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

By utilizing an extreme physiological adaptation known as freeze‐tolerance, Cope's gray treefrog, 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 employ a protein, aquaglyceroporin HC‐3, that mediates transmembrane flux of both water and cryoprotective glycerol to offset cellular lysis/shrinkage. RBCs from cold‐acclimated treefrogs 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 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 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 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 =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). There was no difference in HC‐3 immunofluorescence intensity between the membrane and cytosol in RBCs cultured in CCCM ( P >0.05). 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. Support or Funding Information This research was funded by the Schuellein Chair in the Biological Sciences (CMK), University of Dayton University Honors Program (DLP), and a 2017 APS UGSRF Award to DLP. This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .