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Biotinylation Sites of Tumor Necrosis Factor-α Determined by Liquid Chromatography–Mass Spectrometry
Analytical Biochemistry 298, 181–188 (2001) doi:10.1006/abio.2001.5374, available online at http://www.idealibrary.com on
Biotinylation Sites of Tumor Necrosis Factor-␣ Determined by Liquid Chromatography–Mass Spectrometry Fulvio Magni,* Flavio Curnis,† Laura Marazzini,* Roberto Colombo,‡ Angelina Sacchi,† Angelo Corti,† and Marzia Galli Kienle‡ *Mass Spectrometry Laboratory, IRCCS S. Raffaele, Milan, Italy; †DIBIT, San Raffaele H Scientific Institute, Milan, Italy; ‡Department of Medical Chemistry and Biochemistry, University of Milan Bicocca, Milan, Italy; and §Catholic University Holy Heart of Milan, Milan, Italy
Received February 5, 2001; published online October 18, 2001
Tumor pretargeting with biotinylated antibody/avidin complexes improves the therapeutic index of systemically administered biotin–tumor necrosis factor (TNF) conjugates. Since the number of biotins in this conjugate is known to be critical for activity, we have characterized the structure of different biotin–TNF conjugates, prepared by reaction with D-biotinyl-6aminocaproic acid N-hydroxysuccinimide ester and identified the biotinylation sites by trypsin digestion, reverse-phase chromatography, and electrospray mass spectrometry analyses. The results have shown that N-terminal valine is a preferential biotinylation site at pH 5.8, half of biotins being located on the ␣-amino group of this residue in a conjugate bearing one biotin/trimer (on average). Moreover, evidence has been obtained to suggest that the remaining part of biotins are linked to the ⑀-amino group of lysine 128, 112, and 65, while lysine 11, 90, and 98 were practically unmodified. No evidence of O-biotinylation of serine, threonine and tyrosine was obtained. © 2001 Academic Press Key Words: tumor necrosis factor; mass spectrometry; biotin; tumor therapy; electrospray; HPLC.
Human tumor necrosis factor ␣ (TNF) 1 is a nonglyco homotrimeric protein produced by a large number of cell types and organs in response to various inflammatory and immunological stimuli. Several studies have shown that TNF is endowed with potent antitumor activity in animal models (1). However, the clinical use of TNF as an anticancer drug is limited by severe systemic toxicity (2, 3). In the attempt to overcome this problem, we have recently developed a new strategy based on tumor 1 Abbreviations used: TNF, tumor necrosis factor; TFA, trifluoroacetic acid; ESI-MS, electrospray mass ionization; LC, liquid chromatography.
0003-2697/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.
pretargeting with biotinylated antibody/avidin complexes and systemic administration of biotinylated TNF (4, 5). This pretargeting approach was shown to increase at least five times the antitumor activity of biotinylated TNF in animal models, with no evidence of increased toxicity (6). Studies on the mechanism of action showed that biotin–TNF trimers can slowly dissociate from the targeted cells through trimer–monomer–trimer transition and that the antitumor activity is based mainly on indirect effects, presumably on cells other than the targeted tumor cells (4, 6). We have also shown that nonbiotinylated subunits must be present within TNF trimers for an efficient release of bioactive TNF from the targeting complex. Since the number and the position of biotins in these conjugates is likely critical for the activity, the identification of the product present in the mixture after biotinylation is very useful. Mass spectrometry is a powerful tool to study covalent structure of proteins in conjunction with enzymatic digestion which provides short peptides to be used for additional information on the sequence (7–10). In this work we have characterized the structure of different biotin–TNF conjugates and identified the biotinylation sites, using total and partial trypsin digestion and mass spectrometry analysis. MATERIALS AND METHODS
Preparation of TNF and Biotin–TNF Conjugates Human TNF (5 ⫻ 10 7 U/mg) was produced by recombinant DNA technology. The cDNA coding for TNF (11) was cloned in pET-11b (Novagen, Madison, WI) and used to transform BL21 (DE3) Escherichia coli cells (Novagen). Soluble TNF was recovered from 2-liter cultures by bacterial sonication in 2 mM EDTA, 20 mM Tris–HCl, pH 8.0, followed by centrifugation (15,000g, 20 min, 4°C). The extract was mixed with ammonium sulfate (25% of saturation), left for 1 h at 4°C, and 181
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further centrifuged, as above. The ammonium sulfate in the supernatant was then brought to 65% of saturation, left at 4°C for 24 h, and further centrifuged. The pellet was dissolved in 200 ml of 1 M ammonium sulfate, 50 mM Tris–HCl, pH 8.0, and purified by hydrophobic interaction chromatography on phenyl–Sepharose 6 Fast Flow (Amersham Pharmacia Biotech, Piscataway, NJ) (gradient elution, buffer A: 50 mM sodium phosphate, pH 8.0, containing 1 M ammonium sulfate; buffer B: 20% glycerol, 5% methanol, 50 mM sodium phosphate, pH 8.0). Fractions containing TNFimmunoreactive material (identified by Western blotting) were pooled, dialyzed against 5 mM EDTA, 20 mM Tris–HCl, pH 8.0, and further purified by ionexchange chromatography on DEAE–Sepharose Fast Flow (Pharmacia-Upjohn) (gradient elution, buffer A: 20 mM Tris–HCl, pH 8.0; buffer B: 1 M sodium chloride, 20 mM Tris–HCl, pH 8.0). Fractions containing TNF immunoreactivity were pooled and purified by gel filtration chromatography on Sephacryl-S-300 HR (Pharmacia-Upjohn), preequilibrated and eluted with 150 mM sodium chloride, 50 mM sodium phosphate buffer, pH 7.3. Fractions corresponding to 40,000 –50,000 M r products were pooled and stored frozen at ⫺20°C. Biotin–TNF conjugates with different degrees of biotin incorporation, termed biotin–TNF 1 and biotin– TNF 2, were prepared. Biotin–TNF 1 was obtained by mixing 0.72 ml of TNF (0.41 mg/ml in sodium citrate buffer, pH 5.8) with 0.066 ml of biotin-6-aminocaproylN-hydroxysuccinimide ester (1.82 mg/ml; Societa` Prodotti Antibiotici, Milan, Italy). After 3 h incubation at 22–23°C, the product was mixed with 0.0825 ml of 1 M lysine in water and left to incubate for 1 h. The product was dialyzed against 0.9% sodium chloride and stored at ⫺20°C. Biotin–TNF 2 was prepared in the same way except that 0.1 M sodium borate, pH 8.0, was used as a reaction buffer with 0.066 ml of biotinylating reagent (3 mg/ml in dimethyl sulfoxide). Of note, we observed that different batches of biotin reagent may result in different degrees of biotinylation. HPLC HPLC of biotin–TNF 2 was performed using a C4 reverse-phase column (50 ⫻ 4 mm, Vydac, Hesperia, CA) and a Jasco HPLC system (Jasco Spectroscopic Co. LTD, Tokyo, Japan) equipped with a single pump (Model 880-PU) and a ternary gradient mixer (Model 880-02). The UV detector (Jasco Model 875-UV/VIS) was set at 210 nm. The column was equilibrated with 34% aqueous acetonitrile containing 0.1% TFA. Bound proteins were eluted with a shallow gradient from 34 to 37% acetonitrile over 17 min, starting 3 min after sample injection. Acetonitrile was then increased to 60% in 5 min (flow rate, 1 ml/min). Fractions were collected manually, dried using a SpeedVac System (Savant), and stored at ⫺20°C.
Tryptic Digestion Limited trypsin digestions of wild-type TNF and biotin–TNF 1 were obtained by mixing 0.1– 0.2 mg/ml of each product, previously dialyzed against distilled water, with 0.3 mg/ml trypsin in distilled water (TNF/ trypsin, 20/1 by weight). Aliquots of each mixture were withdrawn after 10, 30, 60, 120, and 480 min of incubation at room temperature and stored at ⫺20°C until analysis by ESI-MS. Complete trypsin digestion of TNF and HPLC-isolated biotin–TNF was obtained by mixing 0.03– 0.2 mg of each product with trypsin in 0.05– 0.2 ml of 50 mM ammonium bicarbonate buffer, pH 8.5 (TNF/trypsin, 50/1 by weight) and by incubating at 37°C for 180 min. The reaction was terminated by freezing the samples at ⫺20°C. ESI Analysis To determine the number of biotins incorporated in each product, biotin–TNF conjugates were dialyzed for 30 – 60 min against distilled water or desalted using ZipTip C18 (Millipore Corp., Bedford, MA) and analyzed by ESI-MS (100 –300 ng of protein) using water: methanol (1:1, v/v) containing 0.1% acetic acid as delivering solvent (flow rate, 3 l/min). Mass spectra were obtained with a Finnigan MAT95 double focusing mass spectrometer. Mass spectrometry conditions were capillary voltage 3.1 kV, heated capillary 250°C and 37 V, tube lens 77 V, skimmer voltage ⫺0.8 V, and octapole voltage 3.4 V. Spectra were recorded from m/z 500 to m/z 2500 at 3 s/decade. System calibration and sensitivity was daily evaluated using 0.5 pmol of apomyoglobin from horse skeletal muscle (Sigma-Aldrich, Milan, Italy). The error in the determination of the molecular mass, calculated from the deconvoluted spectrum of apomyoglobin (16951 Da), was ⫾0.02%. Micro-LC-ESI-MS Peptides deriving from the tryptic digestion of biotin–TNF conjugates were analyzed by micro-LC-ESIMS. About 20 l of each mixture was injected into a Wakosil C18 column (150 ⫻ 1 mm; 5 m; 120 Å, SGE, Rome, Italy), directly connected to the ion source. The column was eluted for 2 min with 5% of acetonitrile, containing 0.025% of TFA, followed by a linear gradient from 5 to 45% over 58 min (flow rate, 50 l/min). Solvent mixing was obtained with a biocompatible tee (HPLC Technologies, Macclesfield-Cheshire, UK). The column was then cleaned by increasing acetonitrile to 80% in 8 min, and reequilibrated for 30 min with the starting solvent before subsequent analysis. In Vitro Cytolytic Assays The bioactivities of TNF and biotin–TNF conjugates were estimated by standard cytolytic assay based on L-M mouse fibroblasts (ATCC CCL1.2) as described (12).
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FIG. 1. Deconvoluted ESI spectra of TNF and of biotin–TNF 1 before (A and C) and after (B and D) partial enzymatic proteolysis for 10 min. Numbers (in italics) above the calculated M r (in bold) of the various biotin–TNF monomers indicate the biotin residues per subunit. Tryptic digestion was carried out as described in the text.
RESULTS AND DISCUSSION
Human TNF contains six lysine residues and one ␣-amino group potentially reactive with D-biotinyl-6aminocaproic acid N-hydroxysuccinimide ester. We have shown previously that TNF can be biotinylated to various extents depending on the pH of the reaction and reagent concentration (4). Optimal activity for in vivo tumor targeting experiments (4) can be achieved with a biotin–TNF 1 bearing one biotin/trimer (on average), prepared at pH 5.8. This conjugate is a mixture of trimers made up by monomers with 0, one, and two biotins. Within trimers with the same number of biotins a high heterogeneity of molecular structure may arise also from combination of subunits bearing biotins in different position. We estimated that the biotin– TNF 1 is made up by trimers without biotin (27%) and
trimers with one (43%), two (24%), and three (5.4%) biotin residues (4). To investigate the biotinylation sites in biotin–TNF we have prepared different conjugates with different contents of biotin residues. These conjugates termed 1 and 2 were prepared and characterized by ESI-MS as described in a previous report (4). In this work we evaluated their molecular masses and that of nonbiotinylated TNF by ESI-MS and of their tryptic maps after enzymatic digestion. Mass spectra were obtained by deconvolution of the raw data by an algorithm implemented into the instrument software. The deconvoluted mass spectrum of TNF 1–157 (Fig. 1A) showed an intense peak corresponding to a molecular mass of 17,349 ⫾ 1.2 Da, in good agreement with the value expected for human TNF monomer, calculated on the
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Peptides Identified by LC-ESI after Tryptic Digestion of TNF and Biotin–TNF 2 Expected Peak
Measured M r
Fragment
Mr
Biotinylated residue
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
2755.4 1240.6 2424.8 907.7 857.4 2851.3 2901.7 2394.2 1396.6 2911.6 3180.2 6514.3 4801.2 5641.2 612.5 3191 3519.4 6853.8 3856.4 5141.3
7–31 33–44 45–65 83–90 91–98 104–128 132–157 66–82–SS–99–103 a 32–44 b 7–32 b 104–131 b 45–SS–103 a,b 45–82–SS–99–103 a 45–82–SS–91–103 a 1–2 104–128 104–131 45–103 104–131 45–82–SS–99–103 a,b
2756 1240 2426 909 857 2852 2902 2395 1397 2912 3181 6515 4803 5642 612 3190 3518 6853 3856 5141
— — — — — — — — — — — — — — Val-1 Lys-112 Lys-112 or Lys-128 Lys-65 or Lys-112 or Lys-128 Lys-112 and Lys-128 Lys-65
Note. The expected molecular mass (M r) was calculated using PAWS software (available at www.proteomics.com) on the basis of the human TNF sequence (7). a Disulfide-linked fragments. b Peptide deriving from incomplete tryptic digestion.
basis of its amino acid sequence (M r ⫽ 17,351). Two additional peaks, corresponding to monomers bearing 1 and 2 biotins were observed in the mass spectrum of biotin–TNF 1 (17,687 and 18,027, respectively, Fig. 1C). Limited digestions of both TNF and biotin–TNF 1 with trypsin were then carried out in distilled water. ESI-MS of TNF after 10 min of digestion revealed the presence of a main peptide with M r 16,675, corresponding to cleavage at Arg-6 (TNF 7–157, Fig. 1B). This peptide was resistant to further proteolysis under these conditions, as it was still observed after 8 h of digestion. When this work was in progress a similar observation about the resistance of TNF to proteolysis was reported (10). Thus, partial proteolysis in distilled water was exploited to study the proportion of biotins on the N-terminus and on the rest of the TNF molecule. At least three peptides with M r 16,675, 17,015, and 17,354 were obtained by partial digestion of biotin–TNF 1 (Fig. 1D), corresponding to residues 7–157 with 0, one, and two biotins (Table 1). Based on the signal intensities in the deconvoluted mass spectra, native, monoand di-biotinylated monomers corresponded to 39.5 ⫾2.8, 45 ⫾2.7, and 15.6 ⫾3.4% (mean ⫾ SD; n ⫽ 4 analysis) of the total before digestion (Fig. 1C) and to 57 ⫾ 3.5, 32.5 ⫾ 3.6, and 8.8 ⫾ 1.2% (mean ⫾ SD; n ⫽ 4 analysis) of total after digestion (Fig. 1D). Thus, the proportion of dibiotinylated monomers was about half
of that measured before the addition of trypsin. The same results was obtained when the relative percentage was calculated based on the intensity of the multicharged ions for each TNF species. Assuming no differences during the ionization process among the various biotin–TNF monomers and considering that the biotin linked to the N-terminal ␣-amino group is lost after limited digestion, these results suggest that about a half of the biotin groups in biotin-TNF 1 are conjugated to the N-terminal Val-1. The signal intensity of peaks after digestion is consistent with the presence of other monomers bearing 1 biotin linked to ⑀-amino groups in the 7–157 region, plus similar compounds deriving from dibiotinylated species that lost the biotinylated N-terminal 1– 6 residue. Similar results were obtained with biotin-TNF 1. Identification of Biotinylation Sites on Biotin–TNF Conjugates To determine the position of the ⑀-amino groups that are accessible and that may react with activated biotin, we have prepared another conjugate using stronger reaction conditions (biotin–TNF 2). Deconvoluted mass spectra obtained by ESI-MS analysis of the product showed the presence of four peaks corresponding to biotinylated monomers with one (17,687 Da), two
SPECTROMETRIC CHARACTERIZATION OF BIOTINYLATED TUMOR NECROSIS FACTOR
FIG. 2.
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HPLC separation (A) and ESI-MS (B–F) identification of biotinylated species present in biotin–TNF 2.
(18,027 Da), and three (18,367 Da) biotin residues and to unmodified TNF (17,349 Da) (4). To facilitate subsequent analysis we attempted to separate the various species present in the mixture by reverse-phase liquid chromatography. HPLC analysis of 0.6 nmol of biotin–TNF 2 revealed the presence of seven major peaks (Fig. 2A, a– g). To avoid mixtures deriving from peak overlapping, fraction collection was limited from the starting point to the peak top (peak a and peak c) or from the top to the end (peak b and peak d). No separation was obtained for peaks e, f, and g which were collected
together. ESI-MS analysis of fractions (Figs. 2B–2F) showed the presence of unmodified protein in Fr-a (rt 4 min) and subunits with one biotin in Fr-b (rt 5 min). Fr-c (rt 7 min) also contained subunits with one biotin while Fr-d (rt 8 min) and Fr-e– g contained TNF subunits with one and two or two and three biotins, respectively. The different chromatographic behavior of TNF molecules containing one biotin residue (fractions b and c; Figs. 2B and 2C) or two biotin groups (fractions d and e; Figs. 2E and 2F) suggests the presence of TNF isomers with the biotin residues located at different positions.
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FIG. 3. Separation and identification of peptides obtained by complete digestion of TNF and biotin–TNF 2 with trypsin. Peptides were separated and identified by micro-LC-ESI (ion chromatogram m/z 400 –2000; left panels). Numbers on the peaks identify peptides as reported in Table 1. Raw ESI spectra of biotinylated peptides (peak 15–20) are shown in the right panels. Numbers above the m/z values (bold) indicate the number of positive charges. Ion chromatograms of fractions f and g were similar to that of fraction e (not shown).
To enable tryptic peptide mapping, HPLC separation was then carried out on a larger amount of biotin-TNF 2 (500 g). When the higher amount of protein was purified by HPLC, however, peaks a and c were combined with peaks b and d, respectively, because of the incomplete chromatographic separation.
The LC-ESI-MS chromatogram obtained with the tryptic digest of Fr-(a⫹b) was very similar to that obtained with the tryptic digest of TNF (Fig. 3), except for peak 15 corresponding to a peptide with M r 612.5 ⫾ 0.2 Da. This value corresponds to that expected (612 Da) for biotin-Val-Arg, a peptide derived from cleavage
SPECTROMETRIC CHARACTERIZATION OF BIOTINYLATED TUMOR NECROSIS FACTOR
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that biotinylation at pH 5.8 has favored biotinylation of the N-terminus.
FIG. 4. Amino acid sequence of TNF. The expected trypsin cleavable sites are shown in bold. Open arrowheads indicate the sites cleaved by trypsin in TNF (ƒ) and the biotinylated ␣- and ⑀-amino groups (2) in biotin–TNF 2 are also shown.
at Arg-2 of biotin–TNF. The HPLC profile of tryptic peptides derived from Fr-(c⫹d) and Fr-e differed from that of Fr-(a⫹b) for the presence of five new peaks in the ion chromatogram (peaks 16 –20 in Fig. 3) which were eluted from 40 to 50 min. No difference was found between the HPLC profile of tryptic peptides derived from fraction e (Fr-e) and those of fractions f and g. Only three of these peaks were present in Fr-(c⫹d) and their intensity increased from Fr-(c⫹d) to Fr-g. The structure of peptides corresponding to peaks 15–20 (Table 1) was identified by comparing the molecular mass of the tryptic fragments determined experimentally by LC-ESI-MS (Fig. 3) with those predicted for the cleavage at the arginine and lysine sites (Fig. 4) and considering that biotinylated lysine are non cleavable by trypsin (13). Thus, fraction (c⫹d) contained TNF subunits with one and two biotins bound to Lys65, Lys-112, or Lys-128 or at the N-terminal Val-1. Fr-e, Fr-f, and Fr-g were similar to Fr-(c⫹d), except for two additional peptides deriving from incomplete tryptic digestion: one with M r 3856, corresponding to residues 104 –131 with two biotins, likely on Lys-112 and Lys-128 and the other with M r 6856, corresponding to residues 45–103 with one biotin. Thus, biotin– TNF 2 is a complex mixture of trimers containing subunits with different number of biotins located in different positions, according to various possible combination. The results suggest that Lys-11, Lys-90, and Lys-98 are not modified under the described biotinylation conditions and/or TNF biotinylated at these ⑀-amino groups is present in a not detectable amount under our analytical conditions. Evaluation of the structure of tryptic peptides also suggests that O-biotinylation of Ser, Thr, and Tyr residues, previously described to occur during biotinylation of other proteins (14), was negligible in TNF. Since the N-hydroxysuccinimide ester is usually more reactive toward an ␣-amino group than an ⑀-amino group at a relatively low pH (15), it is possible
Activity of Biotin–TNF Species To investigate the structure–function relationships of biotin–TNF in the presence of avidin, the cytotoxic activity of Fr-(a⫹b) and Fr-(c⫹d) was then analyzed using a standard L-M cells cytolytic assay, both in the presence and in the absence of 10 g/ml avidin. The activity of these fractions in the absence of avidin (1.0 ⫻ 10 3 and 2.4 ⫻ 10 3 U/mg, respectively) was markedly lower than that of the starting material (4.4 ⫻ 10 7 units/mg). The activity of Fr-(a⫹b) was modestly or not inhibited by avidin, while Fr-(c⫹d) was completely inhibited (data not shown). To check whether the loss of activity after chromatography was attributable to the separation procedure, TNF (5 ⫻ 10 7 U/mg) was dissolved into the HPLC eluent and tested after dilution with cell culture medium. Also this treatment caused a marked loss of activity (several orders of magnitude) indicating that TNF denaturation may occur during and/or after chromatography. However, the finding that the residual activity of molecules bearing biotins on both ␣- and ⑀-amino groups can be completely inhibited by avidin suggests that mild biotinylation is necessary to obtain conjugates that maintain their activity when bound to avidin (4). Since the N-terminal region is located in a flexible segment (16) and is not necessary for receptor binding (17) we speculate that the ␣-amino groups in the N-terminus may represent an ideal position for biotinylation, while the ⑀-amino groups of lysines, more close to the receptor binding site, are likely responsible for the inhibition observed. The present results confirm the usefulness of mass spectrometry in the evaluation of surface labeling at specific site in relation to the protein activity. As a matter of fact this technique has been used to probe conformation changes (18), protein to protein interactions (19, 20), and protein folding (21). In conclusion, this study has shown that the most abundant species in a conjugate that has been proven previously to be active in animal models of tumor pretargeting with avidin is likely made up by TNF monomers with one biotin on the N-terminus, while the rest of biotins are distributed among the ⑀-amino group of Lys-65, Lys-112, and Lys-128. These results may help future development of TNF muteins with improved biotinylation properties. ACKNOWLEDGMENT This work was supported by the Associazione Italiana per la Ricerca sul Cancro (AIRC).
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Biotinylation Sites of Tumor Necrosis Factor-␣ Determined by Liquid Chromatography–Mass Spectrometry Fulvio Magni,* Flavio Curnis,† Laura Marazzini,* Roberto Colombo,‡ Angelina Sacchi,† Angelo Corti,† and Marzia Galli Kienle‡ *Mass Spectrometry Laboratory, IRCCS S. Raffaele, Milan, Italy; †DIBIT, San Raffaele H Scientific Institute, Milan, Italy; ‡Department of Medical Chemistry and Biochemistry, University of Milan Bicocca, Milan, Italy; and §Catholic University Holy Heart of Milan, Milan, Italy
Received February 5, 2001; published online October 18, 2001
Tumor pretargeting with biotinylated antibody/avidin complexes improves the therapeutic index of systemically administered biotin–tumor necrosis factor (TNF) conjugates. Since the number of biotins in this conjugate is known to be critical for activity, we have characterized the structure of different biotin–TNF conjugates, prepared by reaction with D-biotinyl-6aminocaproic acid N-hydroxysuccinimide ester and identified the biotinylation sites by trypsin digestion, reverse-phase chromatography, and electrospray mass spectrometry analyses. The results have shown that N-terminal valine is a preferential biotinylation site at pH 5.8, half of biotins being located on the ␣-amino group of this residue in a conjugate bearing one biotin/trimer (on average). Moreover, evidence has been obtained to suggest that the remaining part of biotins are linked to the ⑀-amino group of lysine 128, 112, and 65, while lysine 11, 90, and 98 were practically unmodified. No evidence of O-biotinylation of serine, threonine and tyrosine was obtained. © 2001 Academic Press Key Words: tumor necrosis factor; mass spectrometry; biotin; tumor therapy; electrospray; HPLC.
Human tumor necrosis factor ␣ (TNF) 1 is a nonglyco homotrimeric protein produced by a large number of cell types and organs in response to various inflammatory and immunological stimuli. Several studies have shown that TNF is endowed with potent antitumor activity in animal models (1). However, the clinical use of TNF as an anticancer drug is limited by severe systemic toxicity (2, 3). In the attempt to overcome this problem, we have recently developed a new strategy based on tumor 1 Abbreviations used: TNF, tumor necrosis factor; TFA, trifluoroacetic acid; ESI-MS, electrospray mass ionization; LC, liquid chromatography.
0003-2697/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.
pretargeting with biotinylated antibody/avidin complexes and systemic administration of biotinylated TNF (4, 5). This pretargeting approach was shown to increase at least five times the antitumor activity of biotinylated TNF in animal models, with no evidence of increased toxicity (6). Studies on the mechanism of action showed that biotin–TNF trimers can slowly dissociate from the targeted cells through trimer–monomer–trimer transition and that the antitumor activity is based mainly on indirect effects, presumably on cells other than the targeted tumor cells (4, 6). We have also shown that nonbiotinylated subunits must be present within TNF trimers for an efficient release of bioactive TNF from the targeting complex. Since the number and the position of biotins in these conjugates is likely critical for the activity, the identification of the product present in the mixture after biotinylation is very useful. Mass spectrometry is a powerful tool to study covalent structure of proteins in conjunction with enzymatic digestion which provides short peptides to be used for additional information on the sequence (7–10). In this work we have characterized the structure of different biotin–TNF conjugates and identified the biotinylation sites, using total and partial trypsin digestion and mass spectrometry analysis. MATERIALS AND METHODS
Preparation of TNF and Biotin–TNF Conjugates Human TNF (5 ⫻ 10 7 U/mg) was produced by recombinant DNA technology. The cDNA coding for TNF (11) was cloned in pET-11b (Novagen, Madison, WI) and used to transform BL21 (DE3) Escherichia coli cells (Novagen). Soluble TNF was recovered from 2-liter cultures by bacterial sonication in 2 mM EDTA, 20 mM Tris–HCl, pH 8.0, followed by centrifugation (15,000g, 20 min, 4°C). The extract was mixed with ammonium sulfate (25% of saturation), left for 1 h at 4°C, and 181
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further centrifuged, as above. The ammonium sulfate in the supernatant was then brought to 65% of saturation, left at 4°C for 24 h, and further centrifuged. The pellet was dissolved in 200 ml of 1 M ammonium sulfate, 50 mM Tris–HCl, pH 8.0, and purified by hydrophobic interaction chromatography on phenyl–Sepharose 6 Fast Flow (Amersham Pharmacia Biotech, Piscataway, NJ) (gradient elution, buffer A: 50 mM sodium phosphate, pH 8.0, containing 1 M ammonium sulfate; buffer B: 20% glycerol, 5% methanol, 50 mM sodium phosphate, pH 8.0). Fractions containing TNFimmunoreactive material (identified by Western blotting) were pooled, dialyzed against 5 mM EDTA, 20 mM Tris–HCl, pH 8.0, and further purified by ionexchange chromatography on DEAE–Sepharose Fast Flow (Pharmacia-Upjohn) (gradient elution, buffer A: 20 mM Tris–HCl, pH 8.0; buffer B: 1 M sodium chloride, 20 mM Tris–HCl, pH 8.0). Fractions containing TNF immunoreactivity were pooled and purified by gel filtration chromatography on Sephacryl-S-300 HR (Pharmacia-Upjohn), preequilibrated and eluted with 150 mM sodium chloride, 50 mM sodium phosphate buffer, pH 7.3. Fractions corresponding to 40,000 –50,000 M r products were pooled and stored frozen at ⫺20°C. Biotin–TNF conjugates with different degrees of biotin incorporation, termed biotin–TNF 1 and biotin– TNF 2, were prepared. Biotin–TNF 1 was obtained by mixing 0.72 ml of TNF (0.41 mg/ml in sodium citrate buffer, pH 5.8) with 0.066 ml of biotin-6-aminocaproylN-hydroxysuccinimide ester (1.82 mg/ml; Societa` Prodotti Antibiotici, Milan, Italy). After 3 h incubation at 22–23°C, the product was mixed with 0.0825 ml of 1 M lysine in water and left to incubate for 1 h. The product was dialyzed against 0.9% sodium chloride and stored at ⫺20°C. Biotin–TNF 2 was prepared in the same way except that 0.1 M sodium borate, pH 8.0, was used as a reaction buffer with 0.066 ml of biotinylating reagent (3 mg/ml in dimethyl sulfoxide). Of note, we observed that different batches of biotin reagent may result in different degrees of biotinylation. HPLC HPLC of biotin–TNF 2 was performed using a C4 reverse-phase column (50 ⫻ 4 mm, Vydac, Hesperia, CA) and a Jasco HPLC system (Jasco Spectroscopic Co. LTD, Tokyo, Japan) equipped with a single pump (Model 880-PU) and a ternary gradient mixer (Model 880-02). The UV detector (Jasco Model 875-UV/VIS) was set at 210 nm. The column was equilibrated with 34% aqueous acetonitrile containing 0.1% TFA. Bound proteins were eluted with a shallow gradient from 34 to 37% acetonitrile over 17 min, starting 3 min after sample injection. Acetonitrile was then increased to 60% in 5 min (flow rate, 1 ml/min). Fractions were collected manually, dried using a SpeedVac System (Savant), and stored at ⫺20°C.
Tryptic Digestion Limited trypsin digestions of wild-type TNF and biotin–TNF 1 were obtained by mixing 0.1– 0.2 mg/ml of each product, previously dialyzed against distilled water, with 0.3 mg/ml trypsin in distilled water (TNF/ trypsin, 20/1 by weight). Aliquots of each mixture were withdrawn after 10, 30, 60, 120, and 480 min of incubation at room temperature and stored at ⫺20°C until analysis by ESI-MS. Complete trypsin digestion of TNF and HPLC-isolated biotin–TNF was obtained by mixing 0.03– 0.2 mg of each product with trypsin in 0.05– 0.2 ml of 50 mM ammonium bicarbonate buffer, pH 8.5 (TNF/trypsin, 50/1 by weight) and by incubating at 37°C for 180 min. The reaction was terminated by freezing the samples at ⫺20°C. ESI Analysis To determine the number of biotins incorporated in each product, biotin–TNF conjugates were dialyzed for 30 – 60 min against distilled water or desalted using ZipTip C18 (Millipore Corp., Bedford, MA) and analyzed by ESI-MS (100 –300 ng of protein) using water: methanol (1:1, v/v) containing 0.1% acetic acid as delivering solvent (flow rate, 3 l/min). Mass spectra were obtained with a Finnigan MAT95 double focusing mass spectrometer. Mass spectrometry conditions were capillary voltage 3.1 kV, heated capillary 250°C and 37 V, tube lens 77 V, skimmer voltage ⫺0.8 V, and octapole voltage 3.4 V. Spectra were recorded from m/z 500 to m/z 2500 at 3 s/decade. System calibration and sensitivity was daily evaluated using 0.5 pmol of apomyoglobin from horse skeletal muscle (Sigma-Aldrich, Milan, Italy). The error in the determination of the molecular mass, calculated from the deconvoluted spectrum of apomyoglobin (16951 Da), was ⫾0.02%. Micro-LC-ESI-MS Peptides deriving from the tryptic digestion of biotin–TNF conjugates were analyzed by micro-LC-ESIMS. About 20 l of each mixture was injected into a Wakosil C18 column (150 ⫻ 1 mm; 5 m; 120 Å, SGE, Rome, Italy), directly connected to the ion source. The column was eluted for 2 min with 5% of acetonitrile, containing 0.025% of TFA, followed by a linear gradient from 5 to 45% over 58 min (flow rate, 50 l/min). Solvent mixing was obtained with a biocompatible tee (HPLC Technologies, Macclesfield-Cheshire, UK). The column was then cleaned by increasing acetonitrile to 80% in 8 min, and reequilibrated for 30 min with the starting solvent before subsequent analysis. In Vitro Cytolytic Assays The bioactivities of TNF and biotin–TNF conjugates were estimated by standard cytolytic assay based on L-M mouse fibroblasts (ATCC CCL1.2) as described (12).
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FIG. 1. Deconvoluted ESI spectra of TNF and of biotin–TNF 1 before (A and C) and after (B and D) partial enzymatic proteolysis for 10 min. Numbers (in italics) above the calculated M r (in bold) of the various biotin–TNF monomers indicate the biotin residues per subunit. Tryptic digestion was carried out as described in the text.
RESULTS AND DISCUSSION
Human TNF contains six lysine residues and one ␣-amino group potentially reactive with D-biotinyl-6aminocaproic acid N-hydroxysuccinimide ester. We have shown previously that TNF can be biotinylated to various extents depending on the pH of the reaction and reagent concentration (4). Optimal activity for in vivo tumor targeting experiments (4) can be achieved with a biotin–TNF 1 bearing one biotin/trimer (on average), prepared at pH 5.8. This conjugate is a mixture of trimers made up by monomers with 0, one, and two biotins. Within trimers with the same number of biotins a high heterogeneity of molecular structure may arise also from combination of subunits bearing biotins in different position. We estimated that the biotin– TNF 1 is made up by trimers without biotin (27%) and
trimers with one (43%), two (24%), and three (5.4%) biotin residues (4). To investigate the biotinylation sites in biotin–TNF we have prepared different conjugates with different contents of biotin residues. These conjugates termed 1 and 2 were prepared and characterized by ESI-MS as described in a previous report (4). In this work we evaluated their molecular masses and that of nonbiotinylated TNF by ESI-MS and of their tryptic maps after enzymatic digestion. Mass spectra were obtained by deconvolution of the raw data by an algorithm implemented into the instrument software. The deconvoluted mass spectrum of TNF 1–157 (Fig. 1A) showed an intense peak corresponding to a molecular mass of 17,349 ⫾ 1.2 Da, in good agreement with the value expected for human TNF monomer, calculated on the
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Peptides Identified by LC-ESI after Tryptic Digestion of TNF and Biotin–TNF 2 Expected Peak
Measured M r
Fragment
Mr
Biotinylated residue
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
2755.4 1240.6 2424.8 907.7 857.4 2851.3 2901.7 2394.2 1396.6 2911.6 3180.2 6514.3 4801.2 5641.2 612.5 3191 3519.4 6853.8 3856.4 5141.3
7–31 33–44 45–65 83–90 91–98 104–128 132–157 66–82–SS–99–103 a 32–44 b 7–32 b 104–131 b 45–SS–103 a,b 45–82–SS–99–103 a 45–82–SS–91–103 a 1–2 104–128 104–131 45–103 104–131 45–82–SS–99–103 a,b
2756 1240 2426 909 857 2852 2902 2395 1397 2912 3181 6515 4803 5642 612 3190 3518 6853 3856 5141
— — — — — — — — — — — — — — Val-1 Lys-112 Lys-112 or Lys-128 Lys-65 or Lys-112 or Lys-128 Lys-112 and Lys-128 Lys-65
Note. The expected molecular mass (M r) was calculated using PAWS software (available at www.proteomics.com) on the basis of the human TNF sequence (7). a Disulfide-linked fragments. b Peptide deriving from incomplete tryptic digestion.
basis of its amino acid sequence (M r ⫽ 17,351). Two additional peaks, corresponding to monomers bearing 1 and 2 biotins were observed in the mass spectrum of biotin–TNF 1 (17,687 and 18,027, respectively, Fig. 1C). Limited digestions of both TNF and biotin–TNF 1 with trypsin were then carried out in distilled water. ESI-MS of TNF after 10 min of digestion revealed the presence of a main peptide with M r 16,675, corresponding to cleavage at Arg-6 (TNF 7–157, Fig. 1B). This peptide was resistant to further proteolysis under these conditions, as it was still observed after 8 h of digestion. When this work was in progress a similar observation about the resistance of TNF to proteolysis was reported (10). Thus, partial proteolysis in distilled water was exploited to study the proportion of biotins on the N-terminus and on the rest of the TNF molecule. At least three peptides with M r 16,675, 17,015, and 17,354 were obtained by partial digestion of biotin–TNF 1 (Fig. 1D), corresponding to residues 7–157 with 0, one, and two biotins (Table 1). Based on the signal intensities in the deconvoluted mass spectra, native, monoand di-biotinylated monomers corresponded to 39.5 ⫾2.8, 45 ⫾2.7, and 15.6 ⫾3.4% (mean ⫾ SD; n ⫽ 4 analysis) of the total before digestion (Fig. 1C) and to 57 ⫾ 3.5, 32.5 ⫾ 3.6, and 8.8 ⫾ 1.2% (mean ⫾ SD; n ⫽ 4 analysis) of total after digestion (Fig. 1D). Thus, the proportion of dibiotinylated monomers was about half
of that measured before the addition of trypsin. The same results was obtained when the relative percentage was calculated based on the intensity of the multicharged ions for each TNF species. Assuming no differences during the ionization process among the various biotin–TNF monomers and considering that the biotin linked to the N-terminal ␣-amino group is lost after limited digestion, these results suggest that about a half of the biotin groups in biotin-TNF 1 are conjugated to the N-terminal Val-1. The signal intensity of peaks after digestion is consistent with the presence of other monomers bearing 1 biotin linked to ⑀-amino groups in the 7–157 region, plus similar compounds deriving from dibiotinylated species that lost the biotinylated N-terminal 1– 6 residue. Similar results were obtained with biotin-TNF 1. Identification of Biotinylation Sites on Biotin–TNF Conjugates To determine the position of the ⑀-amino groups that are accessible and that may react with activated biotin, we have prepared another conjugate using stronger reaction conditions (biotin–TNF 2). Deconvoluted mass spectra obtained by ESI-MS analysis of the product showed the presence of four peaks corresponding to biotinylated monomers with one (17,687 Da), two
SPECTROMETRIC CHARACTERIZATION OF BIOTINYLATED TUMOR NECROSIS FACTOR
FIG. 2.
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HPLC separation (A) and ESI-MS (B–F) identification of biotinylated species present in biotin–TNF 2.
(18,027 Da), and three (18,367 Da) biotin residues and to unmodified TNF (17,349 Da) (4). To facilitate subsequent analysis we attempted to separate the various species present in the mixture by reverse-phase liquid chromatography. HPLC analysis of 0.6 nmol of biotin–TNF 2 revealed the presence of seven major peaks (Fig. 2A, a– g). To avoid mixtures deriving from peak overlapping, fraction collection was limited from the starting point to the peak top (peak a and peak c) or from the top to the end (peak b and peak d). No separation was obtained for peaks e, f, and g which were collected
together. ESI-MS analysis of fractions (Figs. 2B–2F) showed the presence of unmodified protein in Fr-a (rt 4 min) and subunits with one biotin in Fr-b (rt 5 min). Fr-c (rt 7 min) also contained subunits with one biotin while Fr-d (rt 8 min) and Fr-e– g contained TNF subunits with one and two or two and three biotins, respectively. The different chromatographic behavior of TNF molecules containing one biotin residue (fractions b and c; Figs. 2B and 2C) or two biotin groups (fractions d and e; Figs. 2E and 2F) suggests the presence of TNF isomers with the biotin residues located at different positions.
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FIG. 3. Separation and identification of peptides obtained by complete digestion of TNF and biotin–TNF 2 with trypsin. Peptides were separated and identified by micro-LC-ESI (ion chromatogram m/z 400 –2000; left panels). Numbers on the peaks identify peptides as reported in Table 1. Raw ESI spectra of biotinylated peptides (peak 15–20) are shown in the right panels. Numbers above the m/z values (bold) indicate the number of positive charges. Ion chromatograms of fractions f and g were similar to that of fraction e (not shown).
To enable tryptic peptide mapping, HPLC separation was then carried out on a larger amount of biotin-TNF 2 (500 g). When the higher amount of protein was purified by HPLC, however, peaks a and c were combined with peaks b and d, respectively, because of the incomplete chromatographic separation.
The LC-ESI-MS chromatogram obtained with the tryptic digest of Fr-(a⫹b) was very similar to that obtained with the tryptic digest of TNF (Fig. 3), except for peak 15 corresponding to a peptide with M r 612.5 ⫾ 0.2 Da. This value corresponds to that expected (612 Da) for biotin-Val-Arg, a peptide derived from cleavage
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that biotinylation at pH 5.8 has favored biotinylation of the N-terminus.
FIG. 4. Amino acid sequence of TNF. The expected trypsin cleavable sites are shown in bold. Open arrowheads indicate the sites cleaved by trypsin in TNF (ƒ) and the biotinylated ␣- and ⑀-amino groups (2) in biotin–TNF 2 are also shown.
at Arg-2 of biotin–TNF. The HPLC profile of tryptic peptides derived from Fr-(c⫹d) and Fr-e differed from that of Fr-(a⫹b) for the presence of five new peaks in the ion chromatogram (peaks 16 –20 in Fig. 3) which were eluted from 40 to 50 min. No difference was found between the HPLC profile of tryptic peptides derived from fraction e (Fr-e) and those of fractions f and g. Only three of these peaks were present in Fr-(c⫹d) and their intensity increased from Fr-(c⫹d) to Fr-g. The structure of peptides corresponding to peaks 15–20 (Table 1) was identified by comparing the molecular mass of the tryptic fragments determined experimentally by LC-ESI-MS (Fig. 3) with those predicted for the cleavage at the arginine and lysine sites (Fig. 4) and considering that biotinylated lysine are non cleavable by trypsin (13). Thus, fraction (c⫹d) contained TNF subunits with one and two biotins bound to Lys65, Lys-112, or Lys-128 or at the N-terminal Val-1. Fr-e, Fr-f, and Fr-g were similar to Fr-(c⫹d), except for two additional peptides deriving from incomplete tryptic digestion: one with M r 3856, corresponding to residues 104 –131 with two biotins, likely on Lys-112 and Lys-128 and the other with M r 6856, corresponding to residues 45–103 with one biotin. Thus, biotin– TNF 2 is a complex mixture of trimers containing subunits with different number of biotins located in different positions, according to various possible combination. The results suggest that Lys-11, Lys-90, and Lys-98 are not modified under the described biotinylation conditions and/or TNF biotinylated at these ⑀-amino groups is present in a not detectable amount under our analytical conditions. Evaluation of the structure of tryptic peptides also suggests that O-biotinylation of Ser, Thr, and Tyr residues, previously described to occur during biotinylation of other proteins (14), was negligible in TNF. Since the N-hydroxysuccinimide ester is usually more reactive toward an ␣-amino group than an ⑀-amino group at a relatively low pH (15), it is possible
Activity of Biotin–TNF Species To investigate the structure–function relationships of biotin–TNF in the presence of avidin, the cytotoxic activity of Fr-(a⫹b) and Fr-(c⫹d) was then analyzed using a standard L-M cells cytolytic assay, both in the presence and in the absence of 10 g/ml avidin. The activity of these fractions in the absence of avidin (1.0 ⫻ 10 3 and 2.4 ⫻ 10 3 U/mg, respectively) was markedly lower than that of the starting material (4.4 ⫻ 10 7 units/mg). The activity of Fr-(a⫹b) was modestly or not inhibited by avidin, while Fr-(c⫹d) was completely inhibited (data not shown). To check whether the loss of activity after chromatography was attributable to the separation procedure, TNF (5 ⫻ 10 7 U/mg) was dissolved into the HPLC eluent and tested after dilution with cell culture medium. Also this treatment caused a marked loss of activity (several orders of magnitude) indicating that TNF denaturation may occur during and/or after chromatography. However, the finding that the residual activity of molecules bearing biotins on both ␣- and ⑀-amino groups can be completely inhibited by avidin suggests that mild biotinylation is necessary to obtain conjugates that maintain their activity when bound to avidin (4). Since the N-terminal region is located in a flexible segment (16) and is not necessary for receptor binding (17) we speculate that the ␣-amino groups in the N-terminus may represent an ideal position for biotinylation, while the ⑀-amino groups of lysines, more close to the receptor binding site, are likely responsible for the inhibition observed. The present results confirm the usefulness of mass spectrometry in the evaluation of surface labeling at specific site in relation to the protein activity. As a matter of fact this technique has been used to probe conformation changes (18), protein to protein interactions (19, 20), and protein folding (21). In conclusion, this study has shown that the most abundant species in a conjugate that has been proven previously to be active in animal models of tumor pretargeting with avidin is likely made up by TNF monomers with one biotin on the N-terminus, while the rest of biotins are distributed among the ⑀-amino group of Lys-65, Lys-112, and Lys-128. These results may help future development of TNF muteins with improved biotinylation properties. ACKNOWLEDGMENT This work was supported by the Associazione Italiana per la Ricerca sul Cancro (AIRC).
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