Molecular Cloning and Expression of the Pituitary Glycoprotein HormoneN-Acetylgalactosamine-4-O-sulfotransferase

Abstract

The pituitary hormones LH, 1 thyrotropin, and pro-opiomelanocortin bear unique N-linked oligosaccharides that terminate with the sequence SO 4 -4-GalNAc␤1,4GlcNAc␤1,2Man␣ (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-GlcA␤1,3Gal␤1,4GlcNAc (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).

Molecular Cloning and Characterization of Chondroitin-4-O-sulfotransferase-3

“…This recognition is essential for attaining maximal biologic activity in vivo (29). The highly regulated expression of GalNAc-4-ST1 (3,30) in other tissues such as the brain suggests that it will have additional roles that remain to be defined. In addition to their importance for the formation and maintenance of cartilage (31,32), there is also evidence that chondroitin sulfate proteoglycans are important for neural cell adhesion, neurite outgrowth, synaptic plasticity, and regeneration (33)(34)(35)(36)(37).…”

“…The GalNAc-sulfotransferases add sulfate to the C4 hydroxyl of either terminal ␤1,4-linked GalNAc (GalNAc-4-ST1 (3,5) and GalNAc-4-ST2 (6)) or to ␤1,4-linked GalNAc that is substituted at its C-3 hydroxyl with either GlcUA (C4ST-1 (8, 9) and C4ST-3) or IdoUA (D4ST-1 (7)). In contrast, HNK-1 ST adds sulfate to the C-3 hydroxyl of terminal ␤1,3-linked GlcUA (1,2).…”

“…After sedimentation, the precipitates were dissolved in 140 l of H 2 O and separated from any remaining [ 35 S]PAPS and degradation products by passage over BioSpin 6 columns (Bio-Rad). Transfer to GalNAc␤1,4GlcNAc␤1,2Man␣-MCO (19) and GlcUA␤1,3Gal␤1,4GlcNAc␤1,3Gal␤1,4Glc␤1,C 2 H 4 NHC-OCF 3 was assayed as described (2,3). When sulfotransferase bound to Ni 2ϩ -NTA-agarose was analyzed, the reaction was gently mixed every 30 min.…”

“…Sulfotransferase Activity Analysis-Chondroitin-and dermatan-4-sulfotransferase activities were assayed as described (3,8). Each reaction (50 l) contained 50 mM imidazole-HCl, pH 6.8, 2 mM dithiothreitol, 5 ϫ 10 5 cpm [ 35 S]PAPS, 2 M PAPS, and protamine (0.005% (w/v) for chondroitin or 0.05% (w/v) for dermatan), and protease inhibitors (see solubilization buffer above).…”

Peptide-specific Transfer of N-Acetylgalactosamine to O-Linked Glycans by the Glycosyltransferases β1,4-N-Acetylgalactosaminyl Transferase 3 (β4GalNAc-T3) and β4GalNAc-T4

“…pcDNAI-C2GnT1 (27) was provided by Dr. Minoru Fukuda (Burnham Institute for Medical Research, La Jolla, CA), and a plasmid for expression of ␣2,6NeuNAc-transferase (17) was provided by Dr. Karen Colley (University of Illinois, Chicago, IL). pcDNA3.1-GalNAc-4-ST1 and pcDNA3.1-GalNAc-4-ST2 were described previously (13,14). Diplococcal ␤-galactosidase (DP ␤-galactosidase) was prepared by affinity chromatography (28).…”