Abstract
Mucin type O-glycans with core 2 branches are distinct from nonbranched O-glycans, and the amount of core 2 branched O-glycans changes dramatically during T cell differentiation. This oligosaccharide is synthesized only when core 2 beta-1, 6-N-acetylglucosaminyltransferase (C2GnT) is present, and the expression of this glycosyltransferase is highly regulated. To understand how O-glycan synthesis is regulated by the orderly appearance of glycosyltransferases that form core 2 branched O-glycans, the subcellular localization of C2GnT was determined by using antibodies generated that are specific to C2GnT. The studies using confocal light microscopy demonstrated that C2GnT was localized mainly in cis to medial-cisternae of the Golgi. We then converted C2GnT to a trans-Golgi enzyme by replacing its Golgi retention signal with that of alpha-2,6-sialyltransferase, which resides in trans-Golgi. Chinese hamster ovary cells expressing wild type C2GnT and the chimeric C2GnT were then subjected to oligosaccharide analysis. The results obtained clearly indicate that the conversion of C2GnT into a trans-Golgi enzyme resulted in a substantial decrease of core 2 branched oligosaccharides. These results, taken together, strongly suggest that the predominance of core 2 branched oligosaccharides in those cells expressing C2GnT is due to the fact that C2GnT is located earlier in the Golgi than alpha-2,3-sialyltransferase that competes with C2GnT for the common substrate. Furthermore, alteration of Golgi localization renders the chimeric C2GnT much less efficient in synthesizing core 2 branched oligosaccharides, indicating the critical role of orderly subcellular localization of glycosyltransferases.
Highlights
These results, taken together, strongly suggest that the predominance of core 2 branched oligosaccharides in those cells expressing C2GnT is due to the fact that C2GnT is located earlier in the Golgi than ␣-2,3-sialyltransferase that competes with C2GnT for the common substrate
By in situ hybridization of the transcript, it has been shown that immature cortical thymocytes express a substantial amount of C2GnT mRNA, while it was not detected in medullary thymocytes [9]
The conversion of O-glycan structures during thymocyte development may be critical for the apoptotic process in thymus, since such a process is modulated by the presence of O-glycans on thymocytes [10]
Summary
C2GnT, core 2 -1,6-N-acetylglucosaminyltransferase; GST, glutathione S-transferase; PBS, phosphate-buffered saline; PCR, polymerase chain reaction; CHO, Chinese hamster ovary; GalT, -1,4-galactosyltransferase; ST6Gal I, Gal13 4GlcNAc ␣-2,6-sialyltransferase; ␣-ManII, ␣-mannosidase II. Core 2 Branching Enzyme with Altered Golgi Localization distribution of glycosyltransferases that form O-glycans (see Ref. 18). We have first determined that C2GnT is localized in cis to medial-Golgi using antibodies specific for C2GnT. We converted C2GnT into a trans-Golgi enzyme by replacing its domain responsible for Golgi retention with that of Gal134GlcNAc ␣-2,6-sialyltransferase, ST6Gal I [19]. Such altered localization of C2GnT was found to result in altered synthesis of oligosaccharides, demonstrating the importance of the orderly presence of glycosyltransferases
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