Protein tyrosine sulfation, 1993 — an update

C..hemico-Siological

InLeroctionl ELSEVIER

Chemico-Biological Interactions 92 (1994) 257-271

Protein tyrosine sulfation, 1993 - - an update C h r i s t o f N i e h r s t , R o l a n d Beil3wanger, W i e l a n d B. H u t t n e r * Institute for Neurobiology, University of Heidelberg, lm Neuenheimer Feld 364, D-69120 Heidelberg, Germany Received 21 September 1993; revision received 20 December 1993; accepted 3 January 1994

Abstract Sulfation is the most abundant post-translational modification of tyrosine residues and occurs in many soluble and membrane proteins passing through the secretory pathway of metazoan cells. The sulfation reaction is catalysed by tyrosylprotein sulfotransferase, a membrane-bound enzyme of the trans-Golgi-network. Tyrosylprotein sulfotransferase has been purified and its substrate specificity characterized. Tyrosine sulfation has been shown to be important for protein-protein interactions occurring during the intracellular transport of proteins and upon their secretion. Keywords: Tyrosine sulfation; Tyrosylprotein sulfotransferase; Protein-protein interaction

I. Introduction

The last comprehensive reviews on protein tyrosine sulfation date back to 1988 [1,2]. This article reviews the developments in this field during the past 5 years, which concern primarily the enzyme catalysing protein tyrosine sulfation, tyrosylprotein sulfotransferase (TPST), and the physiological significance of this posttranslational modification. 2. Tyrosine-sulfated proteins

Sulfation is the most abundant post-translational modification of tyrosine residues known to occur in multicellular organisms. However, only a small proportion * Corresponding author. tPresent address: DKFZ, D-69120 Heidelberg, Germany 0009-2797/94/$07.00 © 1994 Elsevier Science Ireland Ltd. All rights reserved SSDI 0009-2797(94)03304-Q

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o f the vast n u m b e r o f tyrosine-sulfated proteins have been characterized b e y o n d the status o f a b a n d on a gel. The -~ 40 identified tyrosine-sulfated proteins are listed in Table 1. In line with the intracellular localization o f T P S T in the t r a n s - G o l g i n e t w o r k (see below), all o f these proteins are either secretory or p l a s m a m e m b r a n e proteins. It is now clear from m e t a b o l i c labeling studies that the ratio o f tyrosine sulfate to tyrosine in m e m b r a n e proteins is in the same range as that in secretory proteins and that the greater a b u n d a n c e o f secreted p r o t e i n - b o u n d tyrosine sulfate as c o m p a r e d with m e m b r a n e - a s s o c i a t e d p r o t e i n - b o u n d tyrosine sulfate is p r i m a r i l y due to the higher rate o f synthesis o f the former class o f proteins [3,4]. These studies also suggest the occurrence o f tyrosine sulfate in soluble a n d m e m b r a n e proteins o f intracellular c o m p a r t m e n t s that are p a r t of, or related to, the secretory p a t h w a y (e.g. lysosomes). The experimental a p p r o a c h that has been most c o m m o n l y used to identify tyrosine-sulfated proteins involves m e t a b o l i c sulfate labeling and subsequent tyrosine sulfate analysis o f the proteins o f interest [5]. O t h e r m e t h o d s , which overcome the m a j o r d r a w b a c k o f this a p p r o a c h (the need for living cells) and which can be used to search for the presence o f tyrosine sulfate in unlabeled proteins, have also been

Table 1 Tyrosine-sulfated proteins and their subcellular localisation Protein

Species

Localisation a

Aminopeptidase N A4 Amyloid precursor a-2-Antiplasmin Caerulein Cholecystokinin (CCK) C-Terminal peptide of pro-CCK c~-Choriogonadotropin Chromogranin A (secretory protein I) Chromogranin B (secretogranin I) Cionin Coagulation factor V Coagulation factor VIII Coagulation factor X Complement C4 (a-chain) Dermatan Sulfat (core protein) Entactin/nidogen ~-Fetoprotein Fibrinogen, B- and y-chain Fibromodulin Fibronectin Gastrin Heparin kofactor II Hirudin Immunoglobulin A (u-chain) Immunoglobulin G 2a (7-chain) lmmunoglobulin M (t~-chain)

pig man man frog dog, man, pig man, rat, pig man pig cow, rat ascidian man man cow man, mouse man mouse man several species cow hamster, man, rat several mammals man leech mouse mouse mouse, rat

plm plm sec sec sec sec sec sec sec sec sec sec sec sec sec sec sec sec sec sec sec sec sec sec sec sec

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Table 1 (Continued) Protein

Species

Localisation a

Leukosulfakinin I, I1 Leu-enkephalin B-Lipotropin (pro-opiomelanocortin) a-2-Macroglobulin Maltase-glukoamylase Phyllokinin Procollagen type I11 Procollagen type V Prolactin Secretogranin I1 SG70 Sialoprotein 1I S-Protein/vitronectin Sucrase-isomaltase Thyroglobulin Yolk protein 1 Yolk protein 2 Yolk protein 3

cockroach several mammals cow rat pig frog man chicken sheep cow, rat Volvox mouse man pig mouse, pig, rat fruitfly fruitfly fruitfly

sec sec sec sec plm sec sec sec sec sec sec sec sec plm sec sec sec sec

For references, see text, the previous reviews (Refs. [1,21), and the original Refs. [90-95]. aAbbreviations: sec, secretory; plm, plasma membrane

described (e.g. Refs. [6,7]). Antibodies to tyrosine sulfate would be powerful tools for the detection and analysis of tyrosine-sulfated proteins. Their reliable generation, however, has so far been unsuccessful (Niehrs, Naujoks, Huttner, unpublished data), possibly due to tolerance of the immune system to this widespread epitope. Recently, the increase in protein sequence information, together with the knowledge of the consensus features of tyrosine sulfation (see below), has facilitated the identification of tyrosine-sulfated proteins (e.g., Refs. [8,9]).

3. Tyrosylprotein sulfotransferase 3.1. Occurrence

Tyrosylprotein sulfotransferase (TPST) catalyses the sulfate transfer from 3 ' phosphoadenosine 5 ' - p h o s p h o s u l f a t e (PAPS) to tyrosine residues [21]. Such an enzyme activity was first described in 1983 in PC12 cells [11] and has since been described in m a n y species, tissues and cell lines [I 0,12-28]. Assays by which properties of T P S T have been studied usually involve the incubation of m e m b r a n e extracts in the presence o f detergent with [35S]PAPS and an appropriate sulfate acceptor, such as the r a n d o m copolymer poly(Glu,Ala,Tyr) [13], tubulin [13], or synthetic peptides modelled after known tyrosine sulfation sites [10,14,21,24,29-31]. In X e n o p u s oocytes, the presence of T P S T has recently been demonstrated by a different approach. Secretogranin II, a secretory protein known to undergo tyrosine sulfation [32], was expressed by injection of its c R N A in order to serve as substrate for any T P S T possibly existing already at this stage of development. Indeed, it was

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observed that secretogranin II became tyrosine-sulfated by the oocyte (C. Vannier and W. B. Huttner, unpublished data). In cell and tissue homogenates, TPST has a very low specific activity, in the order of 0.1 pmol/min/mg protein, which makes its quantitation difficult. Therefore, TPST has been characterized after partial enrichment by subcellular fractionation [13,14,21,33]. A remarkable feature of unsolubilized TPST is that it retains activity after treatment of membranes with pH 11.0, which can be conveniently used to further enrich the enzyme. 3.2. Subcellular localization The conclusion that TPST is localized specifically in the trans-Golgi-network [34] has been corroborated by the observations that (i) the TPST protein itself is sialylated [21]; (ii) secretory granule formation, which occurs in the trans-Golgi-network, proceeds directly from the compartment of sulfation [35]; and (iii) TPST, upon treatment of cells with brefeldin A, behaves like a protein residing in the trans-Golginetwork rather than the trans-cisternae of the Golgi complex [36]. In certain cell types, such as adipocytes, tyrosine sulfation may also take place in the medial Golgi, although the evidence supporting this conclusion is weak, resting entirely on the ability of tyrosine sulfation to persist in the presence of monensin [37]. It is unclear whether the retention of TPST in the trans-Golgi involves its immobilization in this compartment, e.g. by anchorage to cytoskeletal elements, or its recycling from a post-Golgi site. Whatever the mechanism, the retention of TPST must be efficient since no significant amounts of TPST have been detected in post-Golgi organelles such as lysosomes, secretory granules and plasma membranes, or secretion fluids such as serum and milk (Ref. [131 and Niehrs and Huttner, unpublished data). On the other hand, TPST activity has recently been detected in platelets [27], and it will be interesting to investigate whether, in these special cells, this reflects TPST present in post-Golgi organelles such as secretory granules or the plasma membrane. 3.3. Membrane association TPST behaves as an integral membrane protein since it is not extracted by 1 M KCI and pH 11 [21] but is solubilized by various detergents [13,21,33] and partitions into the detergent phase upon Triton X-114 phase condensation [38]. Interestingly, a fraction of TPST solubilized from Golgi-enriched membranes partitions into the aqueous phase. This is apparently due to complex formation with a p H I 1-extractable hydrophilic protein [38]. Whether TPST is associated with this factor in vivo is unclear, but it is an attractive hypothesis that the latter may be involved in the Golgi retention of the enzyme. 3.4. Purification and properties Because of the integral nature of TPST as well as its instability after solubilization, several years following its discovery [11] were required before the purification of the protein was achieved [21]. In line with the low specific activity in tissue homogenates, TPST is a rare enzyme, requiring 140 000-fold enrichment from bovine adrenal medulla to be in homogeneous form. Salient to the purification was the ob-

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servation that the binding of the enzyme to a substrate peptide is enhanced in the presence of the cosubstrate PAPS or its analogue PAP. TPST is a sialoglycoprotein of 50-54 kDa apparent molecular weight on SDSPAGE and an apparent S-value of 6. The enzyme has a pH optimum of 6, which is in line with the slightly acidic milieu of the trans-Golgi [39]. Purified TPST requires magnesium ions and, remarkably, fluoride ions for full activity (Niehrs and Huttner, unpublished data). The Km for the cosubstrate PAPS is 1.4/~M. As with most sulfotransferases, TPST is inhibited by PAP (Ref. [40] and Niehrs and Huttner, unpublished data). Vargas et al. have suggested that the sequence of the individual steps in the reaction catalysed by TPST is PAPS binding - peptide binding -- sulfate transfer - sulfopeptide release -- PAP release [40]. TPST is much less susceptible to inhibition by dichloronitrophenol than the cytosolic phenol sulfotransferases [18,25]. The activity of the enzyme in vitro is inhibited by certain lipids such as sphingosine [41], but the physiological relevance of this observation is unclear.

3.5. Regulation Almost nothing is known about the regulation of TPST activity during development, during the cell cycle, or in response to extracellular signals. Addition of 2chloroadenosine, an adenosine receptor agonist, to PC12 cells has been reported to result in a decrease in TPST activity [42], but the signal transduction pathway responsible for this effect remains to be elucidated. Determination of TPST activity in post-mitochondrial supernatants from rat liver and cerebellum suggests that the enzyme activity increases postnatally [20]. However, the opposite conclusion has recently been reported by Vargas et al. [28] who observed a decrease in TPST activity in a brain membrane fraction during rat postnatal development. Interindividual variation in TPST activity in human liver samples has been reported [22], as has a higher level of TPST activity in the gastric mucosa of alcoholics as ccmpared with non-alcoholics [43], but neither the cause of these differences nor their physiological significance have been clarified. It will be important to pursue studies on the regulation of TPST and to relate them to the functional aspects of protein tyrosine sulfation (see below).

4. Substrate specificity of TPST As is discussed below, TPST is a highly selective enzyme and sulfates only one or few tyrosine residues in a typical peptide or protein.

4.1. Synthetic peptide substrates TPST may be conveniently assayed with appropriate synthetic peptides as substrates. The Km for most peptides (8-13 amino acids long) with a single tyrosine sulfation site is in the 10-5 to 10-4 M range ]14,29,30], although lower Km values are observed (see below) for certain peptides containing only one tyrosine residue. It is likely that similar Km values hold true for the larger physiological protein substrates in the lumen of the trans-Golgi-network: the kinetic parameters of leech

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TPST towards full-length hirudin, a 65-residue long polypeptide, were similar to those towards a nonapeptide corresponding to the single tyrosine sulfation site of hirudin [10]. Considerably lower Km values have been reported for synthetic substrates containing multiple potential tyrosine sulfation sites. First, it has been shown that the random copolymer poly(Glu,Ala,Tyr) is a high-affinity substrate for TPST, exhibiting a Km of 0.3 #M [13]. Second, the Km of TPST for a 10-amino acid long peptide containing two adjacent sulfation sites corresponding to those in complement C4 decreased from 17 tzM to 0.4/~M when the sequence was extended by 6 amino acids to include the third adjacent sulfation site of C4 [29]. Third, the Km of 35/~M for a peptide containing the twin sulfation site of the C-terminal peptide of the cholecystokinin (CCK) precursor increased 3-4-fold upon replacement of either one tyrosine residue by phenylalanine [30]. These observations triggered a systematic investigation of the effect of multiple tyrosine sulfation sites by comparing the sulfation of peptides in which the tyrosine sulfation site of chromogranin B (secretogranin I) was repeated 2 and 3 times in tandem [30]. The Km of these peptides was found to decrease exponentially with the number of identical sulfation sites present in the peptides, reaching a value as low as - 4 0 nM for the peptide containing three sulfation sites. Since several proteins exist which contain multiple adjacent sulfation sites - - e.g. C4, cionin, heparin cofactor II and preproCCK - - the increased affinity of TPST for such substrates might be of physiological relevance, e.g. by promoting stoichiometric sulfation. Interestingly, an analogous triple sulfation site peptide in which two of the three tyrosine residues were replaced by phenylalanines still displayed the remarkably low Km of 340 nM [30]. Similarly, Lin et ai., using various peptides corresponding to the triple tyrosine sulfation site of the C4 component of complement in which two of the three tyrosines were replaced by phenylalanine, found Km values between 10 -6 and 10 -5 M provided that each of the three aromatic residues was preceded by an acidic amino acid [31]. One possible interpretation of these observations is that replacing tyrosine by phenylalanine in the context of a proper sulfation site (i.e. when tyrosine is preceded by an acidic amino acid) is not alone sufficient to fully eliminate the binding of a peptide to TPST. 4.2. Structural determinants The sequences surrounding sulfated tyrosine residues are known for a number of proteins. Although there appears to be no single consensus sequence for tyrosine sulfation, all sequences are characterized by common consensus features from positions -5 (N-terminal) to +5 (C-terminal) of the sulfated tyrosine [1,441 which have been successfully used to predict sulfation [8,9]. The hallmark of these proposed features is the presence of acidic amino acids adjacent to the aminoterminal side of the tyrosine. The structural requirements of partially purified and homogenous TPST from bovine adrenal medulla have been experimentally tested with synthetic peptides as substrates [21,30]. The peptides used were modelled after the known or putative tyrosine sulfation sites of preproCCK, chromogranin B (secretogranin I) and vit-

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ronectin. By varying the sequence of these peptides it was found that, indeed, acidic amino acids are the major determinant of the Km for the peptides. Thus, the substitution of a tyrosine-adjacent Asp by Asn leads to an increase of the apparent Km from 21 to 550 I~M in preproCCK peptides. Acidic amino acids seem to enhance the affinity of TPST towards substrates in an incremental manner; thus, it may be expected that proteins which contain clusters of acidic amino acids, like members of the secretogranin family, may be particular good substrates for TPST, but this has not been tested yet. Essentially similar conclusions about the importance of acidic amino acids in tyrosine sulfation sites have been reported by Lin et al. [31]. To investigate whether the amino acids in the immediate vicinity of the tyrosine alone are sufficient to mediate recognition of a peptide by TPST in vivo, an artificial protein, sulfophilin, consisting of 12 heptapeptide repeats corresponding to the identified tyrosine sulfation site of chromogranin B (secretogranin I), was constructed and expressed in fibroblasts. Sulfophilin secreted into the medium was found to contain 12 mol sulfate/mol protein, showing that the structural information contained in the heptapeptide motif is sufficient for stoichiometric tyrosine sulfation to occur in the living cell [451. Although turn-inducing amino acids are frequently found in tyrosine sulfation sites [1], such residues affected the sulfation of synthetic peptides only to minor extent [45]. Possibly, their presence may promote tyrosine sulfation of proteins but not of peptides since the latter are less restrained in adopting an optimal conformation for recognition by TPST. Finally, C-terminal tyrosine residues may serve as substrate for TPST whereas N-terminal tyrosine residues appear to lack some important feature for sulfation, presumably the preceding acidic residues [30]. 4.3. T P S T and tyrosine kinases

The importance of acidic amino acids for sulfation is reminiscent of their role in tyrosine phosphorylation [46]. Indeed, TPST and certain autophosphorylating tyrosine kinases seem to have overlapping substrate specificities since a number of peptides and proteins are modified by both in vitro, e.g. tubulin [13,47], gastrin [48,49] and hirudin [50,51], the random copolymer poly(Glu,Ala,Tyr) [13,52], as well as peptides corresponding to the autophosphorylation site of pp60 v-src (Tyr 416) and the putative phosphorylation site of tubulin [30,53] but not a peptide corresponding to the non-autophosphorylation site of pp60 c'src (Tyr 527) [30,54]. The above findings lend support to the hypothesis that TPST and tyrosine kinases may have evolved from a common ancestor [1]. In contrast to their catalytic similarity, the topologies of these two types of enzymes are different, since tyrosine kinases act in the cytoplasm or on the cytoplasmic side of membranes while TPST acts in the lumen of the trans-Golgi-network. 4.4. T P S T isoenzymes

Species differences in the substrate specificities of leech salivary gland and bovine adrenal medulla TPSTs have been demonstrated in vitro [10]. It would, therefore, not be unexpected if within the same organism, isoenzymes of TPST were found to exist. Indeed, a small number of tyrosine-sulfated proteins (c~-choriogonadotropin

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[55]; cionin [56]; see also Ref. [1] for earlier references) do not conform to the consensus features delineated for the TPST characterized so far, suggesting the presence of additional TPSTs with distinct substrate specificities. Transfection studies (e.g. Ref. [57]) have shown that the degree of sulfation of the foreign protein may vary, depending on the host cell line. It will be important to resolve to which extent such observations reflect differences in the level of expression of the substrate protein or are due to the presence of distinct TPSTs in different cell types.

5. Functional aspects of tyrosine suifation For most tyrosine-sulfated proteins, the biological significance of tyrosine sulfation is unknown. This in part reflects the fact that in the case of a tyrosine-sulfated protein (as opposed to a tyrosine-sulfated peptide), the unsulfated form is not easily available in sufficient amounts to compare its property with the naturally occurring sulfated form. In contrast to peptides, proteins cannot be synthesized chemically, and their desulfation by chemical or enzymatic means is usually destructive or inefficient. Three other approaches, however, have been used to obtain unsulfated proteins for functional studies: (i) the inhibition of tyrosine sulfation in vivo by chlorate, which inhibits PAPS synthesis [58,59], or (in certain systems only) by dichloronitrophenol [60,61]; (ii) the expression of proteins with mutated tyrosine sulfation sites [62,63]; and (iii) the expression of proteins in organisms such as bacteria and yeast that lack tyrosine sulfation [51]. Table 2 summarizes cases in which functional differences between unsulfated and sulfated peptides and proteins have been demonstrated. These indicate a role for protein tyrosine sulfation before as well as after secretion. 5.1. Tyrosine sulfation and intracellular transport o f secretory proteins

Only a subset of secretory proteins are tyrosine-sulfated. These include both conTable 2 Biological effects of tyrosine sulfation Protein

Unsulfated Protein obtained by

Reduced Activity/property due to lack of sulfation

Cholecystokinin

Chemicalpeptide synthesis

Hirudin Leucosulfakinin Gastrin Yolk protein 11

Acid hydrolysis Chemical peptide synthesis Chemical peptide synthesis Site directed mutagenesis of sulfated tyrosine, inhibition of cellular PAPS synthesis Enzymatic hydrolysis Inhibition of cellular PAPS synthesis Site directed mutagenesis of sulfated tyrosine, inhibition of cellular PAPS synthesis

Stimulation of gallbladder contraction and alpha-amylase secretion (260x) Affinity to thrombin (10x) Myotropic activity (104x) Half-life in plasma (5x) Transport rate

Fibronectin Complement C4 Factor VIII

Affinity to fibrin (2x) Hemolytic activity (2x) Binding to von Willebrand factor

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stitutive and regulated secretory proteins (see Table 1), which is consistent with the localization of TPST in the trans-Golgi-network, the compartment in which the constitutive and regulated secretory pathways diverge. In hindsight, the first two proteins found to be tyrosine-sulfated, fibrinogen [64] and gastrin [48], constituted examples of a constitutive and regulated tyrosine-sulfated secretory protein, respectively. It has therefore been obvious ever since the widespread occurrence of protein tyrosine sulfation was discovered [65] that tyrosine suifation cannot be an absolute requirement for protein secretion or a general sorting signal. Rather, with the increasing number of tyrosine-sulfated proteins that were identified as being secretory, it became an attractive possibility that tyrosine sulfation may have more subtle roles in the secretion of certain proteins [1]. Indeed, studies on the secretion of vitellogenin 2 (yolk protein 2) of Drosophila melanogaster have provided compelling evidence that at least in the case of this protein, tyrosine sulfation accelerates its transport from the trans-Golgi-network to the cell surface [621. Other investigators have been unable to detect tyrosine sulfation-dependent differences in secretion for c~2-antiplasmin, complement C4 [66], intestinal aminopeptidase N [61] and type III procollagen [67]. However, it is not clear whether aminopeptidase N is stoichiometrically sulfated. Also, the metabolic labeling in these studies was probably too long to detect subtle differences in secretion kinetics. In line with the above considerations, the sorting of the granins to secretory granules does not seem to be affected by tyrosine sulfation [68]. The same holds true for the sorting of proteins to different plasma membrane domains in polarized cells, as exemplified by aminopeptidase N in intestinal explants [61], although tyrosine-sulfated proteins are predominantly secreted into the apical medium [69]. On the other hand, data consistent with a role of tyrosine sulfation in the intracellular transport of certain secretory proteins have been obtained by Liu and colleagues [70-72]. These investigators have identified, purified and characterized a 175-kDa protein from liver membranes that binds tyrosine sulfate but not tyrosine phosphate. Interestingly, this protein binds to tyrosine-sulfated, but not unsulfated, secretory proteins, and thus may be involved in the intracellular transport of these proteins. 5.2. Tyrosine sulfation and the activity~properties o f secreted proteins

Tyrosine sulfation has long been known to be required for the biological activity of certain regulatory peptides such as caerulein [73], CCK [74] and leucosulfakinin [75]. In addition, the affinity of hirudin to thrombin is increased 10-fold by tyrosine suifation [76]. The structure-function relationship between the presence of a tyrosine sulfate group and biological activity has been most extensively investigated for these peptides. First, in the case of CCK which is 260 times more effective in sulfated than unsulfated form, the substitution of the tyrosine sulfate by a tyrosine phosphate or e-hydroxynorleucine sulfate retains up to 10% of the biological activity, whereas the substitution by serine sulfate yields no activity [73,74]. Second, a non-hydrolyzable tyrosine sulfate ester analogue (L-Phe[p-CH2SO3Na]) retains full biological activity in CCK [77]. Third, in the case of leucosulfakinin II, the presence of the sulfate

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group is essential for its myotropic activity whereas the position of the tyrosine sulfate residue in the sequence seems to be less critical; a synthetic peptide in which the tyrosine sulfate group was shifted one position towards the N-terminus retained 40% of the activity of the parent peptide [78]. Fourth, in the case of hirudin, the - 10-fold greater affinity towards thrombin of the sulfated than unsulfated form was almost fully retained after replacement of the sulfate by phosphate [51]. These studies suggest that for biological activity of these proteins the nature of the side chain and position of the tyrosine sulfate in sequences appear to allow for some flexibility provided that its structure allows the positioning of the sulfate (or sulfate-like) group at a proper distance from the peptide backbone. NMR studies with sulfated and unsulfated CCK26_33 have shown that the introduction of the sulfate group enhances its folding tendency and stabilizes the external orientation of the tyrosine ring in a turn conformation [79]. The three-dimensional structure of the 7-kDa polypeptide hirudin has also been analysed by NMR; the Cterminal 8 amino acids, which include the sulfated tyrosine, form an irregular extended strand which does not interact with the rest of the protein [80]. Recently, the structure of the complex between the C-terminal, tyrosine-sulfated peptide of hirudin and thrombin has been determined at a resolution of 2.2 ]k [81]. This work, an in-depth discussion of which would exceed the scope of this review, shows the structural details of how the tyrosine sulfate residue participates in a specific proteinprotein interaction.

5.3. Tyrosine sulfation and protein-protein interactions: a unifying hypothesis This leads us to a possibly unifying concept of the role of protein tyrosine sulfation, which is that this post-translational modification promotes, and maybe in some instances also prevents, specific protein-protein interactions [82]. The sulfationinduced increase in the binding of complement C4 to complement C 1s [83], fibronectin to fibrin [84] and factor VIII to von Willebrand factor [63] document that the observations with CCK and hirudin - - i.e. a promotion of protein-protein interaction by tyrosine sulfation - - can be extended from peptides and small polypeptides to large proteins. Furthermore, the modulation of secretion kinetics by tyrosine sulfation [62] and the existence of a membrane-bound tyrosine sulfate binding protein [71] are also fully consistent with this unifying concept.

6. Evolutionary aspects of tyrosine sulfation Tyrosine-sulfated proteins have been found in all vertebrates studied as well as in invertebrates including ascidians [56] molluscs [1], annelids [50], and arthropods [75,78,85]. Protein-bound tyrosine sulfate has also been found in the multicellular green alga Volvox [86]. However, protein tyrosine sulfation has not been found in any unicellular organism, such as bacteria, yeast, Dictyostelium discoideum and Paramecium [1]. It will be an important task of future research to elucidate the significance of the apparently concomittant appearance in evolution of protein tyrosine sulfation and multicellularity. Interestingly, the substrate specificity of TPST has been remarkably conserved during evolution. The tyrosine sulfation sites

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of the insect proteins yolk protein 2 [85] and leucosulfakinins [75,87] and of the annelid polypeptide hirudin [50] conform to the consensus features for tyrosine sulfation [1,44,88]. Drosophila yolk protein II transfected into mouse fibroblasts is stoichiometrically sulfated at the correct tyrosine residue [89]. Furthermore, both TPST from leech salivary gland and from bovine adrenal medulla are able to sulfate the same protein and peptide substrates, although they do so with different Km and limax values, suggesting only subtle evolutionary divergence between these TPSTs [10]. The conservation of protein tyrosine sulfation is likely to reflect an important role of this post-translational modification in various biological processes, of which, however, only a few selected cases are presently understood. 7. References 1 W.B. Hunner and P.A. Baeuerle, Protein sulfation on tyrosine, Mod. Cell Biol., 6 (1988) 97-140. 2 W.B. Huttner, Tyrosine sulfation and the secretory pathway, Annu. Rev. Physiol., 50 (1988) 363-376. 3 A. Hille, T. Braulke, K. von Figura and W.B. Huttner, Occurrence of tyrosine sulfate in proteins - - a balance sheet. 1-Secretory and lysosomal proteins, Eur. J. Biochem., 188 (1990) 577-586. 4 A. Hille and W.B. Huttner, Occurrence of tyrosine sulfate in proteins - - a balance sheet. 2. Membrane proteins, Eur. J. Biochem., 188 (1990) 587-596. 5 W.B. Huttner, Determination and occurrence of tyrosine O-sulfate in proteins, Methods Enzymol., 107 (1984) 200-223. 6 A. Bateman, S. Solomon and H.P.J. Bennett, Post-translational modification of bovine proopiomelanocortin, J. Biol. Chem., 265 (1990) 22130-22136. 7 A.H. Johnsen, Nondestructive amino acid analysis at the picomole level of proline-containing peptides using aminopeptidase M and prolidase: application to peptides containing tyrosine sulfate, Anal. Biochem., 197 (1991) 182-186. 8 D. Jenne, A. Hille, K.K. Stanley and W.B. Huttner, Sulfation of two tyrosine-residues in human complement S-protein (vitronectin), Eur. J. Biochem., 185 (1989) 391-395. 9 A. Weidemann, G. Kfnig, D. Bunke, P. Fischer, J.M. Salbaum, C.L. Masters and K. Beyreuther, Identification, biogenesis, and localization of precursors of Alzheimer's disease A4 amyloid protein, Cell, 57 (1989) 115-126. 10 C. Niehrs, W.B. Huttner, D. Carvallo and E. Degryse, Conversion of recombinant hirudin to the natural form by in vitro tyrosine sulfation. Differential substrate specificities of leech and bovine tyrosylprotein sulfotransferases, J. Biol. Chem., 265 (1990) 9314-9318. 11 R.W. Lee and W.B. Huttner, Tyrosine-O-sulfated proteins of PCI2 pheochromocytoma cells and their sulfation by a tyrosylprotein sulfotransferase, J. Biol. Chem.., 258 (1983) 11326-11334. 12 S. Fukui, Y. Numata and I. Yamashina, Comparison of protein sulfation in control and virustransformed baby hamster kidney cells, J. Biochem. (Tokyo), 96 (1984) 1783-1788. 13 R.W. Lee and W.B. Huttner, (Glu 62, Ala 3°, TyrS)n serves as high-affinity substrate for tyrosylprotein sulfotransferase: a Golgi enzyme, Proc. Natl. Acad. Sci. USA, 82 (1985) 6143-6147. 14 F. Vargas, O. Frerot, M.D. Tuong and J.C. Schwartz, Characterization of a tyrosine sulfotransferase in rat brain using cholecystokinin derivatives as acceptors, Biochemistry, 24 (1985) 5938-5943. 15 N. Liu and J.U. Baenziger, In vivo and in vitro tyrosine sulfation of a membrane glycoprotein, J. Biol. Chem., 261 (1986) 856-861. 16 L.I. Fessler, S. Brosh, S. Chapin and J.H. Fessler, Tyrosine sulfation in precursors of collagen V, J. Biol. Chem., 261 (1986) 5034-5040. 17 P.M. Barling, D.J. Palmer and D.L. Christie, Preparation of desulphated bovine fibrinopeptide B and demonstration of its sulphation in vitro by an enzyme system from neuroblastoma-glioma hybrid cells, Int. J. Biochem., 18 (1986) 137-141.

268 18

19 20 21 22 23 24

25 26 27 28 29 30 31 32

33 34 35 36

37 38

39 40

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