466
Bioconjugate Chem. 1991, 2, 466-474
Biotinylated Granulocyte/Macrophage Colony-Stimulating Factor Analogues: Effect of Linkage Chemistry on Activity and Binding Timothy P. Angelotti,'lt Michael F. Clarke,$ Marc A. Longino,s and Stephen G. Emerson$ Department of Pharmacology, Department of Internal Medicine, Division of Hematology and Oncology, and Department of Radiology, University of Michigan Medical School, Ann Arbor, Michigan 48109. Received July 18, 1991
Biotinylated granulocyte/macrophage colony-stimulating factor (GM-CSF) analogues with different linkage chemistries and levels of conjugated biotin were synthesized by reacting recombinant human GM-CSF with sulfosuccinimidyl 6-biotinamidohexanoate or biotin hydrazide/l-[3-(dimethylamino)propyl]-3-ethylcarbodiimide. These chemically reactive forms of biotin produced derivatives biotinylated at amine or carboxyl groups, respectively. Amine-derivatized analogues of 1.2 and 3.8 mol of biotin/mol of protein (N1-bGM-CSF and N4-bGM-CSF)and a carboxyl-modified analogue of 4.6 mol of biotin/mol of protein (C5-bGM-CSF)were synthesized. These analogues were compared to determine the effect of biotinylation on biological activity and GM-CSF receptor binding characteristics. The biotinylated proteins migrated with the same molecular weight as the native, unmodified protein as determined by SDS-PAGE and could be detected by Western blotting with alkaline phosphatase conjugated streptavidin, thus demonstrating the biotin linkage. All three analogues retained full agonist activity relative to the native protein ( E C ~ = O 10-15 pM) when assayed for the stimulation of human bone marrow progenitor cell growth. Cell surface GM-CSF receptor binding was characterized by the binding of the analogues to human neutrophils, with detection by fluorescein-conjugated avidin and fluorescence-activated cell sorting. The N-bGM-CSFs demonstrated GM-CSF receptor specific binding that was displaceable by excess underivatized protein, with the detected fluorescence signal decreasing with increasing biotin to protein molar ratio. In contrast, C5-bGM-CSF binding above background fluorescence could not be detected using this system, suggesting that this derivative could bind to and activate the receptor, but not simultaneously bind fluorescein-conjugated avidin. The amine-derivatized biotinylated GM-CSF analogues retained biological activity, could specifically label cell surface receptors, and may be useful nonradioactive probes with which to study GM-CSF receptor cytochemistry and receptor modulation by flow cytometry.
INTRODUCTION Human granulocyte/macrophage colony-stimulating factor (GM-CSF') is a pleiotropic glycoprotein cytokine that exerts effects on both immature and mature hematopoietic cells. It stimulates not only proliferation and maturation of myeloid progenitor cells but also enhances several physiological functions of mature myeloid-derived cells, including the priming of neutrophil respiratory bursts and antibody-dependent cell-mediated cytotoxicity, and macrophage tumoricidal activity (reviewed in ref 1). Cloning of the cDNA encoding human GM-CSF (2) has made it possible to purify the recombinant protein for study of its in vitro and in vivo effects. Milligram quantities of the pure protein now can be obtained for
* To whom reprint requests should be addressed at Department of Pharmacology, 6322 Medical Science I, Universityof Michigan Medical School, Ann Arbor, MI 48109-0626. + Department of Pharmacology. Department of Internal Medicine. 8 Department of Radiology.
*
'Abbreviations: GM-CSF, granulocyte/macrophagecolonystimulating factor; rhGM-CSF, recombinant human GM-CSF; sulfo-NHS-biotin, sulfosuccinimidyl 6-biotinamidohexanoate; IMDM, EDAC, 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide; Iscove's Modified Dulbecco's Medium; FBS, fetal bovine serum; HABA, 4-hydroxyazobenzene-2'-carboxylicacid; MES, 2-morpholinoethanesulfonicacid;SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; kDa, kilodalton; BCIP, 5-bromo-4-chloro-3-indolyl phosphate; NBT, nitroblue tetrazolium; cpm, counts per minute; FITC-avidin, fluoresceinconjugated avidin; PBS, Dulbecco's phosphate-bufferedsaline (pH = 7.4): Na2P04(4.3mM), KH2P04(1.4mM), KCl(2.7mM), and NaCl (137 mM). 1043-1802f9 112902-0466$02.50f0
chemical modification, and the development of such derivatives has greatly enhanced the characterization of the biochemistry and physiology of this gr' rth factor. Radioiodinated recombinant GM-CSF has been synthesized by many laboratories and utilized to understand the complex interactions of GM-CSF with its cell surface receptor (3-6). Recent cloning of the receptor cDNAs suggests that the low affinity form of the receptor is composed of a single subunit that can be converted to a high-affinity state upon interaction with a second 120kDa protein (6, 7). It is not completely clear, though, which affinity state of the receptor mediates which physiological functions. Radioiodinated GM-CSF has been a useful tool with which to investigate ligand-receptor interactions, but its utility is mostly limited to cell surface and solubilized receptor studies (8). By taking advantage of the high affinity of biotin for avidin or streptavidin (9, IO), development of biotinconjugated protein ligands has enabled many investigators to expand the study of receptors to other functions beyond receptor binding kinetics. These include postreceptor binding events (including receptor internalization), receptor biochemistry, and isolation of viable receptor expressing cells (11-1 7). Radiolabeled ligands are not generally applicable to the study of these events. Recent advances in biotin-conjugation reagents for protein modification at specific amino acid and carboxylate residues (18)have enabled researchers to use biotinylated proteins as tools with which to study how the protein structure effects its function. For some proteins, such as @-nerve growth factor, interleukin-la or -p, parathyroid hormone, and erythropoietin, modification of the protein at specific 0 1991 American Chemical Society
Technical Notes
lysine residues resulted in inactivation of the protein (14, 15, 17, 19, 20),suggesting a role for these amino acid residues in receptor binding or maintenance of protein tertiary structure. We wished to develop a biologically active and chemically stable biotinylated GM-CSF analogue for use in flow cytometric analysis of receptors expressed on cells at low levels. By conjugation of GM-CSF to biotin using different specific amino acid linkage chemistries, we also sought to investigate the role of specific basic and acidic residues on biological activity and binding. In this paper, we describe the synthesis of three biotinylated derivatives of GM-CSF modified at amine- or carboxyl-containing residues. We demonstrated that modification of human GM-CSF with approximately one or four biotins at amine residues or five biotins at carboxyl residues does not diminish biological activity as assessed by the stimulation of enriched bone marrow progenitor cell growth. Detection of the amine-modified derivatives bound to neutrophils using fluoroscein-conjugated avidin (FITC-avidin) and flow cytometry diminished with increasing biotin content. In contrast, the binding of the carboxyl-modified GM-CSF was not perceptible with this system, suggesting that the biotin-modified residues are hidden from detection upon receptor binding or that the protein was not uniformly derivatized using this linkage chemistry. EXPERIMENTAL PROCEDURES
Materials. Yeast- and CHO-derived recombinant human GM-CSF (rhGM-CSF) were the generous gifts of Dr. Steven Gillis (Immunex, Seattle, WA) and Dr. Steven C. Clark (Genetics Institute, Cambridge, MA),respectively. Chemicals used in these experiments were obtained from the following sources: sulfosuccinimidyl 6-biotinamidohexanoate (sulfo-NHS-biotin) and 1-[3-dimethylamino)propyl]-3-ethylcarbodiimide(EDAC),Pierce Chemical Co. (Rockford, IL); d-biotin, Research Organics, Inc. (Cleveland, OH); biotin (long arm) hydrazide, avidin D, and fluorescein-conjugated avidin DCS (cell sorter grade), Vector Laboratories (Burlingame, CA); biotinylated and nonbiotinylated low molecular weight protein markers, Bio-Rad, Inc. (Richmond, CA); streptavidin-conjugated alkaline phosphatase and Iscove's Modified Dulbecco's Medium (IMDM), Gibco-BRL (Gaithersburg, MD); fetal bovine serum, Hyclone Laboratories Inc. (Logan, UT); [3H]thymidine, NEN/Du Pont (Bannockburn, IL);Ficoll Hypaque, Pharmacia Fine Chemicals (Piscataway, NJ); and 4-hydroxyazobenzene-2'-carboxylicacid (HABA), 2-morpholinoethanesulfonic acid (MES), Coomassie Brilliant Blue R250, and all other chemicals, Sigma Chemicals (St. Louis, MO). Cell Source. Peripheral blood granulocytes and bone marrow were obtained following informed consent from normal donors under a protocol approved by the University of Michigan Human Investigation Committee. Protein Concentration. Protein concentration was assayed in duplicate using the BCA Protein Assay Kit (Pierce Chemical Co.) according to the manufacturer's instructions using bovine serum albumin as the standard (21).
Biotin Assay. The biotin content of the GM-CSF derivatives was determined colorimetricallyusing the 4-hydroxyazobenzene-2'-carboxylic acid (HABA) dye-based method of Green (22). In this assay, HABA bound to avidin in solution is displaced by added d-biotin or biotinderivatized proteins, and the decrease in the absorption a t 500 nm (absorption maxima for the avidin-HABA complex) is measured spectrophotometrically. A standard
Bioconjugate Chem., Vol. 2, No. 6, 1991 487
curve was created by plotting the measured change in absorbance against the final concentration of d-biotin added to the cuvette; sequential 2 pL-aliquots of stock d-biotin solutions were added. Biotin derivatized protein solutions were assayed in 20 pL-aliquots. Volumetric changes due to the addition of the biotin or biotin-derivatized GMCSF solutions were taken into account when calculating the concentrations of biotin. The detectable range of biotin was 0.30-30.0 nmol/20-pL aliquot. Dialysis of rhGM-CSF. The yeast-derived rhGMCSF (Immunex) was formulated in a mannitol/sucrose/ Tris buffered solution. Before any chemical reactions could be performed, the Tris buffer (a primary amine) had to be exchanged with a nonprimary amine containing buffer. This was accomplished by dialyzing 500 pg of rhGM-CSF in 1 mL of mannitol/sucrose/Tris buffer against PBS with a Microdialyzer System 500 (Pierce Chemical Co.), according to the maufacturer's instructions. Briefly, the protein solution was dialyzed in three separate aliquots at 4 "C using the bulk flow method, with complete PBS buffer exchanges every hour for the first 5 h, followed by a final 12-h dialysis. The protein recovery was 75.7 % or 378 pg of rhGM-CSF. Biotinylation of GM-CSF via Amine Groups. A fresh 12.5 mM solution of sulfo-NHS-biotin was made in PBS (7.00 mg/10.0 mL). Aliquots were added to four 1.5mL microcentrifuge tubes containing 60 pg (3.0 nmol) of rhGM-CSF (approximate M,= 20 kDa, including variable glycosylation) at molar ratios of 1:1, 2:1,5:1, and 151of sulfo-NHS-biotin:rhGM-CSF in a final volume of 250 p L of PBS (pH = 7.4). The reactions were incubated at 23 "C for 3 h with periodic mixing. Unincorporated sulfoNHS-biotin was removed by dialysis against PBS using the Microdialyzer as described above. Protein and biotin concentrations for each reaction were determined for duplicate 20-pL aliquots as described above. Protein recovery exceeded 90% in all reactions. Biotinylation of GM-CSF via Carboxyl Groups. Two 60-pg aliquots (3.0 nmol) of rhGM-CSF in PBS, were adjusted to pH = 5.0-6.0 by the addition of 25 p L of 300 mM 2-morpholinoethanesulfonic acid (MES) in PBS and placed in 1.5-mL microcentrifuge tubes. Biotin hydrazide was dissolved immediately before use, in dimethylformamide at a concentration of 80 mM (3.0 mg/100pL), diluted 1 : l O with the same solvent, and added to the tubes containing the rhGM-CSF at a 20:l molar excess of biotin hydrazide to GM-CSF. The coupling reagent EDAC was also dissolved at 80 mM in PBS (15.3 mg/l.O mL) and added to the biotin hydrazide/GM-CSF reaction vials a t a 20:l and 200:l molar ratio (EDAC:GM-CSF). The reaction volumes were brought up to 250 p L with the addition of PBS; the final concentration of DMF was 10% v/v. The mixtures were incubated overnight at 23 "C with gentle shaking on a vortex apparatus. The next morning, precipitated material was removed by centrifugation for 10 min at 16 000 rpm. The supernatants were dialyzed as described above to remove the unincorporated chemical reagents, and 20-pL aliquots were assayed for protein and biotin content as before. All proteins were stored at 4 "C. PolyacrylamideGel Electrophoresis. Native and biotinylated GM-CSFs were analyzed by denaturing sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDSPAGE) as described previously by Laemmli (23).Samples of nonbiotinylated low molecular weight markers (l/20 dilution of stock solution from Bio-Rad), native CHOderived GM-CSF, and biotin-derivatized GM-CSF (4.0 pg each) were boiled in sample buffer containing dithiothre-
408
Angelotti et ai.
Bioconlugate Chem., Vol. 2, No. 6, 1991
Table I. Summary of GM-CSF Biotinylation Reactions reaction chemistp reagenbproton ratiob sulfo-NHS-biotin 1:l 2: 1 sulfo-NHS-biotin sulfo-NHS-biotin 5:1 sulfo-NHS-biotin 15:l biotin hydrazide/EDAC 20:1/20:1 biotin hydrazide/EDAC 20:1/200:1
biotin:protein ratio final productc
name
