The human UDP-glucuronosyltransferase isoform UGT1A6 is predicted to be a type I transmembrane protein anchored in the endoplasmic reticulum by a single C-terminal transmembrane domain, followed by a short cytoplasmic tail. This topology is thought to be established through the sequential action of a cleavable Nterminal signal peptide and of a C-terminal stop transfer/anchor sequence. We found that the deletion of the signal peptide did not prevent membrane targeting and insertion of this protein expressed in an in vitro transcription/translation system or in yeast Pichia pastoris. Interestingly, the same results were obtained when the protein was depleted of both the signal peptide and the C-terminal transmembrane domain/cytoplasmic tail sequences, suggesting the presence of an internal topogenic element able to translocate and retain UGT1A6 in the endoplasmic reticulum membrane in vitro and in yeast cells. To identify such a sequence, the insertion of several N-terminal deletion mutants of UGT1A6 into microsomal membranes was investigated in vitro. The data clearly showed that the deletion of the N-terminal end did not affect endoplasmic reticulum targeting and retention until residues 140 -240 were deleted. The signallike activity of the 140 -240 region was demonstrated by the ability of this segment to confer endoplasmic reticulum residency to the cytosolic green fluorescent protein expressed in mammalian cells. Finally, we show that this novel topogenic sequence can posttranslationally mediate the translocation of UGT1A6. This study provides the first evidence that the membrane assembly of the human UGT1A6 involves an internal signal retention sequence.Targeting and insertion of integral membrane proteins involve characteristic topogenic sequences that act to initiate (signal peptide sequence) and terminate (stop transfer sequence) translocation. In mammalian cells, the N-terminal signal peptide generally targets the protein to the endoplasmic reticulum (ER) 1 via a cotranslational pathway by binding to the signal recognition particle, which then interacts with its receptor and releases the signal sequence (1). A second signal recognition event within the ER involving the Sec61 complex, the main constituent of a protein conducting channel, is then required for the subsequent translocation of the nascent polypeptide across the lipid bilayer (2). The elongating polypeptide is then extruded through the membrane until a stop transfer sequence terminates translocation and integrates laterally into the membrane. A number of reports describing overlapping properties of signal sequences, signal anchors, and stop transfer sequences question the nature and specificity of these topogenic sequences. The stop transfer sequence of the IgM heavy chain (3) and the seventh transmembrane segment of band 3 of the erythrocyte anion exchanger (4) are able to initiate the translocation process. Signal anchors can also act as signal sequences as shown for cytochrome b 5 (5) and for the yeast UBC6 transmembrane protein (6). Single...

Objective To assess the variations of galactose‐ β‐1,3‐glucuronosyltransferase I (GlcAT‐I) expression related to the decrease in proteoglycan synthesis mediated by interleukin‐1β (IL‐1β) in rat chondrocytes, and to evaluate the influence of glucosamine on the effects elicited by this proinflammatory cytokine. Methods Rat articular chondrocytes in primary monolayer cultures or encapsulated into alginate beads were treated with recombinant IL‐1β in the absence or presence (1.0–4.5 gm/liter) of glucosamine. Variations of GlcAT‐I and expression of stromelysin 1 (matrix metalloproteinase 3 [MMP‐3]) messenger RNA (mRNA) were evaluated by quantitative multistandard reverse transcriptase–polymerase chain reaction. In vitro enzymatic activity of GlcAT‐I was measured by thin‐layer chromatography, with radiolabeled UDP‐glucuronic acid and a digalactoside derivative as substrates. Proteoglycan synthesis was determined by ex vivo incorporation of Na2‐35SO4. Nitric oxide synthase and cyclooxygenase activities were monitored by the evaluation of nitrite (NO −2) and prostaglandin E2 (PGE2) produced in the culture medium, respectively. Results IL‐1β treatment resulted in a marked inhibition of GlcAT‐I mRNA expression and in vitro catalytic activity, together with a decrease in proteoglycan synthesis. In addition, glucosamine was able to prevent, in a dose‐dependent manner, the inhibitory effects of IL‐1β. In the same way, the amino sugar reduced NO −2 and PGE2production induced by IL‐1β. Finally, the up‐regulation of stromelysin 1 (MMP‐3) mRNA expression by IL‐1β was fully prevented by glucosamine. Conclusion The results of this study suggest that the deleterious effect of IL‐1β on the anabolism of proteoglycan could involve the repression of GlcAT‐I, a key enzyme in the biosynthesis of glycosaminoglycan. Glucosamine was highly effective in preventing these IL‐1β–mediated suppressive effects. The amino sugar also prevented the production of inflammatory mediators induced by the cytokine. This action could account for a possible beneficial effect of glucosamine on osteoarthritic articular cartilage.

We determined whether the two major structural modifications, i.e. phosphorylation and sulfation of the glycosaminoglycan-protein linkage region (GlcA␤1-3Gal␤1-3Gal␤1-4Xyl␤1), govern the specificity of the glycosyltransferases responsible for the biosynthesis of the tetrasaccharide primer. We analyzed the influence of C-2 phosphorylation of Xyl residue on human ␤1,4-galactosyltransferase 7 (GalT-I), which catalyzes the transfer of Gal onto Xyl, and we evaluated the consequences of C-4/C-6 sulfation of Gal␤1-3Gal (Gal2-Gal1) on the activity and specificity of ␤1,3-glucuronosyltransferase I (GlcAT-I) responsible for the completion of the glycosaminoglycan primer sequence. For this purpose, a series of phosphorylated xylosides and sulfated C-4 and C-6 analogs of Gal␤1-3Gal was synthesized and tested as potential substrates for the recombinant enzymes. Our results revealed that the phosphorylation of Xyl on the C-2 position prevents GalT-I activity, suggesting that this modification may occur once Gal is attached to the Xyl residue of the nascent oligosaccharide linkage. On the other hand, we showed that sulfation on C-6 position of Gal1 of the Gal␤1-3Gal analog markedly enhanced GlcAT-I catalytic efficiency and we demonstrated the importance of Trp 243 and Lys 317 residues of Gal1 binding site for enzyme activity. In contrast, we found that GlcAT-I was unable to use digalactosides as acceptor substrates when Gal1 was sulfated on C-4 position or when Gal2 was sulfated on both C-4 and C-6 positions. Altogether, we demonstrated that oligosaccharide modifications of the linkage region control the specificity of the glycosyltransferases, a process that may regulate maturation and processing of glycosaminoglycan chains.

Heparan sulfate proteoglycans (HSPGs), strategically located at the cell-tissue-organ interface, regulate major biological processes, including cell proliferation, migration, and adhesion. These vital functions are compromised in tumors, due, in part, to alterations in heparan sulfate (HS) expression and structure. How these modifications occur is largely unknown. Here, we investigated whether epigenetic abnormalities involving aberrant DNA methylation affect HS biosynthetic enzymes in cancer cells. Analysis of the methylation status of glycosyltransferase and sulfotransferase genes in H-HEMC-SS chondrosarcoma cells showed a typical hypermethylation profile of 3-OST sulfotransferase genes. Exposure of chondrosarcoma cells to 5-aza-2'-deoxycytidine (5-Aza-dc), a DNA-methyltransferase inhibitor, up-regulated expression of 3-OST1, 3-OST2, and 3-OST3A mRNAs, indicating that aberrant methylation affects transcription of these genes. Furthermore, HS expression was restored on 5-Aza-dc treatment or reintroduction of 3-OST expression, as shown by indirect immunofluorescence microscopy and/or analysis of HS chains by anion-exchange and gel-filtration chromatography. Notably, 5-Aza-dc treatment of HEMC cells or expression of 3-OST3A cDNA reduced their proliferative and invading properties and augmented adhesion of chondrosarcoma cells. These results provide the first evidence for specific epigenetic regulation of 3-OST genes resulting in altered HSPG sulfation and point to a defect of HS-3-O-sulfation as a factor in cancer progression.