Abstract
Malignant transformation of rodent cell lines by polyoma virus and by activated ras genes is associated with increased UDP-GlcNAc:Man alpha-R beta-1,6-N-acetylglucosaminyltransferase V (GlcNAc-transferase V) activity and it product -GlcNAc beta 1-6Man alpha 1-6Man beta 1-branched Asn-linked oligosaccharides. In this report, we have compared beta 1-6GlcNAc branching of core O- and N-linked oligosaccharides in three experimental models of malignancy, namely (a) rat2 fibroblasts and their malignant T24H-ras-transfected counterpart; (b) benign SP1 mammary carcinoma cells and two metastic sublines of SP1; and (c) the metastatic MDAY-D2 lymphoma cell line and its poorly metastatic glycosylation mutant KBL-1. In addition to the previously reported increase in GlcNAc-transferase V activity, UDP-GlcNAc:Gal beta 1-3GalNAc alpha-R (GlcNAc to GalNAc) beta-1,6-N-acetylglucosaminyltransferase (core 2 GlcNAc-transferase, EC 2.4.1.102) activity was found to be elevated by 70% in the malignant rat2 and SP1 cell lines while several other glycosyltransferase activities were not significantly different. The action of core 2 GlcNAc-transferase followed by beta 1-4Gal-transferase provides an N-acetyllactosamine antenna that can be extended with polylactosamine (i.e. repeating Gal beta 1-4GlcNAc beta 1-3) provided UDP-GlcNAc:Gal beta-R beta 1-3GlcNAc-transferase (GlcNAc-transferase) (i)) activity is present. Polylactosamine content in microsomal membrane glycoproteins was quantitated by labeling the GlcNAc termini resulting from the action of Escherichia freundii endo-beta-galactosidase with bovine galactosyltransferase/UDP-[3H] Gal. Glycopeptidase F- sensitive and -insensitive fractions were measured to assess the N- and O-linked components. In the SP1 tumor model, the metastatic sublines showed increased core 2 GlcNAc-transferase and GlcNAc-transferase V activities but no change in GlcNAc-transferase (i) activity, yet polylactosamine was increased in both O- and N-linked oligosaccharides. In rat2 cells, down-regulation of GlcNAc-transferase (i) following transformation was associated with decreased polyactosamine even though core 2 GlcNAc-transferase and GlcNAc-transferase V were elevated in the cells. Finally, a 3-fold decrease in GlcNAc-transferase V in KBL-1, the glycosylation mutant of MDAY-D2 cells, resulted in complete loss of polylactosamine in N-linked but no change in O-linked polylactosamine content. These results suggest that, provided GlcNAc-transferase (i) is not limiting, the beta 1-6-branching enzymes core 2 GlcNAc-transferase and GlcNAc-transferase V regulate the levels of polyactosamine in O- and N-linked oligosaccharides, respectively.(ABSTRACT TRUNCATED AT 400 WORDS)
Highlights
The proton assignments summarized in the products by rat2 and rat2-T24-H-ras cells were identical Table I1 for the substrate andproduct were based on analysis to thatof a standardof Gal~l-3(GlcNAc~l-6)GalNAc~~-opfNhpo.monuclear shift correlation spectroscopy (COSY) and
Core 3 has the potential to be extended @w1it-h4Gal, producing an N-acetyllactosaminesequence which couldbe extended furtherwith polylactosamine[26].,since GlcNAc was not added to GalNAca-phenyl, and both rat2 and SP1 cell lines show high levels of /31-3Gal-T activity, this would be expected to lead primarily to core 1 synthesis
We have compared the activities of several glycosyltransferases in three models of malignancy to determine whether increased P1-6GlcNAc branching occurs in the core of 0-linked as well as N-linked oligosaccharides following transformation. rat2 fibroblastsare not tumorigenic when injected into rats oarthymic nude mice but when transformed with activated ras (i.e.v-K-ras or T24H-ras), thceells acquire both tumorigenic and metastatic properties [10, 11]
Summary
Ously reported increase in GlcNAc-transferaVsaectivity, UDP-GlcNAc:GalB1-3GalNAca-R (GlcNAc to GalNAc) 8-1,6-N-acetylglucosaminyltransferase(core Malignant transformationof both murine and humancells found tobe elevated by70%in the malignant rat andis commonly associated with expression of larger N-linked. GlcNAc-transferase (i) activity, yetpolylactosamine loss of GlcNAc-TV activity in the class 3 glycosylation muwas increased in both 0-and N-linked oligosaccha- tants (i.e. KBL-1) of the highly metastatic tumor cell line rides. GalB1-3GalNAc and GalNAc) are commonly associated with "mucin-type" glycoproteins in humancarcinomas [15], transformation-related changes in the biosynthesis of the larger branched 0-linked oligosaccharides have not been examined To address these questions, we have compared glycosyltransferase levels and N- and 0-linkedpolylactosamine content in immortalized rat fibroblasts andtheirT24H-rastransformed counterpart (IO), and in the spontaneous mammary carcinoma cell line SP1, and two metastatic sublines. We have quantitated the polylactosamine content in N- and 0-linked oligosaccharides using a sensitive assay employing E. freundii endo-P-galactosidase and labeling of the resulting GlcNAc termini with UDP-[3H]Gal/bovine milk galactosyltransferase [18].The results suggest that the activities of core 2 GlcNAc-T and GlcNAc-TV, as well as the polylactosamine extension enzyme GlcNAc-T(i),can independently regulate polylactosamine content in 0- and Nlinked oligosaccharides
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