Abstract
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.
Highlights
Golgi-associated glycosyltransferases are type-II integral membrane proteins containing a short N-terminal cytoplasmic (C)2 tail, a single hydrophobic transmembrane (T) domain, a luminal stem (S) region, and the large C-terminal catalytic domain situated in the lumen of the Golgi apparatus [7,8,9]
Primary sequence and hydrophobicity analyses predicted that the xylosyltransferases Xylosyltransferase I (XT-I) and XT-II are, like the majority of eukaryotic glycosyltransferases, type-II transmembrane proteins with a short NH2terminal cytoplasmic tail, a single transmembrane domain, a stem region of variable length, and a large COOH-terminal globular catalytic domain [2, 24]
We have shown that XT-I is secreted into the extracellular space together with proteoglycans [22, 39], and that serum XT-I activity is a confirmed biochemical marker for the determination of fibrotic activity in systemic sclerosis [22, 24]
Summary
Golgi-associated glycosyltransferases are type-II integral membrane proteins containing a short N-terminal cytoplasmic (C) tail, a single hydrophobic transmembrane (T) domain, a luminal stem (S) region, and the large C-terminal catalytic domain situated in the lumen of the Golgi apparatus [7,8,9]. Several studies tried to identify the intracellular compartment where xylosylation occurs; there are data available suggesting a Golgi localization [25, 26], and others propose an activity in the endoplasmic reticulum [27,28,29] None of these analyses used a tagged form of the protein, but they focused on the determination of enzyme activity after separation of the subcellular compartment by centrifugation. The catalytic domain of XT-I is not implicated in their localization, and the region responsible for Golgi targeting is not associated with the activity of the enzyme This is the first report on gene cloning of the full-length human XT-I and the application of GFP-tagged XT-I and XT-II variants to identify the subcellular localization and the signals required for this
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
