Sulfation of thyroglobulin: A ubiquitous modification in vertebrates

Summary Mammalian thyroglobulin is released by thyroid follicle cells as a sulfated glycoprotein; the sulfate residues are mostly linked to tyrosine, but they are also attached to the high-mannose carbohydrate side-chains. To decide whether sulfation of thyroglobulin is confined to mammals, representatives of other vertebrate classes were analyzed for the presence of sulfated thyroglobulin: fish (trout), amphibians (clawed toad) and birds (chicken). Mini-organs were prepared from thyroid tissue and suspended in a ³⁵SO ₄ ^-- -containing culture medium. Light- and electron-microscope autoradiographs prepared from the mini-organs showed that thyroid follicle cells from all species examined incorporate ³⁵SO ₄ ^-- and synthesize a sulfated secretory product which accumulates in the follicle lumen. The Golgi complex was detected as the primary intracellular site of sulfate organification. The ³⁵SO ₄ ^-- -radiolabeled secretory product of all species was shown by polyacrylamide-gel-electrophoretic analyses to consist of thyroglobulin, identified by comparison with biosynthetically ¹²⁵I-labeled thyroglobulin. The results indicate that the sulfation of thyroglobulin is a ubiquitous post-translational modification observed already in the thyroglobulin of lower vertebrates. Our observations suggest that sulfation of thyroglobulin was acquired in the early stages of thyroid evolution.     

Structural analysis of N-linked oligosaccharides from glycoproteins secreted by Dictyostelium discoideum. Identification of mannose 6-sulfate

The N-linked oligosaccharides found on the lysosomal enzymes from Dictyostelium discoideum are highly sulfated and contain methylphosphomannosyl residues (Gabel, C. A., Costello, C. E., Reinhold, V. N., Kurtz, L., and Kornfeld, S. (1984) J. Biol. Chem. 259, 13762-13769). Here we report studies done on the structure of N-linked oligosaccharides found on proteins secreted during growth, a major portion of which are lysosomal enzymes. Cells were metabolically labeled with [2-3H]Man and 35SO4 and a portion of the oligosaccharides were released by a sequential digestion with endoglycosidase H followed by endoglycosidase/peptide N-glycosidase F preparations. The oligosaccharides were separated by anion exchange high performance liquid chromatography into fractions containing from one up to six negative charges. Some of the oligosaccharides contained only sulfate esters or phosphodiesters, but most contained both. Less than 2% of the oligosaccharides contained a phosphomonoester or an acid-sensitive phosphodiester typical of the mammalian lysosomal enzymes. A combination of acid and base hydrolysis suggested that most of the sulfate esters were linked to primary hydroxyl groups. The presence of Man-6-SO4 was demonstrated by the appearance of 3,6-anhydromannose in acid hydrolysates of base-treated, reduced oligosaccharides. These residues were not detected in acid hydrolysates without prior base treatment or in oligosaccharides first treated by solvolysis to remove sulfate esters. Based on high performance liquid chromatography quantitation of percentage of 3H label found in 3,6-anhydromannose, it is likely that Man-6-SO4 accounts for the majority of the sulfated sugars in the oligosaccharides released from the secreted glycoproteins.     

Transport of heparan sulfate into the nuclei of hepatocytes

Monolayer cultures of a rat hepatocyte cell line shown previously to accumulate a nuclear pool of free heparan sulfate chains that are enriched in sulfated glucuronic acid (GlcA) residues (Fedarko, N.S., and Conrad, H.E., (1986) J. Cell Biol. 587-599) were incubated with 35SO4(2-), and the rate of appearance of heparan [35S]sulfate in the nuclei was measured. Heparan [35S]sulfate began to accumulate in the nuclei 2 h after the administration of 35SO4(2-) to the cells and reached a steady state level after 20 h. Heparan [35S]sulfate was lost from the nuclei of prelabeled cells with a t1/2 of 8 h. Chloroquine did not inhibit the transport of heparan sulfate into the nucleus, but increased the t1/2 for the exit of heparan sulfate from the nucleus to 20 h and led to a doubling of the steady state level of nuclear heparan sulfate. Heparan [35S]sulfate which was obtained from the medium or from the cell matrix of a labeled culture and which contained only low levels of GlcA-2-SO4 residues was incubated with cultures of unlabeled cells, and the uptake of the exogenous heparan [35S]sulfate was studied. At 37 degrees C the cells took up proteoheparan [35S]sulfate and transported about 10% of the internalized heparan [35S]sulfate into the nucleus, where it appeared as free chains. The heparan [35S]sulfate isolated from the nucleus was enriched in GlcA-2-SO4 residues, whereas the heparan [35S]sulfate remaining in the rest of the intracellular pool showed a corresponding depletion in GlcA-2-SO4 residues. At 16 degrees C, where endocytosed materials do not enter the lysosomes, the cells also transported exogenous proteoheparan [35S]sulfate to the nucleus with similar processing. Thus, the metabolism of exogenous heparan sulfate by hepatocytes follows the same pathway observed in continuously labeled cells and does not involve lysosomal processing of the internalized heparan sulfate.     

Characterization of novel sequences containing 3-O-sulfated glucosamine in glomerular basement membrane heparan sulfate and localization of sulfated disaccharides to a peripheral domain

Fragmentation of the heparan sulfate chains from bovine glomerular basement membrane (GBM) by hydrazine/nitrous acid treatment followed by NaB3H4-reduction yielded a mixture of six sulfated disaccharides containing D-glucuronic (GlcUA) or L-iduronic acid (IdUA) and terminating in 2,5-anhydro[3H]mannitol (AnManH2), in addition to the nonsulfated component GlcUA beta 1----4AnManH2. Among these products two novel disaccharide units were identified as IdUA alpha 1----4AnManH2(3-SO4) and IdUA(2-SO4)alpha 1----4AnManH2(3-SO4); these accounted for 22% of the total sulfated species indicating that there are 2-3 residues of 3-O-sulfated glucosamine/heparan sulfate chain. The disulfated disaccharide was shown through its release by direct nitrous acid treatment to be situated in a GlcNSO3-IdUA(2-SO4)-GlcNSO3(3-SO4) sequence which is distinct from that in which 3-O-sulfated glucosamine is located in the antithrombin-binding region of heparins. Analyses of heparan sulfate from lens capsule, a nonvascular basement membrane, indicated the absence of sequences containing 3-O-sulfated glucosamine, although otherwise the sulfated disaccharides produced by hydrazine/nitrous acid/Na-B3H4 treatment (GlcUA beta 1----4AnManH2(6-SO4), IdUA alpha 1----4AnManH2(6-SO4), IdUA(2-SO4)alpha 1----4AnManH2 and IdUA(2-SO4)alpha 1----4AnManH2(6-SO4] were the same as from GBM. Examination of the GBM heparan sulfate domains after nitrous acid treatment indicated that the O- as well as N-sulfate groups are clustered in an iduronic acid-rich 10-disaccharide peripheral segment, while the internal region (approximately 20 disaccharides) is composed primarily of repeating GlcUA beta 1----4GlcNAc units. The localization of chain diversity to the outer region may facilitate interactions of the heparan sulfate with other macromolecular components.     

Characterization of a thyroid sulfotransferase responsible for the 3-O-sulfation of terminal beta-D-galactosyl residues in N-linked carbohydrate units

Calf thyroid microsomes were found to contain an enzyme which catalyzes the transfer of sulfate from 3'-phosphoadenosine 5'-phospho[35S]sulfate (PAPS) to C-3 of terminal galactose residues in beta 1----4 linkage to GlcNAc. This sulfotransferase is believed to be involved in the biosynthesis of the recently described Gal(3-SO4) capping groups present in the N-linked oligosaccharides of thyroglobulin (Spiro, R.G., and Bhoyroo, V. D. (1988) J. Biol. Chem. 263, 14351-14358). Assays with various native and modified glycopeptides indicated that the enzyme acted optimally on complex-type carbohydrate units in which beta-linked Gal has been uncovered by desulfation or brought into a terminal position by removal of sialyl and/or alpha-galactosyl residues. With fetuin asialoglycopeptides as acceptors (Km = 0.1 mM) the transfer of sulfate from PAPS (Km = 6.3 microM) had a pH optimum of approximately 7.0, required Mn2+ ions (10-50 mM) and was markedly stimulated by Triton X-100 (0.1%) and ATP (2 mM). The same enzyme apparently sulfated free N-acetyllactosamine (LacNAc; Km = 0.69 mM) and its ethyl glycoside, indicating that it had no absolute requirement for a peptide recognition site. Studies with a number of disaccharides related to LacNAc provided information relating to the specifying role of the beta 1----4 galactosyl linkage and the configuration at C-2 of the sugar to which it is attached. Hydrazine-nitrous acid-NaBH4 treatment of the 35S-labeled products from sulfotransferase action on asialoglycopeptides as well as on the ethyl glycoside of LacNAc yielded the same disaccharide, Gal(3-SO4) beta 1----4 anhydromannitol, as is obtained from a similar treatment of thyroglobulin. Subcellular distribution studies indicated that the PAPS:galactose 3-O-sulfotransferase is located in the Golgi compartment which is consistent with the late occurrence of the requisite beta-galactosylation step. It is proposed that in certain tissues the ultimate nature of the capping groups attached to glycoproteins containing terminal Gal beta 1----4GlcNAc sequences could be the result of a competition between this 3-O-sulfotransferase and sialyl- and/or alpha-galactosyltransferases.     

Presence of sulfate in N-glycosidically linked carbohydrate units of calf thyroid plasma membrane glycoproteins

Calf thyroid slices were found to incorporate [35S] sulfate into two major plasma membrane glycoproteins, which have been previously designated as GP-1 and GP-3 (Okada, Y., and Spiro, R. G. (1980) J. Biol. Chem. 255, 8865-8872). The 35S-glycoproteins were identified on the basis of their characteristic solubility and electrophoretic migration as well as their affinity for Bandeiraea simplicifolia I lectin. After pronase digestion of these glycoproteins, the 35S-label remained associated with the glycopeptides primarily on asparagine-linked carbohydrate units which were released by hydrazinolysis. Examination of the reduced radio-labeled products obtained by nitrous acid cleavage of the hydrazine-liberated oligosaccharides indicated that sulfate esters of N-acetylglucosamine occurred at three locations on the carbohydrate units; two 35S-monosaccharides (2,5-anhydromannitol 4- and 6-sulfate) and one 35S-disaccharide (beta-Gal(1----4)-2,5-anhydromannitol(6-SO4] were formed. The disaccharide is believed to be derived from an internal sulfated N-acetyllactosamine sequence while the monosaccharides most likely originate from 4- and 6-sulfated N-acetylglucosamine residues situated, respectively, at the non-reducing and reducing termini of the oligosaccharide units. Quantitation by NaB[3H]4 reduction of the sulfated saccharides obtained by nitrous acid treatment of hydrazine-released oligosaccharides from unlabeled GP-3 indicated that about 20% of the asparagine-linked carbohydrate units contain sulfate substituents.     

Sulfated N-linked oligosaccharides in mammalian cells. III. Characterization of a pancreatic carcinoma cell surface glycoprotein with N- and O-sulfate esters on asparagine-linked glycans

In the preceding two papers, we described two new classes of sulfated N-linked oligosaccharides isolated from total cellular 35SO4-labeled macromolecules of different mammalian cell lines. The first class carries various combinations of sialic acids and 6-O-sulfate esters on typical complex-type chains, while the second carries heparin and heparan-like sequences. In this study, we have characterized a sulfophosphoglycoprotein of 140 kDa from FG-Met-2 pancreatic cancer cells whose oligosaccharides share some properties of both these classes. The molecule was localized to the cell surface by electron microscopy using a monoclonal antibody (S3-53) and by cell surface 125I-labeling. Metabolic labeling of the cells with radioactive glucosamine, methionine, inorganic sulfate, or phosphate all demonstrated a single 140-kDa molecule. Pulse-chase analysis and tunicamycin treatment indicated the glycosylation of a putative primary translation product of 110 kDa via an intermediate (120 kDa) to the mature form (140 kDa). Digestion with peptide:N-glycosidase F (PNGaseF) indicated a minimum of four N-linked glycosylation sites. PNGaseF released more than 90% of the [6-3H]GlcNH2 label and 40-70% of 35SO4 label from the immunoprecipitated 140-kDa molecule. The isolated oligosaccharides were characterized as described in the preceding two papers. The majority of [6-3H]GlcNH2-labeled molecules were susceptible to neuraminidase. More than 50% of the 35SO4 label was associated with only 5-10% of the 3H-labeled chains. Some of the sulfated chains were partly sialylated molecules with four to five negative charges. Treatment with nitrous acid released about 25% of the 35SO4 label as free sulfate, together with 6% of the [6-3H]GlcNH2 label, indicating the presence of N-sulfated glucosamine residues. Some of these oligosaccharides were degraded by heparinase and heparitinase. Therefore, while they are not as highly charged as typical heparin or heparan chains, they must share structural features that permit recognition by the enzymes. Thus, this 140-kDa glycoprotein contains at least four asparagine-linked chains substituted with a heterogeneous mixture of sulfated sequences. The heterogeneity of these molecules is as extensive as that described for whole-cell sulfated N-linked oligosaccharides in the preceding two papers.     

Sulfated oligosaccharides in human lysosomal enzymes

Cathepsin D, arylsulfatase A and the alpha-chain of beta-hexosaminidase are synthesized in human fibroblasts as sulfated polypeptides. The sulfate is added posttranslationally. Its half-life is less than one-tenth of that of the respective polypeptide chains. The sulfate residues were found on asparagine-linked oligosaccharides sensitive to endoglycosidase F and peptide: N-glycosidase F and resistant to endoglycosidase H. Inhibition of formation of complex type oligosaccharides by 1-deoxy-manno-nojirimycin prevented sulfation, indicating that the sulfate residues were added to complex type oligosaccharides.     

The molecular mechanism of sulfated carbohydrate recognition by the cysteine-rich domain of mannose receptor

The mannose receptor (MR) binds foreign and host ligands through interactions with their carbohydrates. Two portions of MR have distinct carbohydrate recognition properties. One is conferred by the amino-terminal cysteine-rich domain (Cys-MR), which plays a critical role in binding sulfated glycoproteins including pituitary hormones. The other is achieved by tandemly arranged C-type lectin domains that facilitate carbohydrate-dependent uptake of infectious microorganisms. This dual carbohydrate binding specificity enables MR to bind ligands by interacting with both sulfated and non-sulfated polysaccharide chains. We previously determined crystal structures of Cys-MR complexed with 4-SO(4)-N-acetylglucosamine and with an unidentified ligand resembling Hepes (N-[2-hydroxyethyl]piperazine-N'-[2-ethanesulfonic acid]). In continued efforts to elucidate the mechanism of sulfated carbohydrate recognition by Cys-MR, we characterized the binding affinities between Cys-MR and potential carbohydrate ligands using a fluorescence-based assay. We find that Cys-MR binds sulfated carbohydrates with relatively high affinities (K(D)=0.1 mM to 1.0 mM) compared to the affinities of other lectins. Cys-MR also binds Hepes with a K(D) value of 3.9 mM, consistent with the suggestion that the ligand in the original Cys-MR crystal structure is Hepes. We also determined crystal structures of Cys-MR complexed with 3-SO(4)-Lewis(x), 3-SO(4)-Lewis(a), and 6-SO(4)-N-acetylglucosamine at 1.9 A, 2.2 A, and 2.5 A resolution, respectively, and the 2.0 A structure of Cys-MR that had been treated to remove Hepes. The conformation of the Cys-MR binding site is virtually identical in all Cys-MR crystal structures, suggesting that Cys-MR does not undergo conformational changes upon ligand binding. The structures are used to rationalize the binding affinities derived from the biochemical studies and to elucidate the molecular mechanism of sulfated carbohydrate recognition by Cys-MR.     

Enzymatic synthesis of chondroitin sulfate E by N-acetylgalactosamine 4-sulfate 6-O-sulfotransferase purified from squid cartilage

Chondroitin sulfate E (CS-E), a chondroitin sulfate isomer containing GlcAbeta1-3GalNAc(4,6-SO(4)) repeating unit, was found in various mammalian cells in addition to squid cartilage and is predicted to have several physiological functions in various mammalian systems such as mast cell maturation, regulation of procoagulant activity of monocytes, and binding to midkine or chemokines. To clarify the physiological functions of GalNAc(4,6-SO(4)) repeating unit, preparation of CS-E with a defined content of GalNAc(4,6-SO(4)) residues is important. We report here the in vitro synthesis of CS-E from chondrotin sulfate A (CS-A) by the purified squid N-acetylgalactosamine 4-sulfate 6-O-sulfotransferase (GalNAc4S-6ST) which catalyzed transfer of sulfate from 3(')-phosphoadenosine-5(')-phosphosulfate to position 6 of GalNAc(4SO(4)) residues of CS-A and dermatan sulfate (DS). When CS-A was used as an acceptor, about half of GalNAc(4SO(4)) residues, on average, were converted to GalNAc(4,6-SO(4)) residues. Anion exchange chromatography of the CS-E synthesized in vitro showed marked heterogeneity in negative charge; the proportion of GalNAc(4,6-SO(4)) in the most negative fraction exceeded 70% of the total sulfated repeating units. GalNAc4S-6ST also catalyzed the synthesis of oversulfated DS with GalNAc(4,6-SO(4)) residues from DS. Squid GalNAc4S-6ST thus should provide a useful tool for preparing CS-E and oversulfated DS with a defined proportion of GalNAc(4,6-SO(4)) residues.     

A sulfatase specific for glucuronic acid 2-sulfate residues in glycosaminoglycans

Although 2-O-sulfated L-iduronic acid (IdoA) residues have been known to occur in heparin, 2-O-sulfated D-glucuronic acid (GlcA) residues have been reported only recently (Bienkowski, M. J., and Conrad, H. E. (1985) J. Biol. Chem. 250, 356-365). Disaccharides prepared by cleavage of heparin and N-deacetylated chondroitin 6-sulfate with nitrous acid were used to demonstrate a new sulfatase that catalyzed the removal of the 2-O-sulfate substituents from GlcA but not IdoA residues. The deamination products were labeled by NaB3H4 reduction to give disaccharides from heparin and chondroitin sulfate which had reducing terminal 2,5-anhydro-D-mannitol ([3H]AManR) and 2,5-anhydro-D-talitol ([3H]ATalR) residues, respectively. IdoA(2-SO4)-[3H]AManR(6-SO4) from heparin and GlcA(2-SO4)-[3H]ATalR(6-SO4) from chondroitin sulfate were purified for use as substrates. GlcA(2-SO4)-[3H]AManR(6-SO4) was prepared by epimerization of IdoA(2-SO4)-[3H]AManR(6-SO4) with hydrazine at 100 degrees C. Lysosomal enzyme preparations from chick embryo chondrocytes and from two normal human fibroblast cell lines catalyzed the removal of the 2-O-SO4 substituent from the uronic acid residues of IdoA(2-SO4)-[3H]AManR(6-SO4), GlcA(2-SO4)-[3H] AManR(6-SO4), and GlcA(2-SO4)-[3H]ATalR(6-SO4). In contrast, a lysosomal enzyme preparation from a human fibroblast cell line deficient in idurono-2-sulfatase (Hunter's-syndrome), which had no activity on the IdoA(2-SO4)-[3H]AManR(6-SO4), converted GlcA(2-SO4)-[3H]AManR(6-SO4) to a mixture of GlcA-[3H] AManR(6-SO4) and [3H]AManR(6-SO4). This enzyme also converted GlcA(2-SO4)-[3H]ATalR(6-SO4) to a mixture of GlcA-[3H]ATalR(6-SO4) and [3H]ATalR(6-SO4). Digestion of both GlcA(2-SO4)-[3H]AManR(6-SO4) and GlcA(2-SO4)-[3H]ATalR(6-SO4) was inhibited by 35SO2-4 and was arrested at the monosulfated disaccharide stage by 1,4-saccharolactone. The glucurono-2-sulfatase exhibited a pH optimum of 4. The results indicate that there exists a separate sulfatase for the removal of sulfate substituents from C-2 of GlcA residues in glycosaminoglycans.     

Structural elucidation of glycosaminoglycans through characterization of disaccharides obtained after fragmentation by hydrazine-nitrous acid treatment

Hydrazinolysis of glycosaminoglycans to bring about N-deacetylation followed by nitrous acid treatment to effect deaminative cleavage at alternating hexosamine residues has been used to make possible identification and quantitation of disaccharide sequences and position of O-sulfate substitution in nanogram amounts of these polymers. After radiolabeling by NaB3H4 reduction the hydrazine-nitrous acid products were fractionated on Dowex 1 and further resolved by thin-layer chromatography into disaccharides terminating in either sulfated or unsulfated anhydromannitol or anhydrotalitol. Fragmentation of hyaluronic acid, keratan sulfate, chondroitin 4-sulfate, chondroitin 6-sulfate, dermatan sulfate, and heparin yielded a total of 14 disaccharides comprising the major sequences (greater than 1 mol%) occurring in mammalian glycosaminoglycans. Disaccharides representing the predominant variants of the chondroitin sulfates [GlcUA beta 1----3anhydrotalitol(4-SO4) and GlcUA beta 1----3anhydrotalitol(6-SO4)] as well as of dermatan sulfate chains [IdUA alpha 1----3anhydrotalitol(4-SO4) and GlcUA beta 1----3anhydrotalitol(4-SO4)] chains could readily be quantitated by this approach. In the case of heparin a comparison of the disaccharides produced by direct nitrous acid and hydrazine-nitrous acid treatments moreover provided an assessment of the distribution of N-sulfate groups. The characterization of the various disaccharides by Smith periodic acid degradation and glycosidase digestions was facilitated by the preparation and thin-layer chromatographic resolution of the complete series of monosulfated derivatives of anhydromannitol and anhydrotalitol; the sulfate esters were shown to be stable to both the hydrazine and nitrous acid treatments. The high sensitivity of the hydrazine-nitrous acid fragmentation procedure should prove useful in the structural elucidation of cell surface and basement membrane proteoglycans as well as other sulfated glycoconjugates which are present in small amounts.     

Specific inhibition of binding of antistasin and [A103,106,108] antistasin 93-119 to sulfatide (Gal(3-SO4)beta 1-1Cer) by glycosaminoglycans.

Leech-derived antistasin is a potent anticoagulant and antimetastatic protein that binds sulfatide (Gal(3-SO4)beta 1-1Cer) and sulfated polysaccharides. In this study, the synthetic fragment [A103,106,108] antistasin 93-119, which corresponds to the carboxyl terminus, showed specific and saturable binding to sulfatide. Binding was competitively blocked by glycosaminoglycans (GAGs) in the order: dextran sulfate 5000 congruent to dextran sulfate 500,000 greater than heparin greater than dermatan sulfate much greater than chondroitin sulfates A and C. This rank order of inhibitory potency was identical to that observed with whole antistasin. We suggest that residues 93-119 of antistasin represent a critical domain for binding GAGs and sulfated glycolipids.     

Sulfated N-linked carbohydrate chains in porcine thyroglobulin

N-linked carbohydrate chains of porcine thyroglobulin were released by the hydrazinolysis procedure. The resulting mixture of oligosaccharide-alditols was fractionated by high-voltage paper electrophoresis, the acidic fractions were further separated by high-performance liquid chromatography on Lichrosorb-NH2, and analyzed by 500-MHz 1H-NMR spectroscopy and, partially, by permethylation analysis. Of the acidic oligosaccharide-alditols, the following sulfated carbohydrate chains could be identified: NeuAc alpha 2----6Gal beta 1----4GlcNAc beta 1----2Man alpha 1----3[(SO3Na----3)Gal beta 1----4GlcNAc beta1----2-Mana alpha 1----6]Man beta 1----4GlcNAc beta 1----4[Fuc alpha 1----6]GlcNAc-ol and NeuAc alpha 2----6Gal beta 1----4(SO3Na----)0-1 GlcNAc beta 1----2-Man alpha 1----3[NeuAc alpha 2----6Gal beta 1----4(SO3Na----6)1-0GlcNAc beta 1----2Man alpha 1----6]Man beta 1----4GlcNAc beta 1----4[Fuc alpha 1----6]GlcNAc- ol. The sulfated structural elements for porcine thyroglobulin form novel details of N-linked carbohydrate chains. They contribute to the fine structure of these oligosaccharides and are another type of expression of microheterogeneity.     

Sorting and secretion of adrenocorticotropin in a pituitary tumor cell line after perturbation of the level of a secretory granule-specific proteoglycan

A mouse anterior pituitary tumor cell line (AtT-20) that secretes adrenocorticotropin and beta endorphin sorts the proteins it transports to the surface into two exocytotic pathways. AtT-20 cells also synthesize a secretory granule-specific sulfated molecule and secrete it on stimulation (Moore, H.-P., B. Gumbiner, and R. B. Kelly, 1983, J. Cell Biol., 97:810-817). We show here that this molecule is sensitive to proteolysis and that the residual sulfated material co-migrates with a chondroitin sulfate standard on thin-layer electrophoresis. Furthermore, this sulfated molecule is completely sensitive to chondroitinase ABC digestion. Thus the secretory granule-specific sulfated molecule is a proteoglycan with chondroitin sulfate side chains. We examined the role of proteoglycans in the sorting and secretion of adrenocorticotropin in AtT-20 cells by severely decreasing the amount of this vesicle-specific proteoglycan in two ways. First, a xyloside was used to inhibit proteoglycan biosynthesis; second, a variant of the AtT-20 cell line was isolated that synthesized little of the sulfated proteoglycan. In neither case was the sorting or secretion of adrenocorticotropin detectably altered, suggesting that the proteoglycan is not required for these processes.     

A unique nonreducing terminal modification of chondroitin sulfate by N-acetylgalactosamine 4-sulfate 6-o-sulfotransferase

N-Acetylgalactosamine 4-sulfate 6-O-sulfotransferase (GalNAc4S-6ST) transfers sulfate from 3'-phosphoadenosine 5'-phosphosulfate (PAPS) to position 6 of N-acetylgalactosamine 4-sulfate (GalNAc(4SO4)). We previously identified human GalNAc4S-6ST cDNA and showed that the recombinant GalNAc4S-6ST could transfer sulfate efficiently to the nonreducing terminal GalNAc(4SO4) residues. We here present evidence that GalNAc4S-6ST should be involved in a unique nonreducing terminal modification of chondroitin sulfate A (CSA). From the nonreducing terminal of CS-A, a GlcA-containing oligosaccharide (Oligo I) that could serve as an acceptor for GalNAc4S-6ST was obtained after chondroitinase ACII digestion. Oligo I was found to be GalNAc(4SO4)-GlcA(2SO4)-GalNAc(6SO4) because GalNAc(4SO4) and deltaHexA(2SO4)-GalNAc(6SO4) were formed after chondroitinase ABC digestion. When Oligo I was used as the acceptor for GalNAc4S-6ST, sulfate was transferred to position 6 of GalNAc(4SO4) located at the nonreducing end of Oligo I. Oligo I was much better acceptor for GalNAc4S-6ST than GalNAc(4SO4)-GlcAGalNAc(6SO4). An oligosaccharide (Oligo II) whose structure is identical to that of the sulfated Oligo I was obtained from CS-A after chondroitinase ACII digestion, indicating that the terminal modification occurs under the physiological conditions. When CS-A was incubated with [35S]PAPS and GalNAc4S-6ST and the 35S-labeled product was digested with chondroitinase ACII, a 35S-labeled trisaccharide (Oligo III) containing [35S]GalNAc(4,6-SO4) residue at the nonreducing end was obtained. Oligo III behaved identically with the sulfated Oligos I and II. These results suggest that GalNAc4S-6ST may be involved in the terminal modification of CS-A, through which a highly sulfated nonreducing terminal sequence is generated.     

Intracellular transport and tyrosine sulfation of procollagens V

Several tyrosine residues of the extracellular p-collagens V and collagens V are sulfated [Fessler, L. I., Brosh, S., Chapin, S. and Fessler, J. H. (1986) J. Biol. Chem. 261, 5034-5040]. Here, the sulfation of their intracellular precursors, the procollagens V, was studied. A Golgi-enriched subcellular fraction of chick embryo tendon catalyzed the sulfation of tyrosine residues in both endogenous and added, unsulfated procollagens V with the sulfate donor 3'-phosphoadenosine 5'-[35S]phosphosulfate. Intracellular tyrosine sulfation of procollagen V occurred at a point distal to the cis Golgi compartment as judged by change of the N-linked carbohydrate of procollagen V from being endoglycosidase-H-sensitive to being resistant. The time course of the intracellular modifications of procollagen V was determined by incubating tendons with 3H-labeled amino acids and with [35S]sulfate. The pro alpha(V) chains were synthesised in about 10 min and then assembled into unsulfated procollagen V molecules. Tyrosine sulfation occurred 50 min after completion of polypeptide synthesis and the molecules were successively sulfated in the order in which they had been synthesized. The antimicrotubular drug Nocodazole, which disrupts the spatial organization of the Golgi, decreased the time interval between synthesis of procollagens V and sulfation. The sulfated procollagens V were soon secreted and cut to sulfated p-collagens V. Sulfated pro alpha 1(V) chains were cleaved faster than sulfated pro alpha 1'(V) chains. The relationship of sequential protein modification to spatial cellular organization is discussed.     

Synthesis of sulfated phenyl 2-acetamido-2-deoxy-D-galactopyranosides. 4-O-Sulfated phenyl 2-acetamido-2-deoxy-beta-D-galactopyranoside is a competitive acceptor that decreases sulfation of chondroitin sulfate by N-acetylgalactosamine 4-sulfate 6-O-sulfotransferase

We have previously cloned N-acetylgalactosamine 4-sulfate 6-O-sulfotransferase (GalNAc4S-6ST), which transfers sulfate from 3'-phosphoadenosine 5'-phosphosulfate (PAPS) to the C-6 hydroxyl group of the GalNAc 4-sulfate residue of chondroitin sulfate A and forms chondroitin sulfate E containing GlcA-GalNAc(4,6-SO(4)) repeating units. To investigate the function of chondroitin sulfate E, the development of specific inhibitors of GalNAc4S-6ST is important. Because GalNAc4S-6ST requires a sulfate group attached to the C-4 hydroxyl group of the GalNAc residue as the acceptor, the sulfated GalNAc residue is expected to interact with GalNAc4S-6ST and affect its activity. In this study, we synthesized phenyl alpha- or -beta-2-acetamido-2-deoxy-beta-D-galactopyranosides containing a sulfate group at the C-3, C-4, or C-6 hydroxyl groups and examined their inhibitory activity against recombinant GalNAc4S-6ST. We found that phenyl beta-GalNAc(4SO(4)) inhibits GalNAc4S-6ST competitively and also serves as an acceptor. The sulfated product derived from phenyl beta-GalNAc(4SO(4)) was identical to phenyl beta-GalNAc(4,6-SO(4)). These observations indicate that derivatives of beta-D-GalNAc(4SO(4)) are possible specific inhibitors of GalNAc4S-6ST.     

Secretion of 35SO4-labeled proteins from isolated rat hepatocytes

Sulfation is a Golgi-specific modification of secretory proteins. We have characterized the proteins that are labeled with 35SO4 in cultures of rat hepatocytes and studied their transport to the medium. Analysis by polyacrylamide gel electrophoresis showed that of the five most heavily labeled proteins, four had well-defined mobilities--apparent molecular masses of 188, 142, 125, and 82 kDa--whereas one was electrophoretically heterogeneous--apparent molecular mass of 35-45 kDa. Judging by their relatively high resistance to acid treatment, the sulfate residues in the 125- and 35-45-kDa proteins were linked to carbohydrate. Some of the secreted proteins were sialylated. In samples of pulse-labeled cells, there appeared to be no unsialylated forms, indicating that sulfation occurred after sialylation, presumably in the trans Golgi. Kinetic experiments showed that the cellular half-life was the same for all the sulfated proteins--about 8 min--consistent with the idea that transport from the Golgi complex to the cell surface occurs by liquid bulk flow.     

Identification of the site of sulfation of the fourth component of human complement

The alpha-chain of the fourth component of complement (C4) contains tyrosine sulfate (Karp, D.R. (1983) J. Biol. Chem. 258, 12745-12748). Here we have determined the site and stoichiometry of sulfation of C4 secreted by the human hepatoma-derived cell line Hep G2. C4 was labeled with [35S]sulfate and isolated from culture medium by immunoprecipitation. C4 digested with trypsin and chymotrypsin and analyzed by reverse-phase high-performance liquid chromatography contained a single sulfate-labeled peptide. Digestion of C4 with trypsin alone yielded two major sulfate-labeled peptides, suggesting that there may be some sequence variability in C4 near the site of sulfation. Sequential Edman degradation of tryptic peptides labeled with [3H]tyrosine and [35S]sulfate detected tyrosine residues at positions 5, 13, 16, and 18. Chymotrypsin cleaved 5 residues off the NH2-terminal end of tryptic peptides, yielding a peptide with tyrosine at positions 8, 11, and 13. Comparison of the position of tyrosine residues with the reported sequence of C4 identified the sites of sulfation as tyrosine residues at positions 738, 741, and 743 in the alpha-chain of C4. All 3 of these tyrosine residues appeared to be sulfated. When sulfation of C4 was partially inhibited by addition of catechol to culture medium, three different forms of the peptide were resolved by high-performance liquid chromatography, consistent with peptides containing 1, 2, or 3 sulfates. Comparison of the quantities of tyrosine and tyrosine sulfate in C4 which had been labeled with [3H]tyrosine and digested with Pronase also indicated that C4 contained an average of 2-3 residues of tyrosine sulfate/molecule. These results suggest that the biologically active form of the protein is sulfated.