The Functional Glycosyltransferase Signature Sequence of the Human β1,3-Glucuronosyltransferase Is a XDD Motif

Gulberti, Sandrine; Fournel‐Gigleux, Sylvie; Mulliert, Guillermo; Aubry, A.; Netter, Patrick; Magdalou, Jacques; Ouzzine, Mohamed

doi:10.1074/jbc.m207899200

Cited by 27 publications

(19 citation statements)

References 35 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…S5 B and D). It is not uncommon for Golgi-localized enzymes to prefer Mn 2+ as a cofactor (34). The remarkably low K m values are consistent with the idea that these kinases have evolved to function in the lumen of the secretory pathway and/or in the extracellular space, where ATP concentrations are likely to be much lower compared with the cytosol.…”

Section: Resultssupporting

confidence: 66%

Crystal structure of the Golgi casein kinase

Xiao

Tagliabracci

Wen

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

The family with sequence similarity 20 (Fam20) kinases phosphorylate extracellular substrates and play important roles in biomineralization. Fam20C is the Golgi casein kinase that phosphorylates secretory pathway proteins within Ser-x-Glu/pSer motifs. Mutations in Fam20C cause Raine syndrome, an osteosclerotic bone dysplasia. Here we report the crystal structure of the Fam20C ortholog from Caenorhabditis elegans. The nucleotide-free and Mn/ ADP-bound structures unveil an atypical protein kinase-like fold and highlight residues critical for activity. The position of the regulatory αC helix and the lack of an activation loop indicate an architecture primed for efficient catalysis. Furthermore, several distinct elements, including the presence of disulfide bonds, suggest that the Fam20 family diverged early in the evolution of the protein kinase superfamily. Our results reinforce the structural diversity of protein kinases and have important implications for patients with disorders of biomineralization.P rotein phosphorylation is a fundamental mechanism that regulates numerous physiological processes (1, 2). Protein kinases have evolved to function as dynamic switches relaying signals in response to various stimuli by transferring a phosphate from ATP to target proteins (3-5). Most phosphoproteins are found within the cell, residing in the nuclei and cytosol; however, secreted proteins, including casein and other members of the secretory calciumbinding phosphoprotein family, are phosphorylated as well (6). We recently identified a family of kinases that reside in the secretory pathway and function to phosphorylate extracellular substrates (7,8). One member of this family, Fam20C (family with sequence similarity 20, member C), is the physiological casein kinase that phosphorylates multiple secreted proteins within a Ser-x-Glu/pSer motif (7, 9, 10). The importance of this discovery is underscored by the fact that some 75% of the phosphoproteins identified in human serum and cerebrospinal fluid contain phosphate within this motif (11-13). Furthermore, mutations in FAM20C cause Raine syndrome, a deadly osteosclerotic bone dysplasia characterized by generalized osteosclerosis, ectopic calcifications, and characteristic facial features (14-16). Most individuals with Raine syndrome die within a few weeks after birth; however, nonlethal cases with dental abnormalities and clinical features of hypophosphatemia have been reported (17,18). Loss of Fam20C in mice also results in severe bone and tooth anomalies, as well as hypophosphatemia (19)(20)(21).Two other closely related Fam20C paralogs, Fam20A and Fam20B, are present in humans (22). Fam20B is ubiquitously expressed and phosphorylates xylose within the tetrasaccharide linkage region of proteoglycans (23). This phosphorylation event may influence glycosaminoglycan biosynthesis (24). Genetic deletion of Fam20B in mice results in embryonic lethality at E13.5, and mutations in Danio rerio result in reduced cartilage matrix production and skeletal defects (19,25). The substra...

show abstract

Section: Resultssupporting

confidence: 66%

Crystal structure of the Golgi casein kinase

Xiao

Tagliabracci

Wen

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

show abstract

“…GlcAT-I has received much attention because it plays a central role at a branching point common to various GAG chains, and it has been suggested to be rate-limiting in GAG synthesis in Chinese hamster ovary cells (18). We have been deeply involved in structural and functional studies of human GlcAT-I (19)(20)(21). We recently reported that IL-1␤ down-regulates GlcAT-I expression and activity and suggested that this may contribute to the reduced biosynthesis of GAGs observed in chondrocytes after IL-1␤ treatment (22).…”

mentioning

confidence: 99%

Stimulation of proteoglycan synthesis by glucuronosyltransferase-I gene delivery: A strategy to promote cartilage repair

Venkatesan

Barré

Bénani

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

Osteoarthritis is a degenerative joint disease characterized by a progressive loss of articular cartilage components, mainly proteoglycans (PGs), leading to destruction of the tissue. We investigate a therapeutic strategy based on stimulation of PG synthesis by gene transfer of the glycosaminoglycan (GAG)-synthesizing enzyme, ␤1,3-glucuronosyltransferase-I (GlcAT-I) to promote cartilage repair. We previously reported that IL-1␤ down-regulated the expression and activity of GlcAT-I in primary rat chondrocytes. Here, by using antisense oligonucleotides, we demonstrate that GlcAT-I inhibition impaired PG synthesis and deposition in articular cartilage explants, emphasizing the crucial role of this enzyme in PG anabolism. Thus, primary chondrocytes and cartilage explants were engineered by lipid-mediated gene delivery to efficiently overexpress a human GlcAT-I cDNA. Interestingly, GlcAT-I overexpression significantly enhanced GAG synthesis and deposition as evidenced by 35 S-sulfate incorporation, histology, estimation of GAG content, and fluorophore-assisted carbohydrate electrophoresis analysis. Metabolic labeling and Western blot analyses further suggested that GlcAT-I expression led to an increase in the abundance rather than in the length of GAG chains. Importantly, GlcAT-I delivery was able to overcome IL-1␤-induced PG depletion and maintain the anabolic activity of chondrocytes. Moreover, GlcAT-I also restored PG synthesis to a normal level in cartilage explants previously depleted from endogenous PGs by IL-1␤-treatment. In concert, our investigations strongly indicated that GlcAT-I was able to control and reverse articular cartilage defects in terms of PG anabolism and GAG content associated with IL-1␤. This study provides a basis for a gene therapy approach to promote cartilage repair in degenerative joint diseases.osteoarthritis ͉ gene transfer ͉ chondrocyte ͉ glycosyltransferase ͉ glycosaminoglycan

show abstract

“…[19][20][21] With respect to stabilization of the leaving group, inverting (and retaining) GT-A glycosyltransferases (with the exception of C2GnT-L) possess an essential divalent metal ion, coordinated in part by residues in the degenerate DXD sequence motif, that serves to electrostatically counter the negative charge that forms on the leaving group. [22][23][24][25] In contrast, inverting GT-B glycosyltransferases do not seem to possess a conserved means of stabilizing the leaving group, as positively charged side chains, neutral side chains and/or a positive α-helix dipole have all been observed in the vicinity of the scissile bond. 20,[26][27][28][29] Based on the X-ray crystal structure of C2GnT-L both in its apo form and in complex with its acceptor substrate, Galβ1,3GalNAc, 17 we previously showed that although C2GnT-L is a GT-A fold glycosyltransferase, it lacks the characteristic DXD motif and bound metal ion.…”

Section: Introductionmentioning

confidence: 80%