beta1,6 N-acetylglucosaminyltransferase (core 2 GlcNAc-T) expression in normal rat tissues and different cell lines: evidence for complex mechanisms of regulation.
The distribution of the Golgi enzyme beta1, 6-N-acetylglucosaminyltransferase (core 2 GlcNAc-T for short) has been investigated in several tissue and cell systems by combining the potentials of a polyclonal antibody and a novel, sensitive fluorescent enzyme assay. In normal rat tissues, levels of the protein were found to vary and as a general trend did not correlate with enzyme activities. Additionally, we observed tissue-specific core 2 GlcNAc-T forms of various size: 75 kDa (liver), 70 kDa (spleen), 60 kDA (heart), and 50 kDa (heart and lung). These forms might arise from differential protein modifications; alternatively, the smaller form may be a product of proteolytic cleavage, given the presence of a catalytically inactive 50 kDa species in rat serum. Chinese hamster ovary (CHO), MDAY-D2, PSA-5E, and PYS-2 cell lines consistently displayed a 70 kDa enzyme. When induced to retrodifferentiate in the presence of butyrate + cholera toxin, CHO cells exhibited a 21-fold increase in enzyme activity, while protein levels remained constant. A similar trend was observed in the embryonal endoderm cell lines PSA-5E and PYS-2, where an approximately 100-fold difference in core 2 GlcNAc-T activity was found notwithstanding unchanged amounts of the protein and identical mRNA levels, as evidenced by RT-PCR. In contrast, levels of core 2 GlcNAc-T activity in MDAY-D2 cells correlated well with protein expression. Taken together, these observations demonstrate that core 2 GlcNAc-T expression may be subjected to multiple mechanisms of regulation and suggest that in at least some instances (i.e., PSA-5E and PYS-2 cells) expression may be regulated exclusively via posttranslational mechanism(s) of control.
VanderElst
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Department of Cell and Molecular Biology, Section of Biochemistry and Molecular Biology, University of Perugia, 06126 Perugia, Italy.
Datti
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D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D004707:Endoderm / Q000201:enzymology; D015971:Gene Expression Regulation, Enzymologic; D006056:Golgi Apparatus / Q000201:enzymology; D018922:HL-60 Cells / Q000201:enzymology; D006801:Humans; D007527:Isoenzymes / Q000096:biosynthesis; D008099:Liver / Q000201:enzymology; D008168:Lung / Q000201:enzymology; D051379:Mice; D008970:Molecular Weight; D009206:Myocardium / Q000201:enzymology; D017351:N-Acetylglucosaminyltransferases / Q000096:biosynthesis*; D009928:Organ Specificity; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D013154:Spleen / Q000201:enzymology; D014162:Transfection
D007527:Isoenzymes; D011993:Recombinant Fusion Proteins; D017351:N-Acetylglucosaminyltransferases; C053024:beta-1,3-galactosyl-O-glycosyl-glycoprotein beta-1,6-acetylglucosaminyl transferase