Xylose phosphorylation functions as a molecular switch to regulate proteoglycan biosynthesis

Abstract

Most eukaryotic cells elaborate several proteoglycans critical for transmitting biochemical signals into and between cells. However, the regulation of proteoglycan biosynthesis is not completely understood. We show that the atypical secretory kinase family with sequence similarity 20, member B (Fam20B) phosphorylates the initiating xylose residue in the proteoglycan tetrasaccharide linkage region, and that this event functions as a molecular switch to regulate subsequent glycosaminoglycan assembly. Proteoglycans from FAM20B knockout cells contain a truncated tetrasaccharide linkage region consisting of a disaccharide capped with sialic acid (Siaα2-3Galβ1-4Xylβ1) that cannot be further elongated. We also show that the activity of galactosyl transferase II (GalT-II, B3GalT6), a key enzyme in the biosynthesis of the tetrasaccharide linkage region, is dramatically increased by Fam20B-dependent xylose phosphorylation. Inactivating mutations in the GALT-II gene (B3GALT6) associated with Ehlers-Danlos syndrome cause proteoglycan maturation defects similar to FAM20B deletion. Collectively, our findings suggest that GalT-II function is impaired by loss of Fam20B-dependent xylose phosphorylation and reveal a previously unappreciated mechanism for regulation of proteoglycan biosynthesis.secretory kinase | proteoglycan | xylose phosphorylation | Fam20B | GalT-II

Positive Mode LC-MS/MS Analysis of Chondroitin Sulfate Modified Glycopeptides Derived from Light and Heavy Chains of The Human Inter-α-Trypsin Inhibitor Complex*

“…In addition to sulfation, we observed the presence of fucose and phosphate on the CS linkage region of UTI but not in CSF or plasma samples. Because xylose phosphorylation is thought to act as a transitional switch that regulates CS-biosynthesis (19,47), such information may be of value for understanding the structural differences between body fluids. As our analysis was restricted to linkage region glycopeptides we could not assess whether full-length CS-chains carrying phosphate or fucose modifications actually differed in any respect from those only substituted with sulfates.…”

“…Previous studies have made possible the analysis of the average fine structure of chondroitin sulfate (CS) chains facilitating the identification of discrete glycan domains likely involved in ligand interaction (12)(13)(14)(15)(16). The CSPG saccharide linkage region differs significantly from the structure of the rest of the glycan chain and its assembly has been shown to be essential for the regulation of the GAG biosynthesis (17)(18)(19).…”

The secretory pathway kinases

“…Subsequent biochemical work demonstrated that Fam20B phosphorylates the 2-OH on a xylose residue within the conserved tetrasaccharide linkage region of proteoglycans [10]. Xylose phosphorylation markedly stimulates the galactosyltransferase activity of galactosyltransferase II (GalT-II, B3GalT6), an enzyme that adds galactose to the growing tetrasaccharide linkage [25]. Furthermore, xylose phosphorylation appears to be necessary for EXTL2, a polymerase involved in the biosynthesis of heparin sulfate, to transfer a GlcNAc residue to the tetrasaccharide linkage region, which subsequently terminates chain elongation [26].…”

A Single Kinase Generates the Majority of the Secreted Phosphoproteome

“…Although the substrates for Fam20A, Fam198A and Fam198B are unknown, Fam20B phosphorylates a xylose residue within the tetrasaccharide linkage region of proteoglycans (Koike et al, 2009). This phosphorylation markedly stimulates the activity of galactosyltransferase II to promote proteoglycan biosynthesis (Wen et al, 2014). …”