Occurrence of tyrosine sulfate in proteins--a balance sheet. 1. Secretory and lysosomal proteins

1. The abundance of tyrosine sulfate in secretory proteins and in various classes of cellular proteins has been quantified and compared to protein-bound carbohydrate sulfate. 2. HepG2 cells and fibroblasts, two cell types showing only the constitutive pathway of secretion, and PC12 cells, which show both the constitutive and the regulated pathway of secretion, were subjected to pulse-chase and/or long-term labelling with [35S]sulfate and [3H]tyrosine, followed by analysis of proteins in the cells and medium. Under both conditions of labelling, 65-92% of the protein-bound tyrosine sulfate and 44-84% of the protein-bound carbohydrate sulfate were found to be secretory. In HepG2 cells, the frequency of sulfation of tyrosine residues, which can be determined independently from protein abundance and the rate of protein synthesis, was 8-22 times higher in proteins secreted into the medium than in cellular proteins. 3. All cell lines studied contained significant amounts, not only of carbohydrate sulfate, but also of tyrosine sulfate in specific cellular proteins. As shown for fibroblasts, these tyrosine-sulfated proteins were retained within the cells for at least 100 min of chase following a pulse with [35S]sulfate and were almost completely recovered in a light membrane fraction after subcellular fractionation. 4. Lysosomes were found to contain small, but significant, amounts of protein-bound tyrosine sulfate in addition to protein-bound carbohydrate sulfate. Protein-bound tyrosine sulfate in lysosomes reached a peak at 20 min of chase and rapidly disappeared thereafter, whereas protein-bound carbohydrate sulfate accumulated after 20 min of chase. Examination of the known sequences of eleven lysosomal enzymes revealed the presence of potential tyrosine sulfation sites in five of them. 5. Our results show that secretory proteins are the most abundant, but not exclusive, in vivo substrates for tyrosine sulfation and suggest the presence of soluble tyrosine-sulfated proteins in lysosomes and other, as yet unidentified, organelles of the secretory pathway. In the following paper in this journal we describe the abundance of tyrosine sulfate in integral membrane proteins.     

A target-specific approach for the identification of tyrosine-sulfated hemostatic proteins

A simple methodology for the identification of hemostatic proteins that are subjected to posttranslational tyrosine sulfation was developed. The procedure involves sequence analysis of members of the three hemostatic pathways using the Sulfinator prediction algorithm, followed by [³⁵S]sulfate labeling of cultured HepG2 human hepatoma cells, immunoprecipitation of targeted [³⁵S]sulfate-labeled hemostatic proteins, and tyrosine O-[³⁵S]sulfate analysis of immunoprecipitated proteins. Three new tyrosine-sulfated hemostatic proteins—protein S, prekallikrein, and plasminogen—were identified. Such a target-specific approach will allow investigation of tyrosine-sulfated proteins of other biochemical/physiological pathways/processes and contribute to a better understanding of the functional role of posttranslational tyrosine sulfation.     

Tyrosine sulfation of yolk proteins 1, 2, and 3 in Drosophila melanogaster

Protein sulfation was studied in Drosophila melanogaster after in vivo labeling of flies with inorganic [35S]sulfate. After separation of total fly protein by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, proteins with sulfated carbohydrates and proteins containing tyrosine sulfate were found in all the molecular weight ranges analyzed. When female and male fly proteins were compared with each other, the electrophoretic patterns of protein-bound carbohydrate sulfate were found to be similar, whereas those of protein-bound tyrosine sulfate were distinct. The most prominent difference was the exclusive presence in female flies of three major tyrosine-sulfated proteins with apparent molecular masses between 48 and 45 kDa. Radioimmunolabeling after two-dimensional polyacrylamide gel electrophoresis was used to identify these proteins as yolk proteins 1, 2, and 3. Each of the three yolk proteins existed in several isoelectric forms, all of which were sulfated. Since the number of tyrosine residues in the yolk proteins is known, the stoichiometry of tyrosine sulfation could be determined by a novel method and was found to be 2.2, 0.9, and 1.2 mol of tyrosine sulfate per mol of yolk protein 1, 2, and 3, respectively. The present results, together with the recently reported molecular cloning of the yolk protein genes, make the yolk proteins suitable objects for genetic approaches to investigate the biological role(s) of tyrosine sulfation of secretory proteins.     

Tyrosine sulfation: a post-translational modification of proteins destined for secretion?

Protein sulfation was studied in germ-free rats by prolonged in vivo labeling with [35S]sulfate. Specific sets of sulfated proteins were observed in all tissues examined, in leucocytes, and in blood plasma. No protein sulfation was detected in erythrocytes. Analysis of the type of sulfate linkage showed that sulfated proteins secreted into the plasma contained predominantly tyrosine sulfate, whereas sulfated proteins found in tissues contained largely carbohydrate sulfate. This implies some kind of selection concerning the intracellular processing, secretion, turnover or re-uptake of sulfated proteins which is responsible for the enrichment of tyrosine-sulfated proteins in the plasma.     

In vivo expression and stoichiometric sulfation of the artificial protein sulfophilin, a polymer of tyrosine sulfation sites.

To gain insight into the structural requirements for tyrosine sulfation in vivo, we have constructed and expressed an artificial gene encoding a polypeptide substrate for tyrosylprotein sulfotransferase. This gene codes for a protein, referred to as sulfophilin, which consists of a 12-times repeated heptapeptide unit corresponding to the identified tyrosine sulfation site of chromogranin B (secretogranin I), Glu-Glu-Pro-Glu-Tyr-Gly-Glu. The gene was fused to the signal sequence of secretogranin II to direct the sulfophilin protein to the secretory pathway. Stable expression of the artificial gene in NIH 3T3 cells resulted in the secretion of sulfated sulfophilin. Analysis of the stoichiometry of sulfation revealed that each of the 12 tyrosyl residues in sulfophilin was sulfated. Remarkably, up to 50% of the total protein-bound tyrosine sulfate secreted by the cells was contained in sulfophilin. The results indicate that the structural information contained in the heptapeptide motif is sufficient for stoichiometric tyrosine sulfation to occur in the living cell.     

Protein tyrosine sulfation in guinea-pig uterus: estradiol and progesterone effects

Tyrosine sulfation was studied in guinea-pig uterus by in vitro labelling with [35S]sulfate, after estradiol-17 beta (E2) and E2 plus progesterone (P) treatment. [35S]Sulfated tyrosine was identified in tissue and secreted proteins, ranged from 9.3 to 21.0% of total protein sulfation and was higher in secreted proteins than in tissue proteins. Sulfate incorporation into tyrosine increased with hormone treatments. The highest level was found in secreted proteins under the combined effect of E2 plus P. The effect of P may be related to both the increase of cellular uptake of sulfate and the increase of tyrosine sulfation of secreted proteins. These results are consistent with the effect of P on endometrium secretions.     

Sulfation of two tyrosine-residues in human complement S-protein (vitronectin)

Human S-protein (vitronectin) and hemopexin, two structurally related plasma proteins of similar molecular mass and abundance, were analyzed for tyrosine sulfation. Both proteins were synthesized and secreted by the human hepatoma-derived cell line Hep G2, as shown by immunoprecipitation from the culture medium of [35S]methionine-labelled cells. When Hep G2 cells were labelled with [35S]sulfate, S-protein, but not hemopexin, was found to be sulfated. Half of the [35S]sulfate incorporated into S-protein was recovered as tyrosine sulfate. The stoichiometry of tyrosine sulfation was approximately two mol tyrosine sulfate/mol S-protein. Examination of the S-protein sequence for the presence of the known consensus features for tyrosine sulfation revealed three potential sulfation sites at positions 56, 59 and 401. Tyrosine 56 is the most probable site for stoichiometric sulfation, followed by tyrosine 59 which appears more likely to become sulfated than tyrosine 401. Tyrosines 56 and 59 are located in the anionic region of S-protein which has no homologous counterpart in hemopexin. We discuss the possibility that tyrosine sulfation of the anionic region of S-protein may stabilize the conformation of S-protein in the absence of thrombin-antithrombin III complexes and may play a role in its binding to thrombin-antithrombin III complexes during coagulation.     

Sulfation of porcine parathyroid secretory protein I. Detection of tyrosine sulfate

Secretory Protein I (SP-I) is an acidic glycoprotein that is stored and co-secreted with parathormone by parathyroid glands. It has been found to be chemically similar, if not identical, to chromogranin A of the adrenal medulla and to be present in most endocrine cells. In the present study, 35SO4 was shown to be incorporated into SP-I and several other proteins of porcine parathyroid tissue incubated in vitro. The predominant sulfated species secreted to the medium was SP-I. Up to 20% of the tyrosine residues in secreted SP-I were labeled with 35SO4. Both the cellular and secreted forms migrated on sodium dodecyl sulfate gels as a pair of proteins with apparent molecular weights of 82,000 and 78,000. The 82-kDa protein could be converted to the 78-kDa species by treatment with neuraminidase. Sulfate exists in SP-I as tyrosine sulfate based on the identification of this amino acid by thin layer electrophoresis following alkaline hydrolysis. Extracellular Ca2+ (3 mM) greatly suppressed the secretion of 35SO4-labeled SP-I without affecting the intracellular sulfation of the molecule or the secretion of a minor sulfated protein unrelated to SP-I. The ratio of incorporated 35SO4 to 3H-amino-acid was greater in secreted SP-I than in tissue SP-I, suggesting that much sulfation of this protein occurred during or just before secretion.     

In vivo and in vitro tyrosine sulfation of a membrane glycoprotein

A431 cells incorporate 35SO4 into a protein of Mr 61,000 (P61). We examined sulfation of P61 by cells (in vivo) and by a cell-free system (in vitro) which requires only addition of A431 cell membranes and a 3'-phosphoadenosine 5'-phospho[35S]sulfate-generating system prepared from Krebs ascites cells. Sulfate is found exclusively in the form of tyrosine SO4 by two-dimensional high voltage electrophoresis following Pronase digestion. Endoglycosidase F digestion reduces the Mr by 2,000 but does not release the sulfate, indicating that P61 is a glycoprotein but that sulfate is not incorporated into the carbohydrate. Sulfated P61 is not found in the medium from cultured cells and remains associated with the membrane fraction following cell lysis. Treatment of membranes with 0.4 M NaCl, 0.3 M KCl, 15 mM EDTA, or pH 11.0 does not release sulfated P61. P61 is solubilized by Triton X-114 treatment of membranes and partitions into the detergent phase upon warming. Based on these characteristics, we conclude that P61 is an integral membrane protein. Trypsin digestion experiments with intact cells suggest that sulfated P61 is predominantly located in the plasma membrane. This is the first example of an integral membrane protein which is sulfated on tyrosine. The properties of the sulfation reaction are distinct from those reported for secreted proteins and are consistent with the possibility that this modification occurs at the plasma membrane rather than in the Golgi.     

Sulfation of tyrosine residues does not influence secretion of alpha 2-antiplasmin or C4

Sulfation of tyrosine residues is a biosynthetic modification of many secretory proteins. The function of this modification is not known, but it has been proposed that tyrosine sulfate residues may act as sorting signals to direct proteins along the secretory pathway. We tested this hypothesis by examining the effect of sulfation inhibitors on the kinetics of secretion of proteins by HepG2 cells. The inhibitors induced no change in the rate of secretion of alpha 2-antiplasmin and C4 (fourth component of complement), both of which contain tyrosine sulfate residues. Sulfation of tyrosine residues does not contribute to secretion of these proteins.     

The major tyrosine-sulfated protein of the bovine anterior pituitary is a secretory protein present in gonadotrophs, thyrotrophs, mammotrophs, and corticotrophs

The anterior pituitary is a complex secretory tissue known to contain several sulfated macromolecules. In the present study, we identified the major tyrosine-sulfated protein of the bovine anterior pituitary and investigated its cellular and subcellular localization. This protein consisted of two tyrosine-sulfated polypeptides of molecular weight 86,000 and 84,000 that were highly homologous to each other. In agreement with previous biochemical studies, the tyrosine-sulfated protein of Mr 86,000/84,000 was found to be secretory, as it was observed in the matrix of secretory granules by immunoelectron microscopy. Immunofluorescence studies indicated that the tyrosine-sulfated, secretory protein of Mr 86,000/84,000, referred to as TSP 86/84, was present in all endocrine cells except for some somatotrophic cells. Higher levels of immunoreactivity for TSP 86/84 were observed in gonadotrophic and thyrotrophic than in mammotrophic and corticotrophic cells. This appeared to result from the occurrence of TSP 86/84 in all secretory granules of the former cells and in only some secretory granules of the latter cells. We discuss the possibility that TSP 86/84 may have a role in the packaging of several distinct peptides hormones into secretory granules. One, though not the only, possible function of tyrosine sulfation may concern the sorting of this protein in the Golgi complex.     

Tyrosine O-sulfate ester in proteoglycans

Tyrosine O-sulfate residues were detected in the protein core of sulfated proteoglycans. When cultured skin fibroblasts and arterial smooth muscle cells were incubated in the presence of [35S]sulfate, dermatan sulfate proteoglycan and chondroitin sulfate proteoglycan isolated from the culture medium contained tyrosine [35S]sulfate ester which accounted for 0.03%-0.82% of total 35S radioactivity incorporated into the sulfated proteoglycans. This corresponds to a tyrosine sulfation of every second (fibroblasts) and every 10th (smooth muscle cells) dermatan sulfate proteoglycan molecule. [3H]Tyrosine labeling of fibroblast dermatan sulfate proteoglycan gave a similar stoichiometry. However, the relative proportion of tyrosine [35S]sulfate in proteoglycans from arterial tissue was about 10 times higher than in that from cultured arterial cells. Pulse chase experiments with [35S]sulfate revealed that tyrosine sulfation is a late event in the biosynthesis of dermatan sulfate proteoglycan from fibroblasts and occurs immediately prior to secretion. Cultured skin fibroblasts from a patient with a progeroid variant (Kresse et al. 1987, Am. J. Hum. Gen. 41, 436-453) which exhibit a partial deficiency to synthesize dermatan sulfate proteoglycan were shown to form and to secrete a tyrosine-sulfated but glycosaminoglycan-free protein core, thus confirming a selective and independent [35S]sulfate labeling of the protein core.     

Tyrosine sulfation is a trans-Golgi-specific protein modification

The trans-Golgi has been recognized as having a key role in terminal glycosylation and sorting of proteins. Here we show that tyrosine sulfation, a frequent modification of secretory proteins, occurs specifically in the trans-Golgi. The heavy chain of immunoglobulin M (IgM) produced by hybridoma cells was found to contain tyrosine sulfate. This finding allowed the comparison of the state of sulfation of the heavy chain with the state of processing of its N-linked oligosaccharides. First, the pre-trans-Golgi forms of the IgM heavy chain, which lacked galactose and sialic acid, were unsulfated, whereas the trans-Golgi form, identified by the presence of galactose and sialic acid, and the secreted form of the IgM heavy chain were sulfated. Second, the earliest form of the heavy chain detectable by sulfate labeling, as well as the heavy chain sulfated in a cell-free system in the absence of vesicle transport, already contained galactose and sialic acid. Third, sulfate-labeled IgM moved to the cell surface with kinetics identical to those of galactose-labeled IgM. Lastly, IgM labeled with sulfate at 20 degrees C was not transported to the cell surface at 20 degrees C but reached the cell surface at 37 degrees C. The data suggest that within the trans-Golgi, tyrosine sulfation of IgM occurred at least in part after terminal glycosylation and therefore appeared to be the last modification of this constitutively secreted protein before its exit from this compartment. Furthermore, the results establish the covalent modification of amino acid side chains as a novel function of the trans-Golgi.     

Conversion of recombinant hirudin to the natural form by in vitro tyrosine sulfation. Differential substrate specificities of leech and bovine tyrosylprotein sulfotransferases

Hirudin, a tyrosine-sulfated protein secreted by the leech Hirudo medicinalis, is one of the most potent anticoagulants known. The hirudin cDNA has previously been cloned and has been expressed in yeast, but the resulting recombinant protein was found to be produced in the unsulfated form, which is known to have an at least 10 times lower affinity for thrombin than the naturally occurring tyrosine-sulfated hirudin. Here we describe the in vitro tyrosine sulfation of recombinant hirudin by leech and bovine tyrosylprotein sulfotransferase (TPST). With both enzymes, in vitro sulfation of recombinant hirudin occurred at the physiological site (Tyr-63) and rendered the protein biochemically and biologically indistinguishable from natural hirudin. However, leech TPST had an over 20-fold lower apparent Km value for recombinant hirudin than bovine TPST. Further differences in the catalytic properties of leech and bovine TPSTs were observed when synthetic peptides were tested as substrates. Moreover, a synthetic peptide corresponding to the 9 carboxyl-terminal residues of hirudin (which include Tyr-63) was sulfated by leech TPST with a similar apparent Km value as full length hirudin, indicating that structural determinants residing in the immediate vicinity of Tyr-63 are sufficient for sulfation to occur.     

Inhibitors of the sulfation of proteins, glycoproteins, and proteoglycans

Two categories of compounds, substrates of sulfation and sulfate analogs, were tested for the ability to inhibit sulfation of macromolecules secreted by HepG2 cells. Several compounds which most effectively inhibited sulfation without toxic effects on cells were tested for their relative inhibition of sulfation of tyrosine residues (using the fourth component of complement as a model substrate), of N-linked oligosaccharides (alpha 2HS-glycoprotein as substrate), and of proteoglycans. Inhibitors decreased the sulfation of all three classes of substrate, but not always equally. Use of inhibitors from both categories in combination yielded synergistic effects, with more effective inhibition of sulfation and low toxicity. Such combinations of inhibitors should provide a valuable tool for probing the significance of the sulfation of macromolecules.     

Identification and functional importance of tyrosine sulfate residues within recombinant factor VIII.

Sulfated tyrosine residues within recombinant human factor VIII were identified by [35S]sulfate biosynthetic labeling of Chinese hamster ovary cells which express human recombinant factor VIII. Alkaline hydrolysis of purified [35S]sulfate-labeled factor VIII showed that greater than 95% of the [35S]sulfate was incorporated into tyrosine. [3H]Tyrosine and [35S]sulfate double labeling was used to quantify the presence of 6 mol of tyrosine sulfate per mole of factor VIII. Amino acid sequence analysis of thrombin and tryptic peptides isolated from [35S]sulfate-labeled factor VIII demonstrated tyrosine sulfate at residue 346 in the factor VIII heavy chain and at residues 1664 and 1680 in the factor VIII light chain. In addition, the carboxyl-terminal half of the A2 domain contained three tyrosine sulfate residues, likely at positions 718, 719, and 723. Interestingly, all sites of tyrosine sulfation border thrombin cleavage sites. The functional importance of tyrosine sulfation was examined by treatment of cells expressing factor VIII with sodium chlorate, a potent inhibitor of tyrosine sulfation. Increasing concentrations of sodium chlorate inhibited sulfate incorporation into factor VIII without affecting its synthesis and/or secretion. However, factor VIII secreted in the presence of sodium chlorate exhibited a 5-fold reduction in procoagulant activity, although the protein was susceptible to thrombin cleavage. These results suggest that tyrosine sulfation is required for full factor VIII activity and may affect the interaction of factor VIII with other components of the coagulation cascade.     

Tyramine-O-sulfate, in addition to tyrosine-O-sulfate, is produced and secreted by HepG2 human hepatoma cells, but not by 3Y1 rat embryo fibroblasts

The spent media of HepG2 human hepatoma cells and 3Y1 rat embryo fibroblasts labeled with [35S]sulfate, upon ultrafiltration, were analyzed by a two-dimensional thin-layer separation procedure. Autoradiographs of the cellulose thin-layer plate revealed the presence of tyramine-O-[35S]sulfate in addition to tyrosine-O-[35S]sulfate in spent medium from human hepatoma cells. In contrast, only tyrosine-O-[35S]sulfate was observed in spent medium of 3Y1 rat fibroblasts. Using adenosine, 3'-phosphate, 5'-phospho[35S]sulfate as the sulfate donor, sulfotransferase(s) present in HepG2 cell homogenate catalyzed the sulfation of tyramine to tyramine-O-[35S]sulfate, but not the sulfation of tyrosine to tyrosine-O-[35S]sulfate. Endogenous aromatic amino acid decarboxylase present in HepG2 homogenate was shown to catalyze the decarboxylation of [3H]tyrosine to form [3H]tyramine while attempts to use it for the decarboxylation of tyrosine-O-sulfate to form tyramine-O-sulfate were unsuccessful. These results suggest that tyramine-O-sulfate may be derived from the de novo sulfation of tyramine, instead of the decarboxylation of tyrosine-O-sulfate.     

Expression, tyrosine sulfation, and secretion of yolk protein 2 of Drosophila melanogaster in mouse fibroblasts

Tyrosine sulfation is a post-translational modification in the trans Golgi that has been found in all animal species studied. In the preceding paper (Baeuerle, P. A., Lottspeich, F., and Huttner, W. B. (1988) J. Biol. Chem. 263, 14925-14929), we have identified the site of tyrosine sulfation in an insect secretory protein, yolk protein 2 (YP2) of Drosophila melanogaster. In the present report, tyrosine sulfation of this protein was examined after expression in a heterologous mammalian cell system. Mouse fibroblasts, transfected with Drosophila YP2 genomic DNA inserted into the eucaryotic expression vector pSV2, secreted the fly protein in sulfated form. Analyses of Drosophila YP2 produced by the mouse cells showed that the features of sulfation of this protein were identical to those previously determined for YP2 isolated from flies. YP2 secreted from mouse fibroblasts was found to be exclusively sulfated on tyrosine residues. The stoichiometry of tyrosine sulfation was approximately 1 mol of sulfate/mol of YP2. Sulfate was linked to the same tyrosine residue as in YP2 isolated from flies, tyrosine 172. These results show that essential parameters of the tyrosine sulfation reaction are very similar in insects and mammals and thus highly conserved in evolution.     

斑马鱼蛋白酪氨酸硫酸化修饰的检测方法研究

蛋白酪氨酸硫酸化(protein tyrosine sulfation, PTS)是一种重要的翻译后修饰,调控生命活动中多种生理和病理过程,但由于PTS状态不稳定且目前缺乏有效的富集方法,因此在生物样品中难以进行有效地检测。本研究以模式动物斑马鱼(Danio rerio)为研究材料,利用Orbitrap Exploris 480高分辨质谱仪检测了斑马鱼胚胎发育早期总蛋白的酪氨酸硫酸化修饰水平,通过该方法共计检测到26种蛋白(包括膜蛋白、分泌蛋白、胞质蛋白和核蛋白等)存在潜在的29个酪氨酸硫酸化修饰位点。本研究建立了斑马鱼胚胎发育早期蛋白酪氨酸硫酸化修饰的检测方法,为探索生物体蛋白硫酸化修饰的作用机制奠定了技术基础。