Heparin-binding Histidine and Lysine Residues of Rat Selenoprotein P
2001; Elsevier BV; Volume: 276; Issue: 19 Linguagem: Inglês
10.1074/jbc.m010405200
ISSN1083-351X
AutoresRobert J. Hondal, Shuguang Ma, Richard M. Caprioli, Kristina E. Hill, Raymond F. Burk,
Tópico(s)Pharmacological Effects and Assays
ResumoSelenoprotein P is a plasma protein that has oxidant defense properties. It binds to heparin at pH 7.0, but most of it becomes unbound as the pH is raised to 8.5. This unusual heparin binding behavior was investigated by chemical modification of the basic amino acids of the protein. Diethylpyrocarbonate (DEPC) treatment of the protein abolished its binding to heparin. DEPC and [14C]DEPC modification, coupled with amino acid sequencing and matrix-assisted laser desorption ionization-time of flight mass spectrometry of peptides, identified several peptides in which histidine and lysine residues had been modified by DEPC. Two peptides from one region (residues 80–95) were identified by both methods. Moreover, the two peptides that constituted this sequence bound to heparin. Finally, when DEPC modification of the protein was carried out in the presence of heparin, these two peptides did not become modified by DEPC. Based on these results, the heparin-binding region of the protein sequence was identified as KHAHLKKQVSDHIAVY. Two other peptides (residues 178–189 and 194–234) that contain histidine-rich sequences met some but not all of the criteria of heparin-binding sites, and it is possible that they and the histidine-rich sequence between them bind to heparin under some conditions. The present results indicate that histidine is a constituent of the heparin-binding site of selenoprotein P. The presence of histidine, the pKa of which is 7.0, explains the release of selenoprotein P from heparin binding as pH rises above 7.0. It can be speculated that this property would lead to increased binding of selenoprotein P in tissue regions that have low pH. Selenoprotein P is a plasma protein that has oxidant defense properties. It binds to heparin at pH 7.0, but most of it becomes unbound as the pH is raised to 8.5. This unusual heparin binding behavior was investigated by chemical modification of the basic amino acids of the protein. Diethylpyrocarbonate (DEPC) treatment of the protein abolished its binding to heparin. DEPC and [14C]DEPC modification, coupled with amino acid sequencing and matrix-assisted laser desorption ionization-time of flight mass spectrometry of peptides, identified several peptides in which histidine and lysine residues had been modified by DEPC. Two peptides from one region (residues 80–95) were identified by both methods. Moreover, the two peptides that constituted this sequence bound to heparin. Finally, when DEPC modification of the protein was carried out in the presence of heparin, these two peptides did not become modified by DEPC. Based on these results, the heparin-binding region of the protein sequence was identified as KHAHLKKQVSDHIAVY. Two other peptides (residues 178–189 and 194–234) that contain histidine-rich sequences met some but not all of the criteria of heparin-binding sites, and it is possible that they and the histidine-rich sequence between them bind to heparin under some conditions. The present results indicate that histidine is a constituent of the heparin-binding site of selenoprotein P. The presence of histidine, the pKa of which is 7.0, explains the release of selenoprotein P from heparin binding as pH rises above 7.0. It can be speculated that this property would lead to increased binding of selenoprotein P in tissue regions that have low pH. p-hydroxyphenylglyoxal diethylpyrocarbonate trinitrobenzene sulfonic acid matrix-assisted laser desorption ionization-time of flight phenylthiohydantoin fast protein liquid chromatography high pressure liquid chromatography Selenoprotein P is an unusual, extracellular glycoprotein that is the major form of selenium in rat plasma (1Read R. Bellew T. Yang J.-G. Hill K.E. Palmer I.S. Burk R.F. J. Biol. Chem. 1990; 265: 17899-17905Abstract Full Text PDF PubMed Google Scholar, 2Burk R.F. Hill K.E. BioEssays. 1999; 21: 231-237Crossref PubMed Scopus (91) Google Scholar). Its mRNA has 10 UGAs in the open reading frame that specify selenocysteine incorporation (3Hill K.E. Lloyd R.S. Yang J.-G. Read R. Burk R.F. J. Biol. Chem. 1991; 266: 10050-10053Abstract Full Text PDF PubMed Google Scholar). The protein is secreted by the liver, and its mRNA is present in most other tissues, implying that they secrete it as well (4Burk R.F. Hill K.E. J. Nutr. 1994; 124: 1891-1897Crossref PubMed Scopus (159) Google Scholar). An immunohistochemical study has shown that selenoprotein P is strongly associated with endothelial cells (5Burk R.F. Hill K.E. Boeglin M.E. Ebner F.F. Chittum H.S. Histochem. Cell Biol. 1997; 108: 11-15Crossref PubMed Scopus (75) Google Scholar). Thus, selenoprotein P is present in extracellular fluid and bound to cells. There is increasing evidence that selenoprotein P has a role in defense against oxidative injury. Administration of low doses of diquat to selenium-deficient rats causes lipid peroxidation, massive liver necrosis, and death within a few hours (6Burk R.F. Lawrence R.A. Lane J.M. J. Clin. Invest. 1980; 65: 1024-1031Crossref PubMed Scopus (223) Google Scholar). These adverse events can be prevented by injection of a physiological dose of selenium 12 h before administration of the diquat. The selenium-dependent protection correlates with the appearance of selenoprotein P in plasma but not with the appearance of glutathione peroxidase in liver or in plasma (7Burk R.F. Hill K.E. Awad J.A. Morrow J.D. Kato T. Cockell K.A. Lyons P.R. Hepatology. 1995; 21: 561-569PubMed Google Scholar). A subsequent study using this model showed that the initial lesion in the liver was injury of the centrilobular sinusoidal endothelial cells and loss of them within 2 h (8Atkinson J.B. Hill K.E. Burk R.F. Lab. Invest. 2001; 81: 193-200Crossref PubMed Scopus (30) Google Scholar). Endothelial cell loss was followed within an hour by necrosis of the exposed hepatocytes. The results of these studies are compatible with protection by selenoprotein P against diquat-induced oxidative injury of endothelial cells. Two reports have appeared recently suggesting that selenoprotein P has enzymatic activities of an antioxidant nature (9Saito Y. Hayashi T. Tanaka A. Watanabe Y. Suzuki M. Saito E. Takahashi K. J. Biol. Chem. 1999; 274: 2866-2871Abstract Full Text Full Text PDF PubMed Scopus (225) Google Scholar, 10Arteel G.E. Mostert V. Oubrahim H. Briviba K. Abel J. Sies H. Biol. Chem. 1998; 379: 1201-1205PubMed Google Scholar). Selenoprotein P is a heparin-binding protein (11Herrman J.L. Biochim. Biophys. Acta. 1977; 500: 61-70Crossref PubMed Scopus (58) Google Scholar). It binds to heparin-Sepharose columns when applied at pH 7.0, but most of it is eluted when the pH is raised to 8.5. This suggests that histidine residues, which have pKa values of 6.5–7.0, are important in the binding. Elution of bound selenoprotein P with a pH gradient from 7.0 to 8.5 yielded several peaks, suggesting the presence of isoforms of the protein that have differing affinities for heparin (12Chittum H.S. Himeno S. Hill K.E. Burk R.F. Arch. Biochem. Biophys. 1996; 325: 124-128Crossref PubMed Scopus (61) Google Scholar). Two of the eluted peaks contained single proteins that shared the same N terminus. One was the full-length form of selenoprotein P, and the other was a short form that terminated at the second UGA (13Himeno S. Chittum H.S. Burk R.F. J. Biol. Chem. 1996; 271: 15769-15775Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). Other isoforms appear to exist, and all of them bind to heparin (12Chittum H.S. Himeno S. Hill K.E. Burk R.F. Arch. Biochem. Biophys. 1996; 325: 124-128Crossref PubMed Scopus (61) Google Scholar). This report, which presents the first structural data on selenoprotein P, describes experiments carried out to locate its heparin-binding site(s). Site-directed mutagenesis is often used to assess the significance of specific amino acid residues in binding of proteins to heparin. However, expression of animal selenoproteins is generally not possible in bacteria and is even difficult to achieve in animal cells. Thus, site-directed mutagenesis could not be used to study heparin binding by selenoprotein P. The older, but classic, techniques of chemical modification, peptide mapping, and mass spectrometry were used to identify histidine and lysine residues that are responsible for the interaction of selenoprotein P with heparin. p-Hydroxyphenylglyoxal (HPG)1 was from Pierce. DEPC, [14C]DEPC, TNBS, chymotrypsin, trypsin, and heparin-agarose were purchased from Sigma. FPLC and heparin HiTrap columns were obtained from Amersham Pharmacia Biotech. All other reagents were analytical grade or better. For reverse phase HPLC, a Hewlett-Packard XDB-C18 column (2.1-mm inner diameter × 15 cm) was used. For tryptophan emission experiments, a SPEX-Fluorolog-1681 photon-counting fluorometer was used. UV-visible spectroscopy was performed on a Hewlett Packard P8453 spectrophotometer linked to a Hewlett Packard-Vectra XM microcomputer. Selenoprotein P was purified from rat plasma by a simplification of a previously described method (1Read R. Bellew T. Yang J.-G. Hill K.E. Palmer I.S. Burk R.F. J. Biol. Chem. 1990; 265: 17899-17905Abstract Full Text PDF PubMed Google Scholar). A 10-ml immunoaffinity column, made with the monoclonal antibody 8F11 (1Read R. Bellew T. Yang J.-G. Hill K.E. Palmer I.S. Burk R.F. J. Biol. Chem. 1990; 265: 17899-17905Abstract Full Text PDF PubMed Google Scholar), was equilibrated with phosphate-buffered saline containing 0.5 mm EDTA and 0.02% NaN3. Rat plasma, containing a small amount of 75Se-labeled selenoprotein P, was applied to the column. Typically, 50 ml of plasma was applied to the column and then washed with 150 ml of phosphate-buffered saline. The column was washed with 15 ml of 1m NaCl to elute nonspecifically adsorbed plasma proteins. Selenoprotein P was then eluted from the column with 0.1 mglycine, pH 2.8, and collected in 1-ml fractions. The eluted protein was neutralized with 50 μl of 1 m Tris-Cl, pH 9.0, so that the final concentration of Tris-Cl was 50 mm. "Cold" selenoprotein P (without 75Se-labeled plasma added) could be purified using the same procedure. The unlabeled protein was used for labeling with [14C]DEPC, proteolytic digestion, separation by HPLC, and 14C determination by liquid scintillation. Modification by DEPC was performed either on purified protein or on rat plasma. For DEPC modification of selenoprotein P, a final concentration of 6.9 mm DEPC in 0.2 m sodium phosphate buffer, pH 7.0, was used unless otherwise indicated. For modification of lysine residues with TNBS, the final concentration of TNBS was 0.2 mm in 0.2 m sodium carbonate buffer at pH 7.0 or pH 8.5. Modification of arginine residues was done with 1 mm HPG in 0.2 m sodium carbonate, pH 9.0. Four separate columns of heparin-agarose (0.8 ml) were equilibrated with 50 mmsodium bicarbonate, pH 7.0. Purified selenoprotein P (50 μg) was treated for 2 h with either TNBS, DEPC, or HPG, or was not treated. Modification of selenoprotein P with DEPC was done in 0.2m sodium phosphate buffer, pH 7.0. Modification of selenoprotein P with TNBS was done in 0.2 m sodium bicarbonate, pH 8.5. Modification of selenoprotein P with HPG was done in 0.2 m sodium bicarbonate, pH 9.0. After modification, the samples were dialyzed against 4 liters of 50 mm sodium bicarbonate, pH 7.0, for 4 h at 4 °C. After dialysis, each sample was applied to a heparin-agarose column. The columns were each washed with 11 ml of 50 mm sodium bicarbonate, pH 7.0.75Se in the wash was determined by radioactive measurement of 1-ml aliquots of wash in an LKB Compugamma 1282 γ-counter. Carbethoxylation of histidine residues by DEPC causes an increase in absorbance at 240 nm (14Ovadi J. Keleti T. Acta Biochim. Biophys. Acad. Sci. Hung. 1969; 4: 365-378PubMed Google Scholar). Selenoprotein P (50 μg) in 1 ml of 0.1 m sodium phosphate buffer was used as a reference blank, and DEPC was added to an identical sample to give a final concentration of 6.9 mm. The extent of modification, as measured by an increase in absorbance at 240 nm, was monitored over 10 min. The same experiment was carried out with heparin (5 mg/ml) in the solution. The extent of modification of lysine residues was monitored using the lysine-specific reagent, TNBS. When TNBS reacts with the ε-amino group of lysine, an increase in absorbance at 420 nm occurs (15Fields R. Biochem. J. 1971; 124: 581-590Crossref PubMed Scopus (432) Google Scholar). Purified selenoprotein P was modified in 0.1 m sodium phosphate buffer at pH 7 or at pH 8.5. The absorbance at 420 nm was followed for 10 min. Modification of histidine residues by DEPC is dependent upon the pH, since the positively charged imidazolium ion will not react with DEPC. For these studies, 50 μg of selenoprotein P in 1 ml of 0.2m sodium phosphate buffer with pH values from 5.5 to 8.0 was modified with 6.9 mm DEPC, and the extent of modification was followed at 240 nm. For heparin HiTrap chromatography, 0.1-ml aliquots of plasma labeled with 75Se diluted to 1 ml with buffer A (100 mm ammonium acetate, 50 mmTris base, and 1 mm Na2EDTA, pH 7.0) were used. One sample was then modified with 6.9 mm DEPC for 5 min in the starting buffer. A second sample was modified with 6.9 mm DEPC for 5 min in the starting buffer and then treated with 110 volume of 2 m hydroxylamine (adjusted to pH 7.0) and incubated for 30 min at room temperature. Carbethoxylation of histidine residues can be reversed by treatment with hydroxylamine (16Melchior W.B. Fahrney D. Biochemistry. 1970; 9: 251-258Crossref PubMed Scopus (392) Google Scholar). This reversal has previously been used to distinguish modification of histidine residues by DEPC from modification of thiols and other amino groups in proteins (17Miles E.W. Kumagai H. J. Biol. Chem. 1974; 249: 2843-2851Abstract Full Text PDF PubMed Google Scholar). A third sample, used as the control, was not treated with DEPC. The samples were then injected onto 1-ml heparin HiTrap columns attached to a FPLC. The columns were then washed with 10 volumes of buffer A. Selenoprotein P was eluted with a linear gradient from 100% buffer A to 100% buffer B (0.25 ml/min flow rate). Buffer B was identical to buffer A, except that its pH was 8.5. Protein remaining on the column after the pH gradient was eluted with 2m NaCl in buffer B. 400 μg of selenoprotein P in 10 mm Tris-Cl, pH 7.0, was mixed with 2 μg of chymotrypsin, 2 μg of trypsin, or neither and incubated for 3 h at 37 °C. After incubation, the sample was loaded onto a 1-ml heparin HiTrap column equilibrated with 10 mm Tris-Cl, pH 7.0 (buffer C). The column was then washed with 10 ml of buffer C, after which a salt gradient was applied. Buffer D was the same as buffer C except that it contained 1 m NaCl. The flow rate was 0.25 ml/min, and the gradient was set at 2.22% buffer D/ml. A 200-μg sample of selenoprotein P was dissolved in 1 ml of 0.1 msodium phosphate buffer, pH 7.0. [14C]DEPC was added to a final concentration of 6.9 mm (specific activity 1.6 mCi/mmol). The sample was incubated for 5 min at room temperature and then dialyzed against 4 liters of 10 mm NaHCO3buffer for 4 h at 4 °C. This was followed by digestion with 10 μg of chymotrypsin for 5 h at room temperature. The sample was then dried in a SpeedVac to reduce the volume, redissolved in 100 μl of column buffer, and injected onto the HPLC column. The HPLC column was a 2.1-mm inner diameter × 15-cm narrow bore C18 column (XDB-C18). Solvent E consisted of 1 mm sodium phosphate, pH 5.5, 0.1% trifluoroacetic acid. Solvent F consisted of 30% solvent E and 70% acetonitrile. The initial conditions were 95% E, 5% F for 5 min followed by a linear gradient to 100% F in 70 min, followed by a return to the initial conditions over 2 min. Selenoprotein P was also modified in the presence of heparin. Conditions for modification, digestion, and HPLC were the same as above except that the sample contained 5 mg/ml heparin. Plasma (25 μl) containing selenoprotein P radiolabeled in vivo was brought to a volume of 0.5 ml with buffer A, pH 6.9. This sample was applied to a 1-ml heparin HiTrap column and washed with 10 ml of buffer A, pH 6.9, and successively with 10-ml volumes of the same buffer containing 0.0069, 0.069, 0.69, and 6.9 mm DEPC. Then 10-ml of buffer A, pH 6.9, containing 1m NaCl was passed over the column to elute the remaining selenoprotein P. This experiment is shown in Fig. 7 A. Fractions 62–64 shown in Fig. 7 A were combined and extensively buffer exchanged with buffer A, pH 6.9. The buffer-exchanged sample was applied to another heparin HiTrap column as shown in Fig. 7 B and washed with 10 ml of buffer A, pH 6.9. Then 10 ml of buffer B, pH 8.5, was applied, followed by 10 ml of buffer B, pH 8.5, containing 1 m NaCl. 1-ml fractions were collected. Peptides isolated from proteolytic digests by HPLC as described above were sequenced using an Applied Biosystems 492 Procise protein sequencer. MALDI-TOF mass spectrometry analysis was done with a Vestec Voyager Elite Time-of-Flight Mass Spectrometer using an acceleration potential of 20 kV. The instrument was calibrated with insulin as an external calibration standard. 2 μg (∼40 pmol) of full-length selenoprotein P, prepared as peak 2 on a heparin HiTrap column (12Chittum H.S. Himeno S. Hill K.E. Burk R.F. Arch. Biochem. Biophys. 1996; 325: 124-128Crossref PubMed Scopus (61) Google Scholar), was dissolved in 20 μl of 100 mm ammonium bicarbonate. Modified trypsin (0.04 μg) was added to give an enzyme/protein ratio of 1:50, and then the solution was incubated at 37 °C for 18 h. 1 μl of the digested mixture was analyzed using mass spectrometry. 2 μg (∼40 pmol) of full-length selenoprotein P was dissolved as above, and DEPC was added to it at a concentration of 6.9 mm. The reaction was incubated at room temperature for 5 min and then dialyzed against water for 6 h. It was then dried using a SpeedVac and redissolved in 20 μl of 100 mmammonium bicarbonate. Digestion and mass spectrometric analysis was performed as described for the unmodified selenoprotein P. In order to measure the structural integrity of the DEPC-modified protein, tryptophan emission spectra were taken of the modified and native proteins. Excitation was done at 295 nm, and the tryptophan emission was recorded between 300 and 420 nm. Spectra were recorded in 0.2 m sodium phosphate buffer, pH 7.0, at a protein concentration of 25 μg/ml for both native and DEPC-modified selenoprotein P. Heparin binding by proteins is mediated by basic amino acids arranged to form a heparin-binding site. In order to determine which basic amino acids are present in the heparin-binding site(s) of selenoprotein P, the75Se-labeled protein was treated with reagents that selectively modify basic residues. Then the protein was passed over a heparin-agarose column equilibrated with buffer of pH 7.0. It was reasoned that modification of residues in the binding site would interfere with heparin binding of the protein. The amount of selenoprotein P bound to the column was determined by subtracting the amount of 75Se that passed through the column from the amount of 75Se applied to the column. Selenoprotein P was modified with DEPC, TNBS, and HPG in separate experiments. DEPC binds to unprotonated histidine residues, but it can also modify tyrosine, lysine, and cysteine residues (18Miles E.W. Methods Enzymol. 1977; 47: 431-442Crossref PubMed Scopus (836) Google Scholar). TNBS modifies primary amino groups in proteins, especially lysine residues. HPG is highly specific for modifying arginine residues (19Yamasaki R.B. Vega A. Feeney R.E. Anal. Biochem. 1980; 109: 32-40Crossref PubMed Scopus (91) Google Scholar). The results in Table I show that DEPC treatment of selenoprotein P abolished its heparin-binding property at pH 7.0. TNBS treatment blocked most heparin binding. However, modification of selenoprotein P by HPG had only a small effect on the affinity of the protein for heparin. These results implicate histidine and lysine residues in the binding of selenoprotein P to heparin.Table IBinding of chemically modified selenoprotein P to a heparin-agarose columnSelenoprotein P treatment75Se bound to column%None97DEPC3TNBS31HPG86Selenoprotein P was modified with DEPC, TNBS, and HPG as described under "Experimental Procedures." The modified protein was then passed over a heparin-agarose column, and the column was washed with pH 7.0 buffer as described under "Experimental Procedures." Open table in a new tab Selenoprotein P was modified with DEPC, TNBS, and HPG as described under "Experimental Procedures." The modified protein was then passed over a heparin-agarose column, and the column was washed with pH 7.0 buffer as described under "Experimental Procedures." The modification of histidine and tyrosine residues by DEPC can be followed spectrophotometrically. When histidine becomes carbethoxylated, there is a corresponding increase in absorbance at 240 nm (14Ovadi J. Keleti T. Acta Biochim. Biophys. Acad. Sci. Hung. 1969; 4: 365-378PubMed Google Scholar). Modification of tyrosine side chains produces a decrease in absorbance at 278 nm (20Burstein Y. Walsh K.A. Neurath H. Biochemistry. 1974; 13: 205-210Crossref PubMed Scopus (183) Google Scholar). In order to assess histidine and tyrosine modification, purified selenoprotein P was treated with DEPC, and UV spectra were recorded. A progressive increase in absorbance at 240 nm occurred with time, but no decrease took place at 278 nm (spectra not shown). Therefore, modification of histidine occurs, but no evidence was found for modification of tyrosine side chains. When selenoprotein P was modified with DEPC, histidine binding reached saturation in about 2 min at pH 7.0 (Fig.1). When selenoprotein P was modified in the presence of heparin, the increase in absorbance at 240 nm was not as great as seen with the unliganded protein (Fig. 1). This indicates that the presence of heparin prevents DEPC from modifying some of the histidine residues, implicating histidine in the heparin binding. DEPC reacts with unprotonated histidine residues. In order to approximate the pKa of the modifiable histidine residues, the modification of selenoprotein P by DEPC was carried out at varying pH values. The modification of selenoprotein P in sodium phosphate buffer with pH values ranging from 5.5 to 8.0 is shown in Fig. 2. A marked increase in reaction rate occurred when the pH reached 6.8. The data from Fig. 2 indicate that the pKa values of the modified histidine residues are in the range of 6.4–6.8. Peptides that become modified by DEPC treatment of the native protein are predicted to be solvent-exposed and thus might be involved in binding of selenoprotein P to heparin. To determine which peptides of the native protein can be labeled by DEPC, [14C]DEPC was employed. After labeling, the purified selenoprotein P sample was dialyzed to remove excess [14C]DEPC. Then it was digested with chymotrypsin. Peptides were separated by reverse phase HPLC (chromatogram not shown). Peaks that contained 14C were submitted for amino acid sequence analysis. The peptides identified are shown in TableII. Two of them are KHAHL and KKQVSDHIAVY, which correspond to residues 80–84 and 85–95, respectively. Although it has been reported that loss of label is usually observed during the Edman degradation procedure (21Ko Y.-H. Vanni P. Munske G.R. McFadden B.A. Biochemistry. 1991; 30: 7451-7456Crossref PubMed Scopus (17) Google Scholar), Lemaire and colleagues (22Lemaire M. Schmitter J.-M. Issakidis E. Miginiac-Maslow M. Gadal P. Decottignies P. J. Biol. Chem. 1994; 269: 27291-27296Abstract Full Text PDF PubMed Google Scholar) observed an additional peak that did not correspond to any standard PTH-derivative when sequencing a peptide containing a single histidine residue that had been labeled with [14C]DEPC. Such a peak appeared along with a PTH-derivatized histidine residue during its turn in the Edman cycle. Similarly, we were able to identify a secondary peak with a retention time slightly less than PTH-proline that was always associated with DEPC-labeled histidine. A potential interpretation of this result is that some of the carbethoxylated histidine residues survived the sequence analysis and appeared as a new peak in the sequencing chromatogram. These sequencing results (not shown) indicate that His-81, His-83, and His-91 become carbethoxylated upon treatment of the protein with DEPC (Table II).Table IIPeptides Modified with [14C]DEPCResidue numbersSequence 2-aResidues shown in boldface type were modified by DEPC.85–95KKQVSDHIAVY80–84KHAHL106–116TLLNGNKDDFL256–262SLAQRKL2-a Residues shown in boldface type were modified by DEPC. Open table in a new tab We also found evidence for lysine modification by DEPC. Again, the sequence analysis provided the evidence. The PTH-derivatized lysine residues in the peptides in Table II did not elute in the position for PTH-lysine but were shifted so that the retention of the modified lysine was slightly greater than that of PTH-valine. A previous study that used DEPC to modify chick liver glutathioneS-transferase demonstrated that DEPC modified lysine residues and that the N-carbethoxylated PTH-lysine eluted in a similar position to that reported here (23Chang L.-H. Tam M.F. Eur. J. Biochem. 1993; 211: 805-811Crossref PubMed Scopus (16) Google Scholar). Thus, Lys-80, Lys-85, Lys-86, Lys-112, and Lys-261 became modified with DEPC based on the sequencing chromatograms (not shown). A second approach that used MALDI-TOF mass spectrometry for detecting DEPC-labeled peptides was employed. It has been reported that peptides containing carbethoxylated histidine residues can be detected by using MALDI-TOF mass spectrometry (24Glocker M.O. Kalkum M. Yamamoto R. Schreurs J. Biochemistry. 1996; 35: 14625-14633Crossref PubMed Scopus (41) Google Scholar, 25Kalkum M. Pryzbylski M. Glocker M.O. Bioconjugate Chem. 1998; 9: 226-235Crossref PubMed Scopus (44) Google Scholar, 26Krell T. Chackrewarthy S. Pitt A.R. Elwell A. Coggins J.R. J. Peptide Res. 1998; 51: 201-209Crossref PubMed Scopus (19) Google Scholar, 27Dage J.L. Sun H. Halsall H.B. Anal. Biochem. 1998; 257: 176-185Crossref PubMed Scopus (27) Google Scholar). Fig. 3 A shows the mass spectrum of a total tryptic digest of the native full-length isoform of selenoprotein P. 12 peaks with the predicted masses of tryptic peptides could be identified in the spectrum. After treatment with DEPC and subsequent digestion, shifts of 72 mass units, corresponding to the addition of a C(O)OCH2CH3 group to an imidazole nitrogen atom or to an ε-amino group, were detected in eight peptides (Fig. 3 B and Table III).Table IIIPeptides labeled with DEPC and detected by MALDI-TOF mass spectrometryPeak3-aLetters indicate peaks labeled with corresponding letters in Fig. 3.ResiduesPeptide sequenceUnmodified massModified massNo. of residues modifiedm/zobs 3-bm/zobs, the observed mass to charge ratio.m/zcalc3-cm/zcalc, the calculated mass to charge ratio.m/zobs3-dThe number of modifications observed for the most abundant ion is 1. The mass listed in this column corresponds to one modification by DEPC.m/zcalc 3-eThe mass/charge ratio calculated is based on one modification by DEPC.a81–86HAHLKK733.62733.45805.67805.471b233–238HKGQHR762.76762.41834.87834.432c292–297HLIFEK786.92786.45858.68858.471d87–96QVSDHIAVYR1188.011187.621260.201259.641e86–96KQVSDHIAVYR1316.021315.711387.941387.732f178–189TTEPSEEHNHHK1445.951445.641518.011517.662g178–193TTEPSEEHNHHKHHDK1963.251962.882035.222034.902h97–120QDEHQTDVWTLLNGNKDDFLIYDR2937.682937.153009.863009.1523-a Letters indicate peaks labeled with corresponding letters in Fig. 3.3-b m/zobs, the observed mass to charge ratio.3-c m/zcalc, the calculated mass to charge ratio.3-d The number of modifications observed for the most abundant ion is 1. The mass listed in this column corresponds to one modification by DEPC.3-e The mass/charge ratio calculated is based on one modification by DEPC. Open table in a new tab Fig. 4 presents the amino acid sequence of selenoprotein P as derived from its cDNA (3Hill K.E. Lloyd R.S. Yang J.-G. Read R. Burk R.F. J. Biol. Chem. 1991; 266: 10050-10053Abstract Full Text PDF PubMed Google Scholar). Thesolid lines under thesequence coincide with the peptides identified by mass spectrometry of the tryptic digest (Fig. 3 A). Thebroken lines with arrowheads above the sequence indicate the peptides that were detected to be shifted in mass by DEPC treatment of the protein (Fig. 3 B). Residues in boldface typeindicate the DEPC-modified peptides identified by sequencing (TableII). These results demonstrate that at least five stretches of the selenoprotein P sequence are accessible to modification by DEPC in the native protein. These stretches are residues 81–120, 178–193, 233–238, 256–262, and 292–297. These results do not indicate that no other parts of the molecule interact with heparin. The largest peptide detected (residues 190–232 in Fig. 3 A, HGHEHLGSSKPSENQQPGALDVETSLPPSGLHHHHHHHK) represents the sequence between the second and third stretches identified as potential heparin-binding sites. Because of its richness in histidines, this peptide might be expected to bind to heparin. After DEPC modification of the protein, this peptide could not be detected in the trypsin digest (no peak in Fig. 3 B). Peptides on either side of it were detected, so it must have been present in the digest. This result implies that the peptide had been modified by DEPC. It seems likely that the modification impaired the ability of the peptide to be ionized and therefore detected. Thus, this stretch of the sequence would appear to be a candidate for a heparin-binding site along with the adjacent histidine-rich sequences. In order to determin
Referência(s)