Beta1,4-N-acetylgalactosaminyltransferase (GM2 synthase) is released from Golgi membranes as a neuraminidase-sensitive, disulfide-bonded dimer by a cathepsin D-like protease.
Many Golgi membrane-bound glycosyltransferases are released from cells in a soluble form. To characterize this release process, we stably transfected Chinese hamster ovary cells with three myc epitope-tagged forms of cloned beta1, 4-N-acetylgalactosaminyltransferase (GalNAcT); two of these forms resided in the Golgi, while the third was retained in the ER. GalNAcT was released into the culture medium from cells transfected with the Golgi forms but not with the ER form of the enzyme. The medium from cells transfected with the Golgi forms contained disulfide-bonded dimers of GalNAcT, which carried neuraminidase sensitive, complex N-linked carbohydrate chains. This soluble species represented the major degradation product of cellular GalNAcT, which turned over with a half-time of about 1.7 h. The soluble species consisted of a mixture of truncated GalNAcT molecules, the major form of which was produced by cleavage near the boundary between the transmembrane and lumenal domains between Leu-23 and Tyr-24. This cleavage site fits the sequence pattern for sites cleaved by cathepsin D (van Noort, J.M., and van der Drift, A. C.M. (1989) J. Biol. Chem. 264, 14159-14164). These findings suggest that GalNAcT is converted from a membrane-bound to a soluble form as a result of cleavage by a cathepsin D-like protease in a compartment late in the Golgi secretory pathway.
Jaskiewicz
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Department of Biological and Biophysical Sciences and Biochemistry, Health Sciences Center, University of Louisville, Louisville, Kentucky 40292, USA.
Zhu
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Bassi
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Darling
D S
DS
Young
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WW
D000818:Animals; D001665:Binding Sites; D015153:Blotting, Western; D002402:Cathepsin D / Q000378:metabolism*; D002499:Centrifugation, Density Gradient; D006224:Cricetinae; D003412:Cricetulus; D004220:Disulfides / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D006026:Glycoside Hydrolases / Q000378:metabolism; D006056:Golgi Apparatus / Q000201:enzymology*; D007425:Intracellular Membranes / Q000201:enzymology; D017350:N-Acetylgalactosaminyltransferases / Q000378:metabolism*; D009439:Neuraminidase / Q000378:metabolism*; D010053:Ovary / Q000648:ultrastructure; D012995:Solubility; D013379:Substrate Specificity; D014162:Transfection; D000097763:Polypeptide N-acetylgalactosaminyltransferase
D002402:Cathepsin D; D004220:Disulfides; D006026:Glycoside Hydrolases; D017350:N-Acetylgalactosaminyltransferases; D009439:Neuraminidase; D000097763:Polypeptide N-acetylgalactosaminyltransferase; C023220:(N-acetylneuraminyl)-galactosylglucosylceramide N-acetylgalactosaminyltransferase