Involvement of a cysteine protease in the secretion process of human xylosyltransferase I

Abstract

Xylosylation of core proteins takes place in the Golgi-apparatus as the transfer of xylose from UDP-xylose to specific serine residues in proteoglycan core proteins. This initial and rate-limiting step in glycosaminoglycan biosynthesis is catalyzed by human xylosyltransferase I (XT-I). XT-I is proteolytically cleaved from the Golgi surface and shed in its active form into the extracellular space. The secreted, circulating glycosyltransferase represents a serum biomarker for various diseases with an altered proteoglycan metabolism, whereas a physiological function of secreted XT-I is still unknown. To shed light on the secretion process of XT-I and on its biological function, the cleavage site was examined and the group of proteases involved in the cleavage was identified in this study. The peptide mass fingerprint from partly purified secreted XT-I revealed the cleavage site to be localized in the aminoterminal 231 amino acids. The addition of a cysteine protease inhibitor cocktail to cells recombinantly expressing XT-I led to a concentration-dependent shift of enzyme activity towards the cell lysates attended by consistent total intracellular and extracellular XT-I activities. In conclusion, our findings provide a first insight into the XT-I secretion process regulated by a cysteine protease and may contribute to understanding the biological and pathological role of this process.

Activin A-Mediated Regulation of XT-I in Human Skin Fibroblasts

“…The correlation of mRNA expression and enzyme activity increase regarding XT-I has been shown in this and numerous other studies using TGFβ1-treated human dermal and cardiac fibroblasts [26,27,[45][46][47]. Furthermore, we observed, in consistency with previous results in NHDF [25], that the extracellular enzyme activity of untreated cells increased over time due to the protein accumulation in the cell supernatant, while intracellular XT activity remains constant over the test period of 120 h. These findings support the hypothesis that two regulatory mechanisms exist to control enzyme activity in NHDF: One at the transcriptional stage and another operating post-translationally, shedding the Golgi-resident, constitutive active enzyme from the membrane for release to the extracellular space controlling the rate of PG biosynthesis by reducing the cellular enzyme amount [24,29]. In contrast to TGFβ1, activin A treatment of NHDF results in a weaker XYLT1 mRNA expression increase, which can be explained by the autocrine TGFβ1 signalling, which is responsible for sustaining or amplifying the fibrotic responses in the pathogenesis of SSc [7].…”

O-xylosylation in a Recombinant Protein is Directed at a Common Motif on Glycine–Serine Linkers

“…The level of O‐xylosylation was also impacted by the cell line, with HEK‐293 cells having a significantly higher level of xylosylation than CHO cells. Reasons for this could include differences in core protein expression levels, differences in xylosyltransferase activity between cell types, differences in the intracellular and extracellular distribution of xylosyltransferase, availability of key intermediates such as UDP‐xylose, and the presence or absence of certain divalent cations . Our results indicate that the choice of expression system should be considered when manufacturing proteins with GS linkers.…”

Engineering β1,4-galactosyltransferase I to reduce secretion and enhance N-glycan elongation in insect cells

“…Proteolytic cleavage in the stem region of most Golgi glycosyltranferases, including fucosyltransferases (Grabenhorst et al, 1998), N -acetylglucosaminyltransferases (Nakahara et al, 2006), N -acetylgalactosaminyltransferases (El-Battari et al, 2003), xylosyltransferases (Ponighaus et al, 2010), galactosyltransferases (Cho et al, 1997; D’Agostaro et al, 1989), and sialyltransferases (El-Battari et al, 2003; Weinstein et al, 1987), produces subpopulations of soluble, catalytic domains of each enzyme that retain activity. These cleavages reduce Golgi N -glycan processing function by releasing subpopulations of elongating enzymes from Golgi membranes for secretion from the cell (Cho and Cummings, 1997; Grabenhorst et al, 1998; Zhu et al, 1998).…”