Print

Weeraratne, Shyamal Dilhan

Biology

2007

PhD

Glycosyl phosphatidylinositol (GPI) anchored proteins are a class of surface proteins that are attached to the outer leaflet of the plasma membrane via a glyco-lipid anchor. Their function ranges from serving as receptors for ligands, cell adhesion molecules, surface hydrolytic enzymes, and mammalian and protozoan antigens. In Paramecium tetraurelia some of them serve as large surface antigens and also in chemoresponses. Previously it has been shown that disruption of anchoring using an anti-sense expression to PIG-A (an enzyme in the fist step in anchor synthesis), chemoresponses to folate and glutamate in P. tetraurelia are profoundly diminished. This led to the speculation that GPI-anchored components are involved in the above chemosensory pathways. In this work, two sequences encoding the GPI-anchored folate receptor protein in P. tetraurelia are identified. An antibody was developed against the receptor which is used in conjunction with the anti-CRD which recognizes the cleaved anchor to validate that the folate receptor is GPI-anchored. The protein was down regulated using feeding RNAi to show that the folate chemoresponse is specifically diminished. Attraction to other tested stimuli remained at the control level. We correlate the phenotype to reduction in the folate receptor mRNA transcript with semi-quantitative RT-PCR and the loss of folate receptor protein with Western blots. The phenotype is attributed to loss of folate binding in the RNAi cells as shown by instantaneous binding assays. We have examined the distribution of the folate receptor on the cell surface and found that they are not evenly distributed across the surface, but rather cluster near the bases of the cilia reminiscent of the localization pattern of other GPI-anchored proteins. We show this distribution through confocal and deconvolution microscopy using antibodies to the folate chemoreceptor, 5nucleotidase, GPI anchored surface antigens and tubulin. The localization of the folate receptor on the surface is disrupted in RNAi cells. Finally, using a number of bioinformatics tools we developed a method to identify potential GPI anchored proteins in Paramecium tetraurelia genome, based on the presence of sequence motifs and other conserved features. We have identified 153 potential GPI anchored proteins and have categorized them into functional groups. Many of these proteins show similarity to surface antigens.