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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.
Post-translational protein modification by tyrosine sulfation, first discovered in bovine fibrinogen , 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 , change in half-life of proteins in circulation , intracellular transport of secretory proteins , and modulation of extracellular protein-protein interactions . It has been estimated that as much as 1% of an organism’s total proteins may be subjected to tyrosine sulfation . Among a limited number of tyrosine-sulfated proteins identified to date are several blood coagulation factors, including fibrinogen and factors V, VIII, and IX [11–15]. 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 . 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.
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 . 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 . 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 . For protein S, occurrence of γ-carboxylation of glutamic acid residue and β-hydroxylation of asparagine residue had been demonstrated . 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.
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.
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