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Microneme protein 2 (MIC2) and its associated protein M2AP are pivotal for the gliding motility and host cell invasion by Toxoplasma gondii. In our prior work, we showed that M2AP binds specifically to the sixth TSR domain of MIC2, with this interaction mediated dominantly by the hotspot residue H620 situated at the center of TSR6. To delve deeper into the functional significance of H620 and explore the dynamic behavior of Y602, we conducted molecular dynamic (MD) simulations of the Toxoplasma TSR6-M2AP complex, encompassing both wild-type and mutant forms. Our findings underscore the critical role of H620 within TSR6, particularly its hydrogen bond interaction with K72 of M2AP. The H620A mutation disrupts the nearby hydrophobic network while minimally affecting other hydrophilic interactions. Furthermore, our data reveal a highly conserved binding pose between M2AP and TSR6 across different species, consistent with previous trans-genera studies, thereby offering insights for future strategies in infection control development., (© 2024 Wiley Periodicals LLC.)

Pediatric small round cell tumors (PSRCTs) constitute a large proportion of childhood malignancies with overlapping diagnostic and clinical features but radically different therapies. Here, we report a case of 16-year-old male child presenting with diffuse abdominal and mediastinal mass, axillary lymphadenopathy, and pleural effusion. Bone marrow aspirate showed near total replacement by small round malignant cells. The bone marrow biopsy showed interstitial infiltration by malignant cells, which were CD45− CD3− CD20− MIC2+ FLI1+ and diagnosis of Ewing's sarcoma was established. In contrast, flowcytometric immunophenotyping of the bone marrow aspirate showed CD45− cells, which were CD19+ cytCD79a+ CD10+ CD81+ CD38+ HLA-DR+ CD22+ CD20− consistent with B-cell acute lymphoblastic leukemia (B-ALL). The extended immunostaining panel on bone marrow biopsy also showed positivity for cytCD79a, CD10, CD19, and BCL-2, whereas fluorescent in-situ hybridization for EWSR1 gene rearrangement was negative. Thus, a final diagnosis of CD45− FLI1+ MIC2+ B-ALL was established. Rare cases of CD45− B-ALL with immunoreactivity for MIC2 and Friend leukemia virus integration 1 (FLI1) have posed a diagnostic challenge for PSRCTs in the recent past. This case report highlights the role of multimodality approach in establishing a correct diagnosis in CD45− PSRCTs to ensure definitive therapy and better clinical outcome.

CD99 (MIC2; single-chain type-1 glycoprotein) is a heavily O-glycosylated transmembrane protein (32 kDa) present on leukocytes and activated endothelium. Expression of CD99 on endothelium is important in lymphocyte diapedesis. CD99 is a diagnostic marker for Ewing's Sarcoma (EWS), as it is highly expressed by these tumors. It has been reported that CD99 can affect the migration, invasion and metastasis of tumor cells. Our results show that CD99 is also highly expressed in the tumor vasculature of most solid tumors. Furthermore, we found that in vitro CD99 expression in cultured endothelial cells is induced by starvation. Targeting of murine CD99 by a conjugate vaccine, which induced antibodies against CD99 in mice, resulted in inhibition of tumor growth in both a tumor model with high CD99 (Os-P0109 osteosarcoma) and low CD99 (CT26 colon carcinoma) expression. We demonstrated that vaccination against CD99 is safe, since no toxicity was observed in mice with high antibody titers against CD99 in their sera during a period of almost 11 months. Targeting of CD99 in humans is more complicated due to the fact that the human and mouse CD99 protein are not identical. We are the first to show that growth factor activated endothelial cells express a distinct human CD99 isoform. We conclude that our observations provide an opportunity for specific targeting of CD99 isoforms in human tumor vasculature.

CD99 (MIC2; single-chain type-1 glycoprotein) is a heavily O-glycosylated transmembrane protein (32 kDa) present on leukocytes and activated endothelium. Expression of CD99 on endothelium is important in lymphocyte diapedesis. CD99 is a diagnostic marker for Ewing's Sarcoma (EWS), as it is highly expressed by these tumors. It has been reported that CD99 can affect the migration, invasion and metastasis of tumor cells. Our results show that CD99 is also highly expressed in the tumor vasculature of most solid tumors. Furthermore, we found that in vitro CD99 expression in cultured endothelial cells is induced by starvation. Targeting of murine CD99 by a conjugate vaccine, which induced antibodies against CD99 in mice, resulted in inhibition of tumor growth in both a tumor model with high CD99 (Os-P0109 osteosarcoma) and low CD99 (CT26 colon carcinoma) expression. We demonstrated that vaccination against CD99 is safe, since no toxicity was observed in mice with high antibody titers against CD99 in their sera during a period of almost 11 months. Targeting of CD99 in humans is more complicated due to the fact that the human and mouse CD99 protein are not identical. We are the first to show that growth factor activated endothelial cells express a distinct human CD99 isoform. We conclude that our observations provide an opportunity for specific targeting of CD99 isoforms in human tumor vasculature. [ABSTRACT FROM AUTHOR]

Abstract Background Toxoplasma gondii is an obligate intracellular protozoan, causing the important zoonosis toxoplasmosis. This parasite utilizes a unique form of locomotion called gliding motility to find and invade host cells. The micronemal adhesin MIC2 plays critical roles in these processes by binding to substrates and host cell receptors using its extracellular adhesive domains. Although MIC2 is known to mediate important interactions between parasites and host cells during invasion, the specific host proteins interacting with MIC2 have not been clearly identified. In this study, we used a yeast-two-hybrid system to search for host proteins that interact with MIC2. Methods Different adhesive domains of MIC2 were cloned into the pGBKT7 vector and expressed in fusion with the GAL4 DNA-binding domain as baits. Expression of bait proteins in yeast cells was analyzed by immuno-blotting and their autoactivation was tested via comparison with the pGBKT7 empty vector, which expressed the GAL4 DNA binding-domain only. To identify host proteins interacting with MIC2, a mouse cDNA library cloned into a GAL4 activation-domain expressing vector was screened by yeast-two-hybrid using the integrin-like A domain of MIC2 (residues 74–270) as bait. After initial screening and exclusion of false positive hits, positive preys were sequenced and analyzed using BLAST analysis and Gene Ontology Classifications. Results Two host proteins that had not previously been reported to interact with T. gondii MIC2 were identified: they are LAMTOR1 (late endosomal/lysosomal adaptor, MAPK and mTOR activator 1) and RNaseH2B (ribonuclease H2 subunit B). Gene Ontology analysis indicated that these two proteins are associated with many cellular processes, such as lysosome maturation, signaling transduction, and RNA catabolism. Conclusion This study is the first one to report interactions between Toxoplasma gondii MIC2 and two host proteins, LAMTOR1 and RNaseH2B. The data will help us to gain a better understanding of the function of MIC2 and suggest that MIC2 may play roles in modulating host signal transduction and other biological processes in addition to binding host cells.

Lymphoblastic lymphoma (LBL) is a rare hematologic malignancy that originates from immature lymphocytes and usually expresses terminal deoxynucleotidyl transferase (TdT). Here, we report a case of TdT-negative B-LBL. A 71-year-old male patient presented to a hospital with shortness of breath. His chest computed tomography showed a mediastinal mass. Tumor cells did not express TdT but expressed MIC2, which led to LBL diagnosis. MIC2 is a useful marker for LBL diagnosis.

The apicomplexan parasite Toxoplasma gondii must invade host cells to continue its lifecycle. It invades different cell types using an actomyosin motor that is connected to extracellular adhesins via the bridging protein fructose-1,6-bisphosphate aldolase. During invasion, aldolase serves in the role of a structural bridging protein, as opposed to its normal enzymatic role in the glycolysis pathway. Crystal structures of the homologous Plasmodium falciparum fructose-1,6-bisphosphate aldolase have been described previously. Here, T. gondii fructose-1,6-bisphosphate aldolase has been crystallized in space group P22121, with the biologically relevant tetramer in the asymmetric unit, and the structure has been determined via molecular replacement to a resolution of 2.0 Å. An analysis of the quality of the model and of the differences between the four chains in the asymmetric unit and a comparison between the T. gondii and P. falciparum aldolase structures is presented. [ABSTRACT FROM AUTHOR]

Recombinant antigenic proteins of Toxoplasma gondii are alternative source of antigens which are easily obtainable for serodiagnosis of toxoplasmosis. In this study, highly antigenic secretory organellar proteins, dense granular GRA2 and GRA3, rhoptrial ROP2, and micronemal MIC2, were analyzed by bioinformatics approach to express as water- soluble forms of antigenic domains. The transmembrane region and disorder tendency of 4 secretory proteins were predicted to clone the genes into pGEX-4T-1 vector . Recombinant plasmids were transformed into BL21 (DE3) pLysS E. coli, and GST fusion proteins were expressed with IPTG. As a result, GST fusion proteins with GRA225-105, GRA339-138, ROP2324- 561, and MIC21-284 domains had respectively higher value of IgG avidity. The rGST-GRA225-105 and rGST-GRA339-138 were soluble, while rGST-ROP2324-561 and rGST-MIC21-284 were not. GRA231-71, intrinsically unstructured domain (IUD) of GRA2, was used as a linker to enhance the solubility. The rGST-GRA231-71-ROP2324-561, a chimeric protein, appeared to be soluble. Moreover, rGST-GRA231-71-MIC21-284 was also soluble and had higher IgG avidity comparing to rGST-MIC21-284. These 4 highly expressed and water-soluble recombinant antigenic proteins may be promising candidates to improve the serodiagnosis of toxoplasmosis in addition to the major surface antigen of SAG1. [ABSTRACT FROM AUTHOR]

A 22-year-old man presented with tenderness and swelling of the left lateral part of the orbit. Computed tomography revealed a left intraorbital mass measuring 3 cm × 3 cm involving the left lateral wall of the orbit and the greater wing of the left sphenoid bone. Magnetic resonance imaging revealed that the intraorbital mass was extraneuroaxial. During surgery, the tumor was seen to arise from the lateral wall of the orbit and infiltrate into the left temporal muscle. Following the surgery, the patient was administered radiation therapy for the whole cranium and chemotherapy for the residual tumors. However, the tumor recurred, and the patient died about 2 years following the first surgery because the tumor had metastasized to the lung. On light microscopy, the tumor cells were closely packed with uniform, small, and round cells. Immunohistochemical studies showed that the tumor cell membrane stained positive for MIC2. Furthermore, the MIB-1 labeling index was 36.2%. On electron microscopy, small quantities of cytoplasm containing glycogen accumulations without neurosecretory granules and neurofilaments were observed. Based on these results, the tumor was diagnosed to be primary Ewing’s sarcoma. Primary orbital Ewing’s sarcoma of the skull has been considered to be extremely rare, and a review of the literature was performed. [ABSTRACT FROM AUTHOR]

Supratentorial primitive neuroectodermal tumors (S-PNET) that develop in children have recently been classified into two types: central-type PNET (C-PNET), which has been reported over the years, and peripheral-type PNET (P-PNET), which develops intracranially and was referred to as Ewing's sarcoma in the past. P-PNET is fundamentally a malignant tumor, but the patient reported here represents a case of long-term survival from onset without recurrence. At the age of 21 months, a male infant developed a cranial bone deformity and symptoms of high intracranial pressure. A CT scan revealed a cystic tumor attaching to the falx, and cyst drainage operation was immediately performed. The intracranial tumor was then resected. The tumor was an intradural extramedullary tumor, and it was totally excised with the falx attachment. The tumor was initially diagnosed as a neuroblastoma, and postoperative treatment consisted of administration of radiotherapy and chemotherapy using cyclophosphamide and vincristine. Twenty years have now passed without any recurrence. Recent repeated performance of histopathological analysis resulted in a diagnosis of P-PNET. In recent years, studies in molecular biology have demonstrated that P-PNET involves the EWS-FLI1 chimeric gene, and immunohistochemical staining has shown P-PNET to be MIC2 positive. P-PNET also differs from C-PNET with regard to prognosis, and for this reason it is believed that P-PNET and C-PNET should be considered separate entities. That is, in spite of the fact that P-PNET is a malignant tumor, patient survival can be comparatively long. Because P-PNET originates intracranially, it is fundamentally an intradural extramedullary tumor. For this reason, treatment should consist of surgical excision that is as complete as possible, followed by appropriate radiotherapy and chemotherapy. This approach can be expected to result in the patient's long-term survival. [ABSTRACT FROM AUTHOR]

Abstract: The phylum Apicomplexa contains parasites responsible for a variety of diseases including malaria, cryptosporidiosis, and toxoplasmosis. One of the common features of these parasites is that they contain a set of apical organelles whose sequential secretion is required for the invasion of host cells. Microneme proteins are the main adhesins involved in the attachment to the host cell surface by apicomplexans. The microneme protein MIC2, produced by Toxoplasma gondii, is conserved in apicomplexans and serves as a model to understand the first steps of invasion by the phylum. New data about the structure–function relationship of MIC2 reinforce the critical role of this protein in the successful invasion of cells by Toxoplasma and reveal potential therapeutic targets that may be used to control toxoplasmosis. [Copyright &y& Elsevier]

Abstract: The human pseudoautosomal region 1 (PAR1) is essential for the obligatory X–Y crossover in male meiosis. Despite its critical role, comparative studies of human and mouse pseudoautosomal genes have been limited owing to the scarcity of genes conserved between the two species. Human CD99 is a 32-kDa cell surface protein that is encoded by the MIC2 gene localized to the PAR1. Although several sequences such as CD99L2, PBDX, and CD99L1 are related to CD99, its murine ortholog, Cd99, has not yet been identified. Here we report a novel mouse Cd99, designated D4, which shows overall sequence homology to CD99, with the highest conservation between the two genes being found in the transmembrane regions. In addition, the D4 protein displays biochemical characteristics, functional homology, and expression patterns similar to those of CD99. The D4 gene is localized on an autosome, chromosome 4, reflecting a common mapping feature with other mouse orthologs of human PAR1 genes. Furthermore, a phylogenetic analysis of CD99-related genes confirmed that the D4 gene is indeed an ortholog of CD99 and exhibits the accelerated evolution pattern of CD99 orthologs, as compared to the CD99L2 orthologs. On the basis of these findings, we suggest that CD99 belongs to the ancient PAR genes, and that the rapid interspecies divergence of its present sequence and map position is due to a high recombination frequency and the occurrence of chromosomal translocation, supporting the addition–attrition hypothesis for PAR evolution. [Copyright &y& Elsevier]

Apicomplexans such as Toxoplasma gondii actively invade host cells using a unique parasite-dependent mechanism termed gliding motility. Calcium-mediated protein secretion by the parasite has been implicated in this process, but the precise role of calcium signaling in motility remains unclear. Here we used calmidazolium as a tool to stimulate intracellular calcium fluxes and found that this drug led to enhanced motility by T. gondii. Treatment with calmidazolium increased the duration of gliding and resulted in trails that were twice as long as those formed by control parasites. Calmidazolium also increased microneme secretion by T. gondii, and studies with a deletion mutant of the accessory protein m2AP specifically implicated that adhesin MIC2 was important for gliding. The effects of calmidazolium on gliding and secretion were due to increased release of calcium from intracellular stores and calcium influx from the extracellular milieu. In addition, we demonstrate that calmidazolium-stimulated increases in intracellular calcium were highly dynamic, and that rapid fluxes in calcium levels were associated with parasite motility. Our studies suggest that oscillations in intracellular calcium levels may regulate microneme secretion and control gliding motility in T. gondii. [ABSTRACT FROM AUTHOR]

An unusual case of malignant peripheral nerve sheath tumor (MPNST) arising in the posterior mediastinum of a 59-year-old man is reported. Histopathologically, the tumor showed an admixture of a dense proliferation of small round cells resembling a primitive neuroectodermal tumor (PNET) and a pleomorphic spindlle cell sarcomatous area. Abortive rosettes, primitive neural tube-like structures, and a few glandular structures were found in the small round cell area. Small round cells were immunoreactive for neural cell adhesion molecule and synaptophysin, but were not immunoreactive for MIC2 and neuron-specific enolase. Pleomorphic spindle cells were occasionally arranged in a storiform pattern and were diffusely immunoreactive for S-100 protein. The MPNST of small round cell type is distinguishable from PNET by its negative immunoreactivity for MIC2, and the present tumor is assumed to be derived from primitive neuroectodermal cells in the peripheral nerve capable of bidirectional (neuron and Schwann cell) differentiation. [ABSTRACT FROM AUTHOR]

Primitive neuroectodermal tumors (PNET) occur either in the central nervous system (CNS; central PNET, cPNET) or in the peripheral sites (peripheral PNET, pPNET). Recent molecular approaches have been defining a new concept of PNET, that is, the pPNET including Ewing's sarcoma (ES) which expresses MIC2 glycoprotein and shows the specific chimeric gene of EWS-FLI1. The expression of MIC2 and the genetic rearrangement of EWS-FLI1 are considered to be highly specific to the pPNET/ES. This study examined the expression of MIC2 and EWS-FLI1 gene by means of immunohistochemistry and reverse transcriptase–polymerase chain reaction (RT-PCR) on various small round cell tumors originating in the CNS or non-CNS organs. All peripheral PNET tested expressed MIC2 and were positive for EWS-FLI1 (11/11). In contrast, all cPNET and other blastic CNS tumors were negative for MIC2: medulloblastoma (0/3), cerebral PNET (0/2), spinal PNET (0/2), glioblastoma (0/2), retinoblastoma (0/3), and pineoblastoma (0/2). These MIC2-negative tumors were also negative for the chimeric gene product of EWS-FLI1. Interestingly, one PNET originating in the intracranial dura mater was positive for both MIC2 and EWS-FLI1 fusion gene. The results indicate that cPNET lacks any genetic or protein markers, except for a meningeal PNET which falls into the same phenotypic spectrum of pPNET. [ABSTRACT FROM AUTHOR]

We have recently shown that CD99 (MIC2) is differentially expressed during normal early B-cell development in the bone marrow (BM). Since immature B-cell precursors (BCP) are assumed to correspond to some extent to acute lymphoblastic leukaemia (ALL) and non-Hodgkin's lymphoma (NHL) cells with respect to patterns of phenotypic differentiation, we wondered whether the particular maturation-associated expression patterns of CD99 in the normal BCP stages were conserved also in malignant cells. Therefore we compared malignant and physiological B cells from paediatric ALL/NHL and normal BM samples with respect to CD99 expression using selective gating and semi-quantitative flow cytometry. Common-ALLs (n = 45) were similar to their corresponding, very immature BCPs (stage 1) in expressing very high levels of CD99. Most pre-B ALLs (n = 16) were also CD99hi and thus differed from the patterns found in normal cytoplasmic μ-chain+ (cμ+) pre-B cells (stage 2, CD99lo). In particular, we found that those pre-B-ALL cases which were CD34+ also showed higher CD99 expression than the CD34- cases. This prompted us to investigate the levels of CD99 in those rare normal BCPs which also coexpress CD34 and cμ; these cells, which are transitory from stage 1 to stage 2, were found also CD99hi, thus precisely reflecting the patterns of CD34+ pre-B ALLs. The blasts of Burkitt-type B-cell ALL/NHL samples (n = 13) expressed considerably less CD99, similarly to the more differentiated BCP stages 2 (cμ+) and 3 (surface μ-chain+). In summary, we found that paediatric B-lineage malignancies display remarkable synchrony regarding the levels of CD99 expression compared to their putative normal counterparts. [ABSTRACT FROM AUTHOR]

Background. Recent immunocytochemical and cytogenic studies have shown that Ewing's sarcoma and peripheral primitive neuroectodermal tumor (PNET) are probably related neoplasms of common neuroectodermal origin. The purpose of this study was to investigate the morphological characteristics of neural differentiation in these tumors. Methods. We investigated the ultrastructure of 17 cases of Ewing's sarcoma and related tumors in the specimens of 17 patients, and carried out immunohistochemical analysis. Results. Ultrastructurally, the cell process and basal laminae were frequently observed in all cases of an unclassified small round-cell type of malignant peripheral nerve sheath tumor (MPNST-related tumor). In contrast, a poorly developed cell process and fragmented basal lamina were rarely observed in Ewing's sarcoma or in PNET. Cytoplasmic neurosecretory granule-like particles were found in two specimens of Ewing's sarcoma and two of PNET, and were abundant in one MPNST-related tumor specimen. These ultrastructural neural features were not common in Ewing's sarcoma, and were occasionally present in PNET. There was a discrepancy between positivity for immuno-neural markers and ultrastructural neural features. Conclusion. The differential diagnosis between Ewing's sarcoma and PNET was based on the degree of their morphological neural differentiation. Thus, we consider that the diagnosis of PNET should be made on the basis of ultrastructural neural features, taking into account immunohistochemical and histological considerations. [ABSTRACT FROM AUTHOR]

Toxoplasma gondii is an obligate parasite that infects most warm blood animals. Micronemal proteins actively involves in the invasion process, where TgMIC2 and TgM2AP complex plays vital roles. Complete NMR assignments for major fragment of TgM2AP were successfully obtained. [ABSTRACT FROM AUTHOR]

Toxoplasma gondii is an intracellular parasite that causes disseminated infections that can produce neurological damage in fetuses and immunocompromised individuals. Microneme protein 2 (MIC2), a member of the thrombospondin-related anonymous protein (TRAP) family, is a secreted protein important for T. gondii motility, host cell attachment, invasion, and egress. MIC2 contains six thrombospondin type I repeats (TSRs) that are modified by C -mannose and O -fucose in Plasmodium spp. and mammals. Here, using MS analysis, we found that the four TSRs in T. gondii MIC2 with protein O -fucosyltransferase 2 (POFUT2) acceptor sites are modified by a dHexHex disaccharide, whereas Trp residues within three TSRs are also modified with C -mannose. Disruption of genes encoding either POFUT2 or the putative GDP-fucose transporter (NST2) resulted in loss of MIC2 O -fucosylation, as detected by an antibody against the GlcFuc disaccharide, and in markedly reduced cellular levels of MIC2. Furthermore, in 10-15% of the Δ pofut2 or Δ nst2 vacuoles, MIC2 accumulated earlier in the secretory pathway rather than localizing to micronemes. Dissemination of tachyzoites in human foreskin fibroblasts was reduced for these knockouts, which both exhibited defects in attachment to and invasion of host cells comparable with the Δ mic2 phenotype. These results, indicating that O -fucosylation of TSRs is required for efficient processing of MIC2 and for normal parasite invasion, are consistent with the recent demonstration that Plasmodium falciparum Δ pofut2 strain has decreased virulence and also support a conserved role for this glycosylation pathway in quality control of TSR-containing proteins in eukaryotes., (© 2019 Bandini et al.)

Background: Micronemal proteins of the thrombospondin-related anonymous protein (TRAP) family are believed to play essential roles during gliding motility and host cell invasion by apicomplexan parasites, and currently represent major vaccine candidates against Plasmodium falciparum , the causative agent of malaria. However, recent evidence suggests that they play multiple and different roles than previously assumed. Here, we analyse a null mutant for MIC2, the TRAP homolog in Toxoplasma gondii . Methods: We performed a careful analysis of parasite motility in a 3D-environment, attachment under shear stress conditions, host cell invasion and in vivo virulence. Results: We verified the role of MIC2 in efficient surface attachment, but were unable to identify any direct function of MIC2 in sustaining gliding motility or host cell invasion once initiated. Furthermore, we find that deletion of mic2 causes a slightly delayed infection in vivo, leading only to mild attenuation of virulence; like with wildtype parasites, inoculation with even low numbers of mic2 KO parasites causes lethal disease in mice. However, deletion of mic2 causes delayed host cell egress in vitro , possibly via disrupted signal transduction pathways. Conclusions: We confirm a critical role of MIC2 in parasite attachment to the surface, leading to reduced parasite motility and host cell invasion. However, MIC2 appears to not be critical for gliding motility or host cell invasion, since parasite speed during these processes is unaffected. Furthermore, deletion of MIC2 leads only to slight attenuation of the parasite., Competing Interests: Competing interests: No competing interests were disclosed.

Toxoplasma gondii parasites must actively invade host cells to propagate. Secretory microneme proteins have been shown to be important for both gliding motility and active invasion. MIC2-M2AP is a protein complex that is essential for productive motility and rapid invasion by binding to host cell surface receptors. To investigate the architecture of the MIC2 and M2AP complex, we identified the minimal domains sufficient for interaction and solved the NMR solution structure of the globular domain of M2AP. We found that M2AP adopts a modified galectin fold similar to the C-terminal domain of another microneme protein, MIC1. NMR and immunoprecipitation analyses implicated hydrophobic residues on one face of the M2AP galectin fold in binding to the membrane proximal sixth thrombospondin type I repeat domain of MIC2. Our findings provide a second example of a galectin fold adapted for microneme protein-protein interactions and suggest a conserved strategy for the assembly and folding of diverse protein complexes., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

Recombinant antigenic proteins of Toxoplasma gondii are alternative source of antigens which are easily obtainable for serodiagnosis of toxoplasmosis. In this study, highly antigenic secretory organellar proteins, dense granular GRA2 and GRA3, rhoptrial ROP2, and micronemal MIC2, were analyzed by bioinformatics approach to express as water-soluble forms of antigenic domains. The transmembrane region and disorder tendency of 4 secretory proteins were predicted to clone the genes into pGEX-4T-1 vector. Recombinant plasmids were transformed into BL21 (DE3) pLysS E. coli, and GST fusion proteins were expressed with IPTG. As a result, GST fusion proteins with GRA225-105, GRA339-138, ROP2324-561, and MIC21-284 domains had respectively higher value of IgG avidity. The rGST-GRA225-105 and rGST-GRA339-138 were soluble, while rGST-ROP2324-561 and rGST-MIC21-284 were not. GRA231-71, intrinsically unstructured domain (IUD) of GRA2, was used as a linker to enhance the solubility. The rGST-GRA231-71-ROP2324-561, a chimeric protein, appeared to be soluble. Moreover, rGST-GRA231-71-MIC21-284 was also soluble and had higher IgG avidity comparing to rGST-MIC21-284. These 4 highly expressed and water-soluble recombinant antigenic proteins may be promising candidates to improve the serodiagnosis of toxoplasmosis in addition to the major surface antigen of SAG1.

Background: The histological origin of the Ewing's family of tumors (EFT) is still not clear. Since these small cell bone tumors may originate from osteogenic stem cells, the presence of the CD99/MIC2 antigen, known to be overexpressed in EFT, was studied in a human osteoblast cell line in response to differentiation inducers., Methods: The HBA-71 monoclonal antibody directed to the CD99/MIC2 antigen was used to stain a human osteoblast cell line as well as the two EFT cell lines KAL and EW-2 after pretreatment of the cells with the differentiation inducers calcitriol and the histone deacetylase (HDAC) inhibitors sodium butyrate (NaB), sodium phenylacetate (NaPA) as well as N, N'-hexamethylen-bis-acetamide (HMBA). Alkaline phosphatase (ALP) levels were determined as cellular differentiation marker., Results: Significant expression of the CD99/MIC2 antigen, yielding a molecular weight of 32 kD in Western blotting, was found in the human osteoblast cell line. Pretreatment of the osteoblasts with calcitriol and HMBA increased ALP content and suppressed the CD99/MIC2 antigen. Calcitriol had no major effect on CD99/MIC2 expression of both EFT cell lines, but HMBA enhanced ALP activity in KAL cells and downregulated CD99/MIC2. EW-2 cells exhibited reduced levels of both CD99/MIC2 and ALP., Conclusions: This study supports the role of CD99/MIC2 as differentiation antigen of osteoblasts and a Ewing's sarcoma cell line with neuroectodermal phenotype. Response to calcitriol is absent or low in the two EFT cell lines tested.