes and selective MS that the accumulation of one-helix proteins from PSII is light independent and occurs in etioplasts. In contrast, in chloroplasts isolated from light-grown plants, low molecular weight proteins were found to specifically accumulate in PSI and II complexes. Our results demonstrate how plants grown in darkness prepare for the induction of chlorophyll dependent photosystem assembly upon light perception. We anticipate that our investigation will provide the essential means for the analysis of protein assembly in any membrane utilizing low molecular weight protein subunits.

Toward a mechanism for biliprotein lyases: revisiting nucleophilic addition to phycocyanobilin.

Biliprotein lyases attach linear-tetrapyrrolic bilins covalently to apoproteins, which is a prerequisite for the assembly of phycobiliproteins into phycobilisomes, the light-harvesting complexes of cyanobacteria. On the basis of the addition of thiol and imidazole to phycocyanobilin, we propose a generalized lyase reaction mechanism. The adducts contain isomerized phycocyanobilin that can be transferred by the lyase to apoproteins by either back-isomerization, generating phycocyanobilin-containing proteins, or direct transfer, generating phycoviolobilin-containing proteins.

Organelle proteomics.

The proteome of the cell is at the frontier of being too complex for proteomic analysis. Organelles provide a step up. Organelles compartmentalize the cell enabling a proteome, physiology and metabolism analysis in time and in space. Protein complexes separated by electrophoresis have been identified as the next natural level to characterize the organelles' compartmentalized membrane and soluble proteomes by mass spectrometry. Work on mitochondria and chloroplasts has shown where we are in the characterization of complex proteomes to understand the network of endogenous and extrinsic factors which regulate growth and development, adaptation and evolution.

Lil3 assembles as chlorophyll-binding protein complex during deetiolation.

Dark-grown angiosperm seedlings are etiolated and devoid of chlorophyll. Deetiolation is triggered by light leading to chlorophyll dependent accumulation of the photosynthetic machinery. The transfer of chlorophyll to the chlorophyll-binding proteins is still unclear. We demonstrate here that upon illumination of dark-grown barley seedlings, two new pigment-binding protein complexes are de novo accumulated. Pigments bound to both complexes are identified as chlorophyll a and protochlorophyll a. By auto-fluorescence tracking and mass spectrometry, we show that exclusively Lil3 is the pigment-binding complex subunit in both complexes.

Identification and analysis of four candidate symbiosis genes from 'Chlorochromatium aggregatum', a highly developed bacterial symbiosis.

The consortium 'Chlorochromatium aggregatum' currently represents the most highly developed interspecific association between prokaryotes. It consists of green sulfur bacteria, so-called epibionts, which surround a central, motile, chemotrophic bacterium. Four putative symbiosis genes of the epibiont were recovered by suppression subtractive hybridization and bioinformatics approaches. These genes are transcribed constitutively and do not occur in the free-living relatives of the epibiont. The haemagglutinin-like putative gene products of open reading frames (ORFs) Cag0614 and Cag0616 are unusually large and contain repetitive regions and RGD tripeptides. Cag0616 harbours two betagamma-crystalline Greek key motifs. Cag1920 codes for a putative haemolysin whereas the gene product of Cag1919 is a putative RTX-like protein. Based on detailed analyses of Cag1919, the C-terminal amino acid sequence comprises six repetitions of the motif GGXGXD predicted to form a Ca(2+)-binding beta roll. Intact 'C. aggregatum' consortia disaggregated upon the addition of EGTA or pyrophosphate, but stayed intact in the presence of various lectine-binding sugars or proteolytic enzymes. Unlike other RTX toxins, a gene product of Cag1919 could not be detected by (45)Ca(2+) autoradiography, indicating a low abundance of the corresponding protein in the cells. The RTX-type C-terminus coded by Cag1919 exhibited a significant similarity to RTX modules of various proteobacterial proteins, suggesting that this putative symbiosis gene has been acquired via horizontal gene transfer from a proteobacterium.

Cytochrome b6f is a dimeric protochlorophyll a binding complex in etioplasts.

The cytochrome b6f complex is a dimeric protein complex that is of central importance for photosynthesis to carry out light driven electron and proton transfer in chloroplasts. One molecule of chlorophyll a was found to associate per cytochrome b6f monomer and the structural or functional importance of this is discussed. We show that etioplasts which are devoid of chlorophyll a already contain dimeric cytochrome b6f. However, the phytylated chlorophyll precursor protochlorophyll a, and not chlorophyll a, is associated with subunit b6. The data imply that a phytylated tetrapyrrol is an essential structural requirement for assembly of cytochrome b6f.

Localization of 13 one-helix integral membrane proteins in photosystem II subcomplexes.

Photosystem II is a multimeric protein complex of the thylakoid membrane in chloroplasts. Approximately half of the at least 26 different integral membrane protein subunits have molecular masses lower than 10 kDa. After one-dimensional (1D) or two-dimensional (2D) polyacrylamide gel electrophoresis (PAGE) separation, followed by enzymatic digestion of detected proteins, hardly any of these low-molecular-weight (LMW) subunits are detectable. Therefore, we developed a method for the analysis of highly hydrophobic LMW proteins. Intact proteins are extracted from acrylamide gels using a mixture of formic acid and organic solvent, precipitated with acetone, and analyzed by "top-down" mass spectrometry (MS). After offline nanoESI (electrospray ionization) MS, all LMW one-helix proteins from photosystem II were detected. In the four detected photosystem II supercomplexes of Nicotiana tabacum wild-type plants, 11 different one-helix proteins were identified as PsbE, -F, -H, -I, -K, -L, -M, -Tc, -W, and two isoforms of PsbX. The proteins PsbJ, -Y1, and -Y2 were localized in the buffer front after blue native (BN) PAGE, indicating their release during solubilization. Assembled PsbW is detected exclusively in supercomplexes, whereas it is absent in photosystem II core complexes, corroborating the protein's function for assembly of the light-harvesting complexes. This approach will substantiate gel-blot immunoanalysis for localization and identification of LMW protein subunits in any membrane protein complex.

Proteomic comparison of etioplast and chloroplast protein complexes.

Angiosperms grown in darkness develop etioplasts during skotomorphogenesis. It is well known that etioplasts accumulate large quantities of protochlorophyllideoxidoreductase, are devoid of chlorophyll and are the site to assemble the photosynthetic machinery during photomorphogenesis. Proteomic investigation of the membrane protein complexes by Native PAGE, in combination with CyDye labelling and mass spectrometric analysis revealed that etioplasts and chloroplasts share a number of membrane protein complexes characteristic for electron transport, chlorophyll and protein synthesis as well as fatty acid biosynthesis. The complex regulatory function in both developmental states is discussed.

Structural and functional characterization of the trifunctional antibody catumaxomab.

The Triomab family of trifunctional, bispecific antibodies that maintain an IgG-like shape are novel tumor targeting agents. These chimeras consist of two half antibodies, each with one light and one heavy chain, that originate from parental mouse IgG2a and rat IgG2b isotypes. This combination allows cost-effective biopharmaceutical manufacturing at an industrial scale since this specific mouse/rat isotype combination favors matching of corresponding antibody halves during production by means of quadroma technology. Whereas every Triomab family member is composed of an anti-CD3 rat IgG2b half antibody for T cell recognition, the antigen binding site presented by the mouse IgG2a isotype is exchangeable. Several Triomab antibodies have been generated that bind to tumor-associated antigens, e.g., EpCAM (catumaxomab), HER2/neu (ertumaxomab), CD20 (FBTA05), gangliosides GD2/GD3 (Ektomun), on appropriate tumor target cells associated with carcinomas, lymphomas or melanomas. Catumaxomab (Removab) was launched in Europe for treatment of malignant ascites in April 2009. Here, we report the structural and functional characterization of this product. Mass spectrometry revealed an intact mass of 150511 Dalton (Da) and 23717 Da, 24716 Da, 51957 Da and 52019 Da of the reduced and alkylated rat light chain, mouse light chain, rat heavy chain, mouse heavy chain chains, respectively. The observed masses were in agreement with the expected masses based on the amino acid sequence obtained from cDNA sequencing. The glycosylation profile was similar to other human IgG consisting of biantennary oligosaccharides with different numbers of terminal galactose. CD spectroscopy showed mainly beta-sheets secondary structure that is typical for IgG antibodies. Binding measurement revealed the unique trifunctional features of catumaxomab. Other analytical tools were used to evaluate characteristics of catumaxomab preparations, including the presence of isoforms and aggregates.

Identification of the N-termini of NADPH : protochlorophyllide oxidoreductase A and B from barley etioplasts (Hordeum vulgare L.).

The N-termini of the NADPH : protochlorophyllide oxidoreductase (POR) proteins A and B from barley and POR from pea were determined by acetylation of the proteins and selective isolation of the N-terminal peptides for mass spectrometry de novo sequence analysis. We show that the cleavage sites between the transit peptides and the three mature POR proteins are homologous. The N-terminus in PORA is V48, that in PORB is A61, and that in POR from pea is E64. For the PORB protein, two additional N-termini were identified as A62 and A63, with decreased signal intensity of the corresponding N-terminal peptides. The results show that the transit peptide of PORA is considerably shorter than previously reported and predicted by ChloroP. A pentapeptide motif that has been characterized as responsible for binding of protochlorophyllide to the transit peptide of PORA [Reinbothe C, Pollmann S, Phetsarath-Faure P, Quigley F, Weisbeek P & Reinbothe S (2008) Plant Physiol148, 694-703] is shown here to be part of the mature PORA protein.

Phycourobilin in trichromatic phycocyanin from oceanic cyanobacteria is formed post-translationally by a phycoerythrobilin lyase-isomerase.

Most cyanobacteria harvest light with large antenna complexes called phycobilisomes. The diversity of their constituting phycobiliproteins contributes to optimize the photosynthetic capacity of these microorganisms. Phycobiliprotein biosynthesis, which involves several post-translational modifications including covalent attachment of the linear tetrapyrrole chromophores (phycobilins) to apoproteins, begins to be well understood. However, the biosynthetic pathway to the blue-green-absorbing phycourobilin (lambda(max) approximately 495 nm) remained unknown, although it is the major phycobilin of cyanobacteria living in oceanic areas where blue light penetrates deeply into the water column. We describe a unique trichromatic phycocyanin, R-PC V, extracted from phycobilisomes of Synechococcus sp. strain WH8102. It is evolutionarily remarkable as the only chromoprotein known so far that absorbs the whole wavelength range between 450 and 650 nm. R-PC V carries a phycourobilin chromophore on its alpha-subunit, and this can be considered an extreme case of adaptation to blue-green light. We also discovered the enzyme, RpcG, responsible for its biosynthesis. This monomeric enzyme catalyzes binding of the green-absorbing phycoerythrobilin at cysteine 84 with concomitant isomerization to phycourobilin. This reaction is analogous to formation of the orange-absorbing phycoviolobilin from the red-absorbing phycocyanobilin that is catalyzed by the lyase-isomerase PecE/F in some freshwater cyanobacteria. The fusion protein, RpcG, and the heterodimeric PecE/F are mutually interchangeable in a heterologous expression system in Escherichia coli. The novel R-PC V likely optimizes rod-core energy transfer in phycobilisomes and thereby adaptation of a major phytoplankton group to the blue-green light prevailing in oceanic waters.

Intermediate binding of phycocyanobilin to the lyase, CpeS1, and transfer to apoprotein.

The phycobilin: Cysteine-84-phycobiliprotein lyase, CpeS1, catalyzes phycocyanobilin (PCB) and phycoerythrobilin attachment to nearly all cysteine-84 (consensus sequence) binding sites of phycoerythrin, phycoerythrocyanin, phycocyanin and allophycocyanin (Zhao et al. (2007) Proc Natl Acad Sci 104:14300-14305). We now show that CpeS1 can bind PCB, as assayed by Ni(2+) chelating affinity chromatography. Binding is rapid, and the chromophore is bound in an extended conformation similar to that in phycobiliproteins but only poorly fluorescent. Upon addition of apo-biliproteins, the chromophore is transferred to the latter much slower ( approximately 1 h), indicating that chromophorylated CpeS1 is an intermediate in the enzymatic reaction. In addition, imidazole is bound to PCB, as shown by mass spectroscopy of tryptic digests of the intermediate CpeS1-PCB complex.

Sample preparation by in-gel digestion for mass spectrometry-based proteomics.

The proteomic characterization of proteins and protein complexes from cells and cell organelles is the next challenge for investigation of the cell. After isolation of the cell compartment, three steps have to be performed in the laboratory to yield information about the proteins present. The protein mixtures must be separated into single species, broken down into peptides, and, finally, identified by mass spectrometry. Most scientists engaged in proteomics separate proteins by electrophoresis. For characterization and identification of proteomes, mass spectrometry of peptides is the method of choice. To combine electrophoresis and mass spectrometry, sample preparation by "in-gel digestion" has been developed. Many procedures are available for in-gel digestion, which inspired us to review in-gel digestion approaches.

The proteome of the heterocyst cell wall in Anabaena sp. PCC 7120.

Anabaena sp. PCC 7120 is a filamentous cyanobacterium that serves as a model to analyze prokaryotic cell differentiation, evolutionary development of plastids, and the regulation of nitrogen fixation. The cell wall is the cellular structure in contact with the surrounding medium. To understand the dynamics of the cell wall proteome during cell differentiation, the cell wall from Anabaena heterocysts was enriched and analyzed. In line with the recently proposed continuity of the outer membrane along the Anabaena filament, most of the proteins identified in the heterocyst cell-wall fraction are also present in the cell wall of vegetative cells, even though the lipid content of both membranes is different.

Phycobilin:cystein-84 biliprotein lyase, a near-universal lyase for cysteine-84-binding sites in cyanobacterial phycobiliproteins.

Phycobilisomes, the light-harvesting complexes of cyanobacteria and red algae, contain two to four types of chromophores that are attached covalently to seven or more members of a family of homologous proteins, each carrying one to four binding sites. Chromophore binding to apoproteins is catalyzed by lyases, of which only few have been characterized in detail. The situation is complicated by nonenzymatic background binding to some apoproteins. Using a modular multiplasmidic expression-reconstitution assay in Escherichia coli with low background binding, phycobilin:cystein-84 biliprotein lyase (CpeS1) from Anabaena PCC7120, has been characterized as a nearly universal lyase for the cysteine-84-binding site that is conserved in all biliproteins. It catalyzes covalent attachment of phycocyanobilin to all allophycocyanin subunits and to cysteine-84 in the beta-subunits of C-phycocyanin and phycoerythrocyanin. Together with the known lyases, it can thereby account for chromophore binding to all binding sites of the phycobiliproteins of Anabaena PCC7120. Moreover, it catalyzes the attachment of phycoerythrobilin to cysteine-84 of both subunits of C-phycoerythrin. The only exceptions not served by CpeS1 among the cysteine-84 sites are the alpha-subunits from phycocyanin and phycoerythrocyanin, which, by sequence analyses, have been defined as members of a subclass that is served by the more specialized E/F type lyases.

Lyase activities of CpcS- and CpcT-like proteins from Nostoc PCC7120 and sequential reconstitution of binding sites of phycoerythrocyanin and phycocyanin beta-subunits.

Genes all5292 (cpcS2) and alr0617 (cpcS1) in the cyanobacterium Nostoc PCC7120 are homologous to the biliprotein lyase cpcS, and genes all5339 (cpcT1) and alr0647 (cpcT2) are homologous to the lyase cpcT. The functions of the encoded proteins were screened in vitro and in a heterologous Escherichia coli system with plasmids conferring biosynthesis of the phycocyanobilin chromophore and of the acceptor proteins beta-phycoerythrocyanin (PecB) or beta-phycocyanin (CpcB). CpcT1 is a regioselective biliprotein lyase attaching phycocyanobilin exclusively to cysteine beta155 but does not discriminate between CpcB and PecB. The in vitro reconstitutions required no cofactors, and kinetic constants were determined for CpcT1 under in vitro conditions. No lyase activity was found for the lyase homologues CpcS2 and CpcT2, but complexes are formed in vitro between CpcT1 and CpcS1, CpcT2, or PecE (subunit of phycoviolobilin:alpha-phycoerythrocyanin isomerase lyase). The genes coding the inactive homologues, cpcS2 and cpcT2, are transcribed in N-starved Nostoc. In sequential binding experiments with CpcT1 and CpcS1, a chromophore at cysteine 84 inhibited the subsequent attachment to cysteine 155, whereas the inverse sequence generates subunits carrying both chromophores.