The Role of Glycoconjugates in Mediating Human Fertilization and Induction of Fetomaternal Tolerance

Using the hemizona assay (HZA), a in vitro sperm-egg binding assay, we show that specific glycoconjugates known to inhibit immune cell interactions mediated by the selectins, potently block human sperm-egg binding. The selectin ligand sialyl Lewisx inhibits sperm binding in the HZA by 60% at a concentration of 1 mg/ml. Our data indicates that glycodelin-A, a endometrial glycoprotein known to block sperm-egg binding in the HZA at low concentrations expresses unusual fucosylated lacdiNAc type glycans. The fucosylated lacdiNAc type sugars have been previously shown to be 15-20 fold more potent ligands of E-selectin. Glycodelin-S a seminal plasma glycoform of glycodelin-A does not express such unusual glycans and is not contraceptive. These results support our hypothesis that human sperm-egg binding may involve a selectin-like event. Periodate oxidation under conditions that affect only the terminal sugar residues results in a 30-40% loss in sperm binding. Treatment of the ZP with neuraminidase and endo-β-galactosidase results in a 2.5 and 4 fold enhancement in sperm binding respectively. However, sequential treatment of the ZP with neuraminidase and periodate results in 80% decrease in sperm binding. These studies strongly indicate that ZP glycans are essential for mediating human gamete binding. Furthermore, efficient initial human sperm egg binding in vivo may require a prior activation event involving desialylation of the gametes. In this study we provide preliminary evidence that human gametes express the bisecting type glycans. Our studies indicate that these glycans potently inhibit natural killer (NK) cells, the predominant cell type expressed in the uterus during pregnancy. Other glycoconjugates like $\alpha$-fetoprotein, expressed in the uterus during pregnancy, also carry the bisecting-type glycans. Based on these observations we propose that glycoconjugates expressed during pregnancy protect are responsible for mediating feto-maternal tolerance. We refer to this model for as the Human Feto-Embryonic Defense System hypothesis. Finally our data suggests that parasites like schistosomes, filarial worms, and the human immunodeficiency virus may be evading the host\u27s immune responses by a similar by expressing immunosuppressive glycoconjugates on their coats

The detection, treatment, and biology of epithelial ovarian cancer

Ovarian cancer is particularly insidious in nature. Its ability to go undetected until late stages coupled with its non-descript signs and symptoms make it the seventh leading cause of cancer related deaths in women. Additionally, the lack of sensitive diagnostic tools and resistance to widely accepted chemotherapy regimens make ovarian cancer devastating to patients and families and frustrating to medical practitioners and researchers. Here, we provide an in-depth review of the theories describing the origin of ovarian cancer, molecular factors that influence its growth and development, and standard methods for detection and treatment. Special emphasis is focused on interactions between ovarian tumors and the innate and adaptive immune system and attempts that are currently underway to devise novel immunotherapeutic approaches for the treatment of ovarian tumors

MUC16 provides immune protection by inhibiting synapse formation between NK and ovarian tumor cells

<p>Abstract</p> <p>Background</p> <p>Cancer cells utilize a variety of mechanisms to evade immune detection and attack. Effective immune detection largely relies on the formation of an immune synapse which requires close contact between immune cells and their targets. Here, we show that MUC16, a heavily glycosylated 3-5 million Da mucin expressed on the surface of ovarian tumor cells, inhibits the formation of immune synapses between NK cells and ovarian tumor targets. Our results indicate that MUC16-mediated inhibition of immune synapse formation is an effective mechanism employed by ovarian tumors to evade immune recognition.</p> <p>Results</p> <p>Expression of low levels of MUC16 strongly correlated with an increased number of conjugates and activating immune synapses between ovarian tumor cells and primary naïve NK cells. MUC16-knockdown ovarian tumor cells were more susceptible to lysis by primary NK cells than MUC16 expressing controls. This increased lysis was not due to differences in the expression levels of the ligands for the activating receptors DNAM-1 and NKG2D. The NK cell leukemia cell line (NKL), which does not express KIRs but are positive for DNAM-1 and NKG2D, also conjugated and lysed MUC16-knockdown cells more efficiently than MUC16 expressing controls. Tumor cells that survived the NKL challenge expressed higher levels of MUC16 indicating selective lysis of MUC16^lowtargets. The higher csMUC16 levels on the NKL resistant tumor cells correlated with more protection from lysis as compared to target cells that were never exposed to the effectors.</p> <p>Conclusion</p> <p>MUC16, a carrier of the tumor marker CA125, has previously been shown to facilitate ovarian tumor metastasis and inhibits NK cell mediated lysis of tumor targets. Our data now demonstrates that MUC16 expressing ovarian cancer cells are protected from recognition by NK cells. The immune protection provided by MUC16 may lead to selective survival of ovarian cancer cells that are more efficient in metastasizing within the peritoneal cavity and also at overcoming anti-tumor innate immune responses.</p

Characterization of the tumor marker muc16 (ca125) expressed by murine ovarian tumor cell lines and identification of a panel of cross-reactive monoclonal antibodies

<p>Abstract</p> <p>Objectives</p> <p>The ovarian tumor marker CA125 is expressed on human MUC16, a cell surface bound mucin that is also shed by proteolytic cleavage. Human MUC16 is overexpressed by ovarian cancer cells. MUC16 facilitates the binding of ovarian tumor cells to mesothelial cells lining the peritoneal cavity. Additionally, MUC16 also is a potent inhibitor of natural killer cell mediated anti-tumor cytotoxic responses. Extensive studies using human as well as murine ovarian tumor cell models are required to clearly define the function of MUC16 in the progression of ovarian tumors. The major objective of this study was to determine if the murine ovarian tumor cells, MOVCAR, express Muc16 and to characterize antibodies that recognize this mucin.</p> <p>Methods</p> <p>RT-PCR analysis was used for detecting the Muc16 message and size exclusion column chromatography for isolating Muc16 produced by MOVCAR cells. Soluble and cell-associated murine Muc16 were analyzed, respectively, by Western blotting and flow cytometry assays using a new panel of antibodies. The presence of N-linked oligosaccharides on murine Muc16 was determined by ConA chromatography.</p> <p>Results</p> <p>We demonstrate that murine Muc16 is expressed by mouse ovarian cancer cells as an ~250 kDa glycoprotein that carries both O-linked and N-linked oligosaccharides. In contrast to human MUC16, the murine ortholog is primarily released from the cells and cannot be detected on the cell surface. Since the released murine Muc16 is not detected by conventional anti-CA125 assays, we have for the first time identified a panel of anti-human MUC16 antibodies that also recognizes the murine counterpart.</p> <p>Conclusion</p> <p>The antibodies identified in this study can be used in future purification of murine Muc16 and exhaustive study of its properties. Furthermore, the initial identification and characterization of murine Muc16 is a vital preliminary step in the development of effective murine models of human ovarian cancer. These models will aid in the further elucidation of the role that human MUC16 plays in the etiology and progression of ovarian tumors.</p

Identification of Siglec-9 as the receptor for MUC16 on human NK cells, B cells, and monocytes

<p>Abstract</p> <p>Background</p> <p>MUC16 is a cell surface mucin expressed at high levels by epithelial ovarian tumors. Following proteolytic cleavage, cell surface MUC16 (csMUC16) is shed in the extracellular milieu and is detected in the serum of cancer patients as the tumor marker CA125. csMUC16 acts as an adhesion molecule and facilitates peritoneal metastasis of ovarian tumors. Both sMUC16 and csMUC16 also protect cancer cells from cytotoxic responses of natural killer (NK) cells. In a previous study we demonstrated that sMUC16 binds to specific subset of NK cells. Here, we identify the csMUC16/sMUC16 binding partner expressed on immune cells.</p> <p>Results</p> <p>Analysis of immune cells from the peripheral blood and peritoneal fluid of ovarian cancer patients indicates that in addition to NK cells, sMUC16 also binds to B cells and monocytes isolated from the peripheral blood and peritoneal fluid. I-type lectin, Siglec-9, is identified as the sMUC16 receptor on these immune cells. Siglec-9 is expressed on approximately 30-40% of CD16^pos/CD56^dimNK cells, 20-30% of B cells and >95% of monocytes. sMUC16 binds to the majority of the Siglec-9^posNK cells, B cells and monocytes. sMUC16 is released from the immune cells following neuraminidase treatment. Siglec-9 transfected Jurkat cells and monocytes isolated from healthy donors bind to ovarian tumor cells via Siglec-9-csMUC16 interaction.</p> <p>Conclusions</p> <p>Recent studies indicate that csMUC16 can act as an anti-adhesive agent that blocks tumor-immune cell interactions. Our results demonstrate that similar to other mucins, csMUC16 can also facilitate cell adhesion by interacting with a suitable binding partner such as mesothelin or Siglec-9. Siglec-9 is an inhibitory receptor that attenuates T cell and NK cell function. sMUC16/csMUC16-Siglec-9 binding likely mediates inhibition of anti-tumor immune responses.</p

Mesothelin-MUC16 binding is a high affinity, N-glycan dependent interaction that facilitates peritoneal metastasis of ovarian tumors

BACKGROUND: The mucin MUC16 and the glycosylphosphatidylinositol anchored glycoprotein mesothelin likely facilitate the peritoneal metastasis of ovarian tumors. The biochemical basis and the kinetics of the binding between these two glycoproteins are not clearly understood. Here we have addressed this deficit and provide further evidence supporting the role of the MUC16-mesothelin interaction in facilitating cell-cell binding under conditions that mimic the peritoneal environment. RESULTS: In this study we utilize recombinant-Fc tagged human mesothelin to measure the binding kinetics of this glycoprotein to MUC16 expressed on the ovarian tumor cell line OVCAR-3. OVCAR-3 derived sublines that did not express MUC16 showed no affinity for mesothelin. In a flow cytometry-based assay mesothelin binds with very high affinity to the MUC16 on the OVCAR-3 cells with an apparent K(d )of 5–10 nM. Maximum interaction occurs within 5 mins of incubation of the recombinant mesothelin with the OVCAR-3 cells and significant binding is observed even after 10 sec. A five-fold molar excess of soluble MUC16 was unable to completely inhibit the binding of mesothelin to the OVCAR-3 cells. Oxidation of the MUC16 glycans, removal of its N-linked oligosaccharides, and treatment of the mucin with wheat germ agglutinin and erythroagglutinating phytohemagglutinin abrogates its binding to mesothelin. These observations suggest that at least a subset of the MUC16-asscociated N-glycans is required for binding to mesothelin. We also demonstrate that MUC16 positive ovarian tumor cells exhibit increased adherence to A431 cells transfected with mesothelin (A431-Meso(+)). Only minimal adhesion is observed between MUC16 knockdown cells and A431-Meso(+ )cells. The binding between the MUC16 expressing ovarian tumor cells and the A431-Meso(+ )cells occurs even in the presence of ascites from patients with ovarian cancer. CONCLUSION: The strong binding kinetics of the mesothelin-MUC16 interaction and the cell adhesion between ovarian tumor cells and A431-Meso+ even in the presence of peritoneal fluid strongly support the importance of these two glycoproteins in the peritoneal metastasis of ovarian tumors. The demonstration that N-linked glycans are essential for mediating mesothlein-MUC16 binding may lead to novel therapeutic targets to control the spread of ovarian carcinoma

Oxidative Phosphorylation: A Target for Novel Therapeutic Strategies Against Ovarian Cancer

Aerobic glycolysis is an important metabolic adaptation of cancer cells. There is growing evidence that oxidative phosphorylation is also an active metabolic pathway in many tumors, including in high grade serous ovarian cancer. Metastasized ovarian tumors use fatty acids for their energy needs. There is also evidence of ovarian cancer stem cells privileging oxidative phosphorylation (OXPHOS) for their metabolic needs. Metformin and thiazolidinediones such as rosiglitazone restrict tumor growth by inhibiting specific steps in the mitochondrial electron transport chain. These observations suggest that strategies to interfere with oxidative phosphorylation should be considered for the treatment of ovarian tumors. Here, we review the literature that supports this hypothesis and describe potential agents and critical control points in the oxidative phosphorylation pathway that can be targeted using small molecule agents. In this review, we also discuss potential barriers that can reduce the efficacy of the inhibitors of oxidative phosphorylation

Mesothelin, Stereocilin, and Otoancorin are predicted to have superhelical structures with ARM-type repeats

<p>Abstract</p> <p>Background</p> <p>Mesothelin is a 40 kDa protein present on the surface of normal mesothelial cells and overexpressed in many human tumours, including mesothelioma and ovarian and pancreatic adenocarcinoma. It forms a strong and specific complex with MUC16, which is also highly expressed on the surface of mesothelioma and ovarian cancer cells. This binding has been suggested to be the basis of ovarian cancer metastasis. Knowledge of the structure of this protein will be useful, for example, in building a structural model of the MUC16-mesothelin complex. Mesothelin is produced as a precursor, which is cleaved by furin to produce the N-terminal half, which is called the megakaryocyte potentiating factor (MPF), and the C-terminal half, which is mesothelin. Little is known about the function of mesothelin and there is no information on its possible three-dimensional structure. Mesothelin has been reported to be homologous to the deafness-related inner ear proteins otoancorin and stereocilin, for neither of which the three-dimensional structure is known.</p> <p>Results</p> <p>The BLAST and PSI-BLAST searches confirmed that mesothelin and mesothelin precursor proteins are remotely homologous to stereocilin and otoancorin and more closely homologous to the hypothetical protein MPFL (MPF-like). Secondary structure prediction servers predicted a predominantly helical structure for both mesothelin and mesothelin precursor proteins and also for stereocilin and otoancorin. Three-dimensional structure prediction servers INHUB and I-TASSER produced structural models for mesothelin, which consisted of superhelical structures with ARM-type repeats in conformity with the secondary structure predictions. Similar ARM-type superhelical repeat structures were predicted by 3D-PSSM server for mesothelin precursor and for stereocilin and otoancorin proteins.</p> <p>Conclusion</p> <p>The mesothelin superfamily of proteins, which includes mesothelin, mesothelin precursor, megakaryocyte potentiating factor, MPFL, stereocilin and otoancorin, are predicted to have superhelical structures with ARM-type repeats. We suggest that all of these function as superhelical lectins to bind the carbohydrate moieties of extracellular glycoproteins.</p