Carissa M. Krane, Ph.D.
By utilizing an extreme physiological adaptation known as freeze-tolerance, Cope’s gray tree frog, Dryophytes chrysoscelis, freezes and then subsequently thaws up to 65% of its extracellular fluid to survive the winter. During these periods of freezing and thawing, erythrocytes (RBCs) of D. chrysoscelis utilize a protein, aquaglyceroporin HC-3, that facilitates transmembrane flux of both water and cryoprotective glycerol to mediate osmotic adjustments. RBCs from cold-acclimated tree frogs up-regulate HC-3 protein expression, which coincides with more abundant membrane localization and higher levels of glycosylation. However, the functional significance of HC-3 glycosylation on membrane localization and cellular freeze tolerance is currently not known. We hypothesize that anticipatory glycerol accumulation observed in cold-acclimated tree frogs contributes to enhanced post-translational modification of HC-3 via N-linked and O-linked glycosylation, and that HC-3 glycosylation is important in subcellular trafficking of HC-3 from the Golgi to the membrane. RBCs from warm-acclimated D. chrysoscelis were separated into three categories: freshly isolated RBCs (FI), RBCs cultured in complete cell culture media for 48 hours (CCCM), and RBCs cultured in CCCM containing 0.156M glycerol for 48 hours (CCCM+G). Densitometric analyses of immunoblots specific for HC-3 showed a 3.5-fold and 1.9-fold average increase in glycosylated HC-3 (60-120 kDa) from RBCs cultured in CCCM+G as compared to FI RBCs and RBCs cultured in CCCM, respectively. Western blots of RBC proteins treated with PNGase F resulted in a 1.3-fold average decrease in glycosylated HC-3 compared to control proteins. However, protein treatment with the O-Glycosidase and Neuraminidase mix did not appear to change the abundance of glycosylated HC-3, indicating that HC-3 is post-translationally modified via N-linked glycosylation but not O-linked. Additional results were collected using scanning laser confocal microscopy and HC-3 localization was measured in mean fluorescent intensity (arbitrary units) using ImageJ software (N=46 cells per experiment). For RBCs cultured in CCCM+G, immunofluorescence intensity of HC-3 in the plasma membrane was 21.7 times greater than HC-3 immunofluorescence in the cytosol (P0.05). Using an in vitro cell culture system, we have successfully recapitulated cold-acclimated in vivo HC-3 expression patterns by focusing solely on the influence of a glycerol-induced hyperosmotic environment on RBCs of D. chrysoscelis. Thus, a potential correlation between cryoprotective glycerol, increased HC-3 N-linked glycosylation, and enhanced HC-3 membrane localization has been identified.
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Pezzutti, Dante L., "Characterization of the Glycosylation of Aquaglyceroporin HC-3 in Erythrocytes from the Freeze Tolerant Anuran, Dryophytes chrysoscelis" (2018). Honors Theses. 183.