GalNAc-alpha -O-benzyl inhibits sialylation of de Novo synthesized apical but not basolateral sialoglycoproteins and blocks lysosomal enzyme processing in a post-trans-Golgi network compartment.
Glycosylation plays an important role in glycoprotein traffic. Our previous work has shown that long term treatment of mucus-secreting HT-29 cells with GalNAc-alpha-O-benzyl reversibly inhibits sialylation and causes the accumulation of apical glycoproteins in cytoplasmic vesicles. We have analyzed at the biochemical level the effects of GalNAc-alpha-O-benzyl on glycoprotein processing. Both apical and basolateral membrane glycoproteins were sialylated, but GalNAc-alpha-O-benzyl selectively inhibited the sialylation of apical glycoproteins. In addition, lysosomal alpha-glucosidase, which is partially targeted to the apical membrane, was abnormally processed leading to the accumulation of an immature molecular species. Several findings support the conclusion that accumulation of this protein occurs in a post-trans-Golgi network (TGN) compartment: 1) it is partially sialylated; 2) it does not occur when glycoprotein exit from the TGN is blocked at 20 degrees C; 3) upon Triton X-114 partition, it distributes to the aqueous phase, a characteristic that is acquired in a post-TGN compartment; and 4) its appearance is inhibited when cells are cultured in the presence of NH(4)Cl. The processing of cathepsin D was also found to be affected by GalNAc-alpha-O-benzyl treatment. In conclusion, GalNAc-alpha-O-benzyl selectively inhibits sialylation of apical glycoproteins and perturbs lysosomal enzyme processing; these effects occur in a post-TGN acidic compartment and are reminiscent of the alterations found in sialic acid storage diseases.
Ulloa
F
F
Unitat de Biologia Cel.lular i Molecular, Institut Municipal d'Investigació Mèdica, Universitat Pompeu Fabra, carrer Dr. Aiguader, 80, 08003 Barcelona, Spain.
Franci
C
C
Real
F X
FX
D000116:Acetylgalactosamine / Q000031:analogs & derivatives* / Q000494:pharmacology; D001593:Benzyl Compounds / Q000494:pharmacology*; D002451:Cell Compartmentation; D002462:Cell Membrane / Q000378:metabolism; D006056:Golgi Apparatus / Q000187:drug effects* / Q000201:enzymology; D019073:HT29 Cells; D006801:Humans; D008247:Lysosomes / Q000187:drug effects* / Q000201:enzymology; D019158:N-Acetylneuraminic Acid / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D012795:Sialoglycoproteins / Q000378:metabolism*; D000520:alpha-Glucosidases / Q000378:metabolism
D001593:Benzyl Compounds; D012795:Sialoglycoproteins; C080739:benzyl-alpha-N-acetylgalactosamine; D000520:alpha-Glucosidases; D019158:N-Acetylneuraminic Acid; D000116:Acetylgalactosamine