Glycodelin A : A Novel Immunoregulatory Lectin Of The Female Reproductive Tract : Molecular Mechanism Of GdA-Induced Apoptosis In Activated T Cells
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Glycodelin is a 162 amino acid secreted glycoprotein classified as a member of the lipocalin (carriers of small hydrophobic molecules) superfamily based on the presence of lipocalin signature motifs in its primary sequence. The protein has several isoforms which are expressed by various primate tissues, predominantly reproductive tissues. These isoforms are products of the same gene and hence have the same primary sequence; however, they are differentially glycosylated depending on tissue origin. The individual glycodelin isoforms perform very varied functions, which are largely dictated or modulated by the specific glycans on the molecule. Glycodelin A (GdA) is the major glycodelin isoform of the female reproductive tract; and is subclassified as an immunocalin (lipocalins with immunological function) due to its ability to modulate immune responses. Diverse activities have been associated with GdA; pertaining to determination of cell fate, tissue differentiation and significantly, immunomodulation towards fetal-allograft tolerance. The fetus expresses paternal allo-antigens and would be regarded as non-self or foreign by the maternal immune system. However, several synergistic mechanisms of immunomodulation at the fetal-maternal interface establish tolerance towards fetal antigens, protecting it from rejection. GdA is secreted by the uterine endometrium under progesterone induction, and is therefore the most abundant progesterone-regulated secretory glycoprotein of the uterus at the time of implantation and early pregnancy. GdA has been shown to have immunomodulatory activity targeting innate, humoral and cellular responses. It is inhibitory to T cell and B cell proliferation, and NK cell activity. It stimulates the Th2-type cytokine profile, and inhibits interleukins IL-2 and IL-1 production from mitogenically stimulated lymphocytes and mononuclear cell cultures. It has been reported from our laboratory that GdA induces apoptosis in activated T cells. GdA has also been shown to be inhibitory to B cells and monocytes. Clinical studies correlate subnormal levels of GdA with implantation Synopsis failure, habitual abortion and recurrent miscarriage. Due to its pleiotropic nature namely its diverse activities on different immune cell types; its spatio-temporal restriction of expression by progesterone; and its indispensable requirement for successful pregnancy; GdA is being increasingly recognized as a mechanism towards fetal allograft tolerance. Our laboratory has focused on the T cell inhibitory activity of GdA, with particular emphasis on T cell apoptosis. This study was aimed at delineating the molecular mechanism of GdA-induced apoptosis in activated T cells. Previous results from our laboratory have revealed that GdA-induced apoptosis is caspase dependent; and is not initiated by the extrinsic pathway involving Fas/death receptor signailing or initiator caspase 8. In this thesis, we present evidence that GdA triggers the intrinsic apoptotic program in T cells. Characterization of the apoptotic program initiated by GdA is presented in Chapter 1. We observe that GdA treatment triggers a stress response leading to decrease in mitochondrial transmembrane potential, which indicates mitochondrial membrane permeabilization (MMP). GdA-induced apoptosis can also be blocked by inhibition of caspase 9, the initiator caspase for the intrinsic program. The kinetics of mitochondrial depolarization precede onset of DNA fragmentation in both peripheral blood T cells and Jurkat cells treated with GdA. We also observe caspase 2 activation downstream of the mitochondria. Overexpression of the antiapoptotic protein Bcl-2 is sufficient to protect from GdA-induced cellular stress indicating that the apoptotic program can be reversed upstream of the mitochondria. Further, our studies reveal that stress signaling by GdA is not mediated by any of the canonical second messengers of stress signaling, namely, reactive oxygen species; the stress activated protein kinases JNK, p38 MAPK and ERK; intracellular calcium or ceramide. It has been reported that GdA desensitizes T cell receptor (TCR) signaling by decreasing the stability of TCR-triggered phosphoproteins, probably by its association ith the transmembrane tyrosine phosphatase CD45. TCR-desensitization would result in decreased proliferation and cytokine secretion, and has been postulated as the mechanism of T cell-inhibition by GdA. We have tested this theory and Chapter 2 provides evidence that the apoptogenic activity of GdA is not a consequence of its ability to blunt TCR-signaling. Further, GdA-induced apoptosis does not depend on components of the TCR signal cascade namely CD45, the kinase Lck and CTLA4, molecules that are proven transducers of apoptotic signals to the mitochondria in response to diverse stress stimuli. GdA triggers apoptosis in the CD45 deficient cell line J45.01 with similar kinetics of MMP and DNA fragmentation as with Synopsis wildtype cells, demonstrating that CD45 is not the determinant receptor for apoptosis on cells. We also observe that GdA is inhibitory to T cells stimulated with phorbol ester and calcium ionophore, which bypasses TCR-proximal signaling events; and that GdA treatment does not interfere with early T cell activation as evidenced from induction of the activation marker CD69. Thus, GdA initiates mitochondrial stress mediated apoptosis in T cells by a pathway that is distinct and independent from the TCR-coupled signaling pathway. This study presents a novel mode of immunosuppression for GdA and highlights the ability of GdA to suppress the immune response by more than one mechanism. Cell surface glycoproteins undergo alterations in their carbohydrate profiles upon T cell activation and differentiation, and this has a significant role to play in lymphocyte fate and function. One such global alteration in cell surface glycans is a difference in sialylation upon T cell activation and differentiation. While activated T cell have a lesser degree of sialylated surface glycoproteins as compared to naïve T cells, memory T cells are sialylated to a higher extent, and Th2 cells have more cell surface sialic acids than Th1 cells. As GdA is capable of triggering apoptosis in activated T cells, we investigated the requirement of cell surface glycans for differential recognition of T cell subsets by GdA, the results for which are detailed in Chapter 3. We observe that the activity of GdA could be competed out by asialofetuin and not fetuin, suggesting that GdA recognizes terminal galactose residues on asialofetuin glycans, which would be masked by sialic acids in case of fetuin glycans. This assumption was confirmed as the free sugars lactose and galactose, but not annose, could also competitively inhibit GdA activity. We also demonstrate that the lectin-activity of dA is calcium independent, typical of mammalian galectins. Thus, our results reveal GdA to be a novel galactose-specific lectin of the female reproductive tract. This carbohydrate specificity of GdA is responsible for its apoptotic activity on T cells. The selectivity of GdA towards activated T cells is a result of increased exposure of terminal galactose residues on activated T cell surface receptors, as demonstrated by staining of naïve and stimulated T cells with Fluorescent lectin-conjugates of different carbohydrate specificities. We also demonstrate hat GdA shows specificity towards N-liked glycans on cell surface glycoproteins. This is evident from the use of glycan processing inhibitors, which prevent addition of galactose to the core glycan on the nascent polypeptide chain. We observe that inhibition of processing of N-glycans, and not O-glycans, render cells resistant to GdA. Incidentally, we observe that another property of GdA, namely its ability to induce Synopsis epithelial differentiation and apoptosis in the breast cancer cell line MCF-7, is also due to ts galactose-specific lectin activity. It is therefore probable that the diverse functions ssociated with GdA are a consequence of its ability to recognize different glycoprotein receptors on different cell types. We can thus draw a comparison for GdA with the galectins, which are the prototype beta-galactoside binding mammalian lectins with diverse roles in determining cell fate and apoptosis, especially in the immune system. In fact, the immune-related activities of GdA are almost identical to the effects of galectin-1 on the immune system. Galectin-1 has also very recently been shown to play a significant role in fetal-tolerance. This raises a strong possibility of shared receptors for GdA and galectin-1 on the T cell surface, resulting from a shared calcium-independent recognition property for complex glycans with terminal galactose residues. Two predominant galectin-1 receptors on T cells are the glycoproteins CD45 and CD7. We have already observed that though GdA may recognize CD45, this association does not mediate its apoptotic activity. We therefore examined the possibility of the activation-induced glycoprotein CD7 as receptor for GdA. Our experiments reveal that the apoptotic activity of GdA on different T cell lines is dependent on the degree of CD7 expression by these cell lines. Notably, the CD7 negative lymphoma cell line HuT78 was completely resistant to GdA. To confirm CD7 as receptor, we obtained a cell line HuT78.7 in which CD7 expression has been restored by stable transfection. We observed that these CD7 positive cells now responded to GdA comparable to Jurkat cells, and GdA-induced apoptosis in these cells could be completely competed out with asialofetuin, not fetuin. To summarize, our study identifies GdA as a novel pregnancy-related galectin-like lectin of the female reproductive tract, which triggers mitochondrial stress and apoptosis in activated T cells. GdA shares receptors on T cells with galectin-1 due a common carbohydrate recognition property. We identify CD7 as a molecular target for GdA on activated T cells, capable of mediating the apoptotic signal. However, it is likely that GdA also recognizes other galectin receptors on T cells, as it is capable of inhibition by more than one mechanism. This underscores the requirement for redundant mechanisms indispensable for establishment and maintenance of successful pregnancy.
- Biochemistry (BC)