Cloning and Recombinant Expression of Active Full-length Xylosyltransferase I (XT-I) and Characterization of Subcellular Localization of XT-I and XT-II

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 ␣-mannosidase II, which is a known enzyme of the Golgi cisternae. Furthermore, a co-localization of native human XT-I and ␣-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.

Biosynthesis of Chondroitin and Heparan Sulfate in Chinese Hamster Ovary Cells Depends on Xylosyltransferase II

“…This co-expression suggests that these enzymes must act on different substrates or differ in kinetics toward the same substrates or are differentially compartmentalized in the cell. The latter appears to not be the case for HEK-293 cells since both enzymes appear localized to the cis-Golgi or medial Golgi compartments (29). Differences in substrate specificity or enzyme kinetics may help explain the observed expression patterns.…”

“…In addition, both enzymes appear localized to the same cellular compartment (29). However, the inability of XylT2 to have activity in vitro and the contrasting expression levels between XylT1 and XylT2 suggests that the biology of these enzymes is complex.…”

“…et al (29) found that XylT1 is secreted, and Gotting et al (13) showed that CHO-K1 cells secrete XylT activity, which we now know to be XylT2. In addition, both enzymes appear localized to the same cellular compartment (29).…”

Functional UDP-xylose Transport across the Endoplasmic Reticulum/Golgi Membrane in a Chinese Hamster Ovary Cell Mutant Defective in UDP-xylose Synthase

“…The proteoglycan xylosyltransferases have been shown to be Golgi-localized (49), but evidence exists for co-translational xylosylation (50). Although evidence has been presented for UXS in the Golgi (28), our tagged constructs appear only in the ER.…”

The Xylosyltransferase I Gene Polymorphism c.343G>T (p.A125S) Is a Risk Factor for Diabetic Nephropathy in Type 1 Diabetes

“…Furthermore, it was discussed that the stem region is an important part of the signal sequence required for the Golgi localization of the enzyme. The localization of XT-I in the Golgi compartment is crucial for the proper function of this enzyme, since the xylosylation of the core proteins occurs in the early Golgi apparatus (24). The importance of the alanine residue at position 115 is supported by a phylogenetic comparison of the xylosyltransferase sequences of mus musculus, pan troglodytes, canis familiaris, Xenopus laevis, and other species.…”