Bioconjugate Chem. 2009, 20, 1459–1473
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Carbodiimide EDC Induces Cross-Links That Stabilize RNase A C-Dimer against Dissociation: EDC Adducts Can Affect Protein Net Charge, Conformation, and Activity Jorge P. Lo´pez-Alonso,† Fernando Diez-Garcı´a,† Josep Font,§ Marc Ribo´,§ Maria Vilanova,§ J. Martin Scholtz,| Carlos Gonza´lez,† Francesca Vottariello,‡ Giovanni Gotte,‡ Massimo Libonati,‡ and Douglas V. Laurents*,† Instituto de Quı´mica Fı´sica “Rocasolano” (C.S.I.C.), Serrano 119, E-28006, Madrid, Spain, Dipartimento di Scienze Morfologico-Biomediche, Sezione di Chimica Biologica, Facolta` di Medicina e Chirurgia, Universita` di Verona, Strada Le Grazie 8, I-37134, Verona, Italy, Laboratori d’Enginyeria de Proteı¨nes, Departament de Biologı´a, Facultad de Cie`ncies, Universitat de Girona, Campus Montilivi, 17071 Girona, Spain, and Department of Medical Biochemistry, Texas A&M University School of Medicine, College Station, Texas 77478. Received September 16, 2008; Revised Manuscript Received June 17, 2009
RNase A self-associates under certain conditions to form a series of domain-swapped oligomers. These oligomers show high catalytic activity against double-stranded RNA and striking antitumor actions that are lacking in the monomer. However, the dissociation of these metastable oligomers limits their therapeutic potential. Here, a widely used conjugating agent, 1-ethyl-3-(3-dimethylaminoisopropyl) carbodiimide (EDC), has been used to induce the formation of amide bonds between carboxylate and amine groups of different subunits of the RNase A C-dimer. A cross-linked C-dimer which does not dissociate was isolated and was found have augmented enzymatic activity toward double-stranded RNA relative to the unmodified C-dimer. Characterization using chromatography, electrophoresis, mass spectrometry, and NMR spectroscopy revealed that the EDC-treated C-dimer retains its structure and contains one to three novel amide bonds. Moreover, both the EDC-treated C-dimer and EDCtreated RNase A monomer were found to carry an increased number of positive charges (about 6 ( 2 charges per subunit). These additional positive charges are presumably due to adduct formation with EDC, which neutralizes a negatively charged carboxylate group and couples it to a positively charged tertiary amine. The increased net positive charge endowed by EDC adducts likely contributes to the heightened cleavage of double-stranded RNA of the EDC-treated monomer and EDC-treated C-dimer. Further evidence for EDC adduct formation is provided by the reaction of EDC with a dipeptide Ac-Asp-Ala-NH2 monitored by NMR spectroscopy and mass spectrometry. To determine if EDC adduct formation with proteins is common and how this affects protein net charge, conformation, and activity, four well-characterized proteins, ribonuclease Sa, hen lysozyme, carbonic anhydrase, and hemoglobin, were incubated with EDC and the products were characterized. EDC formed adducts with all these proteins, as judged by mass spectrometry and electrophoresis. Moreover, all suffered conformational changes ranging from slight structural modifications in the case of lysozyme, to denaturation for hemoglobin as measured by NMR spectroscopy and enzyme assays. We conclude that EDC adduct formation with proteins can affect their net charge, conformation, and enzymatic activity.
INTRODUCTION Bovine pancreatic ribonuclease A (RNase A) is a 124-residue monomeric enzyme that catalyzes the breakdown of singlestranded RNA with specificity for pyrimidines at the 3′-side of the cleavage site. RNase A forms oligomers by a threedimensional domain swapping mechanism (1). Dimers can be formed when two monomers swap their N-terminal R-helices (2) or their C-terminal β-strands (3). These dimers are called the N-dimer and C-dimer, respectively. Higher oligomers form by combinations of N-terminal or C-terminal swapping or both (4-7). This oligomerization can be induced by lyophilization from 40% glacial acetic acid (1) or incubation at elevated temperature at high concentration in water (8) or in alcohol/ water solution (9). The oligomerization of RNase A has been shown to endow the protein with a marked increased catalytic activity against * To whom correspondence should be addressed: dlaurents@ iqfr.csic.es. † C.S.I.C. ‡ Universita` di Verona. § Universitat de Girona. | Texas A&M University School of Medicine.
double-stranded poly(A) · poly(U) or double-stranded viral RNA (dsRNA) (10) and, even more remarkably, a potent and selective toxicity in ViVo against human tumors (11). The activity on dsRNA and the cytotoxicity of RNase A oligomers increases with their size (12). All RNase oligomers formed are metastable and dissociate over time into monomers. The dissociation of the larger oligomers into smaller ones is markedly faster than the disassociation of the dimers into monomers. The lifetime of the dimers can be as long as months in optimal conditions, namely, 4 °C, pH 7, in phosphate buffer. However, their lifetime is considerably lower at physiological temperatures. RNase S, the product of the mild digestion of RNase A into two fragments which remain associated by noncovalent interactions, forms a 3D domain-swapped C-dimer (13). By conjugation of the small fragment with DNA or PNA, it is possible to form semisynthetic RNase S monomers and C-dimers with modulated activities (14). However, the study and application of semisynthetic RNase S dimers is impeded by their rather strong tendency to dissociate. Therefore, the addition of cross-links to stabilize RNase A or RNase S oligomers could maintain their novel activity and increase their in ViVo antitumor activity. In this paper, we have selected 1-ethyl-3-(3-dimethylaminoisopropyl) carbodiimide (EDC), which induces the formation
10.1021/bc9001486 CCC: $40.75 2009 American Chemical Society Published on Web 07/16/2009
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of isopeptide bonds between nearby amine and carboxylate groups, as a suitable cross-linking reagent for RNase oligomers. We have identified suitable conditions for obtaining cross-linked RNase A C-dimers. Using these conditions, we have produced and purified cross-linked RNase A C-dimers and determined their enzymatic activities against ssRNA and dsRNA. We also isolated and characterized RNase A monomers carrying EDC adducts which result from a side reaction. The EDC adducts endow RNase A monomers with an increased positive charge and an augmented ribonucleolytic activity on dsRNA. EDC is a popular reagent for cross-linking two proteins or for activation of carboxylate groups for reaction with other molecules containing amines. The formation of carbodiimide adducts to proteins is well-documented in the literature up until 1995 (15-18), but the effects of these adducts on protein conformation and function is relatively unexplored. Therefore, a second objective of this work has been to determine how EDC adducts affect protein stucture and activity. To this end, we studied the reaction of EDC with the dipeptide Ac-Asp-AlaNH2, and we reacted four well-characterized proteins: ribonuclease Sa (RNase Sa), hen egg white lysozyme, carbonic anhydrase, and hemoglobin with EDC. Using modern biophysical techniques, we show that EDC forms adducts with all four proteins and affects their net charge, conformation, and enzymatic activities to varying degrees.
EXPERIMENTAL PROCEDURES Materials. Bovine pancreatic ribonuclease A (RNase A) was obtained from Sigma and further purified by cation-exchange chromatography as described previously (4). Ribonuclease Sa was expressed in E. coli and purified as previously described (19). An Asp-Ala dipeptide with the N- and C-termini blocked by acetyl and amide groups, respectively, was purchased from GenScript Corp. It is over 97% pure, and its identity was confirmed by mass spectrometry and NMR spectroscopy. The best grades of hen egg white lysozyme, bovine carbonic anhydrase II, and human hemoglobin were obtained from Sigma and were used without further purification. Yeast single-stranded RNA, synthetic double-stranded poly(A) · poly(U), and 4-methylumbelliforyl D-D-N,N′,N′′-triacetylchitotrioside were purchased from Sigma. Sodium dihydrogen phosphate and sodium hydrogen phosphate were obtained from Merck and MES was from Sigma. Sodium azide (99%) was from Panreac. Doubledistilled water was deionized using a Milli-Q system. Glacial acetic acid (99.9%) was from Quimipur. 1-Ethyl-3-(3-dimethylaminoisopropyl)carbodiimide (EDC) and N-hydroxysulfosuccinimide (sulfo-NHS) were purchased from Pierce. Structure Analysis and pKa Prediction. The atomic structures of the RNase A C-dimer (pdb: 1f0v) (3) and N-dimer (pdb: 1a2w) (2) were examined using the program Mage (v 6.30) to measure distances between amine groups and carboxylate groups. The program MolMol was used to calculate the percent solvent-accessible surface of carboxylate oxygen atoms in the RNase A C-dimer (20). pKa values in the RNase A C-dimer were predicted using the empirical approach implemented in the program Propka (21). Oligomer Formation. RNase A oligomers were obtained by incubation in 40% glacial acetic acid/60% water for 1 h, followed by lyophilization (1), and purified as previously described (4). Briefly, lyophilized samples were dissolved in sodium phosphate buffer (0.065 M, pH 6.70) containing 0.02% sodium azide, and the C-dimer of RNase A was isolated by cation exchange chromatography with a Source 15S HR 16/10 column using a gradient of 0.09-0.20 M sodium phosphate, pH 6.7, which also contained 0.02% sodium azide. The flow rate was 1.2 mL per min. All chromatography procedures were preformed at room temperature. After isolation, the C-dimer
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was concentrated using Centriplus concentrators with an Mr cutoff of 3 kDa or 10 kDa. EDC Reaction. A series of trial reactions were performed at various concentrations of EDC (6 to 180 mM), RNase A (0.2 to 0.7 mg/mL), NaCl (0 to 0.5 M), and different pH values (4.0 to 7.0) with a 2 h reaction time at room temperature. The yield of cross-linked C-dimer was determined by integrating the dimer and monomer peaks after heating the sample to dissociate noncross-linked C-dimer. Using the paper printout of the chromatogram, integration was performed manually by cutting out the peaks corresponding to cross-linked C-dimer and monomer and weighing them on a high-precision balance. The most satisfactory yields of cross-linked C-dimer were obtained using a concentration of C-dimer of 0.5 mg/mL, in 100 mM MES at pH 5.0 with 68 mM EDC. These same conditions and reactant concentrations were used for the reaction of EDC with RNase Sa, hen lysozyme, carbonic anhydrase, and hemoglobin. Some RNase A reaction trials included 5 mM sulfo-NHS. EDC (∼100 mM) reactions with the dipeptide Ac-Asp-Ala-NH2 (8 mM) in 20 mM MES buffer were carried out at 25 °C and pH 5.5-5.8 and monitored by NMR spectroscopy and mass spectrometry. Product Isolation and Characterization. Superdex 75 gel filtration columns, equilibrated with 0.2 M sodium phosphate buffer at pH 6.7 was used at room temperature at a flow rate of 0.5 mL/min to purify and characterize the RNase A reaction products. Further purification and characterization of the product species was performed using cation exchange chromatography using a Mono S column. After reaction with EDC, RNase Sa, hen lysozyme, carbonic anhydrase, and hemoglobin were desalted using a PD-10 column (GE Healthcare) and concentrated using Centricon concentrators with a Mr cutoff of 10 kDa. Gel Electrophoresis. Denaturing SDS polyacrylamide gel electrophoresis (PAGE) was used for analysis (22).Ten percent polyacrylamide gels containing SDS were run at 20 mA for 90 min at room temperature. Cathodic PAGE under nondenaturing conditions was performed according to ref 23 with the slight modification of using β-alanine/acetic acid buffer at pH 4.0 as described previously (6). The 12.5% polyacrylamide gels were run at 20 mA for 100-120 min at 4 °C. For both types of gels, fixing and staining were performed with 12.5% trichloroacetic acid and 0.1% Coomassie brilliant blue. Densitometry and molecular weight estimations were performed using the GeneTools computer program (v 3.06) from SynGene/SynOptics Ltd., Cambridge, England. Mass Spectrometry. Matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectra were registered with an Applied Biosystems Voyager System 6214 mass spectrometer as previously described (24). Peptide Mapping using Proteolysis and HPLC/Electrospray Mass Spectrometry. The position of the EDC modified carboxylate groups in RNase A was characterized by proteolysis with subtilisin and trypsin. First, RNase A or EDC modified RNase A was cleaved with subtilisin. The amount of subtilisin added was 1/100th (w/w) that of RNase A. The solution is then incubated at room temperature at pH 8.0 in 25 mM ammonium acetate buffer. This buffer was chosen because it is one of the least interferring salts for mass spectrometry. Small amounts of KOH were added over the course of 3-31/2 h to maintain the pH close to 8.0. The reaction was performed at room temperature. After this time, the pH of the solution was reduced to
