The pituitary hormones LH, 1 thyrotropin, and pro-opiomelanocortin bear unique N-linked oligosaccharides that terminate with the sequence SO 4 -4-GalNAc1,4GlcNAc1,2Man␣ (1-6).The terminal GalNAc-4-SO 4 is recognized by a receptor, the Man/GalNAc-4-SO 4 receptor (7-9), which is located in hepatic endothelial cells and controls the circulatory half-life of LH and other glycoproteins bearing this structure (10, 11). A short circulatory half-life in conjunction with the regulated release from storage granules in the gonadotroph produces the episodic rise and fall seen in circulating LH levels (12, 13). This is essential for efficient activation of the LH receptor in the ovary and testis due to the down-regulation and internalization of the activated receptor by -arrestin (14). Sequential actions of a hormone-specific 1,4 GalNAc-transferase and a GalNAc-4-sulfotransferase are required for the synthesis of GalNAc-4-SO 4 on LH and other glycoproteins (13,(15)(16)(17)(18)(19). Three key functions have been conserved throughout vertebrate evolution: 1) the peptide recognition determinant utilized by the GalNAc-transferase, 2) the GalNAc-transferase itself, and 3) the GalNAc-4-sulfotransferase. As a result, Nlinked structures terminating with 1,4-linked GalNAc-4-SO 4 occur on glycoprotein hormones from all vertebrate species (20). GalNAc-transferase and GalNAc-4-sulfotransferase activities with the same properties as those expressed in the pituitary have been detected in other tissues and cells (21). Furthermore, a number of other glycoproteins bearing N-and O-linked structures terminating with 1,4-linked GalNAc-4-SO 4 have been described (2,5,(22)(23)(24)(25)(26). Thus, oligosaccharides terminating with GalNAc-4-SO 4 may fulfill a number of different roles in vivo.Recently the HNK-1 sulfotransferase (HNK-1 ST), a glucuronyl-3-sulfotransferase that mediates synthesis of the HNK-1 epitope SO 4 -3-GlcA1,3Gal1,4GlcNAc (27) was cloned (28, 29). The HNK-1 structure is found in the central and peripheral nervous systems on glycolipids and glycoproteins involved in neural recognition and synaptogenesis (Ref. 30; for reviews, see Refs. 31 and 32). Expression of the HNK-1 structure is spatially and temporally regulated, suggesting an important role in axon outgrowth (33). It is a predominant autoantigen in demyelinating diseases of the peripheral nervous system (34). Recently this glycan has been recognized to be a crucial player in synaptic plasticity involving inhibitory interneurons in the hippocampus (35). The sulfate group of the HNK-1 glycan is essential for its function (36, 37).
The Ewing Sarcoma Family Tumors (ESFT) consist of the classical pathologic entities of Ewing Sarcoma and peripheral Primitive Neuroectodermal Tumor. Occurring largely in the childhood through young adult years, these tumors have an unsurpassed propensity for metastasis and have no defined cell of origin. The biology of these aggressive malignancies centers around EWS/FLI1 and related EWS/ETS chimeric transcription factors, which are largely limited to this tumor class. Much progress has been made in the identification of a network of loci whose expression is modulated by EWS/FLI1 and its congeners. To date, little progress has been made in reconstructing the sequence of direct and indirect events that produce this network of modulated loci. The recent identification of GLI1 as an upregulated target of EWS/ETS transcription factors suggests a target which may be a more central mediator in the ESFT signaling network. In this paper, we further define the relationship of EWS/FLI1 expression and GLI1 upregulation in ESFT. This relationship is supported with data from primary tumor specimens. It is consistently observed across multiple ESFT cell lines and with multiple means of EWS/FLI1 inhibition. GLI1 inhibition affects tumor cell line phenotype whether shRNA or endogenous or pharmacologic inhibitors are employed. As is seen in model transformation systems, GLI1 upregulation by EWS/FLI1 appears to be independent of Hedgehog stimulation. Consistent with a more central role in ESFT pathogenesis, several known EWS/FLI1 targets appear to be targeted through GLI1. These findings further establish a central role for GLI1 in the pathogenesis of Ewing Tumors.
We have identified and characterized an N-acetylgalactosamine-4-O-sulfotransferase designated chondroitin-4-sulfotransferase-3 (C4ST-3) (GenBank TM accession number AY120869) based on its homology to HNK-1 sulfotransferase (HNK-1 ST). The cDNA predicts an open reading frame encoding a type II membrane protein of 341 amino acids with a 12-amino acid cytoplasmic domain and a 311-amino acid luminal domain containing a single potential N-linked glycosylation site. C4ST-3 has the greatest amino acid sequence identity when aligned with chondroitin-4-O-sulfotransferase 1 (C4ST-1) (45%) but also shows significant amino acid identity with chondroitin-4-O-sulfotransferase 2 (C4ST-2) (27%), dermatan-4-O-sulfotransferase 1 (29%), HNK-1 ST (26%), N-acetylgalactosamine-4-O-sulfotransferase 1 (26%), and N-acetylgalactosamine-4-Osulfotransferase 2 (23%). C4ST-3 transfers sulfate to the C-4 hydroxyl of 1,4-linked GalNAc that is substituted with a -linked glucuronic acid at the C-3 hydroxyl. The open reading frame of C4ST-3 is encoded by three exons located on human chromosome 3q21.3. Northern blot analysis reveals a single 2.1-kilobase transcript. C4ST-3 message is expressed in adult liver and at lower levels in adult kidney, lymph nodes, and fetal liver. Although C4ST-3 and C4ST-1 have similar specificities, the highly restricted pattern of expression seen for C4ST-3 suggests that it has a different role than C4ST-1.We and others have recently reported the cloning and functional characterization of members of the HNK-1 family of sulfotransferases. These include human HNK-1 sulfotransferase (HNK-1 ST) 1 itself (1, 2), GalNAc-4-ST1 (3-5), GalNAc-4-ST2 (5, 6), dermatan-4-sulfotransferase-1 (D4ST-1) (7), chondroitin-4-sulfotransferase-1 (C4ST-1) (8, 9), and chondroitin-4-sulfotransferase-2 (C4ST-2) (8). With the exception of HNK-1 ST itself, which transfers sulfate to the C-3 hydroxyl of terminal glucuronic acid in the sequence GlcUA1,3Gal1,4GlcNAc-R to produce the HNK-1 epitope SO 4 -3-GlcUA1,3Gal1,4GlcNAc-R, the members of this family of sulfotransferases are all GalNAc-4-sulfotransferases. GalNAc-4-ST1 and GalNAc-4-ST2 both transfer sulfate to the C-4 hydroxyl of terminal 1,4-linked GalNAc on N-linked oligosaccharides such as those found on the glycoprotein hormones lutropin and thyrotropin (10, 11). Whereas C4ST-1, C4ST-2, and D4ST-1 are also GalNAc-4-O-sulfotransferases, they only transfer sulfate to the C-4 hydroxyl of internal 1,4-linked GalNAc moieties within the repeating disaccharide sequences found in chondroitin and dermatan (7-9). We now report the cloning of another member of this family of sulfotransferases. Like C4ST-1, this new sulfotransferase, chondroitin-4-sulfotransferase-3 (C4ST-3) transfers sulfate to the C-4 hydroxyl of 1,4-linked GalNAc that is flanked by GlcUA residues in chondroitin. In contrast to C4ST-1, C4ST-3 is labile at 37°C and has a restricted distribution, suggesting that it may have a unique biological role in vivo. EXPERIMENTAL PROCEDURES Molecular Cloning of a cDNA Encoding Human Chon...
I-Glucosidase can be located after nondenaturing polyacrylamide gel electrophoresis by incubating the gel with 0.1% esculin and 0.03% ferric chloride. The esculetin released from esculin by I-glucosidase action reacts with ferric ion to produce a black band, corresponding to the j-glucosidase, against the transparent background.
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