Feb 14, 1994 - NH4HCO3 (pH 8.5); protein concentrations were about 2 mg/ml (Bio-Rad protein assay kit with bovine serum albumin as standard).
Proc. Nati. Acad. Sci. USA Vol. 91, pp. 5868-5872, June 1994
Biochemistry
Disulfide linkages in the in vitro refolded intermediates of recombinant human macrophage-colony-stimulating factor: Analysis of the sulfhydryl alkylation of free cysteine residues by fast-atom bombardment mass spectrometry M. 0. GLOCKER*t, B. ARBOGAST*, R. MILLEY*, C. COWGILLt§, AND M. L. DEINZER*§ *Department of Agricultural Chemistry, Oregon State University, Corvallis, OR 97331; and tChiron Corporation, 4560 Horton Street, Emeryville, CA 94608
Communicated by F. W. McLafferty, February 14, 1994 (received for review November 11, 1993)
rhM-CSF is produced in E. coli as insoluble and inactive inclusion bodies. Isolated inclusion bodies can be reduced, denatured, and diluted under appropriate conditions to initiate folding into an active molecule (7). The kinetics of rhM-CSF refolding have been studied by SDS/PAGE and rhM-CSF bioactivity assays (7), but only limited structural information has been obtained on the refolding intermediates. Fast-atom bombardment mass spectrometry (FAB-MS) has been used for the identification of disulfide linkages (8, 11, 12), and trapping of in vitro refolding protein intermediates is well documented (13). The dynamics of disulfide-bond formation and folding of insulin was described after FAB-MS was used for the analysis of digests (14). In the present study the time course for disulfide-bond formation during refolding of rhM-CSF was investigated by FAB-MS analysis for disappearance of the alkylated cysteine residues in proteolytic peptides of refolding intermediates.
Fast-atom bombardment mass spectrometry ABSTRACT was used to follow the time course of disulfide bond formation during in vitro refolding of recombinant human macrophagecolony-stimulating factor. The content of iodoacetamidealkylated half-cystines in proteolytic peptides of trapped refolding intermediates collected at 0, 6, 17, 24, and 72 hr was determined under reducing conditions. Size-exclusion highperformance liquid chromatography analyses of the collected alkylated samples indicate that aggregated monomer proceeded through a nonaggregated monomer to an intermediate dimer and finally to the fully folded and active dimer. Underalkylation was first detected by fast-atom bombardment mass spectrometry in 17-hr samples at Cys'57 and Cys'59' and this corresponded to the first sample containing dimer. Analyses of intermediates from subsequent time points indicated a decrease in alkylated sulfhydryls, and at 72 hr no alkylated peptide was detected. Early samples containing only monomer showed no evidence of disulfide bonds, and the occurrence of disulfide shuffling at the monomer stage could be ruled out under the highly reducing conditions used for refolding. Biological activity was not detectable in early samples but increased to 3.6% after 24 hr of refolding and to 86% of maximum at the 72-hr time point.
MATERIALS AND METHODS In Vitro Refolding of rhM-CSF and Trapping of Intermediates. Highly purified mature rhM-CSF dimer (mature rhMCSF) was stored frozen at an A280 of 22 in 5 mM sodium citrate (pH 6.5). A thawed aliquot was diluted 1:1 in a 16 M urea slurry containing 0.1 M Tris'HCl (pH 8.5), 10 mM dithiothreitol, and 25 mM EDTA. The sample was incubated at room temperature for 3 hr and then diluted 1:11 into prechilled (4°C) 50 mM Tris HCl, pH 8.5/1 mM EDTA/0.4 mM glutathione (GSH)/0.2 mM oxidized glutathione (GSSG). Final monomer concentration was 1.4 mg/ml. Aliquots were removed at given time points up to 8 days and blocked by addition of iodoacetamide (1 mM). Dimerization in each aliquot was estimated by size-exclusion (SE)-HPLC in 20 mM sodium phosphate (pH 7.0)/0.2 M ammonium sulfate with a DuPont GF-250 monitored at A214. The flow rate was 1 ml/min for 20 min. Biological activity was determined by the M-NSF-60 cell culture assay (15). Reversed-Phase (RP)-HPLC Purification of rhM-CSF Refolding Intermediates and Mature rhM-CSF. An Altex model 322 gradient liquid chromatograph equipped with a Waters 486 tuneable absorbance detector set at 220 nm was used. Linear gradients consisted of 0.1% trifluoroacetic acid (TFA) in water as solvent A and 0.08% TFA in acetonitrile (CH3CN) as solvent B at a flow rate of 1 ml/min. Protein was purified by RP-HPLC on a Vydac C4 column (30 nm, 10 ,um, 4.6 x 250 mm). Samples from the refolding time course were chromatographed starting with 10%6 solvent B. After 5 min the
Macrophage-colony-stimulating factor (M-CSF) is a cytokine of interest because of its activity as an antiinfective in immunocompromised patients (1, 2). It is a hematopoietic growth factor that stimulates the survival, proliferation, differentiation, and function of mononuclear phagocytes (3, 4). Three human cDNA clones have been isolated and expressed as active protein from cells containing a single M-CSF gene (5, 6), and this indicates that distinct M-CSF species are produced through alternative mRNA splicing. The coding sequences reveal polypeptide precursors 256 aa (a), 554 aa (P,), and 438 aa (y). The three species contain the same 149 N-terminal residues and 75 C-terminal residues (postulated to be transmembrane sequences) with internal sequences of variable length. The recombinant human M-CSF (rhM-CSF) studied here was cloned and expressed from Escherichia coli as a truncated form (aa 4-218) of the longest cDNA clone (M-CSFI3) (7). rhM-CSF contains nine disulfide bonds, three of which participate in intermolecular bridges resulting in a 49-kDa homodimer. We recently reported the locations of the disulfide linkages (Fig. 1B) in rhM-CSF,8 (9). X-ray analysis of a smaller clone (aa 4-158), rhM-CSFa, has confirmed the assignments and reveals that the dimer is formed by end-toend disulfide linking of two four-helical bundles in which the helices run up-up-down-down (10).
Abbreviations: rhM-CSF, recombinant human macrophage-colonystimulating factor; FAB, fast-atom bombardment; GSH, (reduced) glutathione; SE, size-exclusion; RP, reversed-phase; TFA, trifluoroacetic acid. tPresent address: Faculty of Chemistry, University of Konstanz, P.O. Box 5560, 78434 Konstanz, Germany. §To whom reprint requests should be addressed.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. 5868
Biochemistry: Glocker et A
Proc. Natl. Acad. Sci. USA 91 (1994)
A 10
SEYCSHM 50
EQLKDPVCYL LJ 90
QELSLRLKSC LJ
130
FNETKNLLDK LJ
20
IGSGHLQSLQ 60
KKAFLLVQDI LJLJ
100
FTKDYEEHDK
140
DWNIFSKNCN
LT L1
Li
170
YPKAIPSSDP L] K]
210
SLLPGEQPLH
Li
LJ
180
ASVSPHQPLA 220
TVDPGSAKQR
L]
30 RLIDSQMETS 70
MEDTMRFRDN 110
ACVRTFYETP
40 CQITFEFVDQ 80 TPNAIAIVQL
120 LQLLEKVKNV
150
LJ L]
160
NSFAECSSQD WTKPDCNCL LT LT KJ KJ 200 190 PSMAPVAGLT WEDSEGTEGS P
B C7
I~
C31
031
C48
C90
~
090
0102 0102
C139
C90
C102
C139
0139
C146 C157 C159
C7
C31
C~48
Thermolysin (EC 3.4.24.4, Sigma) (1 A4, 1 mg/ml) in 50 mM NH4HCO3 (pH 8.5) was added to 8 /4 of protein solution (5 or 2 mg/ml) in 50 mM NH4HCO3 (pH 8.5) (ES, 1:40 or 1:16). Proteinase K (EC 3.4.21.14, Sigma) (1 /4, 1 mg/ml) in 50 mM NH4HCO3 (pH 8.5) was added to 8 A4 of protein solution (5 or 2 mg/ml) from refolding intermediates in 50 mM NH4HCO3 (pH 8.5) (ES, 1:40 or 1:16). In the case of mature rhM-CSF, 5 /1 of the major BrCN cleavage fragment (aa 66-183) in 50 mM NH4HCO3 (pH 8.5) (4 mg/ml) was digested with thermolysin or proteinase K (E/S, 1:20). All digestions were carried out at 37°C for 3 hr. FAB-MS Analysis of Proteolytic Digests. Aliquots (2 /4) were mixed on the probe with 2 ,l of dithiothreitol/ dithioerythritol (5:1, wt/wt). FAB-MS was carried out on a Kratos MS-50 instrument at a resolution of 900 using 7- to 8-keV Xe sputtering beams from an Ion-Tech gun. The acceleration potential was 8 keV and the postacceleration potential was 25 keV.
RESULTS The refolding reaction was initiated by dilution of denatured and reduced monomer in a buffer of high reducing potential (Q0.4 mM residual dithiothreitol SH plus 0.4 mM GSH plus 0.5 mM protein SH/0.4 mM GS- = 3.3).1 The half-time for dimer formation was =32 hr as compared to 2.5 hr under ideal conditions (7). This highly reducing environment was selected to slow down possible monomer oxidation (7). Aliquots were taken at various time points and treated with iodoacetamide, and the blocked intermediates were purified and partially fractionated by RP-HPLC (Fig. 2) before protease digestion. Analysis of early times of refolding (0-6 hr) showed no dimer formation or biological activity. Under SE-HPLC conditions, almost all of the material traveled as a hydrophobic monomer aggregate at the void volume (peak M, Fig. 3). When SDS was included in the buffer, the peak shifted to the position of nonaggregated monomer. Peak M probably represents an extended form which aggregates during aqueous sizing analysis. RP-HPLC purification of the 0-hr sample gave one homogeneous peak (Fig. 2A, fraction 1), and peptide mapping of fraction 1 proved that all cysteines were completely alkylated (see Tables 2-4). The mass spectrum of Lys-C-derived peptide L12, for example, gave one peak with m/z 3108, which means that Cys'39, Cys46', Cys157, and Cys159 in this peptide were completely alkylated. Thus, both reduction and alkylation of the protein initially went to completion and the protein remained reduced for 6 hr. After 17 hr of refolding, 9o dimer was detected by SEHPLC (Table 1). The dimer D' (Fig. 3) was eluted before the mature dimer. Moreover, 16% of the sample components had a retention volume indicative of the nonaggregated monomer M' (Fig. 3). This sample had almost no activity (Table 1). Protein was collected as one peak after RP-HPLC purification (Fig. 2B, fraction 2) and digested with Lys-C. The sample contained protein populations with some underalkylation. The dominant peak in the spectrum of L12 corresponded to the fully alkylated peptide (m/z 3108), but peaks with m/z 3051 and m/z 2994 revealed underalkylation of one and two cysteines, respectively. All other cysteine-containing peptides were fully alkylated (Table 2). The mass spectrum of a thermolysin digest of fraction 2 showed a peak with m/z 1083 for alkylated T1 (aa 134-142). This result indicated that Cys139 was completely alkylated
~~~~~~~~~~~~~~~~~~~~~~~~~.......
.(
......
5869
C146 C157C159 ~~~~~~~~~~~~~~~~~~~~~~~~......
FIG. 1. (A) Amino acid sequence of rhM-CSF (7). Selected cleavage sites for Lys-C (L), proteinase K (K), and thermolysin (T) are shown below the amino acid sequence. (B) Schematic representation of the rhM-CSF dimer (9).
concentration was increased over 30 min to 60% B and then held constant for 10 min. Protein-containing fractions were collected, pooled, and dried in a Speedvac concentrator (Savant). The 0-hr (Fig. 2A, fraction 1) and 17-hr (Fig. 2B, fraction 2) samples were dissolved in 50 /4 of 50 mM NH4HCO3 (pH 8.5) to give a protein concentration of 5 mg/ml. The 24-hr (Fig. 2C, fractions 3-6) and 72-hr (Fig. 2D, fractions 7-9) samples were dissolved in 30 /4 of 50 mM NH4HCO3 (pH 8.5); protein concentrations were about 2 mg/ml (Bio-Rad protein assay kit with bovine serum albumin as standard). Mature rhM-CSF was chromatographed starting with 10%6 solvent B. After 5 min B was increased over 60 min to 60%o and then held constant for 10 min. The major protein fraction (>90%) (Fig. 2E, fraction 10) was dried and dissolved in 500 /4 of a 6:4 mixture of 0.1% TFA in water and 0.08% TFA in CH3CN, giving a protein concentration of 4.5 mg/ml. Cyanogen Bromide (BrCN) Cleavage of Mature rhM-CSF and Separation of the Cleavage Products. A solution (10 /4) of 5 M BrCN in CH3CN was added to 500 Al of protein solution (4.5 mg/ml, 46 nmol) in a 6:4 mixture of 0.1% TFA in water and 0.08% TFA in CH3CN. After 20 hr at 25°C in the dark, 500 /4 of water was added and excess BrCN was removed. The volume was adjusted to 200 /l and the sample was subjected to RP-HPLC. After 1 min at 10% B the concentration was raised over 65 min to 60%o B. The major protein fraction was eluted after 62 min. Buffer Exchange After BrCN Reaction. After BrCN cleavage the major protein fragment was concentrated with care to avoid precipitation. The sample was transferred to a Centricon (Amicon; Mr cutoff, 10,000). The buffer was exchanged three times at 4°C by adding 300 / of 50 mM NH4HCO3 (pH 8.5) and reducing the volume to =100 /4. The final volume was adjusted to 300 ul (protein, 4 mg/ml). Enzymatic Digestion of rhM-CSF Refolding Intermediates. Lys-C endopeptidase (EC 3.4.21.50; Wako chemicals) (1 /4, 1 mg/ml) in 50 mM NH4HCO3 (pH 8.5) was added to 11 /4l of protein solution (5 or 2 mg/ml) in 50 mM NH4HCO3 (pH 8.5) [enzyme/substrate (E/S) ratio, 1:55 or 1:22 (wt/wt)].
IThese values were calculated, not determined experimentally. Dithiothreitol was used to reduce the protein initially; its residual sulfbydryl concentration in the refolding reaction after dilution was calculated as 0.9 mM dithiothreitol SH mM.
-
0.5 mM protein SH
=
0.4
5870
Biochemistry: Glocker et al. A
O
B
hr
1
17 hr 2
C
6
L
II
6
I1
E
4
Final
3
10
KX Retention time
FIG. 2. RP-HPLC of refolding intermediates at 0, 17, 24, and 72 hr of refolding (A-D respectively) and of purified mature rhM-CSF
(E).
and, therefore, not connected through a disulfide linkage (Table 3). Thermolysin-derived peptide T2 (aa 143-159) showed a dominant peak with m/z 2016 for the fully alkylated peptide and peaks with m/z 1959 and 1901 revealed the presence of singly and doubly underalkylated peptide (Table
Time,
min
FIG. 3. SE-HPLC analysis of alkylated rhM-CSF refolding intermediates. M, aggregated monomer which is eluted at the void volume; M', monomer intermediate; D', dimer intermediate; D, mature dimer.
Proc. Natl. Acad. Sci. USA 91 (1994) Table 1. Intermediates and bioactivity of rhM-CSF during folding
Time, aggregated % dimer % monomer hr (M) (D' + D) (M') bioactivity
