GalNAc-α-O-benzyl Inhibits Sialylation ofde Novo Synthesized Apical but Not Basolateral Sialoglycoproteins and Blocks Lysosomal Enzyme Processing in a Post-trans-Golgi Network Compartment

doi:10.1074/jbc.M000510200

GalNAc-α-O-benzyl Inhibits Sialylation ofde Novo Synthesized Apical but Not Basolateral Sialoglycoproteins and Blocks Lysosomal Enzyme Processing in a Post-trans-Golgi Network Compartment*

From the Unitat de Biologia Cel.lular i Molecular, Institut Municipal d'Investigació Mèdica, Universitat Pompeu Fabra, carrer Dr. Aiguader, 80, 08003 Barcelona, Spain

Abstract

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-α-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-α-O-benzyl on glycoprotein processing. Both apical and basolateral membrane glycoproteins were sialylated, but GalNAc-α-O-benzyl selectively inhibited the sialylation of apical glycoproteins. In addition, lysosomal α-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 °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 NH₄Cl. The processing of cathepsin D was also found to be affected by GalNAc-α-O-benzyl treatment. In conclusion, GalNAc-α-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.

Footnotes

↵* This work was supported in part by Grant SAF97-0085 from Comisión Interministerial de Ciencia y Tecnologı́a, Grant SGR-00433 from Comissió Interdepartamental de Recerca i Tecnologia (Generalitat de Catalunya), and a grant from the Mizutani Foundation for Glycoscience.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵‡ Recipient of a predoctoral fellowship from the MUTIS Program.
↵§ To whom correspondence should be addressed. Tel.: 34-93-2211009; Fax: 34-93-2213237; E-mail: preal@imim.es.
Published, JBC Papers in Press, April 5, 2000, DOI 10.1074/jbc.M000510200
↵2 F. Ulloa and F. X. Real, manuscript in preparation.
Abbreviations:

BG

GalNAc-α-O-benzyl

AAG

acid α-glucosidase

APN

aminopeptidase N

DPP-IV

dipeptidylpeptidase IV

gp

glycoprotein

M6

HT-29 cells selected in 1 μm methotrexate

MAL

Maackia amurensis lectin

PNA

peanut agglutinin

SASD

sialic acid storage disease

SI

sucrase-isomaltase

TGN

trans-Golgi network

FBS

fetal bovine serum

DMEM

Dulbecco's modified Eagle's medium

PBS

phosphate-buffered saline

mAb

monoclonal antibody

PAGE

polyacrylamide gel electrophoresis

endo H

endoglycosidase H

ST

sialyltransferase
- Received January 24, 2000.
- Revision received April 3, 2000.
The American Society for Biochemistry and Molecular Biology, Inc.