From the a Department of Pharmaceutical Chemistry and Mass Spectrometry Facility, University of California, San Francisco, CA 94143; c Department of Biological Chemistry, Weizmann Institute of Science, Rehovot 76100, Israel; d Institute for Neuroscience and f Department of Biochemistry and Biophysics, University of California, San Francisco, CA 94143; and h Department of Physiology and Biophysics, University of California, Irvine, CA 92697
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ABSTRACT |
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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 (T3 and T4) 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 Golgi-associated 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 O4 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.
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MATERIALS AND METHODS |
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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 semi-preparative C18 column. Sulfonation of Ser (16) was achieved by dissolving 20 µmol of peptide in 1 ml of trifluoroacetic acid and reacted with 50 µl of chlorosulfonic acid (ClSO3H) at room temperature for 20 min. The reaction was terminated by adding 200 µl of H2O. 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 427943 (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 (MGSSKSKPKDPSQRRR) 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 749943 were deleted from the construct by generating a PCR fragment with an XhoI site after residue 748 to create the pc6-myrRor2MH vector. No unintentional mutations were detected in any of the constructs.
Protein Expression and Purification
Human embryonic kidney 293T cells were cultured in Dulbeccos modified Eagles 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 LipofectAMINETM 2000 (Invitrogen) according to the manufacturers 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 µl of the same solution. Cells were pelleted by centrifugation at 5,000 x 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 Na3VO4) and cleared by centrifugation at
14,000 x 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 µl) with 15 µl 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 MnCl2; 10 mM dithiothreitol; 1 mM Na3VO4). Beads were then incubated in kinase buffer containing 100 µM ATP (20 µl reaction volume) for 30 min at 30 °C. Reactions were stopped by the addition of 5 µl of 6x SDS-PAGE loading buffer (300 mM Tris-HCl, pH 6.8; 30% (v/v) glycerol; 10% (w/v) SDS; 6 mM dithiothreitol; 0.12% (w/v) bromphenol blue).
Beta-elimination Conditions
ß-elimination of H2SO4 from synthetic sulfopeptides was accomplished using 25 mM Ba(OH)2, employing a 1-h incubation at 37 °C. Equimolar (NH4)2SO4 was then added in order to precipitate the excess Ba2+. 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 PepMap 75-µm x 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 350 nl/min. Approximately 1/10 of each digest (1 µl) 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-µm x 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-µm 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).
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RESULTS |
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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 ( = 15 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: 47SSISPGVYQQLSSSGITDFK66, 131KVIDELASSK140, 147LAGLQDEIGSLR158, 159ELIVTYESQAK169, 304YASQLNQLR312, 340NAAYAELATR349, and 428TLVEQAIGTQSK439. (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 8HTTNV[I/L]SMFR17 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).
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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).
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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 backbone 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 ß-elimination producing a loss of 98 amu forming the corresponding dehydroalanyl residue. This latter behavior is well documented in the literature (2228). In addition, as expected we have determined that sulfono-threonyl analogs behave similarly to their sulfono-seryl analog molecules (data not shown).
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Finally, it is of interest to note that chemical base-catalyzed ß-elimination has been used extensively in combination with mass spectrometry for the analysis of serine and threonine phosphorylation (2931) and O-glycosylation (3234). 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 ß-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 b3 and y7 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.
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DISCUSSION |
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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 ß-elimination of the elements of phosphoric acid (98 Da) from Ser- and Thr-modified peptides occurs as a favored dissociation process (2228). 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 (3841).
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 ß-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 ß-elimination is performed. In addition, it has been reported that a small percentage of unmodified Ser residues also display water loss under commonly used ß-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.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Published, MCP Papers in Press, January 29, 2004, DOI 10.1074/mcp.M300140-MCP200
1 The abbreviations used are: PAPS, 3'-phosphoadenosine-5'-phosphosulfate; CID, collision-induced dissociation; ECD, electron capture dissociation; ESI, electrospray ionization; MS, mass spectrometry; LC, liquid chromatography; HPLC, high-performance LC; PEB, protein extraction buffer; FT, Fourier transform; QqoaTOF, quadrupole selection, quadrupole collision cell orthogonal acceleration time-of-flight.
* Financial support was provided by National Institutes of Health National Center for Research Resources Grant RR 01614 (to A. L. B.), the USA-Israel Binational Science Foundation (to M. F. and A. L. B.), and by the Eotvos Scholarship of the Hungarian Scholarship Board (to Z. D.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
b Current address: Proteomics Research Group, Biological Research Center of the Hungarian Academy of Sciences, H-6726, Szeged, Temesvari krt. 62, Hungary
e Current address: Department of Biochemistry, Southwestern Medical Center, University of Texas, Dallas, TX 75390-9050
g Current address: Department of Pathology, Stanford University Medical School, Stanford, CA 94305-5324
i To whom correspondence should be addressed: Department of Pharmaceutical Chemistry, University of California, 513 Parnassus Avenue, San Francisco, CA 94143-0446. Tel.: 415-476-5641; Fax: 415-476-0688; E-mail: alb{at}itsa.ucsf.edu
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REFERENCES |
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