Cloning and recombinant expression of active full-length xylosyltransferase I (XT-I) and characterization of subcellular localization of XT-I and XT-II.
Xylosyltransferase I (XT-I) catalyzes the transfer of xylose from UDP-xylose to serine residues in proteoglycan core proteins. This is the first and apparently rate-limiting step in the biosynthesis of the tetrasaccharide linkage region in glycosaminoglycan-containing proteoglycans. The XYLT-II gene codes for a highly homologous protein, but its physiological function is not yet known. Here we present for the first time the construction of a vector encoding the full-length GFP-tagged human XT-I and the recombinant expression of the active enzyme in mammalian cells. We expressed XT-I-GFP and various GFP-tagged XT-I and XT-II mutants with C-terminal truncations and deletions in HEK-293 and SaOS-2 cells in order to investigate the intracellular localization of XT-I and XT-II. Immunofluorescence analysis showed a distinct perinuclear pattern of XT-I-GFP and XT-II-GFP similar to that of alpha-mannosidase II, which is a known enzyme of the Golgi cisternae. Furthermore, a co-localization of native human XT-I and alpha-mannosidase II could also be demonstrated in untransfected cells. Using brefeldin A, we could also show that both xylosyltransferases are resident in the early cisternae of the Golgi apparatus. For its complete Golgi retention, XT-I requires the N-terminal 214 amino acids. Unlike XT-I, for XT-II, the first 45 amino acids are sufficient to target and retain the GFP reporter in the Golgi compartment. Here we show evidence that the stem regions were indispensable for Golgi localization of XT-I and XT-II.
Institut für Laboratoriums- und Transfusionsmedizin, Herz- und Diabeteszentrum Nordrhein-Westfalen, Universitätsklinik der Ruhr-Universität Bochum, 32545 Bad Oeynhausen, Germany.
Sylvia
S
Schön
Christian
C
Prante
Claudia
C
Bahr
Joachim
J
Kuhn
Knut
K
Kleesiek
Christian
C
Götting
D020126:Brefeldin A / Q000737:chemistry; D002460:Cell Line; D003001:Cloning, Molecular; D003513:Cycloheximide / Q000494:pharmacology; D015971:Gene Expression Regulation, Enzymologic; D006025:Glycosaminoglycans / Q000737:chemistry; D006056:Golgi Apparatus / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D010430:Pentosyltransferases / Q000737:chemistry* / Q000235:genetics*; D010957:Plasmids / Q000378:metabolism; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D011994:Recombinant Proteins / Q000737:chemistry*; D000097608:UDP Xylose-Protein Xylosyltransferase
D020126:Brefeldin A; D003513:Cycloheximide; D006025:Glycosaminoglycans; D049452:Green Fluorescent Proteins; D010430:Pentosyltransferases; D011500:Protein Synthesis Inhibitors; D011994:Recombinant Proteins; D000097608:UDP Xylose-Protein Xylosyltransferase