Tyrosine sulfation: an increasingly recognised post-translational modification of secreted proteins

New Biotechnology  Volume 25, Number 5  June 2009

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Review

Tyrosine sulfation: an increasingly recognised post-translational modification of secreted proteins Martin J. Stone1, Sara Chuang1, Xu Hou1, Menachem Shoham2 and John Z. Zhu3 1

Department of Biochemistry and Molecular Biology, Monash University, Clayton, VIC 3800, Australia Department of Biochemistry, School of Medicine, Case Western Reserve University, USA 3 Interdisciplinary Program in Biochemistry, Indiana University, Bloomington, IN 47405, USA 2

The post-translational sulfation of tyrosine residues occurs in numerous secreted and integral membrane proteins and, in many cases, plays a crucial role in controlling the interactions of these proteins with physiological binding partners as well as invading pathogens. Recent advances in our understanding of protein tyrosine sulfation have come about owing to the cloning of two human tyrosylprotein sulfotransferases (TPST-1 and TPST-2), the development of novel analytical and synthetic methodologies and detailed studies of proteins and peptides containing sulfotyrosine residues. In this article, we describe the TPST enzymes, review the major techniques available for studying the presence, location and function of tyrosine sulfation in proteins and discuss the biological functions and biochemical interactions of several proteins (or protein families) in which tyrosine sulfation influences the protein function. In particular, we describe the detailed evidence supporting the importance of tyrosine sulfation in the cellular adhesion function of P-selectin glycoprotein ligand-1, the leukocyte trafficking and pathogen invasion functions of chemokine receptors and the ligand binding and activation of other G-protein-coupled receptors by complement proteins, phospholipdis and glycoprotein hormones. Contents Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Tyrosylprotein sulfotransferases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Discovery and cloning . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Expression patterns. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Biological effects of TPST gene disruption . . . . . . . . . . . . . . . . . . . . . . . . . . Sequences of TPSTs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Enzymatic mechanism and structure–function relationships . . . . . . . . . . . . Target specificity. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Methods for studying the presence, location and function of tyrosine sulfation Bioinformatic prediction of tyrosine sulfation sites . . . . . . . . . . . . . . . . . . . Metabolic labelling and amino acid analysis . . . . . . . . . . . . . . . . . . . . . . . . Inhibition of sulfation and removal of sulfate . . . . . . . . . . . . . . . . . . . . . . . Anti-sulfotyrosine antibodies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Mass spectrometry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Sulfotyrosine-containing peptides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Corresponding author: Stone, M.J. ([email protected]) 1871-6784/$ - see front matter ß 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.nbt.2009.03.011

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Review

Tyrosine-sulfation of P-selectin glycoprotein ligand-1 (PSGL-1) in leukocyte adhesion . . . . . . . . . . . . . . . . Function of PSGL-1. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Importance of amino-terminal region . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Tyrosine sulfation of the amino-terminal region . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Structural basis of sulfotyrosine recognition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Role of chemokine receptor sulfation in leukocyte trafficking . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Chemokine and chemokine receptor families . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Studies of tyrosine sulfation in intact receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Studies of chemokine receptor peptides. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Role of chemokine receptor sulfation in pathogen recognition. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Chemokine receptors in HIV infection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Role of tyrosine sulfation in interaction of CCR5 with HIV-1 gp120 . . . . . . . . . . . . . . . . . . . . . . . . . . . Role of tyrosine sulfation in the interaction of Duffy antigen and receptor for chemokines (DARC) with malarial parasite Plasmodium vivax . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Sulfation of other G-protein-coupled receptors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Tyrosine sulfation of GPCRs in the immune system . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Tyrosine sulfation of glycoprotein hormone receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Concluding remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Introduction The complete sequencing of numerous genomes and the development of proteomics have led to renewed appreciation of both the prevalence and the functional significance of post-translational modifications of proteins. This review focuses on one such modification, the O-sulfation of tyrosine residues to give the modified amino acid sulfotyrosine (sulfoTyr; Figure 1). The natural occurrence of tyrosine sulfation was first discovered in 1954 in a peptide derived from bovine fibrinogen [1]. During the 1960s, several small peptide hormones were found to contain sulfoTyr [2–5]. Subsequently, in the 1980s, sulfated proteins were observed in secreted proteins from several organisms, including fruit fly [6], rat [7,8], cow [8,9] and human [10]. In recent years, interest in tyrosine sulfation has surged owing, partly, to: (1) the discovery that tyrosine sulfation plays crucial roles in human physiology and pathology; (2) cloning of the human tyrosylprotein sulfotranferases (TPSTs), the enzymes that catalyse tyrosine sulfation; and (3) the development of increasingly powerful methods for identification and characterisation of sulfoTyr-containing

FIGURE 1

The structure of sulfoTyr. 300

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proteins. A recent search of the UniProtKB database (http:// www.uniprot.org/) found 376 known tyrosine-sulfated proteins, including 46 human proteins, the majority of which are peptide hormones, enzymes, extracellular matrix proteins, blood coagulants or anticoagulants, complement proteins or proteins that function in leukocyte trafficking and adhesion. However, it seems likely that tyrosine sulfation remains to be identified in numerous additional proteins. Several recent reviews have discussed various aspects of tyrosine sulfation [11–15]. In this article, we review the current state of knowledge regarding TPSTs, describe the major techniques available for studying tyrosine sulfation and discuss in detail the roles that tyrosine sulfation plays in the functions of several crucial human proteins. The latter sections focus on the cellular adhesion function of P-selectin glycoprotein ligand-1 (PSGL-1), the leukocyte trafficking and pathogen invasion functions of chemokine receptors and the binding and activation functions of other G-protein-coupled receptors.

Tyrosylprotein sulfotransferases Discovery and cloning Cell free tyrosine O-sulfation was initially demonstrated utilising fractionation of cell lysate from rat pheochromocytoma PC12 cells. The activity was attributed to a previously unknown enzyme dubbed tyrosylprotein sulfotransferase (TPST) [16]. TPST activity has since been shown to occur in other cells and tissues [17–19]. Further fractionation experiments demonstrated the protein sulfation activity was in the Golgi membrane fraction [16]. Subsequent work has since shown tyrosine sulfation to be a trans-Golgispecific modification and that TPSTs are integral membrane proteins with the catalytic domain luminally orientated [20,21]. TPST activity has been purified from bovine adrenal medulla [22], liver tissue [23] and salivary glands [24]. SDS-PAGE analysis of purified TPST from bovine adrenal medulla found a doublet at 50– 54 kDa [22], suggesting the presence of isoenzymes of TPST [22]. In 1998, Ouyang et al. used affinity for a sulfation target peptide to

isolate TPST protein from rat liver and then used the sequences of derived peptides to identify the TPST-1 genes from human and mouse EST libraries [25]. Shortly afterwards the gene for human TPST-2 was also cloned [26,27]. Orthologues for TPST-1 and TPST-2 have also been found in many other species, both vertebrate and invertebrate, supporting the widespread occurrence and importance of tyrosine O-sulfation [12], although Drosophila melanogaster lacks a second TPST gene [12].

Expression patterns The incidence of the human TPST-1 and TPST-2 gene expression in 20 human tissues, determined from RNA levels, is shown in Figure 2. Whilst it appears that both TPST-1 and TPST-2 are expressed in most tissues, the two isoforms exhibit different patterns of expression. For example, TPST-1 is expressed at substantially higher levels than TPST-2 in testis, whereas TPST-2 is expressed more strongly in the blood, trachea, thyroid gland and several other organs. The existence of two similar enzymes with differential expression in a range of tissues suggests that the two enzymes may have specific biological functions and possibly different protein target specificities.

Biological effects of TPST gene disruption Knockout mouse studies have been carried out to assess the biological roles of TPSTs. Targeted disruption of the TPST-1 gene has shown that disruption of one allele at the TPST-1 locus has no significant effect, and that TPST-1+/ mice appear to be normal, have normal fertility and growth curves [28]. However, TPST-1/ mice showed reduced postnatal body weight, suggested to be due to a change in feeding behaviour or digestion as a result of the inability

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to sulfate two known sulfated gastrointestinal peptide hormones (gastrin and CCK), or potentially other unknown TPST-1 substrates [28]. Disruption of the TPST-2 gene also showed no significant effect in TPST-2+/ mice, which appeared normal and had normal fertility [29]. In comparison to TPST-1 deficient mice, TPST-2/ mice also showed reduced postnatal body weight, but reached normal body weight at 10 weeks of age [29]. The most significant difference between TPST-1/ and TPST-2/ is the reduced fertility of TPST2 knockout mice. Whilst TPST-2/ males appear to have normal sperm in terms of number, morphology and motility in normal media, these mice were virtually infertile [29]. TPST-2/ sperm demonstrated an impaired ability to ascend the female genital tract, decreased motility in viscous media and impaired fertilisation ability in vitro [29]. This indicated that there were proteins that required TPST-2 for sulfation for normal male reproductive function. Most recently a double knockout was developed to investigate the importance of tyrosine sulfation in vivo. However, although the double knockout mice were born alive, most died in the early postnatal period as a result of cardiopulmonary insufficiency [30]. The mice that survived the postnatal period subsequently displayed primary hypothyroidism [30]. These results indicated that proteins sulfated by TPSTs were required for both normal pulmonary function at birth and normal thyroid function postnatally. Furthermore, the ability of mice to survive upon homozygous disruption of a single TPST gene but not upon deletion of both TPST genes suggests that there is some redundancy (or compensation) in TPST activity. The result also supports the proposal that TPST-1 and TPST-2 are the only TPSTs in the mouse. In summary, the results of the genetic knockout studies underscore the vital importance of tyrosine sulfation for a range of crucial biological functions.

FIGURE 2

Tissue expression of human TPST-1 and TPST-2 as indicated by analysis of expressed sequence tag (EST) count. Data were obtained from the UniGene EST Profile Viewer Web site of the National Center for Biotechnology Information on August 28, 2008 (http://www.ncbi.nlm.nih.gov/UniGene/). www.elsevier.com/locate/nbt

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Sequences of TPSTs On the basis of their gene sequences, human TPST-1 and TPST-2 are predicted to have 370 and 377 amino acids, respectively, with corresponding molecular masses of 42.2 and 41.9 kDa [26]. A sequence alignment of human TPST-1 and TPST-2 with each other and with several orthologues is shown in Figure 3. Human TPST-1 and TPST-2 have 63% amino acid sequence identity with each other but there is substantially higher sequence identity (>90%) between each human enzyme and its corresponding enzyme in other mammalian species. This pattern of sequence conservation

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suggests that duplication of the TPST gene occurred before evolutionary divergence of the mammalian species but that the sequences were subsequently highly conserved, possibly due to functionally important differences between the sequences of the two TPST isoforms. On the basis of sequence analysis, both TSPT-1 and TPST-2 are predicted to have type II transmembrane topology with a short 8-residue N-terminal cytoplasmic domain, a 17residue transmembrane domain and a luminal catalytic domain (Figure 3B) [12,31]. Each human enzyme has two potential Nlinked glycosylation sites and six conserved cysteine residues in

Review FIGURE 3

(A) Sequence alignment of human TPST-1 and TPST-2 with each other and with several orthologues of TPST-1; the sequence of Drosophilia TPST has been truncated at position 417 out of 499 residues. Residues in the putative transmembrane (TM) region are underlined and those in the proposed stem region are shown in italics. Cysteine residues are in bold type and the 50 -phosphosulfate binding motif is boxed. Symbols on the bottom row indicate invariant residues (*), conservative substitutions (:) and semi-conservative substitutions (.). The alignment was conducted using Clustal W 2.0 [120]. (B) Schematic representation of human TPST domain structure and positions of conserved cysteine residues in the luminal domain. 302

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Fig. 3. (Continued ).

the luminal domain (Figure 3B), suggesting the possible formation of three disulfide bonds [12,31]. The sequence conservation between TPST1 and TPST-2 is not evenly distributed throughout the sequence (Figure 3A). Instead, it is particularly high (77%) over residues 69–352, presumably representing the majority of the catalytic domain. The region from the luminal end of the putative transmembrane helix (residue 26) to the beginning of the conserved region (residue 68) has remarkably low sequence identity between the two isoforms, suggesting that this region may be an unstructured ‘stem’ (Figure 3B) [12]. Alternatively, it is possible that these residues, in addition to the few variant residues across the conserved region, play a role in regulating the specificity of the two TPST enzymes for different substrates.

Enzymatic mechanism and structure–function relationships The reaction catalysed by TPSTs is shown in Figure 4. TPSTs employ 30 -phosphoadenosine 50 -phosphosulfate (PAPS) as the sulfate donor [13]. The sulfate is transferred from the 50 -phosphosulfate group of PAPS to the phenol of a tyrosine residue of a peptide or protein to form an O-sulfated tyrosine and adenosine

30 ,50 -diphosphate (30 ,50 -ADP) [12]. Although the detailed catalytic mechanism has not been elucidated, it is proposed to follow the sequential steps: (1) binding of PAPS; (2) binding of the target protein; (3) sulfate transfer; (4) release of the sulfated target and (5) release of 30 ,50 -ADP [32]. Both TPST-1 and TPST-2 are optimally active at slightly acidic pH (6.5 and 6.0, respectively) and both are strongly stimulated by Mn2+ and inhibited by Ca2+ [31]; curiously, TPST-2 is also stimulated by Mg2+ whereas TPST-1 is not. Km values for various peptide substrates vary from the low micromolar to low millimolar range [31]. The three-dimensional structures have not been reported for TPSTs from human or any other species. However, there are a number of structures of enzymes that catalyse transfer of sulfate to hydroxyl groups on non-protein molecules, including phenols, oestrogen, sugars and cholesterol. Considering that all known sulfotransferases utilise PAPS as the sulfate donor, comparisons of TPST sequences with the sequences of these other sulfotransferases may provide some insights into TPST structure and structure–function relationships. For example, in mouse oestrogen sulfotransferase (mEST) crystal structures, two structural motifs www.elsevier.com/locate/nbt

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Review FIGURE 4

Tyrosine O-sulfation catalysed by TPSTs. TPSTs catalyse the transfer of a sulfate group from 30 -phosphoadenosine-50 -phosphosulfate (PAPS), to tyrosine residues, resulting in sulfoTyr and adenosine 30 ,50 -diphosphate.

were found to be involved in the binding of 50 -phopshosulfate and the 30 -phosphate groups of PAPS; the motifs are designated 50 -PSB and 30 -PB, respectively [33]. As shown in Figure 5, the sequence of the 50 -PSB motif of mEST aligns extremely well with sequences in the luminal domains of human TPSTs. Two residues in the 50 -PSB motif of mEST (Lys48 and Thr51) are thought to be involved in catalysis [34]. Thr51 is retained (as Thr82 or Thr81) in the human TPST sequences, whereas there is a conservative substitution of Lys48 by Arg79 in human TPST-1 and Arg78 in human TPST-2. The high sequence similarity in this region strongly suggests that these residues of the TPST enzymes are directly involved in the chemistry of sulfate group transfer. On the contrary, although the TPSTs have been suggested to contain a 30 -PB motif corresponding to that of mEST [12], there is no sequence in human TPST-1 or TPST-2 with high identity to the 30 -PB sequence of mEST. Moreover, the overall sequence identity of TPSTs with mESTs is rather low (4–12%), so it is unclear whether they share the same overall fold. A more detailed understanding of the structure–function relationships of TPSTs will require determination of the three-dimensional structure of at least one of these enzymes.

FIGURE 5

Sequence alignment of the 50 -PSB motif of mEST with sequences in the luminal domains of human TPSTs. Boxed residues indicate the conserved motif, which interacts with the 50 -phosphosulfate group of PAPS. Sequences aligned are human TPST-1 (hTPST-1), human TPST-2 (hTPST-2) and mouse oestrogen sulfotransferase (mEST). The alignment was conducted using Clustal W 2.0 [120]. 304

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Target specificity Unlike some post-translational modifications (e.g. N-linked glycosylation), the occurrence of sulfation on a particular Tyr residue is not related in a simple way to the sequence in which that Tyr residue is located. Nevertheless, analysis of sequences with known sulfated tyrosines has identified certain common characteristics that appear to favour sulfation. These features are summarised in Table 1. One of the major criteria appears to be the presence of acidic amino acid residues [27,35]. Studies of the sulfation of synthetic peptides have determined that the presence of acidic residues in the vicinity of the sulfated tyrosine is a structural requirement [32,36]. In general, either aspartic or glutamic acid is found adjacent to the sulfated tyrosine, with additional acidic residues located within five amino acid positions away from the sulfated tyrosine [35]. In addition to acidic residues, Huttner et al. found that tyrosine sulfation sites contained strong turn inducing amino acids such as proline or glycine, or several weaker turn inducing amino acids such as aspartic acid, serine or asparagine within seven amino acid positions from the sulfated tyrosine [35]. Subsequent studies found that turn inducing amino acids were not definitive in predicting sulfation [36,37]. Although the same studies found that small amino acids were generally positioned 1–2 amino acids upstream of the sulfated tyrosine, this was hypothesised to be required for the sharp turning of the tyrosine upon binding to TPST [32,37]. Alternatively, the requirement for sulfated tyrosine to be sterically unhindered could be a constraint imposed by the structure of the target binding sites on the TPST enzymes. Further supporting the need for a sterically unhindered target, sulfated tyrosine residues are generally well separated from both disulfide bonds and N-linked glycosylation sites [35]. For example, in mouse IgG2a, inhibition of N-linked glycosylation allows for sulfation to occur, suggesting that the N-linked oligosaccharides hinder the binding of the tyrosine to TPSTs [32,38]. In summary, although the sequence requirements for

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TABLE 1

Consensus sequence characteristics for tyrosine sulfation sites Sequence feature

Position from Tyr

Description

Acidic residue

5

Acidic amino acid adjacent to Tyr at N-terminal end and at least three acidic amino acids within five residues of Tyr

Small residue

7

More than one of Pro or Gly OR more than two of Ser, Asn and Asp

Disulfide bond

7

No cysteines within seven residues of Tyr

Sugar

7

No glycosylation sites within seven residues of Tyr

Hydrophobic residue

5

Fewer than three hydrophobic residues within five residues of Tyr

TABLE 2

Method/reagent Metabolic labelling

Review

Common methods for studying protein sulfation Information content or application 35

SO4

2

Presence of sulfate

Amino acid analysis

Presence of sulfotyrosine

Inhibition of ATP-sulfurylase with ClO3 or SeO42

Presence of sulfate Function of sulfation

Sulfate removal using arylsulfatase

Presence of sulfotyrosine Function of tyrosine sulfation

Anti-sulfotyrosine antibodies

Detection of sulfotyrosine (especially in new proteins) Purification of sulfotyrosine-containing proteins Quantitation of sulfotyrosine in proteins

Mass spectrometry

Presence of sulfate Position(s) of sulfation

Synthetic peptides

Function of specific sulfotyrosine residues Structural/mechanistic studies

tyrosine sulfation are not absolute, several factors have been identified that either favour or disfavour sulfation of nearby tyrosine residues. As discussed below, these factors have allowed the development of bioinformatic approaches for prediction of sulfation sites. The sequence determinants for tyrosine sulfation discussed above are presumably based, at least partly, on the selectivity of the TPST enzymes. Mishiro et al. have reported kinetic constants for the two human TPSTs with a variety of peptide substrates [31]. Interestingly, although both enzymes can act upon the same set of substrates, there are some notable differences in specificity. For example, the catalytic efficiency (Vmax/Km) of TPST-2 is 15-fold higher than that of TPST-1 for sulfation of a peptide from chemokine receptor CCR2, whereas TPST-2 is 2.6-fold less efficient than TPST-1 for sulfation of a peptide from CCR8. Interestingly, these differences in efficiency include contributions from both underlying kinetic parameters, suggesting that they are not solely a consequence of substrate affinity but also indicate substantial variation in the reactivity of different substrates bound to the enzyme active site. With most substrates tested, TPST-2 was a more efficient enzyme than TPST-1.

Methods for studying the presence, location and function of tyrosine sulfation A thorough understanding of the biology of tyrosine sulfation requires: (1) identification and isolation of proteins containing sulfoTyr; (2) determination of the positions of sulfation and the

degree of sulfation at each position; and (3) characterisation of the functional roles of the sulfate groups. A variety of techniques have been utilised to achieve these goals (summarised in Table 2), although a number of challenges still remain.

Bioinformatic prediction of tyrosine sulfation sites In theory, if the sequence features controlling tyrosine sulfation were sufficiently well understood, it would be possible to both identify the proteins containing sulfoTyr residues and predict the locations of those residues by computational analysis of protein sequence databases. Although this is challenging owing to the large variability of sulfation sequences, there has been considerable success. Initially, Rosenquist et al. applied a computational test examining the common set of criteria listed in Table 1, and found high accuracy in predicting known sulfated proteins, correctly selecting 100% of proteins from a dataset containing known sulfoTyr-containing proteins [35,37]. However, this analysis excluded proteins that lack acidic amino acids near the tyrosine. Subsequently, the same group developed a position-specific scoring matrix in an attempt to increase the accuracy of prediction [39]. However, this approach did not accommodate for insertions and deletions within a target site. To address such issues, Monigatti et al. have developed the Sulfinator software tool [40]. This program employs four different Hidden Markov Models, each designed to recognise a sulfated tyrosine within a different type of sequence environment: (1) near the N-terminus of a sequence; (2) near the C-terminus of a www.elsevier.com/locate/nbt

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Review FIGURE 6

Two-step reaction pathway for the biosynthesis of the universal sulfate donor PAPS from two molecules of ATP and one inorganic sulfate ion. In the first step, the sulfate ion displaced pyrophosphate from one ATP molecule to give adenosine-50 -phosphosulfate (APS). In the second step, a phosphate group in transferred from ATP to the 30 position of APS, to give 30 -phosphoadenosine-50 -phosphosulfate (PAPS).

sequence; (3) near the centre of a relatively long (>25 amino acid) sequence; and (4) in a sequence containing multiple tyrosine residues. Remarkably, Sulfinator was found to correctly predict 98% of validated tyrosine sulfation sites and 98% of validated non-sulfated tyrosines. The program can be accessed online via the ExPASy web interface (http://ca.expasy.org/tools/sulfinator/). Finally, whilst sophisticated bioinformatics tools are an important advance, it is important to bear in mind that proteins that do not pass through the cell’s secretory pathway will not encounter the TPSTs and therefore are not expected to contain sulfated tyrosine residues, even if they contain sequences capable of being sulfated by TPSTs.

Metabolic labelling and amino acid analysis Several methods for studying protein tyrosine sulfation are built upon an understanding of the biochemical pathways giving rise to sulfation. In an elegant review a half-century ago, Lipmann [41] summarised the experiments of his laboratory and others that defined the biosynthesis of adenosine-30 -phosphate-50 -phosphosulfate (PAPS), the universal high-energy sulfate donor used in cells for sulfation of small molecule phenols, glycans and proteins alike (Figure 6). PAPS is biosynthesised in two steps from two 306

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molecules of ATP and one inorganic sulfate ion. In the first step, catalysed by ATP-sulfurylase, the sulfate ion displaces pyrophosphate from ATP, yielding adenosine-50 -phosphosulfate (APS). However, this reaction is thermodynamically unfavourable. The energy increase of the first reaction is overcome by coupling to a second reaction, catalysed by APS kinase, in which the 30 -hydroxyl group of APS is phosphorylated by the second molecule of ATP, yielding the product PAPS and the by-product ADP. In addition the pyrophosphate by-product of the first reaction is hydrolysed by pyrophosphatase releasing additional energy and thereby favouring APS production from sulfate over the reverse reaction. Considering that the sulfate in tyrosine-sulfated proteins is derived from inorganic sulfate, it is possible to incorporate 35Senriched sulfate by metabolic sulfate labelling. The labelled protein can then be detected by autoradiography or separated from other cellular components and identified by detection of 35S. This approach is relatively straightforward for proteins expressed in cell culture under which conditions the level of unlabelled sulfate can be minimised. However, in vivo the sulfate labelling is more difficult owing to dilution of the label. A difficulty in interpretation of such experiments arises because 35S-sulfate will be incorporated into proteins not only as sulfoTyr but also potentially as

sulfated sugars. In order to distinguish between these possibilities it may be possible to either inhibit glycoslyation or remove sulfated sugars from the produced protein. However, a more convincing way to unequivocally demonstrate the presence of sulfoTyr is to hydrolyse the protein and observe 35S-labelled sulfoTyr by coelution with chromatographic standards. As demonstrated by Wilkins et al. [42] this can be accomplished by reversed phase HPLC under conditions that distinguish between tyrosine, sulfoTyr, sulfate and a range of sulfated sugar residues. It is important to note that acidic conditions, such as those commonly used for protein hydrolysis, will readily hydrolyse the sulfate group from sulfoTyr. However, peptide hydrolysis without cleavage of the sulfate monoester can be achieved under harsh alkaline conditions, for example, 1.0 M sodium hydroxide at 110 8C for 24 h [42]. Similarly, HPLC (or other chromatographic separations) should be performed under neutral or alkaline conditions to avoid loss of sulfate from sulfoTyr.

Inhibition of sulfation and removal of sulfate To confirm the presence of sulfate in a protein of interest and to determine the functional role of the sulfate group, it is often useful to either inhibit protein sulfation or to remove the sulfate group from the expressed protein. Inhibition of sulfation can be achieved using sulfate analogues that inhibit ATP-sulfurylase, thereby preventing biosynthesis of PAPS [43]. Selenate has been described as a specific inhibitor of ATP-sulfurylase [44] but Pouyani and Seed found selenate to be too toxic to be used in transient expression experiments using COS cells [45]. On the contrary, chlorate (at concentrations ranging from 1 mM to 10 mM) has been found to effectively inhibit sulfation of both proteins and proteoglycans in cell culture without affecting cell growth or protein synthesis [46,47]. Moreover, sulfation resumed upon removal of chlorate from the growth medium, presumably indicating reversible inhibition of ATP-sulfurylase by chlorate [47]. Because PAPS is the sulfate donor for a variety of substrates, the loss of protein sulfation or activity upon treatment with chlorate could result from inhibition of either tyrosine or glycan sulfation. Therefore, it is useful to complement this approach with experiments in which the sulfate groups of sulfoTyr residues are specifically removed from the protein. This can be accomplished using the Aerobacter aerogenes arylsulfatase enzyme, which is available commercially and catalyses desulfation of sulfoTyr without releasing sulfate from various sulfated glycans [42,48]. However, Bundgaard et al. have reported that arylsulfatase digestions can be very inefficient [48], so the results of such experiments should be interpreted cautiously. In particular, the loss of sulfation (or associated function) upon arylsulfatase treatment can be taken as strong supporting evidence for the presence (or role) of tyrosine sulfation, but the lack of such evidence should not be interpreted to indicate the lack of tyrosine sulfation. Similarly, Kehoe et al. have shown that a commercial preparation of abalone sulfatase causes not only protein desulfation but also some proteolysis [49]. Thus, loss of function upon treatment with this sulfatase is not necessarily indicative of sulfoTyr being present or playing a functional role.

Anti-sulfotyrosine antibodies One of the most powerful approaches for analysis and isolation of specific tyrosine-sulfated proteins is the use of antibodies that

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recognise sulfoTyr within the relevant defined peptide sequence. Budgaard et al. have noted that sulfopeptides of at least 10 residues are required to raise high-titre antibodies and have described detailed methods for obtaining such antibodies and for their application in radioimmunoassay [14]. However, antibody identification and immunoassay development can be expensive and requires significant investment of time. More generally, it would be useful to have antibody reagents that recognise sulfoTyr independent of the surrounding peptide sequence. Obtaining such non-specific antibodies has not been possible by conventional animal immunisation methods, presumably because the animals’ immune systems are highly tolerant of sulfoTyr since it is a common protein modification. It has been noted that this is a general problem in obtaining antibodies against post-translational modifications [49]. To overcome this challenge, two groups have recently used in vitro phage display methods to identify antisulfoTyr antibodies [49,50]. Both groups used large phage display libraries of single-chain Fv fragments but subsequently elaborated the selected fragments into full-length IgG. The antibody PSG2, isolated by Hoffhines et al. [50], was able to recognise sulfoTyr in the third position of a wide variety of test pentapeptides; binding was observed in 81% of peptides tested with strong binding in 43% of peptides. Although binding was favoured by Asp or Glu within two residues of the sulfoTyr residue and disfavoured by Lys immediately after the sulfoTyr residue, indicating some sequence dependence, this should not be a major drawback in the usefulness of PSG2 because most natural sulfoTyrs occur close in sequence to acidic amino acids. Importantly, PSG2 did not recognise peptides lacking sulfoTyr (including those with tyrosine in the corresponding position) and was effective in distinguishing between Tyrsulfated and non-sulfated forms of various proteins in Western blots. In an independent study, Kehoe et al. [49] used two different phage display strategies to identify the same antibody that was selective for a variety of sulfoTyr-containing proteins over their non-sulfated forms in both ELISA and Western blot assays. Both PSG2 and the Kehoe et al. antibody were selective for sulfoTyr over phophotyrosine within a peptide sequence and both bound to sulfoTyr monomer very weakly in comparison to sulfoTyr-containing peptides. PSG2 was also unable to bind a variety of sulfated glycan structures. In summary, these two new antibodies are expected to be powerful research tools for identification of new proteins containing sulfoTyr, for distinguishing proteins containing sulfated tyrosine from those containing sulfated glycans and for monitoring the sulfation status of known proteins.

Mass spectrometry In recent years mass spectrometry has become the method of choice for identification and localisation of many post-translational modifications in proteins, particularly when high-throughput is required, for example, in proteomics projects. Typically, proteins are proteolytically digested to produce a set of component peptides that are then subjected to mass spectral analysis. The application of mass spectrometry to peptides containing sulfoTyr has been discussed in detail elsewhere [11,15]. Here, we briefly summarise the key issues. Under standard conditions used for positive ion Matrix-Assisted Laser Desorption Ionisation (MALDI) or Electrospray Ionisation (ESI) mass spectrometry, ionisation of proteins and peptides containing sulfoTyr leads to desulfation. www.elsevier.com/locate/nbt

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This is a consequence of the lability of the sulfate group under the acidic sample conditions used as well as ‘in-source decay’ induced by the energy of the ionisation sources. However, if spectra are obtained in the negative ion mode (with basic sample conditions) and the energy of the ionisation source is reduced, sulfate groups can sometimes be left intact. Even if ionisation is accomplished with the sulfate group intact, subsequent gas-phase manipulations of the sulfated ions can be problematic. Standard peptide fragmentation by collision-induced dissociation (CID) is not effective because loss of SO3 from the sulfate group occurs more readily than peptide dissociation. Whilst this loss of SO3 (80 Da) can, in itself, be useful evidence for the presence of a sulfate group on the peptide, identification of the sulfation position cannot be achieved by CID. Recently, however, a variety of methods have been developed in which radical peptide ions are produced by capture or transfer of electrons to the ionised peptide in the gas phase, then the radical ions undergo peptide fragmentation without loss of side chain groups. Application of these methods to sulfopeptides should yield fragmentation patterns containing information about not only the presence but also the location of sulfate groups in the peptides. Thus, although sulfopeptides are particularly challenging for standard mass spectrometry analysis, careful choice of sample conditions, ionisation parameters and fragmentation methods may provide detailed information on the sulfation of specific proteins and perhaps even enable large scale proteomic analysis of this post-translational modification, especially if combined with isolation methods utilising anti-sulfoTyr antibodies (vide supra).

Sulfotyrosine-containing peptides Whilst mass spectrometry may be useful in identifying the positions of sulfoTyr residues in proteins, determining the functional roles of the sulfate groups on these residues remains very difficult. The traditional approach has been to mutate the putatively sulfated tyrosine residue to phenylalanine [51–53]. The loss of sulfation upon mutation is good supporting evidence for the mutated residue being sulfated in the wild-type protein, but the influence of such mutations on function could be due to either the loss of the sulfate group or the amino acid change. Since individual sulfate groups cannot generally be manipulated in an intact protein without also affecting sulfation at other sites, it is desirable to use site-specifically sulfated peptides to model the functional influence of sulfation at specific positions. Comparison of target-binding or functional inhibition by peptides with tyrosine versus sulfoTyr at specific positions may provide evidence supporting the functional role of tyrosine sulfation in the corresponding natural proteins [54–58]. Such peptides are also useful for structural studies [59,60]. Synthesis of site-specifically sulfated peptides by solid-phase methods requires incorporation of the sulfoTyr residue bearing a side chain protecting group that can withstand subsequent steps in the synthesis and then be removed without hydrolysis of the sulfate itself. Because sulfoTyr and its derivatives are acid-labile, synthesis should be performed using Fmoc-protected amino acids rather than tBoc-protected amino acids to avoid repeated acid deprotection steps. The typical protected form used is the sodium salt, that is, the sulfoTyr is added in the form of FmocTyr(SO3Na)OH [61,62]. However, the reaction of this protected amino acid can be slow and even the mild acid conditions required for resin 308

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cleavage can often cause partial hydrolysis of the sulfate group. Consequently, yields are low so obtaining peptides with multiple sulfoTyr residues is extremely difficult [49,62]. Recently, Simpson and Widlanski have described the protection of the sulfoTyr side chain with acid-stable alkyl protecting groups such as the neopentyl group [63]. This group is stable to the conditions of Fmocbased peptide synthesis and has recently been used to incorporate one or two sulfoTyr residues at specific positions in a peptide with high yields [121]. We anticipate that this approach will facilitate future studies of the functional and structural roles of specific sulfoTyr residues in proteins.

Tyrosine-sulfation of P-selectin glycoprotein ligand-1 (PSGL-1) in leukocyte adhesion Function of PSGL-1 One of the best-characterised proteins in which sulfoTyr plays a functional role is P-selectin glycoprotein ligand-1 (PSGL-1), which is involved in leukocyte-mediated inflammation. A major hallmark of inflammation is the migration of circulating leukocytes into tissues. An early phase in the extravasation of leukocytes involves weak adhesion between the leukocytes and the endothelial surface of the blood vessel. This adhesion is mediated by specific adhesion molecules extending from the surfaces of the two cells. In the case of neutrophil extravasation, adhesion involves binding between P-selectin expressed on endothelial cells and PSGL-1 on neutrophils [64]. Mature PSGL-1 is a 361 amino acid protein that contains 15 heavily O-glycosylated mucin repeats, a single membrane-spanning segment and a 69-residue cytoplasmic tail. PSGL-1 exists predominantly as disulfide-linked homodimers and its P-selectin binding is dependent upon appropriate glycosylation [64]. In addition, the amino-terminal 20 amino acids of mature PSGL-1 are unusually rich in acidic amino acids (five Glu and two Asp) and include three tyrosine residues that are potential sulfation sites (Figure 7).

Importance of amino-terminal region The functional role of the amino-terminal region of mature PSGL1 has been thoroughly studied. Pouyani and Seed found that deletion of the first 20 amino acid residues of mature PSGL-1 abolished its ability to bind P-selectin [45]. Independently, De Luca et al. [65] observed that treatment of neutrophils or soluble PSGL-1 with the cobra metalloproteinase mocarhagin abolished Pselectin binding activity. Analysis of mocarhagin-treated PSGL-1 suggested that the protease had removed the first 10 amino acids from the N-terminus of mature PSGL-1 and this cleavage specificity was confirmed using a synthetic peptide corresponding to PSGL-1 residues 3–17. These experiments indicated that the amino-terminal region of PSGL-1 is crucial for P-selectin binding.

FIGURE 7

The amino-terminal 20 amino acids of mature PSGL-1. The three sulfated tyrosine residues are in bold type, the seven acidic residues are underlined and the O-glycosylated threonine residue is indicated by an arrow.

This conclusion was supported by the observation that an antibody directed against the first 15 residues of PSGL-1 also inhibits binding to P-selectin [65]. Moreover, Sako et al. showed that a fusion protein consisting of the first 19 amino acids of PSGL-1 linked to an immunoglobulin G (IgG) Fc domain (dubbed 19.Fc) was capable of binding to P-selectin [66]. Similarly, Pouyani and Seed reported that addition of the first 16 residues of PSGL-1 to the mucin CD43 introduced P-selectin binding activity to this molecule [45]. Interestingly PSGL-1 binding to E-selectin was not affected by treatment with mocarhagin [65] and 19.Fc showed only very weak binding to E-selectin [66], suggesting that the amino-terminal region of PSGL-1 is an important determinant for selectin-binding specificity as well as enhancing affinity for Pselectin itself. In apparent disagreement with this conclusion, Goetz et al. reported that the first 19 amino acids of PSGL-1 can support attachment and rolling of microspheres on CHO cell monolayers expressing either P-selectin or E-selectin [67].

Tyrosine sulfation of the amino-terminal region Considering the importance of the amino-terminal region of PSGL-1 and the presence of putative tyrosine sulfation sites in this region, studies have investigated the presence and function of sulfation in this region. Peptides corresponding to the aminoterminal region can be sulfated in vitro using either TPST-1 or TPST-2 [26]. Wilkins et al. [42] observed that 35S-sulfate was metabolically incorporated into PSGL-1 and that hydrolysis of the labelled PSGL-1 with strong base released 35S-sulfoTyr. Furthermore, enzymatic desulfation of PSGL-1 using Aerobacter aerogenes arylsulfatase decreased the ability of PSGL-1 to bind P-selectin [42]. These results indicated that PSGL-1 contains sulfoTyr residues that are required for high affinity P-selectin binding. Two independent studies showed that P-selectin binding by PSGL-1 or by fusion proteins containing the N-terminal region of PSGL-1 was severely reduced either by metabolic inhibition of sulfation using chlorate or by simultaneous mutation of the three tyrosine residues near the amino terminus of PSGL-1 [45,66]. Sako et al. also observed that sulfoTyr could be released from the first 19 amino acids of PSGL-1 fused to IgG Fc [66]. In addition, Pouyani and Seed showed that inhibition of sulfation using chlorate greatly reduced the attachment and rolling of PSGL-1-expressing cells on P-selectincoated coverslips under conditions of shear stress [45]. Leppa¨nen et al. used an elegant enzymatic synthesis strategy to prepare an Nterminal peptide from PSGL-1 containing sulfation on all three Tyr residues as well as complex O-glycosylation on Thr-16 [68]. Tyrosine-sulfation of this peptide was essential for binding to Pselectin. Finally, Snapp et al. have isolated a monoclonal antibody (KPL1) that recognises the amino-terminal region of PSGL-1 in a sulfation-dependent manner [69]. KPL1 bound to PSGL-1 on a variety of peripheral blood mononuclear cells but only bound B cells at low levels. The authors suggested that B cells might regulate PSGL-1 sulfation in a manner that reduces their migratory capacity. In summary, there is a wealth of evidence that one or more of the three Tyr residues in the amino-terminal region of PSGL-1 is sulfated and that sulfation is important for function. The roles of sulfation at specific tyrosine residues in PSGL-1 have been analysed using peptides isolated from tissue culture cell lines and using synthetic peptides. Somers et al. [70] showed that the predominant form of the fusion protein between PSGL-1 residues 1–

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19 and IgG Fc (19.Fc; see above), expressed in CHO cells, incorporates sulfate groups on all three Tyr residues in the N-terminal region (Tyr-5, 7 and 10) but that there are minor forms containing two, one or no sulfate groups in addition to O-linked glycosylation on Thr-16; the mono-sulfated and bis-sulfated forms are each mixtures of up to three different regioisomers. After isolation of the corresponding Nterminal peptides, surface plasmon resonance analysis indicated that the triply sulfated peptide bound to P-selectin with identical affinity to soluble PSGL-1 (Kd  0.8 mM), the bis-sulfated form bound with only slightly lower apparent affinity (Kd  3 mM), and that the mono-sulfated and non-sulfated forms had significantly lower apparent affinities (Kd values of 12 mM and 31 mM, respectively). Leppa¨nen et al. prepared a series of synthetic peptides with sulfate groups specifically incorporated at each possible combination of the three Tyr residues and then used enzymatic methods for O-glycosylation of Thr-16 [54]. Consistent with the previous study, the resulting triply sulfated peptide bound to soluble Pselectin with Kd  0.65 mM. Removal of the sulfate group on Tyr5 or Tyr-10 reduced the affinity by approximately twofold to fourfold whereas removal of the Tyr-7 sulfate reduced affinity by almost an order of magnitude. In summary, it appears that PSGL-1 is expressed with combinations of sulfate groups on all three Tyr residues and that all contribute to P-selectin binding affinity.

Structural basis of sulfotyrosine recognition Structural insights into the roles of the sulfate groups in PSGL-1 have been provided by the X-ray crystal structure of the triply sulfated N-terminal glycopeptide bound to the lectin domain of Pselectin in the context of a tandem lectin-EGF domain construct [70]. The ordered region of the peptide includes sulfoTyr-7 and sulfoTyr-10. Unsurprisingly, these sulfoTyr residues bind to a region of positive electrostatic potential on the lectin domain. Consistent with its 10-fold influence on binding affinity, the Tyr7 sulfate group forms four direct hydrogen bonds to side chain or backbone groups on the lectin domain as well as one watermediated hydrogen bond to the protein backbone. Presumably, the non-polar interactions observed between the aromatic ring of Tyr-7 and several protein side chains function to position the sulfate group appropriately for these interactions. Residue sulfoTyr-10 is located within a region of the PSGL-1 peptide that forms a turn structure. This structure allows, the aromatic ring of sulfoTyr10 to pack against the side chains of two leucine residues in the peptide (Leu-8 and Leu-13), thus positioning the sulfate group for salt bridge formation with an arginine side chain in the protein. In contrast to the well-defined interactions of sulfoTyr-7 and sulfoTyr-10, sulfoTyr-5 was disordered in the crystal despite its contribution to P-selectin binding affinity [54]. This suggested that it may not be possible for all three sulfoTyr residues to simultaneously form specific interactions with the lectin domain. Instead, Somers et al. suggested that the crystal structure may represent one of several possible modes of interaction in which different sulfoTyr residues interact with the binding site on P-selectin [70].

Role of chemokine receptor sulfation in leukocyte trafficking Chemokine and chemokine receptor families Considering the crucial role of tyrosine sulfation for the leukocyte adhesion function of PSGL-1, it is particularly interesting that www.elsevier.com/locate/nbt

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another group of proteins central to leukocyte trafficking, the chemokine receptors, also appear to depend on tyrosine sulfation for optimal binding of their chemokine ligands and for biological function. Chemokines (chemotactic cytokines) are a group of 40 small (8–10 kDa) soluble proteins, with conserved three-dimensional structure, that are released by a variety of tissues to recruit leukocytes and maintain homeostasis [71,72]. Chemokines function by binding to and activating chemokine receptors, a family of 20 G-protein-coupled receptors expressed on the surfaces of leukocytes. Receptor activation induces surface expression of integrins, thus promoting firm adhesion to the endothelial surface, and morphological changes that allow the leukocytes to move through the endothelial layer into the tissues [71]. Chemokines are classified on the basis of the spacing of the first two of four conserved cysteine residues in their sequences. In the two predominant subfamilies, CC chemokines have two adjacent cysteines whereas CXC chemokines have one intervening residue between the two cysteines (X6¼Pro). Other types of chemokines include XC and CX3C. Chemokines are named either according to a systematic nomenclature system (e.g. CCL1, CCL2 etc. for CC chemokines; CXCL1, CXCL2 etc. for CXC chemokines) [73,74] or according to common names (e.g. CCL11 is eotaxin). The classification and systematic nomenclature of chemokine receptors [73,74] is based upon the classification of their chemokine ligands. For example, CC chemokine receptor 3 (CCR3) is classified as a CC receptor because it responds to CC type chemokines such as eotaxin.

Studies of tyrosine sulfation in intact receptors Chemokine receptors are integral membrane proteins that are generally expressed at low levels, thus making detailed biochemical characterisation difficult. Nevertheless, it has become clear that tyrosine sulfation within the amino-terminal regions of chemokine receptors (the region preceding the first transmembrane segment)

plays an important role in their interactions with cognate chemokines. All known chemokine receptors contain tyrosine residues in their N-terminal sequences. Moreover, bioinformatic comparisons of these sequences to sequences containing known sulfated and non-sulfated tyrosines predicted that many of the tyrosine residues in chemokine receptor N-terminal regions are sulfated [75]. Table 3 shows the sequences of the N-terminal regions of the human chemokine receptors. The majority of tyrosine residues in these sequences are either adjacent to or within 3–4 residues away from one or more Asp or Glu residues as well as containing other features predictive of tyrosine sulfation sites (vide supra). The prediction that Tyr residues in these regions are sulfated is well supported by numerous experimental studies, as outlined for several examples below. The receptor CXCR3 binds to its cognate chemokines CXCL10/ IP-10 and CXCL11/I-TAC with high affinity (IC50 values of 0.06 and 0.5 nM, respectively) and contains two potential tyrosine sulfation sites within the sequence ENFSSSY27DY29GENESDS. When Colvin et al. mutated Tyr-27 to Phe, the result was a complete loss of detected binding, and a 5–10-fold reduction in chemotaxis [76]. The mutant Tyr-29 ! Phe retained 20% of WT binding affinity to CXCL10, and retained 10% of WT chemotaxis. However, the Tyr-27 ! Ala/Tyr-29 ! Ala double mutant displayed no binding and no chemotaxis for CXCL9/Mig, CXCL10 or CXCL11. These results clearly indicate the importance of Tyr-27 and Tyr-29 in function but do not directly implicate tyrosine sulfation. To investigate the potential roles of sulfation, Colvin et al. studied the incorporation of 35S sulfate into wild type and mutant receptors. The CXCR3 single mutants Tyr-27 ! Phe and Tyr-29 ! Phe showed a reduction in 35S sulfate labelling relative to wild-type receptor and the Tyr-27 ! Phe/Tyr-29 ! Phe double mutant showed no labelling. It should be noted that the level of surface expression was not affected by these mutants [76].

TABLE 3

Sequences of the N-terminal regions of the human chemokine receptors Receptor subfamily

Chemokine Receptor

Amino acid sequence N-terminal regiona

References b

CCR

CCR1 CCR2 CCR3 CCR4 CCR5 CCR6 CCR7 CCR8 CCR9 CCR10

METPNTTEDYDTTTEFDYGDATPCQKVNE MLSTSRSRFIRNTNESGEEVTTFFDYDYGAPCHKFDVK MTTSLDTVETFGTTSYYDDVGLLCEKADTR MNPTDIADTTLDESIYSNYYLYESIPKPCTKEGIK MDYQVSSPIYDINYYTSEPCQKINVK MSGESMNFSDVFDSSEDYFVSVNTSYYSVDSEMLLCSLQEV MDLGKPMKSVLVVALLVIFQVCLCQDEVTDDYIGDNTTVDYTLFESLCSKKDV MDYTLDLSVTTVTDYYYPDIFSSPCDAELIQ MTPTDFTSPIPNMADDYGSESTSSMEDYVNFNFTDFYCEKNNV MGTEATEQVSWGHYSGDEEDAYSAEPLPELCYKADVQ

[75] [52,75,119] [52,75] [75] [52,60,75,79]

CXCR1 CXCR2 CXCR3 CXCR4 CXCR5 CXCR6

MSNITDPQMWDFDDLNFTGMPPADEDYSPCMLETET MEDFNMESDSFEDFWKGEDLSNYSYSSTLPPFLLDAAPCEPESLE MVLEVSDHQVLNDAEVAALLENFSSSYDYGENESDSCCTSPPCPQDFSL MEGISIYTSDNYTEEMGSGDYDSMKEPCFREENA MNYPLTLEMDLENLEDLFWELDRLDNYNDTSLVENHLCPATEGP MAEHDYHEDYGFSSFNDSSQEEHQDFLQFSKVFLPCMYLVVF

[75] [75] [52,75,76] [52,75,78,81]

[53]

CXCR

CX3CR

CX3CR1

MDQFPESVTENFEYDDLAEACYIGDIV

XCR

XCR1

MESSGNPESTTFFYYDLQSQPCENQAWVFATLATTVLYCLVFLLS

DARC

hDARC

MGNCLHRAELSPSTENSSQLDFEDVWNSSYGVNDSFPDGDYDANLEAAAPCHSCNLLDDS

a

Sequences for each receptor extend from the amino terminus to six residues after the first conserved Cys. b References cited are those in which tyrosine sulfation has been either predicted or experimentally demonstrated.

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[52,75,77]

[51]

CX3CR1 has one known chemokine ligand, fractalkine and two tyrosine residues in its N-terminal region, Tyr-14 and Tyr-22 (Table 3). Mutation of Tyr-14 to Phe resulted in as much as a 100-fold decrease in the affinity of CX3CR1 for fractalkine, measured by surface plasmon resonance. Interestingly, mutation of Tyr-22 to Phe had very little effect on the binding of fractalkine, although the Tyr-22 ! Phe mutant did display a reduction in cell adhesion [53]. Again, the surface expression of the receptor was not significantly different from WT. It is noteworthy that Tyr-22 of CX3CR1 is located immediately following the first cysteine residue, which is likely to form a disulfide bond to the third extracellular loop of the receptor. Thus, it should not be surprising, on the basis of the criteria discussed above, if sulfation of Tyr-22 does not occur. In this light it is also interesting to note that the majority of Tyr residues in the amino-terminal regions of chemokine receptors are well separated from the conserved cysteine residue (Table 3). Mouse CCR8 N-terminus has a sequence of 1-MDYTMEPNVTMTDYYPD-17, which is similar to its human counterpart with the exception that in human CCR8 there are three consecutive Tyr residues (see Table 3). Gutierrez et al. mutated all three tyrosine residues in the N-terminal region of mouse CCR8. The Tyr-3 ! Phe mutant showed no measurable difference from WT, but the Tyr-14 ! Phe, Tyr-15 ! Phe and Tyr-14 ! Phe/Tyr15 ! Phe mutants all displayed large reductions in binding affinity [77]. Farzan et al. have studied the presence of tyrosine sulfation in the N-terminal regions of both CXCR4 and CCR5 and have investigated the role of sulfation in chemokine binding [52,78]; the importance of sulfation in these receptors for HIV infection will be discussed in detail later. CXCR4 has three potential N-terminal sulfation sites (Tyr-7, Tyr-12 and Tyr-21). Mutation of Tyr-21 to Phe, resulted in a large reduction in sulfate incorporation, whereas simultaneous mutation of Tyr-7 and Tyr-12 resulted in only a slight reduction in sulfation compared with the wild-type receptor, indicating that Tyr-21 is more highly sulfated than the other two tyrosine residues [78]. It was also demonstrated that Tyr21 ! Phe and the triple mutant (Tyr-7 ! Phe/Tyr-12 ! Phe/Tyr21 ! Phe) both had >50% reduction in SDF1a binding, compared with 30% reduction in binding for the double mutant (Tyr7 ! Phe/Tyr-12 ! Phe) [78]. These results indicate that Tyr-7 and/or Tyr-12 play(s) a functional role but do not distinguish between sulfated and non-sulfated forms of the receptor with regard to their functions. Using similar techniques as for CXCR4, Farzan et al. also showed that of the four tyrosine residues (at positions 3, 10, 14 and 15) in the N-terminal region of CCR5, Tyr-3 and one other Tyr are sulfated [52]. This finding is interesting in comparison to an in vitro study of TPST-catalysed sulfation of a peptide containing residues 2–18 of CCR5. Seibert et al. found that Tyr-14 and Tyr15 are selectively sulfated first by TPST-1 and TPST-2, whereas Tyr3 is sulfated last [79]. The contrast between these two studies suggests that in the cell there may be other factors in addition to TPST that determine the sulfation pattern.

Studies of chemokine receptor peptides In the studies of intact receptors discussed above, the combination of Tyr-Phe mutations and sulfate labelling data strongly suggests

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that sulfated tyrosine residues contribute to the chemokine-binding function of chemokines receptors. Nevertheless, as discussed in the methodology section above, such studies do not directly distinguish between the functional role of the sulfate group and the contributions of the tyrosine residue itself. For this reason, and in order to gain structural insights into the functions of the sulfoTyr residues, there has been substantial interest in studying sulfoTyr-containing peptides derived from the N-terminal regions of chemokine receptors. Typically, chemokines bind to their receptors with low nanomolar affinities. Nevertheless, numerous studies have shown that peptides bearing the N-terminal sequences of chemokine receptors can often bind to the cognate chemokines of those receptors with measurable affinities, typically with Kd values in the range 5– 500 mM [60,80–82]. Considering that these peptides usually contain the putative sulfated tyrosine residues, comparison of sulfated and non-sulfated forms of these peptides allows the influences of sulfate groups to be distinguished from those of the tyrosine residues themselves and also allows the effect of sulfation at different positions to be assessed. To date, studies of such comparisons have been reported for the receptors CXCR4, CCR5 and CCR3. Veldkamp et al. showed that sulfation of Tyr-21 of CXCR4 (1–38) enhances the affinity of this peptide for the chemokine SDF1a approximately threefold [81]. Duma et al. used NMR to compare binding of the chemokine RANTES by non-sulfated and doubly sulfated (Tyr-10, Tyr-14) forms of a peptide derived from residues 1–25 of CCR5 [60]. The non-sulfated form showed a typical binding curve and yielded a fitted Kd of 168 mM. However, the doubly sulfated peptide bound essentially stoichiometrically. Although the latter binding data were fit to a Kd of 1.2 mM, determination of such a tight binding constant under the conditions of the experiment (200 mM RANTES) is extremely difficult. Nevertheless, the primary data clearly indicate a Kd value lower than 20 mM, suggesting that the two sulfate groups enhance binding affinity by at least eightfold. In a recent study [121], we have compared binding of the chemokine eotaxin by a non-sulfated form, two singly sulfate forms (Tyr-16 versus Tyr-17), and a doubly sulfated (Tyr-16, Tyr-17) form of a peptide derived from residues 8–23 of receptor CCR3. Interestingly, sulfation of Tyr-17 enhances eotaxin binding affinity by a factor of 7, whereas sulfation of Tyr-16 (or double sulfation) enhances affinity by at least a factor of 28. It will be interesting to explore whether mechanisms exist to regulate the extent and pattern of chemokine receptor sulfation as a possible way to control chemokine binding affinity. The ability of sulfoTyr-containing peptides to bind to chemokine in vitro provides an opportunity to explore the structural basis of sulfoTyr recognition. To date, no structures have been reported for sulfated N-terminal receptor peptides bound to chemokines. However, the studies of binding affinity discussed above have all utilised 2D NMR to monitor the chemokine–peptide interactions and have thereby provided some information about the likely sulfoTyr binding site on the chemokine. These data are most readily interpreted with reference to the structure of the chemokine interleukin-8 (IL-8) bound to a non-sulfated peptide analogue related to the N-terminal sequence of the IL-8 receptor CXCR1 [80]. Although it is not sulfated, the peptide encompasses residues 9–29 of CXCR1 and thus includes the one potential sulfation site (Tyr-27). The peptide binds in a groove formed by the ‘N-loop’ and www.elsevier.com/locate/nbt

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b2-b3 turn elements of IL-8. The NMR experiments with sulfated peptides and other chemokines are generally consistent with the formation of binding interactions in this same region of the chemokine. Veldkamp et al. found that the backbone amide resonances of H25, R41 and R47 in SDF-1a were greatly perturbed by the sulfate group of the CXCR4 peptide [81]. Duma et al. proposed H23, K25, K45 and K47 as a possible sulfate binding site of RANTES [60]. We observed that sulfation of Tyr-16 in the CCR3 peptide greatly perturbed the amide groups of L23, T43 and K47 in eotaxin study [121]. Residues Arg-47 of RANTES and SDF-1a and the equivalent Lys-47 of eotaxin are located within the b2–b3 turn. Taken together, the data suggest that this residue may form a conserved salt bridge to a receptor sulfoTyr residue. In summary, the studies discussed above clearly show that sulfation of chemokine receptors occurs in vivo, that the sulfated tyrosine residues are functionally important and that the sulfate groups themselves contribute to chemokine-binding affinity. These studies also indicate that not all tyrosine residues are sulfated to the same extent and that some sulfoTyr residues are more important than others for ligand recognition and biological activity. Further studies of chemokine receptors are needed to fully explore the structural role of tyrosine sulfation, the role of differential receptor sulfation in chemokine recognition and the possibility that receptor sulfation plays a role in selective binding of different chemokines.

Role of chemokine receptor sulfation in pathogen recognition Chemokine receptors in HIV infection In addition to their functions in mediating leukocyte trafficking, chemokine receptors also play important roles in two major infectious diseases, AIDS and malaria. In the pathogenesis of AIDS, chemokine receptors act as coreceptors for human immunodeficiency virus type 1 (HIV-1). In the mid-1990s, Feng et al. identified a host cell coreceptor for HIV-1 that, in combination with the receptor CD4, binds to the viral gp120 envelope glycoprotein, leading to membrane fusion and virus entry [83]. This coreceptor for HIV-1 later turned out to be the chemokine receptor CXCR4 [84]. Soon afterwards another chemokine receptor, CCR5, was also found to mediate HIV entry into the host cells [85,86]. Although there are reports that other chemokine receptors may also function as HIV coreceptors [87], CCR5 and CXCR4 are the only HIV coreceptors so far demonstrated to be of physiological significance [88]. Between the two phenotypes of HIV, the R5 phenotype that uses CCR5 as coreceptor and the X4 phenotype that uses CXCR4, the R5 is by far the predominant form in the early infection of HIV [89]. Although the detailed molecular mechanism of HIV infection is still not fully understood, it is believed that the HIV envelope glycoprotein complex, consisting of three heterodimers each of which includes a gp120 glycoprotein attached to a gp41 transmembrane glycoprotein, binds initially to CD4 on the host cell. This binding induces a conformational rearrangement in gp120 leading to the exposure of a previously inaccessible domain, which binds to CCR5 or CXCR4 [90]. Binding to the coreceptor induces another significant conformational change, uncovering the fusion domain of gp41, which then links the viral membrane and host cell membrane to promote their fusion and viral entry [89]. 312

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Strongly supporting the physiological importance of CCR5, it has been found that homozygotes of 32-base pair deletion in the CCR5 gene, CCR5D32, which codes for truncated dysfunctional CCR5 receptor, show immunity to HIV acquisition [91]. In addition, a number of chemokine proteins have been shown to inhibit HIV infection [89,92], presumably by reducing CCR5 availability to HIV gp120. Indeed, CCR5 inhibitors and anti-CCR5 antibodies have been explored as therapeutic and prophylactic strategy for HIV infection (reviewed by [93]). Recently, one non-competitive CCR5 inhibitor, Maraviroc, has been approved for clinical use in treatment-experienced patients [94].

Role of tyrosine sulfation in interaction of CCR5 with HIV-1 gp120 The elements in CCR5 responsible for binding to HIV-1 gp120 are located in the N-terminal region and the second extracellular loop [95]. In the N-terminal region there are four tyrosine residues (Tyr3, Tyr-10, Tyr-14 and Tyr-15), all located close in sequence to acidic residues (see Table 3). Scanning mutagenesis has shown that these tyrosines and acidic residues are crucial for the entry of R5 HIV-1 into host cells [96,97]. Post-translational O-sulfation of tyrosine residues in CCR5 Nterminus is crucial for CCR5 to function as a coreceptor for HIV. Inhibiting tyrosine sulfation with chlorate results in decreased binding affinity between CCR5 and gp120/CD4 complexes without affecting CCR5 expression in cells transfected with CCR5 [52]. In addition, site-directed mutagenesis of the sulfated tyrosine residues in the CCR5 N-terminus compromises the efficiency of viral entry [52]. In vitro binding studies between gp120/CD4 complexes and CCR5 N-terminal domain peptides also show that sulfation of the tyrosine residues in CCR5 N-terminus is required for gp120/CD4 binding [56]. Although all the four tyrosine residues in CCR5 N-terminus can be sulfated, sulfation of Tyr-10, Tyr14 and Tyr-15 appears to be more important than sulfation of Tyr-3 for facilitating HIV entry [52]. Other studies using CCR5 N-terminal peptides show that sulfation of Tyr-10 and Tyr-14 is sufficient to mediate the binding between CCR5 and gp120/CD4 complexes in vitro [56]. Notably, tyrosine sulfation has also been observed in a number of CD4-induced anti-gp120 antibodies at their antigen binding sites, and the association of these antibodies with gp120 is dependent on their sulfate moieties [98], suggesting that tyrosine sulfation may also contribute to the recognition of HIV by the immune system. At present we have only limited understanding of the structural basis for binding of HIV gp120 to the sulfoTyr residues of CCR5. By examination of the binding between CCR5 and a number of HIV variants, Rizzuto et al. have shown that the CCR5 N-terminus binds to a highly conserved bridging sheet structure in gp120 that is formed upon CD4 binding [99]. More recently, Huang et al. have used NMR to show that CCR5 N-terminal peptide (residues 2–15, sulfated at Tyr-10 and Tyr-14) adopts a predominantly a-helical conformation when bound to the CD4-gp120 complex [59]; this structure was obtained using the transferred NOE approach that does not provide information about the specific interactions with the binding partners. Nevertheless, the authors were able to obtain insights into sulfoTyr recognition by the gp120/CD4 complex from the crystal structure of a complex between a tyrosine-sulfated anti-gp120 antibody, 412d, and the gp120/CD4 complex [59]. Two

sulfoTyr residues in 412d, Tyr-100 and Tyr-100c, are involved in the interactions with gp120. However, the two sulfoTyr residues bind to gp120 in distinct manners by interacting with different crucial residues in gp120. A rearrangement occurs in the third variable (V3) loop of gp120 upon binding to 412d to facilitate the interactions with the sulfoTyrs of 412d, resulting in an increase in the rigidity of V3 [59]. Molecular docking simulations using the crystallographic structure of the gp120/CD4 complex derived from the 412d/gp120/CD4 complex and the structure of the CCR5 peptide determined by NMR suggest that sulfated Tyr-14 of CCR5 and sulfated Tyr-100c of 412d interact in a similar manner with the same domain of gp120. Therefore, by interacting with a few conserved residues in the coreceptor binding site on gp120, sulfated Tyr-14 of CCR5 may induce a conformational change in the V3 loop of gp120 that facilitates HIV entry [59]. The molecular mechanisms remain unknown by which sulfation at the other tyrosine residues of CCR5 contributes to gp120 binding and HIV entry. Interestingly, although at least one tyrosine residue in the N-terminal region of CXCR4 is also sulfated, tyrosine sulfation of CXCR4 does not seem to play a major role in the entry of CXCR4dependent HIV [78]. The molecular basis for this significant difference between CCR5 and CXCR4 is yet to be resolved.

Role of tyrosine sulfation in the interaction of Duffy antigen and receptor for chemokines (DARC) with the malarial parasite Plasmodium vivax The Duffy protein was first characterised as a blood group antigen responsible for the expression of Fya and Fyb alloantigens [100]. The name DARC is derived from the later observation that the Duffy antigen binds to chemokines [101]. However, unlike other chemokine receptors, DARC shows rather low ligand selectivity by binding chemokines of both CC and CXC classes [102]. In addition, although the sequence of DARC is consistent with the expected topology of a seven-transmembrane receptor [103], DARC lacks the intracellular Asp-Arg-Tyr (DRY) motif that is associated with G-protein signaling so does not directly induce G-protein-coupled signal transduction upon ligand binding [104]. Instead, DARC may be involved in chemotaxis by regulating the function of other chemokine receptors [105].

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In addition to its chemokine-binding properties, DARC functions as the erythrocyte receptor for the malarial parasite Plasmodium vivax [106]. DARC is absolutely required for P. vivax invasion; Fyab individuals who do not express DARC are resistant to P. vivax invasion [106]. Invasion of P. vivax merozoites into erythrocytes begins with the recognition of DARC by a Duffy-binding protein (DBP) on P. vivax [107]. DBP contains a highly conserved N-terminal cysteine-rich domain, the Duffy-binding like (DBL) domain [108]. The central region of this DBL domain is likely to be the binding site for DARC [109]. On the contrary, the domain for DBP binding has been mapped to the N-terminal region of DARC. Peptides from the DARC N-terminal region inhibit erythrocyte invasion of P. vivax [110]. In addition, antibodies against the N-terminus of DARC inhibit erythrocyte invasion by the rodent DARC-dependent P. yoelii [111], suggesting that blocking the DBP binding domain in DARC N-terminus is adequate to inhibit P. vivax infection. Similar to chemokine receptors, the N-terminus of human DARC contains two tyrosine residues adjacent to acidic residues (Tyr-30 and Tyr-41) (Table 3). Metabolic labelling experiments with wild-type DARC and Tyr ! Phe mutants have shown that both Tyr-30 and Tyr-41 can be sulfated [51]. Tyr-41 plays a crucial role in binding to P. vivax DBP (PvDBP). The authors show that association of PvDBP-expressing cells with erythrocytes is abolished by the Tyr-41 ! Phe mutation of DARC [51]. In addition, soluble peptides of sulfated DARC N-terminus are capable of blocking the binding of PvDBP to erythrocytes [111]. Interestingly, the binding of DARC to chemokines is also dependent on the sulfated tyrosine residues. However, different tyrosine residues appear to be important for binding to different chemokines. Mutation of Tyr41 to Phe substantially reduces association of DARC with the chemokines MCP-1, RANTES and MGSA but not to IL-8, whereas mutation of Tyr30 to Phe substantially reduces binding to IL-8 but does not influence binding to the other chemokines [51]. Thus, it appears that sulfation of both positions is likely to be required for full biological function. Owing to the lack of structural knowledge on DARC, the molecular basis for the involvement of tyrosine sulfation in DARC binding to either PvDBP or to chemokines is yet to be elucidated.

TABLE 4

Other sulfated G-protein-coupled receptors Receptor

Function

Sulfation sequence motif 174

184

References 188

C3a-anaphylatoxin chemotactic receptor (C3aR)

Receptor activation by binding of C3a inflammatory peptide leads to complement activation, stimulation of chemotaxis, leukotriene production, platelet aggregation and superoxide anion production

HNRCGY KFGLSSSLDY PDFY GDPLEQGFQDY317Y318NLGQF

[11]

C5a-anaphylatoxin chemotactic receptor (C5aR)

Broad spectrum complement activation triggered by binding of C5a inflammatory peptide

TTPDY11GHY14DDKDTLD

[115]

Type 1 Sphingosine 1-phosphate receptor (S1P1R)

Receptor activation by ligand binding is essential for proper T cell traffic

D-Y19-V/G/-N-Y22-D

[113]

Follicle-stimulating hormone receptor (FSHR)

Receptor activation by hormone binding is essential for normal reproductive function in both sexes

F-D/E-Y335

[117]

Luteinising hormone receptor (LHR)/chorionic gonadotropin receptor (CGR)

Receptor activation by ligand binding is necessary for normal hormonal function during reproduction

Y385-D/E-Y

[117]

Thyroid-stimulating hormone receptor (TSHR)

Activation by hormone binding is essential for proper endocrine function in the thyroid gland

Y385-D/E-Y

[117]

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Sulfation of other G-protein-coupled receptors Overview

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In addition to its function in chemokine receptors, sulfation of tyrosine residues plays an important regulatory role in a variety of other G-protein-coupled receptors (GPCRs) as well as some nonGPCR receptors. This section focuses on the tyrosine-sulfated GPCRs listed in Table 4. The common feature of all these receptors is the presence of a large ectodomain, usually located at the Nterminus upstream of the first transmembrane helix. Hormone binding takes place on the ectodomain and is mediated by the presence of sulfoTyr residues. This post-translational modification leads to increased affinity of the agonist to its receptor. Conversely, mutagenesis of ‘sulfatable’ tyrosines decreases the binding of the agonist to its cognate receptor. Sulfation creates a negative charge on the surface of the ectodomain, which leads to stronger electrostatic interactions with positively charged moieties on the ligand. Tyrosine sulfation in these GPCRs is crucial for proper signaling. It is therefore not surprising that defects in tyrosine sulfation have been implicated in several disease states.

Tyrosine sulfation of GPCRs in the immune system An immune response is triggered and propagated by communication between immunologically relevant receptors and their cognate ligands on mononuclear cells. This kind of signaling is sometimes regulated by tyrosine sulfation. Over 60 immune system proteins have been shown to contain ‘sulfatable’ tyrosine residues [112]. Of these, the C3a-anaphylactic receptor (C3aR), C5a-anaphylactic receptor (C5aR) and the type 1 sphingosine 1phosphate receptor (S1P1R) are GPCRs, as shown in Table 4 [11,112,113]. C3a and C5a anaphylatoxins are proteins involved in complement activation and released at the site of an inflammation. Their biological function is exerted by binding to and activating their cognate receptors C3aR and C5aR [114]. This interaction plays a prominent role in autoimmune diseases, such as rheumatoid arthritis, psoriasis and acute respiratory distress syndrome [115]. The N-terminal extracellular domain of C5aR contains two tyrosine residues adjacent to acidic residues. Sulfation was demonstrated by incorporation of [35S]sulfate into C5aR expressed on the surface of HEK293T cells. Sulfation of Tyr 11 and Tyr 14 was found to be crucial for the interaction with C5a, as demonstrated by site-directed mutagenesis of these tyrosine residues. A tyrosine-sulfated peptide corresponding to residues 7–28 of C5aR but not the unsulfated peptide inhibits the binding of C5a to C5aR [115]. In contrast to C5aR the C3aR lacks the tyrosine-rich N-terminal ectodomain. Instead it has a large second extracellular loop consisting of 172 residues, which has been implicated in C3a binding [116]. Five tyrosine residues in this loop are sulfated but only sulfoTyr 174 is crucial for binding C3a, as shown by site-directed phenylalanine mutagenesis of the tyrosine residues in this loop [116]. Sphingosine 1-phosphate (S1P) binds to its cognate receptor S1P1R to mediate T cell chemotaxis and T cell transmigration of lymph nodes [113]. S1P1R contains two sulfoTyr residues in its Nterminal ectodomian at positions Tyr 19 and Tyr 22 with flanking aspartic acid residues (Table 4). Sulfation was demonstrated by incorporation of [35S]sulfate into wild-type S1P1R and to a lesser degree into a S1P1R mutant that had phenylalanine in position 22 314

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[113]. Lack of sulfation at these tyrosine residues impairs highaffinity binding of S1P, as well as S1P-mediated effects on T cell function. For example, the chemotactic response was impaired in the mutant as well as in wild-type S1P1R in the presence of the sulfation inhibitor sodium chlorate. Moreover, preincubation of cells with arylsulfatase, which removes more than 85% of sulfate from tyrosine residues, suppressed chemotaxis by 80–95% [113].

Tyrosine sulfation of glycoprotein hormone receptors The family of glycoprotein hormone receptors encompasses the thyroid-stimulating hormone receptor (TSHR), luteinising hormone receptor (LHR)/choriogonadotropin receptor (CGR) and the follicle-stimulating hormone receptor (FSHR). This rhodopsin-like GPCR subfamily is characterised by a large N-terminal ectodomain located before the first transmembrane helix [117]. The 359–414 residue long ectodomain, consisting of two cysteine clusters and 11 leucine-rich repeats, contains the hormone-binding sites. Specific contacts between the hormones and their cognate receptors involve some of the leucine-rich repeats [118]. A conserved Tyr-Asp-Tyr sequence motif in the hinge region of the ectodomain close to the first transmembrane helix contains sulfoTyr residues that confer high-affinity binding of all glycoprotein hormones to their cognate receptors. The presence of sulfoTyrs was demonstrated by incorporation of [35S]sulfate into TSHR. The Phe-Asp-Tyr and Tyr-Asp-Phe mutants exhibited less uptake of [35S]sulfate and the double mutant Phe-Asp-Phe did not show any uptake [118]. The functional role of sulfoTyr 385 was explored by TSH-mediated accumulation of cyclic AMP [118]. Wild type TSHR and the Tyr-Asp-Phe mutant were responsive to TSH stimulation whereas the Phe-Asp-Tyr mutant was completely unresponsive. This shows that the first sulfoTyr in this motif at position 385 is required for biological function. Moreover, a Glu-Asp-Tyr mutant was also inactive, indicating that the effect is specific to sulfoTyr and not just an electrostatic effect requiring a negatively charged residue at position 385. The Tyr-Asp-Tyr/Tyr-Glu-Tyr motif is also present in LHR (Table 4). In FSHR the motif is altered to Phe-Asp-Tyr but there is an additional tyrosine three residues upstream [118]. Mutant LHR and FSHR were made in which phenylalanine residues were substituted for tyrosine residues, and the functional consequences were measured in transiently transfected COS cells. Similar to the situation in TSHR the Phe-Glu-Tyr mutant was inactive whereas the Tyr-Glu-Phe variant was active. In FSHR mutation of the tyrosine following the conserved aspartate (Phe-Asp-Phe) resulted in decreased function and mutation of the upstream tyrosine had no effect. However, the function of the Phe-Asp-Phe mutant can be restored to wild-type level by introduction of a tyrosine before the conserved aspartate (Tyr-Asp-Phe). This indicates some flexibility in the required location of the sulfoTyr residue in FSHR [117]. These results indicate that tyrosine sulfation plays an important role in high-affinity binding of all three glycoprotein hormones to the corresponding receptors. The similarity of these interactions in all three receptors suggests this to be a non-specific hormone– receptor interaction on top of specific contacts of the hormones with the leucine-rich domains of their cognate receptors [118]. Several basic residues in the a-subunit common to all three glycoprotein hormones may form non-specific contacts with sulfoTyr residues on the receptors [117]. It is conceivable that the interac-

tions with sulfoTyr in the hinge region trigger conformational changes that contribute to transmembrane signaling to activate the receptors.

Concluding remarks The examples discussed above demonstrate that tyrosine sulfation plays crucial roles in both normal physiology as well as pathological processes. The TPST knockout mice demonstrate that tyrosine sulfation is essential in vivo for normal development, growth and fertility. Similarly, the analyses of PSGL-1, chemokine receptors and other GPCRs show that sulfation of specific tyrosine residues is essential for normal cellular adhesion, leukocyte trafficking, immune function and glycopeptide hormone activity. Moreover, the discoveries that both HIV and P. vivax invade target cells by interacting with sulfoTyr-containing regions of receptors underscore the importance of this post-translational modification in human disease. Despite the substantial recent advances in our knowledge and understanding of protein tyrosine sulfation, a number of important questions remain. How widespread is tyrosine sulfation in the human proteome? Presumably, numerous tyrosine-sulfated proteins remain to be identified. This is likely to be addressed in the near future by proteomic application of recently developed mass spectrometry and affinity-based purification methods. Proteomic studies may also start to determine the extent to which tyrosinesulfation of specific proteins varies between different tissues. If so, it

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will be interesting to determine the functional consequences of such variation and whether it is dynamically controlled. Once new sulfated proteins have been identified, elucidation of the physiological roles played the post-translational modification will still be challenging, requiring a combination of genetic approaches (e.g. knockout and mutant knockin mice) as well as cell-based and in vitro studies. It will be useful to develop agents such as antibodies that can distinguish in vivo between the sulfated and non-sulfated forms of specific proteins. Similarly, it will be helpful to develop technologies for making homogeneous tyrosine-sulfated proteins, which can then be used in biological experiments. Such proteins and their peptide analogues will also be applied to determine the structural basis by which tyrosine-sulfated proteins are recognised by their binding partners. Such studies are likely to assist efforts to develop both drugs and vaccines again HIV and malaria. In summary, we expect that research into tyrosine sulfation over the coming few years will significantly enhance our understanding of many important physiological processes and will have a substantial influence on therapeutic and preventative strategies against human disease.

Acknowledgements We thank Professors Chris King and Ted Widlanski for helpful discussions. This work was supported by grants to MJS from the National Science Foundation (MCB-0212746) and the Australian Research Council (DP0881570) and by a fellowship to XH from Monash University.

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