J. Biol. Chem., Vol. 263, Issue 24, 11736-11742, 08, 1988
Remodeling of a rat hepatocyte plasma membrane glycoprotein. De- and reglycosylation of dipeptidyl peptidase IV
W Kreisel, C Hanski, TA Tran-Thi, N Katz, K Decker, W Reutter and W Gerok
Klinikum Albert-Ludwigs-Universitat, Freiburg, Federal Republic of Germany.
The present paper demonstrates the terminal de- and reglycosylation of a
rat hepatocyte plasma membrane glycoprotein, dipeptidyl peptidase IV (DPP
IV). Cultured hepatocytes were used in pulse-chase experiments with
[3H]L-fucose and [14C]N-acetyl-D-mannosamine as markers for terminal
carbohydrates, [3H]D-mannose as marker of a core-sugar, and
[35S]L-methionine for labeling the protein backbone. Membrane DPP IV was
immunoprecipitated with a polyclonal antibody which bound selectively at 4
degrees C to the cell-surface glycoprotein. The times of maximal labeling
of hepatocyte plasma membrane DPP IV were 6-9 min for [3H]L-fucose, 20 min
for [3H]D-mannose, and 25 min for [35S]L- methionine. When antibodies were
bound to cell-surface DPP IV at 4 degrees C, the immune complex remained
stable for more than 1 h after rewarming to 37 degrees C, despite ongoing
metabolic and membrane transport processes. This was shown by pulse
labeling with [35S]L- methionine at 37 degrees C, followed by cooling to 4
degrees C, and addition of antibody against plasma membrane DPP IV. During
rewarming, the radioactivity in the complex remained constant. In a similar
experiment with [3H]L-fucose, the radioactivity in the immune complex
declined rapidly, indicating a defucosylation of the plasma membrane
glycoprotein. Using the same experimental design with [3H]D-mannose, the
radioactivity in the immune complex remained constant, showing that the
core-sugar D-mannose is not cleaved from the membrane glycoprotein.
Terminal reglycosylation (refucosylation and resialylation) was
demonstrated as follows. Hepatocytes were maintained at 37 degrees C in a
medium supplemented with tunicamycin in order to block the de novo
synthesis of N-glycosidically bound carbohydrate chains. At 4 degrees C the
antibody against DPP IV bound only to cell surface glycoprotein. During the
rewarming period at 37 degrees C, radioactivity from [3H]L- fucose and
[14C]N-acetyl-D-mannosamine became incorporated into the immune complex.
This indicates a fucosylation and sialylation of the glycoprotein
originally present at the cell surface. The mechanisms whereby terminal de-
and reglycosylation of plasma membrane glycoproteins may occur during
membrane recycling are discussed.
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