Characterization of the Glycosylation of Aquaglyceroporin HC-3 in Erythrocytes from the Freeze Tolerant Anuran, Dryophytes chrysoscelis

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Characterization of the Glycosylation of Aquaglyceroporin HC-3 in Erythrocytes from the Freeze Tolerant Anuran, Dryophytes chrysoscelis

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Cope’s gray treefrog, Dryophytes chrysoscelis, is a freeze-tolerant anuran that uses glycerol as a cryoprotectant. In erythrocytes of D. chrysoscelis, transmembrane glycerol flux is likely facilitated through the aquaglyceroporin, HC-3. Previous research demonstrated that erythrocytes from cold-acclimated treefrogs up-regulate HC-3 protein expression, membrane localization, and glycosylation. Thus, we hypothesize that anticipatory glycerol accumulation observed in cold-acclimated treefrogs 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 to the membrane. Densitometric analyses of immunoblots specific for HC-3 showed a 3.5-fold and 1.9-fold average increase in glycosylated HC-3 from RBCs cultured with the addition of glycerol (CCCM+G) as compared to Freshly Isolated RBCs (FI) and RBCs cultured in CCCM alone, 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 O-Glycosidase and Neuraminidase did not change the abundance of glycosylated HC-3. 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=4-6 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 (P<0.05). In contrast, immunofluorescence intensity of HC-3 in the cytosol was 3.2 times greater than HC-3 immunofluorescence in the membrane for FI RBCs (P<0.01). Through the use of an in vitro cell culture system, we have recapitulated cold-acclimated in vivo HC-3 expression patterns through the addition of a glycerol-induced hyperosmotic environment to warm-acclimated erythrocyte cell cultures of D. chrysoscelis. Thus, in addition to its osmoregulatory role, glycerol may also influence the N-linked glycosylation and membrane trafficking of HC-3.

Publication Date

4-18-2018

Project Designation

Honors Thesis

Primary Advisor

Carissa M Krane

Primary Advisor's Department

Biology

Keywords

Stander Symposium poster

Comments

Presenter: Dante Laurenti Pezzutti

Characterization of the Glycosylation of Aquaglyceroporin HC-3 in Erythrocytes from the Freeze Tolerant Anuran, Dryophytes chrysoscelis

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