PMCCPMCCPMCC

Search tips
Search criteria 

Advanced

 
Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
Anal Biochem. Author manuscript; available in PMC 2010 July 1.
Published in final edited form as:
PMCID: PMC2695991
NIHMSID: NIHMS108194

A Target-specific Approach for the Identification of Tyrosine-sulfated Hemostatic Proteins

Summary

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

Keywords: Hemostasis, tyrosine sulfation, post-translational protein modification

Post-translational protein modification by tyrosine sulfation, first discovered in bovine fibrinogen [1], is now known to have a widespread occurrence among proteins of multicellular eukaryotic organisms [2,3]. This unique protein modification has been implicated in the alteration of biological activities of proteins [4,5], proteolytic processing of bioactive peptides [6], change in half-life of proteins in circulation [7], intracellular transport of secretory proteins [8], and modulation of extracellular protein-protein interactions [9]. It has been estimated that as much as 1% of an organism’s total proteins may be subjected to tyrosine sulfation [10]. Among a limited number of tyrosine-sulfated proteins identified to date are several blood coagulation factors, including fibrinogen and factors V, VIII, and IX [1115]. In view of the abundance of tyrosine-sulfated proteins that remain unidentified, it is possible that some other members of the blood coagulation pathway, as well as those involved in anticoagulation and fibrinolysis, may also be subjected to tyrosine sulfation.

Amino acid sequences encompassing sulfated tyrosine residues are known for a number of proteins and peptides [16,17]. Comparison of these sequences had revealed that a consensus feature is the presence of acidic amino acid (Asp and Glu) residues within −5 (N-terminal) and +5 (C-terminal) of the sulfated tyrosine [16,17]. Turn-inducing amino acids (e.g., Pro and Gly) are also found near (from −7 to +7) the sulfated tyrosine [16,17]. Based on these sequence features, a software tool, dubbed “Sulfinator” (available at http://www.expasy.org/tools/sulfinator/), has been developed to aid in the prediction of potential tyrosine sulfation sites in proteins [18]. While Sulfinator may enable users to conveniently identify potential tyrosine sulfation sites in proteins, it is not a guarantor for the identified proteins to be actually sulfated in cells. Therefore, we attempted in the present study to develop a simple methodology for the identification and verification of new tyrosine-sulfated proteins.

Using hemostatic proteins as a model, we first performed a sequence analysis of members of the three hemostatic pathways using the Sulfinator prediction algorithm. Table 1 shows the potential tyrosine sulfation sites identified for three previously reported tyrosine-sulfated hemostatic proteins, (fibrinogen, Factor V, and heparin cofactor II) and three not-yet-identified but potentially tyrosine-sulfated proteins (protein S, prekallikrein, and plasminogen). To verify whether these three latter proteins are indeed subjected to post-translational tyrosine sulfation, we used the HepG2 human hepatoma cells that are known to produce many plasma proteins for which there are antibodies commercially available. HepG2 cells were labeled with [35S]sulfate (0.3 mCi/ml; 1Ci=37 GBq) in sulfate-free minimum essential medium (prepared by omitting streptomycin sulfate and replacing magnesium sulfate with magnesium chloride) without serum. After an 18-hour incubation, the labeling medium was collected and a protease inhibitor cocktail was immediately added to prevent protein degradation. For immunoprecipitation, 1-ml aliquots of the labeling medium were incubated individually with 50 μg each of antibodies against, respectively, fibrinogen, Factor V, heparin cofactor II, protein S, prekallikrein, and plasminogen. After an overnight incubation on ice, 50 μl of Protein G-Sepharose CL-4B was added to each sample and the mixture was agitated by rotation at 4°C for 30 min. Protein G-Sepharose bound with the immune complex was subsequently brought down by centrifugation, washed three times with phosphate-buffered saline, and placed in the SDS sample buffer for the subsequent SDS-polyacrylamide gel electrophoresis. After the electrophoresis, the gel was stained with Coomassie blue, destained, dried, and subjected to autoradiography. Panel (A) of Figure 1 shows the autoradiograph taken from the dried gel. All six proteins, as indicated by arrow heads on their respective electrophoretic lanes, were found to be [35S]sulfated. The radioactive bands corresponding to these proteins were assigned based on their molecular weights: fibrinogen (Mr of Bβ subunit 55,000), Factor V (Mr 330,000), Heparin cofactor II (Mr 66,000), protein S (Mr 70,000), prekallikrein (Mr 88,000), and plasminogen (Mr 106,900). It is noted that nonspecific radioactive bands were also observed on different electrophoretic lanes (Panel (A), Figure 1). These could be due to proteins that interacted with specific proteins being immunoprecipitated or protein G-Sepharose gel beads.

Figure 1
Immunoprecipitation of known and potentially tyrosine-sulfated hemostatic proteins (A), and two-dimensional thin-layer analysis of the Pronase Hydrolysates of [35S]sulfate-labeled protein S (B), prekallikrein (C), and plasminogen (D)
Table 1
Potential tyrosine sulfation sites of the plasma proteins identified using Sulfinatora

To further examine the chemical nature of the bound [35S]sulfate, the radioactive bands corresponding to the three potentially tyrosine-sulfated proteins, protein S, prekallikrein, and plasminogen, were located by autoradiograph and excised from the dried gel, and subjected to Pronase hydrolysis, followed by a two-dimensional thin-layer separation combining high-voltage electrophoresis and thin-layer chromatography (TLC), based on a procedure previously established [19]. As shown in Panels (B), (C), and (D) of Figure 1, the autoradiographs taken from the TLC plates used for the two-dimensional separation of the Pronase hydrolysates of [35S]sulfate-labeled protein S, prekallikrein, and plasminogen clearly revealed the presence of tyrosine O-[35S]sulfate. The additional radioactive spots detected on the three autoradiographs are likely due to the carbohydrate-bound [35S]sulfate also present in these three proteins.

Plasminogen, which is a central component in the fibrinolytic system, had previously been shown to undergo post-translational modifications by O-glycosylation and N-glycosylation [20]. Prekallikrein, a precursor of kallikrein responsible for cleaving kininogen to generate bradykinin as well as activating several coagulation factors such as Factors XII and VII, had also been reported to be subjected to N-glycosylation [21]. For protein S, occurrence of γ-carboxylation of glutamic acid residue and β-hydroxylation of asparagine residue had been demonstrated [22]. Our current results showing the tyrosine sulfation of protein S, prekallikrein, and plasminogen imply that these different post-translational modifications may collectively contribute to the functioning of these three hemostatic proteins.

In conclusion, the abundance and distribution of tyrosine-sulfated proteins have remained poorly understood due to the lack of suitable analytical methods and tools. In this communication, we reported a simple methodology for the identification of new tyrosine-sulfated hemostatic proteins. The same approach may be employed to identify new tyrosine-sulfated proteins involved in other biochemical/physiological pathways/processes.

Acknowledgments

This work was supported in part by a National Institutes of Health grant GM085756 and a startup fund from College of Pharmacy, The University of Toledo.

Footnotes

Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

References

1. Bettelheim FR. Tyrosine-O-sulfate in a peptide from fibrinogen. J Am Chem Soc. 1954;76:2838–2839.
2. Niehrs C, Beisswanger R, Huttner WB. Protein tyrosine sulfation, 1993 - an update. Chem Biol Interact. 1994;92:257–271. [PubMed]
3. Moore KL. The biology and enzymology of protein tyrosine O-sulfation. J Biol Chem. 2003;278:24243–24246. [PubMed]
4. Jensen SL, Holst JJ, Nielsen OV, Rehfeld JF. Effect of sulfation of CCK-8 on its stimulation of the endocrine and exocrine secretion from the isolated perfused porcine pancreas. Digestion. 1981;22:305–309. [PubMed]
5. Brand SJ, Andersen BN, Rehfeld JF. Complete tyrosine-O-sulphation of gastrin in neonatal rat pancreas. Nature. 1984;309:456–458. [PubMed]
6. Rosa P, Hille A, Lee RWH, Zanini A, De Camilli P, Huttner WB. Secretogranins I and II: two tyrosine-sulfated secretory proteins common to a variety of cells secreting peptides by the regulated pathway. J Cell Biol. 1985;101:1999–2011. [PMC free article] [PubMed]
7. Pauwels S, Dockray GJ, Walker R. Comparison of the metabolism of sulfated and unsulfated heptadecapeptide gastrin in humans. Gastroenterology. 1987;92:1220–1225. [PubMed]
8. Hille A, Rosa P, Huttner WB. Tyrosine sulfation: a post-translational modification of proteins destined for secretion? FEBS Lett. 1984;117:129–134. [PubMed]
9. Kehoe JW, Bertozzi CR. Tyrosine sulfation: a modulator of extracellular protein-protein interactions. Chemistry & Biology. 2000;7:R57–R61. [PubMed]
10. Baeuerle PA, Huttner WB. Tyrosine sulfation of yolk proteins 1, 2, and 3 in Drosophila melanogaster. J Biol Chem. 1985;260:6434–6439. [PubMed]
11. Liu MC, Yu S, Sy J, Redman CM, Lipmann F. Tyrosine sulfation of proteins from the human hepatoma cell line HepG2. Proc Natl Acad Sci USA. 1985;82:7160–7164. [PubMed]
12. Farrell DH, Mulvihill ER, Huang SM, Chung DW, Davie EW. Recombinant human fibrinogen and sulfation of the gamma’ chain. Biochemistry. 1991;30:9414–9420. [PubMed]
13. Hortin GL. Sulfation of tyrosine residues in coagulation factor V. Blood. 1990;76:946–952. [PubMed]
14. Michnick DA, Pittman DD, Wise RJ, Kaufman RJ. Identification of individual tyrosine sulfation sites within factor VIII required for optimal activity and efficient thrombin cleavage. J Biol Chem. 1994;269:20095–20102. [PubMed]
15. Bond M, Jankowski M, Patel H, Karnik S, Strang A, Xu B, Rouse J, Koza S, Letwin B, Steckert J, Amphlett G, Scoble H. Biochemical characterization of recombinant factor IX. Semin Hematol. 1998;35:11–17. [PubMed]
16. Hortin G, Folz R, Gordon JI, Strauss AW. Characterization of sites of tyrosine sulfation in proteins and criteria for predicting their occurrence. Biochem Biophys Res Commun. 1986;141:326–333. [PubMed]
17. Huttner WB, Baeuerle PA. Protein sulfation on tyrosine. Mol Cell Biol. 1988;6:97–140.
18. Monigatti F, Gasteiger E, Bairoch A, Jung E. The Sulfinator: predicting tyrosine sulfation sites in protein sequences. Bioinformatics. 2002;18:769–770. [PubMed]
19. Liu MC, Lipmann F. Decrease of tyrosine-O-sulfate-containing proteins found in rat fibroblasts infected with Rous sarcoma virus or Fujinami Sarcoma virus. Proc Natl Acad Sci USA. 1984;81:3695–3698. [PubMed]
20. Rudd PM, Woods RJ, Wormald MR, Opdenakker G, Downing AK, Campbell ID, Dwek RA. The effects of variable glycosylation on the functional activities of ribonuclease, plasminogen and tissue plasminogen activator. Biochim Biophys Acta. 1995;1248:1–10. [PubMed]
21. Lu HS, Hsu YR, Narhi LO, Karkare S, Liu FK. Purification and characterization of human tissue prekallikrein and kallikrein isoforms expressed in Chinese hamster ovary cell. Protein Expr Purif. 1996;8:227–237. [PubMed]
22. Ploos van Amstel HK, van der Zanden AL, Reitsma PH, Bertina RM. Human protein S cDNA encodes Phe-16 and Tyr 222 in consensus sequences for the post-translational processing. FEBS Lett. 1987;222:186–190. [PubMed]