Bioconjtgate Chem. 1992, 3, 404-492
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Identification and Characterization of a Nucleotide Binding Site on Recombinant Murine Granulocyte/Macrophage-Colony Stimulating Factor Michael A. Doukas,’p+ Ashok J. Chavan,* Cecelia Gas,+Thomas Boone,s and Boyd E. Haley* Colleges of Medicine and Pharmacy, Lucille P. Markey Cancer Center, University of Kentucky and Veterans Affairs Medical Center, Lexington, Kentucky 40536-0093, and Amgen, Inc., Amgen Center, Thousand Oaks, California 91320. Received March 26, 1992
Granulocytelmacrophage-colonystimulating factor (GM-CSF) is a regulatory cytokine important in the proliferative and functional activation of hematopoietic cells. It belongs to a family of 20 kDa or less acidicglycoprotein moleculesfound in a broad range of Lellular sources. On the basis of the previously reported nucleotide-binding properties of interleukin-2 (IL-21, atrial natriuretic factor (ANF), and glucagon, the interaction of GM-CSF with nucleotides was investigated. Using radiolabeled 8-miand T PApdA, ) the putative biological doadenosine-containing photoprobes of ATP ( [ T - ~ ~ P I - ~ N ~ A alarmone ( [p’-32Pl-8N3Ap&), we have identi‘ed a nucleotide binding site on recombinant murine GM-CSF (rmGM-CSF). Specificity of bindilig was demonstrated by saturation and competition experiments. Saturation of photoinsertion by [ T - ~ ~ P I - ~ N ~and A T [/3’-32Pl-8N3Ap& P occurs with apparent Kd)s of 10 and 0.7 pM, respectively. Using an immobilized Fe3+ affinity chromatography technique, developed specifically for the isolation of photolabeled peptides, a single radiolabeled peptide was isolated. It was identified as amino acids 5-14 near the N-terminus of GM-CSF. This peptide region has been shown in previous studies to be critical for biological activity. Also consistent with this observation is our finding that the photolabeled GM-CSF has lost most, if not all, of its biological activity, as determined by a cellular proliferation assay.
INTRODUCTION
Granulocyte/macrophage-colonystimulating factor (GMCSF)’ has a broad range of proliferative, differentiating, and activating effects upon inflammatory cells and their precursors (1,2). While original work focused primarily on neutrophils, monocytes, and their precursors, the activities of GM-CSF are now known to include effects upon the eosinophil and basophil lineages. GM-CSF in vitro has additive effects (to IL-5) on eosinophil colony formation (3) and in clinical trials provokes a profound eosinophilia and neutrophilia (4). This cytokine also activates the mature eosinophils and neutrophils with regard to cellular activities (2). GM-CSF also promotes basophil production in liquid culture (5). This broad range of activities for GM-CSF supports the view that this cytokine is central, or certainly contributory, to most inflammatory processes. Receptor binding of GM-CSF appears to be mediated by ~tleast two membrane glycoproteins (aand 0) which together bind GM-CSF with high affinity and transduce a biolagical signal (6) On some cell lines, however, there ~~
~
* Author
~
to whom correspondence should be addressed: Hematology/Oncology Section (lll-E/CDD), Veterans Affairs Medical Center, 2250 Leestown Road, Lexington, KY 405111093. Phone No. (606) 281-4956; Fax No. (606) 257-1020. + College of Medicine, University of Kentucky and Veterans Affairs Medical Center. College of Pharmacy, University of Kentucky Medical Center. Amgen, Inc. Abbreviations used GM-CSF, granulocyte/macrophage colony stimulating factor; IL, interleukin; ANF, atrial natriuretic factor; 8-N3ATP, &midoadenosine triphosphate; &NsGTP, 8-azidoguanosine triphosphate; 8-N3Ap&, 8-midoadenosine 5/,5”‘-P1,P-tetraphosphate adendsine; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; CBB, Coomassie Brilliant Blue; TNF, tumor necrosis factor.
*
are only low affinity (a) GM-CSF receptors, which apparently do not transduce a signal and whose function is unclear at this time (7).Indirect evidence as to the activities of the receptors in subsequent signal-transduction events confirms the importance of protein kinase activation (8) and specifically tyrosine phosphorylation (9). While experimentation has revealed parts of the puzzle regarding receptor physiology and subsequent signal transduction, no clear view has yet emerged as to the signaling events triggered by cytokines which lead to cellular proliferation and specific gene transcription. The mature murine GM-CSF consists of a polypeptide chain of 124 amino acids with a great deal of hydrophobicity and conformational stability (10). The three-dimensional structure of GM-CSF has been predicted by molecular modeling techniques (11). The initial report on the crystal structure of the human GM-CSF indicates that it crystallizes with two molecules in the asymmetric unit (12, 13).
As an energy source for certain cellular signaling mechanisms, nucleotides are critical in the regulation of biological systems. Much of our knowledge concerning regulation of these systems comes from research utilizing analogs of the various nucleotides. Nucleotide photoaffinity probes have been very effectively used for this purpose (14, 15). A possible role for nucleotides in the mechanism of action of small peptide hormones and cytokines has been suggested by demonstration of specific nucleotide binding sites on IL-2, ANF, and glucagon. IL-2 has high affinity for ATP and NAD+ (16),whereas ANF and glucagon has high affinity for GTP (17, 18). In the present study, utilizing the nucleotide photoaffinity probes [y-32Pl-8N3ATPand [p-32Pl-8N3Ap&, we have detected, characterized, and identified a nucleotide binding site on GM-CSF. We describe the detection and specificity of binding and the identification of the peptide region of the 0 1992 American Chemical Society
Nucieotlde-Binding Properties of rmGM-CSF
GM-CSF moleculewhich is involved in nucleotide binding. The specificity and very low concentrations at which nucleotide binding occurs, and the loss of biological activity of GM-CSF with covalently attached nucleotide, indicate a possible physiological role for these novel interactions. EXPERIMENTAL PROCEDURES
Materials. rmGM-CSF (Escherichia coli derived) was kindly provided by Amgen, Inc., and was >95% pure by SDS-PAGE and reverse-phase HPLC analysis. Protein molecular weight standards were obtained from Bio-Rad. All other reagents were analytical grade and obtained from Sigma or Aldrich. H332P04was from ICN. Synthesis of Nucleotide Photoaffinity Probes. [y-32P]-8N3ATPand [y-32Pl-8N3GTP(5-30 mCi/pmol) were synthesized using the previously published procedures (14,19). [j3'-32Pl-8N3Ap4A (5-15 mCi/pmol) was synthesized as described elsewhere (20). Photolabeling of rmGM-CSF. Samples containing 0.2 pg of rmGM-CSF in 40-60 pL of photolysis buffer (20 mM NaHzP04, pH 4.5) were incubated at 4 "C in Eppendorf tubes with photoprobe for 15 s, followed by a 45-5 irradiation at 4 "C with a hand-held 254-nm UV lamp (intensity = 7200 pW/cm2). The reaction was quenched by addition of a protein-solubilizing mixture consisting of 10% SDS, 3.6 M urea, 162 mM dithiothreitol, pyronin Y (tracking dye), and 20 mM Tris (pH 8.0). For protection studies, rmGM-CSF was first incubated for 60 s at 4 "C with the competitor, followed by incubation with photoprobe for 15 s at 4 "C and photolysis for 45 s at 4 "C. For the multiple photolysis experiments, 0.2 pg of rmGMCSF was incubated sequentially with either 16.7, 12.5, and 10 pM [8'-32Pl-8N3Ap4Aor 167, 125, and 100 pM [y-32Pl-8N&TP for 15 s each at 4 OC and photolyzed for 45 s between each sequential addition of the probe a t 4 "C. The samples photolyzed once, twice, and three times were analyzed separately by SDS-PAGE to determine the percent photoincorporation after each step. The percent modification of cytokine was calculated by converting the cpm in gel pieces to mCi. Then, by making use of specific activity of the probe used, picomole of probe present with cytokine in the gel piece was calculated. Knowing the picomole of cytokine used for the experiment and assuming a single binding site on each cytokine molecule, the ratio of picomole of probe to picomole of cytokine afforded the percent modification. SDS-Polyacrylamide Gel Electrophoresis. Solubilized protein samples were subjected to electrophoresis in a 10% polyacrylamide separating gel with a 4 % stacking gel according to the method of Laemmli (21). Gels were either stained with Coomassie Brilliant Blue R, destained overnight, and dried on a slab gel dryer or fixed and dried without staining. Gels were subjected to autoradiography and 32Pincorporation was quantified by excision of the labeled protein bands and liquid scintillation counting utilizing a Packard Minaxi or Tri-Carb scintillation counter (counting efficiency, 99% for 32P). Isolation of the Photolabeled Tryptic Peptides of rmGM-CSFPhotolabeledwith [@'-32P]-8N3Ap4A. (a) Photolabeling and Trypsin Digestion. Two hundred microgramsof rmGM-CSF was incubated with photoprobe (cytokine to probe ratio of 1:5, sp act. = 3.9 mCi/pmol) for 45 s at 4 "C and photolyzed for 90 8 at 4 "C. This was followed by a second incubation with nonradioactive photoprobe (cytokine to probe ratio of 1:5) for 45 s at 4 "C and photolysis for 90 s at 4 "C. The cytokine was precipitated by adding an equal volume of ice-cold 7%
Bioconlugate Chem., Vol. 3, No. 6, 1992 485
perchloric acid. Trypsin digestion was done according to a procedure described elsewhere (22). The photolabeled cytokine was carboxamidomethylated on cysteine residues with iodoacetamide and then digested with modifiebi trypsin (5% w/w and 5% w/w after 3 h) for 18 h a t 37 "C. (b) Immobilized Fe3+Affinity Chromatography. In a plastic column 300 pL of resin, iminodiacetic acid-epoxy activated Sepharose 6B fast flow (Sigma), was washed successively with 10 mL of water, 10 mL of 50 mM ferric chloride, 20 mL of water, 15 mL of buffer A (100 mM ammonium acetate, pH 8.0),10 mL of buffer A containing 0.5 M NaC1,lOmL of buffer A, 10mL of buffer A containing 4 M urea, and 10 mL of buffer A. The tryptic digest from part (a) above was diluted with buffer A and passed through the resin very slowly. More than 90% of the radioactivity was retained on the resin. The resin was then successively washed with 15 mL of buffer A, 10 mL of buffer A containing 0.5 M NaC1,lO mL of buffer A, 10 mL of buffer A containing 4 M urea, and 10 mL of buffer A. Finally the photolabeled peptides were eluted with 10mL of buffer A containing 5 mM K2HP04. During washings 1.5-mL fractions were collected and assayed for radioactivity. Greater than 90 % of the radioactivity was eluted with the phosphate wash. About 5 % eluted with the other washes. (c) Reverse-Phase High-Performance Liquid Chromatography of Photolabeled Peptides. The fractions containing radioactivity from the metal affinity chromatography were pooled, concentrated, and analyzed by reversephase HPLC using an Aquapore RP-300 C8 column (220 X 4.6 mm, Brownlee Labs). Absorbance was monitored using a diode-array spectral detector. The gradient used was 0.1 % TFA at 0 min, 0.13'% TFA at 5 min, 0.1 % TFA in 70% acetonitrile at 65 min at 0.5 mL/min, and 0.5-mL fractions were collected. The fractions were assayed for radioactivity. The fractions containing radioactivity and indicating UV absorbance at 214 nm were subjected to amino acid sequence analysis using Applied Biosystems 477A pulse liquid protein sequencer with on-line PTH identification at the University of Kentucky Macromolecular Sequencing Facility. Isolation of the Photolabeled Tryptic Peptide of rmGM-CSFPhotolabeledwith [y-32P]-8NsATP.The procedure described above for the isolation of photolabeled peptide was followed for 150 pg of rmGM-CSF which was photolabeled twice with [y-32Pl-8N3ATP as described earlier (cytokine to probe ratio of 1:5, sp act. = 8.0 mCi/ pmol). Bioassay. The 8-N3Ap4A photolabeled rmGM-CSF samples were prepared by incubating 5 pg of cytokine with 16.5 pM probe for 45 s at 4 "C and then photolyzing for 90 s at 4 "C. This was followed by two more additions of 12.5 and 10pM probe and photolysis. The control sample was photolyzed for 4.5 min before the probe was added in three additions of 16.5, 12.5, and 10 pM, respectively. FDCP-1D cells were seeded (1X 104/well)into 96 well microtiter plates which contained rmGM-CSF standards or photolabeled samples in 1:2 serial dilutions starting at 200 ng/mL. The assay mixture in a final volume of 200 pL/well was then incubated for 48 h at 37 "C in a 5% COZ humidified incubator and pulsed with [3Hlthymidine (1 pCi/well) for the final 4 h. The contents of the wells were harvested onto glass-fiber filters and PHI thymidine incorporated into DNA was determined. Digestionof the Photolabeledand Nonphotolabeled rmGM-CSF with Trypsin and Chymotrypsin. Two micrograms of the cytokine were photolabeled with 1p M [@"32P]-8N3Ap&. To this was added 5 % w/w trypsin or chymotrypsin followedby incubation at room temperature
Doukas et el.
Bioconlugete Chem., Vol. 3, No. 6, 1992
488
Table I. Effect of pH on the Photoincorporation of the Photoprobe into rmGM-CSF % 32P incorpO % 32Pincorp [8'-32Pl [ - p P ][8'-32Pl[y32PlpH 8N3ApA 8NATP pH 8N3ApJ 8N3ATP 4.0 4.5 5.0 5.5 6.0
94 f 6 100 91 f 7 65 f 2 49 f 1
87 f 8 100 92 f 6 92 f 4 52f 1
6.5 7.0 7.5 8.0 8.5
39f7 30f 1 20f9 1113 8f3
60f5 67f 8 60f9 51f 12 51f9
Mean f SEM of two experiments.
for 1h. Two micrograms of the nonphotolabeled cytokine was also treated with trypsin or chymotrypsin in an identical fashion. The proteolyzed samples were separated by 12% SDS-PAGE. The gel was dried on a transparent cellophane sheet. Autoradiography exposure was for 12 h. The CBB-stained protein bands from the gel and the autoradiogram were quantified by LKB 2202 Ultroscan laser densitometer. Boronate Affinity Chromatography. rmGM-CSF (50 pg) was photolabeled sequentially three times with [6'-32P]-8N3Ap& as described earlier. The unbound nucleotide was removed by gel filtration on G-25. The photolabeled cytokine was applied to 3 mL of Affi-Gel601 (Bio-Rad) equilibrated with 250 mM ammonium acetate, pH 8.8,and purified as described elsewhere (23). The equivalent amounts of cytokine present in unbound fractions (fractions 2-5 pooled) and bound fractions (fractions 41-49 pooled) in Figure 8A were determined by reverse-phase HPLC analysis and assayed for biological activity using I3H1thymidine uptake assay as described elsewhere in this paper. RESULTS
Table 11. Effect of Various Divalent Metal Ions and Metal Ion Chelators on [@'-32P]-8N3ApdA Photoincorporation into rmGM-CSF metal ion/ % 32P metal ion/ 7% 32P chelator at 2 mM incorpO chelator at 2 mM incorpa control 100 COC12 58 f 13 MgCh 112 f 20 CUCl2 37 f 14 MnClz 72 f 9 EDTA 119 f 23 CaClz 107 f 19 EGTA 65 k 21 ZnClz 71 f 4 a Mean f SEM of four experiments. 1.50
t
0,25L-0.00 0
5
10
[j'32P]-8N3Ap.+A
15
20
&M)
Figure 1. Saturation of [j3'-32Pl-8N3Apa photoincorporation into rmGM-CSF: rmGM-CSF (0.2rg) in 40 rL of photolysis buffer was incubated with increasing concentrations of the photoprobe (sp act. = 10.9 mCi/pmol) for 15 sat 4 OC, photolyzed for 45 s a t 4 "C, and subjected to 10% SDS-PAGE analysis and autoradiography. 32Pincorporation was quantified by cutting out appropriate protein bands and determining radioactivity by liquid scintillation counting. The result shown is representative of two trials.
reactive intermediate generated by photolysis and rearrangement of the nitrene. Characterizationof Nucleotide Binding and SpecThe effect of various metal ions and chelators on [@'ificity. In order to investigate the nucleotide binding 32Pl-8N3Apdphotolabeling is summarized in Table 11. properties of rmGM-CSF, it was photolabeled with the nucleotide photoaffinity probes [y-32Pl-8N3ATP,[ Y - ~ ~ P I - Mg2+and Ca2+increased the photoinsertion to a small extent whereas Mn2+,Zn2+,Co2+,and Cu2+decreased it BNaGTP, and [@'-32Pl-8N3Ap4A.The latter is an analog to varying degrees. EDTA increased the photoinsertion of Ap4A, the putative biological alarmone. The photowhereas EGTA decreased it. The concentration of metal labeling was optimized for pH, buffer, temperature, ions present in the rmGM-CSF supplied was not deterpreincubation time, and time of photolysis. Although mined. significant labeling was observed over a wide pH range, To show the specificity of nucleotide interaction, the the optimum photolabeling in 20 mM NaH2P04 buffer was at pH 4.5 (Table I). At pH values above 4.5 the photolabeling should be saturable and protected by the photoinsertion of h/3'-32Pl-8N3ApA was greatly reduced parent compound a t physiologically relevant concentrawhereas that with [y-32Pl-8N&TPwas not greatly affected tions. In the case of [@'-32Pl-8N3Ap4A the saturation of and seemed to have leveled off at pH 7.0. The photolalabeling was achieved at approximately 10 WMconcenbeling was optimized for preincubation time and time of tration with an apparent Kd of approximately 0.7 pM photolysis. rmGM-CSF exhibited rapid binding in that (Figure 1). At a saturating concentration of 8-N3Ap& a short preincubation time of 15 s and photolysis for 45 (15 pM), a minimum of 39 f 5% of the cytokine was modified. s a t 4 "C seemed to give maximum photolabeling (data not shown). Protection studies with a wide variety of nucleotides at 100 pM concentration showed that the best competitor The UV light did not have any major effect on rmGMfor 8 - N d p d binding and photoinsertion was A p d (Table CSF. Pre-exposure to UV light for up to 2.5 min and 111). When rmGM-CSF was photolabeled with 0.7 pM subsequent photolabeling yielded photoincorporation [p'J2P1-8N3Ap4A in the presence of increasing concensimilar to the control labeling (data not shown). Also, trations of A p d , close to 90 % protection of photolabeling prephotolysis did not affect biological activity (data not was achieved at 200 pM A p d with half-maximal protection shown). Photoincorporation was dependent upon UV light since in the absence of light less than 0.2% 32P incorpoat 30 pM concentration (Figure 2). ration was observed as compared to control (data not Weak protection against [@'-32Pl -8N3Apd photoinsershown). This also proves the absence of phosphorylation tion by tripolyphosphate and pyrophosphate was also under these conditions. A 45-5 photolysis of the probe noted with half-maximal protection occurring at approxprior to the immediate addition of rmGM-CSF resulted imately 100 pM (data not shown), indicating a role for a in less than 1%of labeling compared to control (data not polyphosphate moiety in the binding site. This was shown). This indicated the absence of pseudoaffinity confirmed by the sequential reduction of the inhibitory labeling that may result due to any long-lived chemically effect of adenosine nucleotides with fewer phosphate
Bloconjugete Chem., Vol. 3, No. 8, 1932 487
NucleotMe-Blndlng Properties of rmGM-CSF ’--
I
I
r)
AP4A
0
OLW
50
100
150
200
250
300
350
400
ATP &M)
Figure 2. Protection of [p-32P]-8N3ApAphotoincorporation into rmGM-CSF by Ap& rmGM-CSF (0.2pg) was incubated in photolysis buffer with indicated concentrations of A p A for 60 s at 4 OC then with 0.7 pM of the photoprobe (sp act. = 11.0 mCi/pmol) for 15 s and photolyzed. SDS-PAGE analysis and determination of 32Pincorporation was done as in Figure 1.The result shown is representative of three trials. Table 111. Protection of 0.7 pM [j?-ST]-8NsAp,A Photoincorpation into rmGM-CSF by Various Competitors 7% 32P competitor % 32P competitor incorpa incorpa at 100 pM at 100 pM 73 f 16 100 GTP no competitor 40 f 9 NAD+ 78 f 17 AP4A 65 f 4 CTP 93 10 ATP 86 10 UTP 87 f 8 ADP 100 f 14 TTP 85 f 10 AMP 94 f 6 adenosine
Figure 4. Protection of [yJ2P]-8N3ATP photoincorporation into rmGM-CSF by ATP: rmGM-CSF (0.2 pg) in 40 p L of photolysis buffer was incubated with indicated concentrations of ATP for 60 s a t 4 OC and then with either 2.0 pM photoprobe (A)or 20 pM photoprobe ( 0 )(sp. act. = 17.6 mCi/pmol) for 15 s a t 4 OC and photolyzed for 45 s a t 4 OC. SDS-PAGE analysis and determination of 32Pincorporation was done as in Figure 1. The result shown is representative of four trials.
*
Mean
f SEM
of three experiments. 0.0 0
50
100
150
200
250
300
[ Y ~ ~ P ] - S N , G T P (&I)
Figure 5. Saturation of [ T - ~ ~ P I - ~ N ~photoincorporation GTP
U
2.0 0.0
f4 0
I
50
100
150
200
250
300
350
400
[r32P] -8N3ATP (pM)
Figure 3. Saturation of [y-32P]-8NaTP photoincorporation into rmGM-CSF: rmGM-CSF (0.2 rg) in 40 p L of photolysis buffer was incubated with increasing concentrations of photoprobe (sp act. = 5.3 mCi/wmol) for 15 s a t 4 OC and photolyzed for 45 s a t 4 OC. SDS-PAGE analysis and determination of 32P incorporation was done as in Figure 1. The result shown is representative of two trials.
groups (Table 111). Additionally, a comparison of the protective effect on [j3’-32P]-8N3Ap4A photoinsertion by Ap3A,A p d , and Ap& at 100pM levels indicated that the polytetraphosphate gave a maximum protective effect. A p d decreased [/3’-32Pl-8N&p4A photoinsertion by 50 % while ApA and A p d both gave a 75% decrease (data not shown). With [ T - ~ ~ P I - ~ N ~ A saturation TP, of labeling was achieved at approximately 100 pM concentration with an apparent Kd of 10 pM (Figure 3). At the saturation concentration of 100 pM, 43 f 5% of the cytokine was modified. When rmGM-CSF was photolabeled with 2 0 ~ [rJ2P1M 8N3ATP in the presence of increasing concentrations of ATP, 70% of the photoincorporation was protected by
into rmGM-CSF rmGM-CSF (0.2 pg) in 40 p L of photolysis buffer was incubated with increasing concentrations of photoprobe (sp act. = 12.8mCi/pmol) for 15 s at 4 “C, photolyzed for 45 s at 4 OC, and subjected to SDS-PAGE analysis and autoradiography. S2Pincorporation was quantified as in Figure 1. The result shown is representative of two trials.
200pM ATP with half-maximal protection being achieved at approximately 100 pM ATP (Figure 4). When this protection experiment was repeated with 2 pM [rJ2P18N3ATP,which is well below the saturation concentration, a reproducible increase of 16 f 7% in photoincorporation was observed at 10-15pM ATP in each of four experiments (Figure 4). This indicates a possible cooperative allosteric effect involving higher order ligand-protein interactions. To increase the level of photoinsertion, rmGM-CSF was photolabeled in three sequential steps as described in the -8NdTP, sequenexperimental procedures. With [r-32Pl tial photolyses with 167,125,and 100 pM probe resulted in 38%, 58%,and 60% total modification of the cytokine after one, two, and three photolysis events, respectively. With [/3’J2P1-8N3Ap4A, sequential photolyses with 16.7, 12.5,and 10 pM probe resulted in 38%, 41%, and 44% total modification of the cytokine, respectively (data not shown). rmGM-CSF was also photolabeled with [ T - ~ ~ P I - ~ N ~ GTP. The labeling was determined to be similar to that with 8-NsATP. The photolabeling was saturable at approximately 100-125 pM with an apparent Kd of 25-30 pM (Figure 5). GTP and ATP protected 8-N3GTP photoinsertion to the same extent (data not shown).
Doukas et el.
Bloconlugate Chem., Vol. 3, No. 8, 1992
1
A
+ 4.0
8
Fraction No.
Figure 6. Radioactivity elution profile for Fe3+ affinity chromatography of tryptic digest of rmGM-CSF photolabeled with [@'-32Pl-8N&p&. 200 pg of rmGM-CSF was photolabeled with the photoprobe, digested with trypsin, and loaded onto a 300-pL Fe3+ affinity column as described in Experimental Procedures. (a) Tryptic digest loaded, washed with (b) buffer A, (c) buffer A containing 0.5 M NaC1, (d) buffer A, (e) buffer A containing 4 M urea, (0 buffer A, and (g) buffer A containing 5 mM KzHP04. The fractions collected (1.5 mL) were assayed for radioactivity by liquid scintillation counting. Table IV. Resistance of Photolabeled rmGM-CSF to Trypsin and Chymotrypsin Digestion % remaining chymotrypsin trypsin Oh lh Oh lh
nonphotolyzed photolyzed
protein protein cpm
100 100 100
22 19
100 100
60
100
6 3 23
Proteolysis of Photolabeled rmGM-CSF. The photolabeled rmGM-CSF was more resistant to proteolysis with trypsin and chymotrypsin than the unmodified material (Table IV). With trypsin, while only 19%of CBBstained protein band remained, 60 % of the radioactivity was still undigested after 1h. With chymotrypsin, 3 % of the CBB-stained protein band remained whereas 23 % of radioactivity remained. All of our studies, including those done under denaturing conditions, indicated that proteolysis of photolabeled rmGM-CSF took longer than expected when compared to the nonphotolabeled material. We were able to digest the photolabeled protein with protease K. However, this resulted in a complex mixture of peptides which could not be easily resolved and was not used to identify the photolabeled peptide. We observed that carboxamidomethylation of rmGM-CSF cysteine residues, followed by prolonged digestion with a more active modified trypsin (Promega), proved to be the most successful procedure for generating small peptides. Immobilized Metal Affinity Chromatography. The use of immobilized Fe3+ affinity chromatography was recently developed in our laboratory to isolate photolabeled peptides (20).When the tryptic digest of the photolabeled rmGM-CSF was passed through the Fe3+affinity column, the bulk of the tryptic peptides were in the flow through (Figure 7A). However, greater than 90% of the radioactivity was retained on the column (Figure 6). To elute any peptides bound to the resin other than through phosphate-Fe3+ interaction, the column was washed with 100 mM ammonium acetate containing salt (0.5 M NaC1) and 4 M urea. These washes resulted in elution of less than 5% of the eluted radioactivity. However,when these washes were analyzed by reverse-phase HPLC, the presence of nonlabeled peptides was observed (data not shown). The photolabeled peptides were eluted with a 5 mM potassium phosphate wash as indicated by the elution of
f
IJ
73.0 x d
i
,I
h
J
0
0
10
20
SO min
40
50
60
Figure 7. Reverse-phase HPLC analysis of fractions collected from Fe3+affinity chromatography: (A) UV elution profile of reverse-phase HPLC analysisof flow-through fractions1-10 from Figure 6, and (B) UV and radioactivityelution profile of reversephase HPLC analysis of fractions 42-46 from Figure 6 a~ described in experimental procedures. The peak indicated by the arrow was subjected to amino acid sequence analysis. Table V. Amino Acid Sequence Analysis of the Photolabeled Tryptic Peptides of rmGM-CSF
amino acid (pmol)
radioactivity (Figure 6). The radioactivity obtained in the phosphate wash represented approximately 90% of the eluted radioactivity and 30-50% of the loaded radioactivity. When the phosphate wash from the Fe3+affinity column was analyzed by reverse-phase HPLC, as described in the Experimental Procedures, two radioactive peaks containing approximately 50 % each of the total radioactivity were obtained (Figure 7B). The first peak, which was in the flow through volumes, did not contain any peptide. The second peak which eluted a t 31 min, was subjected to amino acid sequence analysis and gave data consistent for a peptide corresponding to residues 5-14 (SPITVTRPWK) located near the N-terminus of rmGM-CSF (Table V). Approximately 5-15 % of the loaded radioactivity eluted with the peptide. The site of modification could not be determined with certainty. Based on the picomole recovery, the site of modification may be on the residue number 12, proline. The picomole recovery of tryptophan residue could not be determined since diphenylurea coeluted with it during sequencing. Picomole recovery of peptide upon sequence analysis was in excess of 70% of
NucleotMaBlndlng Properties of rmGM-CSF
Bioconjugate Chem., Voi. 3, No. 6, 1992 A
489
I
6.0 m
I
4.0
0 X
I
a
2.0
0
0.0 0
10
20
30
40
50
Fraction No.
! E
e-* 0--0 A-A
Stancard Modified Unmocified
1
5.0~
~
e-*
L*T\
A-A
Unbound Bound
4.0..
e
‘g 1 .o
12.5
0.4 rm GM -CSF (ng,”
0.01 2 L)
Figure 8. Bioassay of the rmGM-CSF photolabeled with (A) 8-NsATP and (B) 8-N3ApA: 5 pg of rmGM-CSF was photolabeled with the photoprobe and assayed for biological activity using [3H]thymidine uptake assay as described in Experimental rmGM-CSF photoProcedures; ( 0 )standard rmGM-CSF, (0) labeled with the photoprobe, (A)rmGM-CSF photolyzed in the absence of photoprobe and then photoprobe added to it. Each point is mean of three observations.
that used for sequencing based on the specific activity of the photoprobe used. In one experiment two radioactive peaks with corresponding peptides were obtained during HPLC. Besides the peak at 31 min, indicated in Figure 7B, the second was obtained at 36 min. Amino acid sequence analysis revealed it to be a longer version of the peptide obtained at 31 min (Le.residues 5-20, SPITVTRPWKHVEAIK). The other UV-absorbing peaks eluting after 40 min in Figure 7B were either due to some peptides being nonspecifically adsorbed or some non-peptide UV-absorbing material being eluted from the resin in the phosphate wash. This was confirmed since these peaks were also present in a control experiment where rmGM-CSF was photolyzed in the absence of probe. Additionally the peak at 31 min in Figure 7B which was due to the photolabeled peptide was absent in the control experiment. This provided additional evidence for the peptide sequenced being the photolabeled peptide. When this peptide isolation procedure was repeated using [y-32Pl-8N3ATP,the same peptide consisting of residues 5-14 was isolated (Table V), indicating that both the probes were binding to the same domain. Bioassay of Photolabeled rmGM-CSF. The initial cell proliferation bioassays were conducted on the rmGMCSF photolabeled sequentially three times with 8-N3Ap& and 8-NsATP as described in the Experimental Procedures, without removing the unbound nucleotide, since we could photomodify it over 40%. The assays indicated that the photolabeled rmGM-CSF was much less active than the control. In the case of 8-NATP, the half-maximal cell proliferation, or unit activity, for the photolabeled sample was obtained at approximately 0.3 ng/mL compared to 0.04 ng/mL for the control reflecting a difference of approximately 3.5 dilutions, which would be equivalent to approximately 85 % modification of the cytokine (Figure
10.0
A‘
0.312 “34-CSF
0.0075
0.00015
(Arbitrary Units/mL)
Figure 9. (A) Separation of photolabeled rmGM-CSF from nonphotolabeled r m G M - C S F using b o r o n a t e affinity chromatography: rmGM-CSF (50 pg) was photolabeled sequentially three times with [@’J2P]-8N3Ap,A and purified on AffiGel 601 as described in Experimental Procedures. (B) Bioassay of rmGM-CSF purified by boronate affinity chromatography: Biological activity of rmGM-CSF present in unbound fractions (fractions 2-5 pooled) and bound fractions (fractions 41-49 pooled) from boronate affinity chromatography was determined by [3H]thymidine uptake assay as described in the Experimental Procedures. Each point is mean of three observations.
8A). The covalent linkage of the photoprobe was essential for this inactivation since rmGM-CSF containing nonphotolyzed probe did not show this effect. When the bioassay was conducted with rmGM-CSF photolabeled sequentially three times with 8-N3Ap&, the unit activity indicated a difference of approximately 1.5dilutions, which would be equivalent to approximately 65 7% modification of the cytokine (Figure 8B). To confirm that photolabeling inactivated rmGM-CSF, 50 pg of the cytokine was photolabeled sequentially three times with [P’-32P]-8N3Ap,Aand the unbound nucleotide was removed by gel filtration on G-25. Taking advantage of the two ribose rings present on ApdA, the photolabeled rmGM-CSF was separated from the nonphotolabeled rmGM-CSF by boronate affinity chromatography on AffiGel 601as described in the Experimental Procedures. Since the recovery of rmGM-CSF from the boronate affinity resin was low, the equivalent amount of cytokine present in unbound and bound fractions (Figure 9A) was determined by reverse-phase HPLC analysis (data not shown). The nonphotolabeled rmGM-CSF was in the flow through (unbound, fractions 2-5 pooled) whereas the photolabeled cytokine stayed attached until released by a pH change (bound, fractions 41-49 pooled). The unbound fractions contained most of the nonphotolabeled rmGM-CSF, whereas the bound fractions, which contained most of the radioactivity and hence most of the photolabeled cytokine showed a difference in unit biological activity of approximately six dilutions as shown in Figure 9B. DISCUSSION
Many cytokines and growth factors such as GM-CSF are of appreciable molecular weight, greater than that
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which might be expected to be necessary for receptor interaction alone. On the basis of the observation that IL-2 (16),ANF (IT),and glucagon (18)possessed specific nucleotide binding sites, we decided to study the possible nucleotide binding properties of the growth factor GMCSF. To show the specificity of a nucleotide binding site using photoaffinity probes the photolabeling should be saturable and be protected by the natural compound at physiologically relevant concentrations. rmGM-CSF was initially photolabeled with [ T - ~ ~ P I - ~ N ~and A T[y-32Pl-8N3GTP. P Photolabeling with [rJ2P1-8N3ATP was saturable at approximately 100 pM with an apparent Kd of 10 pM. In the presence of increasing concentrations of ATP, 70% of the photolabeling with 20 pM [y-32Pl-8N3ATP was protected by 200 pM ATP with half-maximal protection being achieved at approximately 100 pM ATP. Photolabeling with [r-32Pl-8N3GTPwas saturable at approximately 100-125 pM with an apparent Kd of 25-30 pM. GTP at a concentration of 500 pM afforded approximately 90% protection of 10 pM [y-32Pl-8N3GTPphotolabeling. Overall, GTP exhibited similar affinity. NAD+ was a poor protector of either 8-NsATP or 8-N3GTP photolabeling, indicating a different kind of nucleotide binding site compared to IL-2 (affinity for both ATP and NAD+)and ANF and glucagon (affinity specific for GTP). The photoincorporation with these probes was light dependent, indicating the absence of phosphorylation. Photolysis of the probe prior to incubation with protein resulted in lees than 2 % of photoincorporation compared to control, indicating the absence of pseudoaffinity labeling resulting from any long-lived chemically reactive intermediate. To obtain binding site peptide purification and to develop a procedure for purifying the photolabeled GMCSF from the nonphotolabeled GM-CSF we photolabeled rmGM-CSF with [8’-32Pl-8N3Ap4A. It is a photoaffinity probe of a biological stress-related nucleotide Ap4A (24). This dinucleotide binds boronate affinity resin by virtue of two ribose rings possessing cis-hydroxyl groups as in the case of NAD+ (23 and references therein). The lower apparent Kd value of 0.7 pM and saturation at 10 pM obtained for this analog compared to [y-32Pl-8N3ATP prompted us to study the binding properties with 8-N3A p d in detail. Protection experiments indicated that 90% of the photolabeling was protected by approximately 200 pM Ap4A with half-maximal protection at approximately 30 pM Ap4A. Protection of 8-NsAp4A photolabeling with other nucleotides indicated decreased protection in the order of ATP, ADP, AMP, and adenosine. In addition, tripolyphosphate and pyrophosphate also afforded 50% protection at 100 pM concentration, indicating that the phosphate binding region is also contributing to the specificity of this nucleotide binding domain. The divalent metal ions did not show any significant effect on photolabeling. Mn2+, Zn2+, Co2+, and Cu2+ showed an inhibitory effect on photoinsertion. Photoincorporation increased 7 % in the presence of Ca2+and decreased 35% in the presence of EGTA. When rmGM-CSF was photolabeled with 2 pM [ T - ~ ~- P I 8N3ATP, which is below the saturation concentration, an increase in photoincorporation was observed at low (1015 pM) concentrations of competing ATP. This could be interpreted as cooperative binding as observed with multisubunit enzymes (25). GM-CSF is a monomeric protein ( I ) , and no data on higher order (quaternary) structures in solution are known to us. However, in preliminary crystal structure work on hGM-CSF, crys-
Doukas et al.
tallization occurs with two molecules in the asymmetric unit (12, 13). The possibility of higher order structures of GM-CSF molecules in solution could explain what appears to be a cooperative effect of 8-NsATP binding with the addition of low concentrations of ATP. GMCSF may be like IL-6 and TNF where dimers and trimers are apparently the dominant forms in solution (26,27). When the photolabeled rmGM-CSF was subjected to proteolysis for the purpose of generating small peptides, it exhibited considerable resistance compared to nonphotolabeled samples. This has also been observed in our study of nucleotide binding properties of other cytokines (unpublished results). Standard trypsin and chymotrypsin were unsuccessful in digesting the photolabeled rmGMCSF to an appreciable extent, The covalently attached nucleotide appears to interfere with the digestion procedure. This result may be indicative of a possible protective role for nucleotide interaction invivo. Use of a more active, modified trypsin (Promega) achieved successin generating small peptides. The problems encountered in isolating photolabeled peptides, especially with reverse-phase HPLC are welldescribed (28). The problems stem either from lability of the photoinserted bond and/or the lability of N-glycosidic bond to HPLC. Also, the phosphates which carry the radioactive tag may be lost during the purification procedure. To overcome these problems, recent efforts in this laboratory have concentrated on using a mild purification procedure prior to reverse-phase HPLC for the isolation of photolabeled peptides. Boronate affinity chromatography and to some extent anion exclusion chromatography have been useful for this purpose (23,291. However, we still lacked an effective procedure for isolating peptides photolabeled with nucleoside triphosphates. Photolabeling with 8-NsAp4A was tested assuming that it would be able to interact with the ATP binding site and could be used to provide us with a procedure for isolating photolabeled peptide using boronate affinity chromatography. The observed low apparent Kd for 8-NsAp4A was not expected. Boronate affinity chromatography did afford a satisfactory resolution of the intact photolabeled rmGMCSF from the nonphotolabeled rmGM-CSF. However, it was not very effective in terms of material recovered. Also, the separation of smaller photolabeled peptides from nonphotolabeled peptides was not as good as obtained using the following procedure. Immobilized Fe3+ affinity chromatography has been previously used for the isolation of phosphoproteins and phosphopeptides (30). Recently, using a modification of these procedures we have developed metal affinity chromatography for the isolation of photolabeled peptides (20). This procedure is based on the principle that the photolabeled peptides carrying photoinserted phosphates will form a complex with transition-state metals immobilized on a Sepharose support. The complex is relatively stable to salt washes, as well as to urea washes, which can be used to dissociate peptides retained nonspecificallydue to ionic, electrostatic, or hydrophobic interactions. Using this method, in combination with reverse-phase HPLC, we selectively isolated a heavily radiolabeled (3.4 X lo4cpm/ 10 pL of 0.5 mL fraction), single peptide consisting of residues 5-14 near the N-terminus of GM-CSF and suggest that this domain is involved in nucleotide binding. During this study and several other in this laboratory we have determined that the radiolabel retained with the photolabeled peptide during reverse-phase HPLC is dependent
NucleotldaBlndlng Properties of rmGMCSF mGM-CSF
(5-13)
S P I T V T R P W
T2
(161-169) T P V D V T C P W
T4
(160-168)
T6
(161-169) T P V D V T C P W
T P V D V T C P W
Figure 10. Amino acid sequence comparison of the photolabeled peptide of rmGM-CSF with other nucleotide-binding proteins. The method used for sequencecomparison was that of Needleman and Wunsch (31)as implemented by Dayhoff (32)and Doolittle (33). The scan was done using a Dayhoff MDM-78 matrix with a bias of 60 and a gap penalty of 60.
upon the flow rate. Approximately 80% more of the radiolabel was lost at 1mL/min compared to 0.5 mL/min flow rate. Also, photolysis with [y-32Pl-8N3ATPresulted in the isolation of the same peptide indicating that both the probes were interacting with the same region on GM-CSF. We isolated only one peptide in the case of 8 - N A p dwhich can potentially interact with two binding regions due to the presence of two adenine rings. This indicates that this binding domain has higher affinity for the 8-azidoadenine ring than for the adenine ring. The site of modification could not be determined with certainty. Strictly on the basis of the picomole recovery of residues, proline may be the site of photoinsertion. When this peptide sequence was analyzed for sequence homology with other proteins by scanning Swiss prot 16 database using the PC-Genes program, it showed sequence homology with several nucleotide binding proteins (Figure 10). Of interest was the approximately 84% sequence homology with single-stranded DNA binding protein from bacteriophage T2, T4, and T6. This sequence comparison was also carried out in the case of our IL-2 work where the ATP/NAD+ binding domain has been identifieda2 The peptide regions in the nucleotide binding domain on IL-2 showed greater than 90% sequence homology with sequences on several nucleotide binding proteins, the most interesting one being the sequence on subunit 1fromBordetellapertussis,which has been implicated in the NAD+ binding and ADP ribosylating activity. A similar sequence was also observed in cholera toxin. The three-dimensional structure of GM-CSF has been predicted by molecular modeling techniques (11). The preliminary crystallographic examination confirms the presence of four a helices (12,131. The areas of the GMCSF molecule critical to bioactivity have been described by a variety of techniques. Small N-terminal and C-terminal deletions do not adversely affect activity, nor does glycosylation (34,35). Regions critical to bioactivity are arrayed throughout the molecule. On the basis of scanning-deletion analysis, residues 18-22 near the N-terminus of mGM-CSF were determined to be important for biological activity (36). On the basis of mutagenesis studies, residues 11-15 near the N-terminus have also been implicated in biological activity (37). The nucleotide binding domain identified by photoaffinity labeling lies within or in close proximity to this critical region near the N-terminus. Most neutralizing antibody epitopes reported are in the C-terminus half of the human GM-CSF molecule, leaving certain biologically critical regions in the N-terminus portion with no receptor binding function described (38,39). We suggest, due to the specificity and avidity of
* (a) Campbell, S. R., and Haley, B. E., manuscript under preparation. (b) Campbell, S.R. (1991)Ph.D. Thesis, Department of Biochemistry, University of Kentucky.
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the binding site described, that this nucleotide interaction is critical for GM-CSF signal transduction in a novel fashion. Possible candidate roles include modulation of receptor affinity after internalization, protection of GMCSF against proteolytic digestion, endosomal trafficking of internalized GM-CSF (the optimum pH of 4.5 of nucleotide binding is interesting in this regard), or possible catalytic activity. We tested the bioactivity of rmGM-CSF photolabeled with either 8-N3Apd or 8-N3ATP in a cell-proliferation assay using [3H]thymidine uptake. The photolabeled samples, without removal of any unbound nucleotide, indicated decreased bioactivity compared to a control sample as well as to a sample which was prephotolyzed, photoprobe being subsequently added to it. In the case of 8-N3Ap4A the difference in bioactivity was equivalent to approximately 1.5 dilutions, which would be equivalent to approximately 65 % modification of the cytokine. The percent modification determined by quantifying 32P incorporation after SDS-PAGE separation was 44 % .This is a minimal percent due to inherent loss of peptide and instability of the photolabel to SDS-PAGE and gel staining and drying procedures. In the case of 8-N3ATP the difference in bioactivity was equivalent to approximately 3.5 dilutions, which would be equivalent to approximately 85% modification of the cytokine. The percent modification observed based on SDS-PAGE analysis in this case was 60 5%. The percent modification in the case of 8-N3A p d was lower compared to that in the case of 8-NsATP. This is probably due to the non-azido adenine ring staying intact after photolysis of 8-NsApdA and being able to act as a competitor for the second and third addition and photolysis of 8-N3ApdA. The inactivation of the photolabeled rmGM-CSF was further confirmed by partial purification of the photolabeled cytokine from the non-photolabeled cytokine using boronate affinity chromatography. The bound material and the unbound material indicated a difference of approximately 6 dilutions in bioactivity, indicating that more than 90% of rmGM-CSF in the bound fractions was modified. The bioactivity result would be consistent with the hypothesis that the critical region near the N-terminus, for which no clear function is yet ascribed, is being interfered with by the covalent attachment of nucleotide analog. However, the possibility of inability of covalently modified GM-CSF molecule to bind to the receptor does exist. Such a possibility of modulation of receptor binding via nucleotide binding site occupancy is being investigated. The observation of this nucleotide binding site opens new avenues for exploring the physiology of cytokine signal transduction which is currently not fully understood. ACKNOWLEDGMENT
We thank the National Institutes of Health (Grant GM 35766), the Lexington Clinic Foundation (B.E.H.), and the Association for Medical Research (M.A.D.) for their generous financial support. LITERATURE CITED
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Registry No. GM-CSF, 83869-56-1.
