O-Sulfonation of Serine and Threonine

Protein sulfonation on serine and threonine residues is described for the first time. This post-translational modification is shown to occur in proteins isolated from organisms representing a broad span of eukaryote evolution, including the invertebrate mollusk Lymnaea stagnalis, the unicellular malaria parasite Plasmodium falciparum, and humans. Detection and structural characterization of this novel post-translational modification was carried out using liquid chromatography coupled to electrospray tandem mass spectrometry on proteins including a neuronal intermediate filament and a myosin light chain from the snail, a cathepsin-C-like enzyme from the parasite, and the cytoplasmic domain of the human orphan receptor tyrosine kinase Ror-2. These findings suggest that sulfonation of serine and threonine may be involved in multiple functions including protein assembly and signal transduction.

Protein sulfonation on serine and threonine residues is described for the first time. This post-translational modification is shown to occur in proteins isolated from organisms representing a broad span of eukaryote evolution, including the invertebrate mollusk Lymnaea stagnalis, the unicellular malaria parasite Plasmodium falciparum, and humans. Detection and structural characterization of this novel post-translational modification was carried out using liquid chromatography coupled to electrospray tandem mass spectrometry on proteins including a neuronal intermediate filament and a myosin light chain from the snail, a cathepsin-C-like enzyme from the parasite, and the cytoplasmic domain of the human orphan receptor tyrosine kinase Ror-2. These findings suggest that sulfonation of serine and threonine may be involved in multiple functions including protein assembly and signal transduction.

Molecular & Cellular Proteomics 3:429 -443, 2004.
Sulfonation occurs as a common enzymatic modification of endogenous substances including proteins, carbohydrates, catecholamines, and estrogenic steroids as well as xenobiotic chemicals (1). Sulfonation refers to the transfer of the sulfonate group (SO 3 Ϫ1 ) from 3Ј-phosphoadenosine-5Ј-phosphosulfate (PAPS), 1 the only known sulfonate donor (2), and can occur through several types of linkages, such as esters and anhydrides (O-sulfonation), amides (N-sulfonation), and thioesters (S-sulfonation), of which O-sulfonation is the most prominent (3). The transfer of SO 3 Ϫ1 to a hydroxyl or phenolic acceptor (O-sulfonation) generates a sulfono-derivative, and this reaction has commonly been referred to as sulfation rather than the more accurate O-sulfonation.
The majority of cellular sulfonation is of the O type and occurs primarily on polysaccharides, steroids, catecholamines, and thyroid hormones (1). These reactions are catalyzed by the soluble cytosolic sulfotransferases and appear to alter their bioactivity. For example, estrogen, testosterone, and thyroid hormones (T 3 and T 4 ) can interact with their respective receptors to regulate transcription, whereas their sulfate-containing moieties cannot. Furthermore, the half-life of these compounds in blood is significantly shorter than that of their conjugated counterparts, suggesting that sulfonation maintains these compounds in an inactive state ready for rapid deployment by the removal of the sulfonyl group.
While the cytosolic sulfotransferases conjugate cell-permeable or intracellular compounds, the membrane-bound Golgiassociated sulfotransferases are primarily responsible for sulfonation of extracellular proteins via a co-or post-translational mechanism. The membrane-bound sulfotransferases are responsible for the sulfonation of various glycosaminoglycans, such as heparin and heparan sulfate. Additionally, these enzymes catalyze the direct sulfonation of proteins on the O 4 position of tyrosine residues (4). It is one of the last modifications to occur during protein transiting the trans-Golgi and thus has been found almost exclusively on secreted and plasma membrane proteins of all metazoan species examined. In addition, there is a large body of evidence that this modification is present usually at the interface of interacting proteins and hence is known to modulate extracellular protein-protein interactions. In humans, protein tyrosine sulfonation has been implicated in proteins of the vasculature and hemostasis. Examples include the mediation of inflammatory leukocyte adhesion, chemokine receptors, and modulation of the blood coagulation cascade (5). Significantly, only tyrosine residues have been described as sites for O-sulfonation within proteins, and O-sulfonation of proteins has not previously been shown to occur within the cytosol. Several tyrosyl protein sulfotransferases (6,7) and arylsulfatases (8) present in the trans-Golgi have been described, but unlike tyrosine phosphorylation/dephosphorylation (9) there is no evidence of dynamic regulation of tyrosine sulfonation (4,5). Until now, only widespread modification of tyrosine has been observed (10,11).
In this report, we describe the discovery and structural characterization of O-sulfonation of both serine and threonine residues in proteins of diverse class and function isolated from eukaryotes spanning the range from a unicellular parasite to humans. These include a neuronal intermediate filament protein and a myosin light chain from the snail (Lymnaea stagnalis), a cathepsin-C-like protein from the protazoan malaria parasite (Plasmodium falciparum), and cytoplasmic constructs of the human orphan receptor tyrosine kinase, Ror2. The presence of this new post-translational protein modification was detected and characterized by on-line high-performance liquid chromatography (HPLC) tandem electrospray mass spectrometry from proteins isolated by SDS-PAGE.

MATERIALS AND METHODS
Protein Isolation-Proteins isolated from L. stagnalis nerve axoplasm were subjected to two-dimensional PAGE screening. Differentially expressed protein spots were visualized by a mass spectrometric-compatible silver stain, excised, digested in-gel with trypsin (donatello.ucsf.edu/ingel.html), and analyzed by liquid chromatography (LC) collision-induced dissociation (CID) mass spectrometry (MS) (see below). Tryptic peptide sequences were deduced from interpretation of the CID spectra measured (12).
Proteins isolated from P. falciparum extracts were obtained by affinity isolation of total cellular extracts treated with the general cysteine protease activity-based probe, DCG-04. This probe covalently modifies papain family cysteine proteases and allows their direct isolation by virtue of a biotin tag on the probe. The detailed protocol for the purification is outlined elsewhere (13,14). Isolated proteins were separated on SDS-PAGE gels followed by excision and in-gel digestion prior to analysis by tandem mass spectrometry. The protease found to contain a site for O-sulfonation was identified by sequence analysis using tandem mass spectrometry and was subsequently found to match a sequence in the P. falciparum database (locus IDMal12P1.457) that has high homology to the human cathepsin-C protease.
Peptide Synthesis-The peptide LAGLQDEIGSLR was synthesized using optimized 0.25-mmol-scale Fastmoc chemistry (15) on an Applied Biosystems 433 automated peptide synthesizer (Applied Biosystems, Foster City, CA) on Rink amide methylbenzhydrylamine copoly-(styrene-divinylbenzene) resin (Novabiochem, La Jolla, CA) with a substitution value of 0.65 mmol/g and N ␣ -Fmoc-protected amino acids (Novabiochem). After acidiolytic deprotection (15) of side chain-protecting groups and cleavage of the peptide from the resin, the crude peptide was purified by reversed-phase HPLC on a semipreparative C 18 column. Sulfonation of Ser (16) was achieved by dissolving 20 mol of peptide in 1 ml of trifluoroacetic acid and reacted with 50 l of chlorosulfonic acid (ClSO 3 H) at room temperature for 20 min. The reaction was terminated by adding 200 l of H 2 O. The sulfonated peptide was purified by reversed-phase HPLC, and the final product was characterized by electrospray ionization (ESI) CID MS.
Ror2 Vector Construction-The transmembrane and cytoplasmic domains of human Ror2 were amplified by RT-PCR from total RNA isolated from human SH-SY5Y cells. The cDNA, encompassing residues 427-943 (17) with an XhoI site in place of the stop codon, was subcloned into pcDNA6-Myc/His-A (Invitrogen, San Diego, CA) to add a carboxyl-terminal Myc/His tag resulting in the plasmid termed pc6-Ror2cytoMH. To target the Ror2 construct to the inner surface of the membrane, the chicken c-Src myristylation signal (MGSSKSKP-KDPSQRRR) was added to the amino terminus starting at residue 432 using the unique SgrAI site within the myristylation sequence, creating the pc6-myrRor2cytoMH vector. Residues 749 -943 were deleted from the construct by generating a PCR fragment with an XhoI site after residue 748 to create the pc6-myrRor2⌬MH vector. No unintentional mutations were detected in any of the constructs.
Protein Expression and Purification-Human embryonic kidney 293T cells were cultured in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum and 1% penicillin/streptomycin solution (Invitrogen). Approximately 800,000 cells were seeded per 60-mm dish and transiently transfected using LipofectAMINE™ 2000 (Invitrogen) according to the manufacturer's recommended protocol. Cells were incubated for 48 h prior to harvesting. Cells were washed twice with ice-cold phosphate-buffered saline and scraped into 750 l of the same solution. Cells were pelleted by centrifugation at ϳ5,000 ϫ g for 1 min, and supernatant was removed by aspiration. Cells were lysed in protein extraction buffer (PEB) (50 mM Tris-HCl, pH 7.4; 150 mM NaCl; 1 mM EGTA; 1% (v/v) Nonidet P-40; 0.25% (w/v) sodium deoxycholate; 2% protease inhibitor mixture (P-8340, Sigma, St. Louis, MO); 50 mM NaF; 1 mM sodium pyrophosphate; 1 mM Na 3 VO 4 ) and cleared by centrifugation at ϳ14,000 ϫ g for 10 min at 4°C. Myc/His-tagged proteins were immunoprecipitated by incubating 1 mg of each sample (diluted in PEB to 500 l) with 15 l of agarose-conjugated anti-myc antibody (clone 9E10; Santa Cruz Biotechnology, Santa Cruz, CA) overnight at 4°C. Agarose beads were washed twice with PEB and once in kinase buffer (20 mM HEPES, pH 7.55; 10 mM MnCl 2 ; 10 mM dithiothreitol; 1 mM Na 3 VO 4 ). Beads were then incubated in kinase buffer containing 100 M ATP (20 l reaction volume) for 30 min at 30°C. Reactions were stopped by the addition of 5 l of 6ϫ SDS-PAGE loading buffer (300 mM Tris-HCl, pH 6.8; Beta-elimination Conditions-␤-elimination of H 2 SO 4 from synthetic sulfopeptides was accomplished using 25 mM Ba(OH) 2 , employing a 1-h incubation at 37°C. Equimolar (NH 4 ) 2 SO 4 was then added in order to precipitate the excess Ba 2ϩ . The precipitate was pelleted by centrifugation, and the supernatant was used for further analysis.
Tandem Mass Spectrometry-The digests and synthetic peptides were analyzed by nano HPLC-ESI-QqoaTOF-MS using an Ultimate HPLC system equipped with a FAMOS autosampler and a C18 Pep-Map 75-m ϫ 150-mm column (Dionex-LC-Packings, San Francisco, CA). Solvent A was 0.1% formic acid in water, and solvent B was 0.1% formic acid in acetonitrile, at a flow rate of ϳ350 nl/min. Approximately 1/10 of each digest (1 l) was injected at 5% B, then the organic content of the mobile phase was increased linearly to 50% over 30 min. The column effluent was directed to a QSTAR Pulsar tandem mass spectrometer (Applied Biosystems/MDS Sciex, Toronto, CA). During the elution of the peptides 1-s MS acquisitions were followed by 5-s CID experiments for computer-selected precursor ions in information-dependent acquisition mode. The collision energy was set according to the mass value and charge state of the precursor ion. The CID mass spectra were interpreted manually.
Accurate Mass Measurement-The tryptic digest of another modified L. stagnalis protein was analyzed by capillary HPLC-ESI-Fourier transform (FT) MS using a Surveyor HPLC pump interfaced to an LTQ-FT mass spectrometer (both from Thermo-Finnegan, San Jose, CA). Separation was performed using a 150-m ϫ 10-cm C18 column (Micro Tech Scientific, Sunnyvale, CA). Solvent A was 0.1% formic acid, and solvent B was 0.1% formic acid in acetonitrile, and the gradient was 2% B for the first 5 min, then a gradient to 40% B over the next 40 min, followed by a gradient up to 90% B over the next 10 min at a flow rate of 800 nl/min. Spraying was from an uncoated 15-m ID spraying needle (New Objective, Woburn, MA). All MS data was acquired in the ion cyclotron cell with ion injection amounts into the cyclotron optimized by monitoring ion counts in the linear trap prior to injection into the cyclotron.
Bioinformatic Tools-Because the L. stagnalis genome has not been sequenced, protein class and function assessment was carried out with the aid of a variety of bioinformatics including database homology search engines (18) such as MS-Pattern (prospector.ucsf. edu) and MS-Blast (dove.embl-heidelberg.de/Blast2/msblast.html).

RESULTS
As part of an ongoing effort to identify proteins involved in nerve regeneration, two-dimensional PAGE analyses of neuronal axoplasm from the freshwater snail L. stagnalis were carried out (19). Differentially expressed proteins were subjected to in-gel tryptic digestion followed by reversed-phase chromatographic separation and mass spectral analysis of Ser-modified peptide in Fig. 5  the digest mixture. Peptide sequences were deduced by de novo interpretation of their CID mass spectra and used to query genomic databases to assign tentative homologies in sequenced genomes where possible. This effort revealed a number of protein spots with extensive homology to intermediate filament proteins from other mollusks, including Helix (20) and Aplysia (21). During the course of carrying out de novo sequencing of these particular proteins, a number of digest components were discovered from analysis of an LC-ESI-CID-MS experiment that displayed identical mass values for their entire CID sequence ion series (viz. identical fragmentation patterns), but eluted with significantly different chromatographic retention times (⌬ ϭ 1-5 min) and possessed different protonated molecular masses. In fact, all of the later-eluting components of these identical fragmentation pattern pairs displayed an 80-Da increment in their measured molecular mass. Careful analysis of these LC-CID-MS spectra revealed seven such tryptic peptides in one particular digest of a protein homologous to intermediate filament proteins: 47 SSISPGVYQQLSSS-GITDFK 66 , 131 KVIDELASSK 140 , 147 LAGLQDEIGSLR 158 , 159 ELIVTYESQAK 169 , 304 YASQLNQLR 312 , 340 NAAYAELAT-R 349 , and 428 TLVEQAIGTQSK 439 . (Note that the Ile/Leu assignments and sequence positions indicated here are based on cDNA sequence information obtained later). In another digest, the modified peptide 8 HTTNV[I/L]SMFR 17 was observed. This protein was identified as myosin light chain and the sequence positions are assigned based on the observation of the N-terminal peptide in the digest (see Table I).
In order to explore these observations in more detail, the chromatographic ion extraction profiles corresponding to the mass values of the modified peptides and their unmodified counterparts were retrieved from the former entire digest LC-MS dataset. This result is presented in Fig. 1 for one specific peptide, LAGLQDEIGSLR, and its modified analog, m/z ϭ 636.35 (upper panel) and 676.35 (lower panel), respectively.
Based on these observations, it was clear that we had detected the presence of several analogs having identical peptide sequences modified by a moiety of ⌬M ϭ 80 Da. As is readily seen from the nominal mass spectrum (inset in Fig.  1, lower panel), these modified analogs are stable during ESI under ambient conditions and any, even small, loss observed from the parent ion is 80 Da, rather than the 98 Da that represents the characteristic behavior observed for serine and threonine phosphorylation. These characteristics suggested that the identity of the modification in question must be a sulfono-moiety, rather than the isobaric phospho-modification. This reasoning is consistent with results described in earlier studies on protein phosphorylation (22)(23)(24)(25)(26)(27)(28). The CID spectra corresponding to the components detected in Fig. 1 are presented in Fig. 2. Similar to the reported behavior of tyrosine-sulfonated peptides (29), this peptide (see lower panel) underwent a rearrangement reaction that resulted in gas-phase elimination of SO 3 during the CID experiments, thus making the modified molecules indistinguishable from their unmodified counterparts simply by inspection of their fragmentation pattern.
In addition to the freshwater snail, similar experiments car-ried out on digests derived from proteins isolated from both the malaria parasite, P. falciparum, and from human embryonic kidney 293T cells have revealed peptide analogs that are covalently modified on both serine and threonine residues. In the case of P. falciparum, one of the proteins was detected using a suicidal substrate probe for cysteine protease activity (13,14). It was identified as a remote homolog of cathepsin-C based on remote homology searches using de novo sequences obtained from tandem mass spectrometry. For example, the CID spectrum of one of its tryptic peptides, 7-O-sulfono-RIEVALTK, is shown in Fig. 3. It should be noted that this modified peptide eluted chromatographically later than its unmodified counterpart as well. In this chromatographic system, phosphopeptides also behave in a similar fashion. Again, the sequence ion series are virtually identical (see middle and lower panels). This finding was confirmed by synthesis of this peptide and its modified analogs by subsequent measurements that showed comparable mass spectral behavior (data not shown).  Table II. Finally, during mass spectrometric characterization of the myristyl juxtamembrane construct of the tyrosine kinase receptor Ror2, the peptide 465 LKEISLSAVR 474 was observed together with two additional chromatographically distinct, later-eluting peaks representing modified species (see middle inset of Fig. 4). CID analyses of each of these produced fragmentation spectra identical to the unmodified species (see middle and lower panels of Fig. 4). These results suggest that this particular peptide exists as two modified isobars at Ser 469 or Ser 471 .
Although phosphate and sulfate moieties have the same nominal mass, the difference between the two species is sufficient to be distinguished using a mass spectrometer able to measure mass with the appropriate mass accuracy (sulfate addition ϭ 79.9568; phosphate addition ϭ 79.9663). The 9.4 mmu mass difference represents 9.4 ppm deviation between the differently modified species for a 1-kDa peptide. Thus, one would desire mass accuracy reliable within 5 ppm for unambiguous assignment. Hence, a tryptic digest of the myosin light chain homolog from L. stagnalis was analyzed by FT-MS. The mass measurements obtained for representative components are presented in Table I. Five entries correspond to unmodified peptides from this protein along with one that is modified by either sulfonation or phosphorylation. As can be seen from accurate mass measurement values in this table, the experimental measurement value for this modified peptide fits very well with the accurate mass value calculated for a sulfonated peptide. This experimental mass value does not fit for a putative phosphorylated species at the same mass accuracy observed for all the other peptides, thereby establishing this modification to be sulfonation unambiguously. Obviously for higher-molecular-mass species higher mass accuracy would be required, i.e. for a modified peptide of ϳ2000 Da within 2 ppm.  Table II. In order to provide even further evidence that the observed modifications of these proteins are in fact due to O-sulfonation, a series of peptides were synthesized for components identified from L. stagnalis. For example, to compare the chromatographic retention and mass spectral fragmentation behavior, the sequence LAGLQDEIGSLR was synthesized as such together with its phospho-and sulfonoseryl-analogs. These synthetic peptides were studied by LC-MS/MS under similar conditions to those employed during analyses of the original gel plug digests. The chromatographic elution observed for this sulfono-modified peptide occurred later than its unmodified counterpart as observed in the original experiment (vida supra) (data not shown). The CID spectra of both the synthetic sulfono-and phospho-species are shown in Fig.  5. These spectra establish that sulfonated peptides preferentially eliminate the modification upon deposition of sufficient vibronic energy to induce dissociation of the peptide back-bone bonds, causing a loss of 80 amu from the parent ion. Hence, they produce low-energy CID sequence ion series indistinguishable from the corresponding unmodified molecules; whereas, the phospho-seryl analog undergoes partial ␤-elimination producing a loss of 98 amu forming the corresponding dehydroalanyl residue. This latter behavior is well documented in the literature (22)(23)(24)(25)(26)(27)(28). In addition, as expected we have determined that sulfono-threonyl analogs behave similarly to their sulfono-seryl analog molecules (data not shown).
In addition, analogs of another peptide, YASQLNQLR (observed modified in the L. stagnalis intermediate filament protein digest), were prepared that were sulfonated on either the tyrosyl or seryl residues. The mass spectral behavior of both sulfono-seryl versus sulfono-tyrosyl is shown in Fig. 6 together with that for this unmodified sequence. These spectra show that tyrosyl sulfonation is significantly more labile than seryl. As was anticipated the CID sequence ion series for The upper panel shows the CID spectrum of the unmodified peptide, with an inset displaying the ion extraction profile of its doubly charged ion. The middle and lower panels show the CID spectra of the two modified peptides sulfated at one Ser residue each, that eluted in two chromatographically distinct peaks (see inset). Asterisks indicate gas-phase elimination of the modifying group. Predicted sequence ions are listed in Table II. these three peptide analogs are identical (data not shown).
Thus the preferential elimination of the sulfo moiety to form the corresponding unmodified residue in CID analysis prevents determination of the site(s) of modification when there is more than one potential site present in any given sequence.
Finally, it is of interest to note that chemical base-catalyzed ␤-elimination has been used extensively in combination with mass spectrometry for the analysis of serine and threonine phosphorylation (29 -31) and O-glycosylation (32)(33)(34). Therefore, we attempted to employ the same strategy on our synthetic sulfopeptides to ascertain whether this approach might be useful for determination of sulfonation sites. This experiment resulted in essentially complete ␤-elimination for the synthetic sulfono-seryl peptide, YASQLNQLR, as established by interpretation of the CID spectrum of this product as shown in Fig. 7. The presence of sequence ions b 3 and y 7 confirm the formation and presence of 3-dehydroalanyl residue, demonstrating that this approach could be employed to determine sites of serine and threonine O-sulfonation. DISCUSSION We have observed the occurrence of sulfonation as a posttranslational modification for serine and threonine residues in a diverse range of proteins throughout the spectrum of eukaryotic evolution. From this work, it is shown that particular behavior is observed using tandem mass spectrometry that is characteristic of aliphatic sulfono-peptides that permits distinction of this modification from phosphorylation of the same residues in isobaric peptides. Based on these observations in three separate species, it is clear that protein sulfonation on aliphatic amino acids is of widespread occurrence.
The quantitative elimination of phospho-functions does not occur either during ESI or under low-energy collisional activation. Thus far the only observation of extensive gas-phase dephosphorylation during ESI involved the highly reactive phospho-histidine species (35). Therefore, the observations described above appear to be diagnostic for the presence of a sulfono-moiety (36). Extensive previous studies have shown that Tyr-sulfonated peptides usually display some sulfo-moiety loss even during MS acquisition (37). Our results suggest that serine and threonine sulfonation are more stable than their tyrosyl counterpart in MS. However, the difference in stability is not sufficient to reliably differentiate between aromatic and aliphatic sulfonation.
The fragmentation behavior of phospho-peptides has been studied extensively for over a decade. It is well known that ␤-elimination of the elements of phosphoric acid (-98 Da) from Ser-and Thr-modified peptides occurs as a favored dissociation process (22)(23)(24)(25)(26)(27)(28). The CID fragmentation pattern of sulfated peptides is characteristically different, with elimination of the sulfono-moiety (-80 Da) preceding any peptide backbone fragmentation, making modification site determination impossible from CID data. In the future, this obstacle may be overcome with the use of electron capture dissociation, wherein nonergodic fragmentation cleaves the peptide backbone while leaving labile amino acid modifications intact (38 -41).
Of course, using electron capture dissociation (ECD) fragmentation, sulfonation will be indistinguishable from phosphorylation. However, we show that one can also distinguish between the two modifications on the basis of accurate mass measurement. Therefore, discrimination of sulfonation using FT-MS holds the most promise for characterizing this modification and its sites of occupancy using the ECD technique. Also, we have an indication that base-catalyzed ␤-elimination can be used to determine sulfonation sites. Several approaches employing Michael additions after elimination have been published for enriching or assisting in the identification of modification sites. DTT has been added to provide a tag for enrichment of GlcNAc-modified peptides (34). This approach is also applicable to serine and threonine phosphorylation and sulfonation. Addition of 2-aminoethanethiol also introduces a thiol group that can be used for enrichment, as well as a modification-specific enzymatic cleavage site (31,42). However, it has to be noted that one cannot distinguish between phosphorylation, sulfation, or O-glycosylation once ␤-elimina-  Table II. tion is performed. In addition, it has been reported that a small percentage of unmodified Ser residues also display water loss under commonly used ␤-elimination conditions (43). Thus, this approach would be most appropriate for assigning the exact site(s) of modification once the presence of sulfation has been established.
Another potential discriminatory method that could be used to differentiate between phosphorylation and sulfonation is by performing fragmentation of the species in negative ion mass spectrometry. Phosphorylation produces a characteristic negative ion at m/z 79 (25), whereas the sulfo-group forms an equivalent negative ion fragment at m/z 80 (28).
The identification of serine/threonine sulfonations in three proteins from very different organisms suggests that this modification is widespread and may occur ubiquitously in all eukaryotes. Moreover, the three modified proteins we report are targeted to distinct cell compartments, cytoplasm (Lymnaea intermediate filament), location unknown (Plasmodium cathepsin-like), and plasma membrane (human Ror2), suggesting that serine/threonine sulfonation occurs both in the endoplasmic reticulum continuum and the cytoplasm.
There are a number of sulfotransferases known, and they can be divided into two main groups: a membrane-bound class that is found in the Golgi and is involved in the modification of proteoglycans and polysaccharides (including the enzymes involved in tyrosine derivatization) and a soluble cytoplasmic class that modifies small molecules, such as estrogen (1). PAPS is the sulfate group donor utilized by these sulfotransferases for all sulfations described thus far (2,4,5). Therefore, although we do not yet know which enzyme(s) might sulfate serine/threonine in proteins, one or more of the already known sulfotransferases is the most likely candidate. We cannot, however, rule out that a different as yet unidentified enzyme is involved and conceivably a different donor compound. Similar considerations apply to any sulfatases, assuming that these modifications are physiologically reversible.
Finally, we should note that this study does not identify a function for any of the observed modifications. O-Sulfonation of tyrosine is thought to aid in protein-protein interactions (4), and a similar function could be true for the aliphatic modifications.