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http://www.abbs.info e-mail:[email protected] ISSN 0582-9879 ACTA BIOCHIMICA et BIOPHYSICA SINICA 2003, 35(2): 122-126 CN 31-1300/Q |
Influence
of A7-B7 Disulfide Bond Deletion on the Refolding and Structure of Proinsulin
LIU
Ying, TANG Jian-Guo*
College of Life Science, Peking University, Beijing 100871, China )
Abstract To probe the role of [A7-B7] disulfide bond in the structure and folding of proinsulin, [A7, B7Ser]-proinsulin was prepared. The differences in the in vitro refolding, oxidation of free thiol groups, circular dichroism (CD) spectra, antibody and receptor binding assays, and sensitivity to tryptic digestion between the mutant and the wild type proteins were studied. The deletion of [A7-B7] disulfide bond in proinsulin resulted in a significant decrease of α-helix content of the molecule and a great increase in sensitivity to tryptic digestion. The [A7-B7] disulfide bond deleted proinsulin showed a great loss of its receptor binding activity. The in vitro refolding study indicated that the rate of the oxidation of free thiol groups in the mutant was a little slower at the later stage as compared to the native molecule, but the deletion of [A7-B7] disulfide bond had little effect on the refolding yield. A possible proinsulin folding pathway way was proposed. During the folding, the intra-A chain disulfide bond forms first and very fast. The formation of the [A20-B19] disulfide bond is more crucial than [A7-B7] disulfide bond and forms very likely earlier than the latter.
Key
words
disulfide bond; in vitro
refolding; proinsulin; secondary structure
Disulfide bond in proteins usually serves as
a major element for structural stabilization. Formation of the disulfide bond
also has important impact on protein folding pathway. The folding pathways of
several proteins containing disulfide bonds, such as bovine pancreatic trypsin
inhibitor (BPTI)[1, 2], RNase A[3-5] and insulin-like
growth factor I (IGF-I)[6-8], have been widely studied. Insulin is a
protein containing three disulfide bonds, i.e., one intra-A chain [A6-A11], two
inter-chain [A7-B7] and [A20-B19]. In the biosynthesis of insulin, the three
disulfide bonds are formed in an intramolecular manner. Much progress in the
study of the folding pathway of insulin precursor has been made recently. It
was demonstrated that the intra-A chain disulfide bond forms first in the
folding pathway of insulin precursor[9] and a putative folding
pathway of insulin precursor has been proposed[10]. Des-B30-[A7,
B7Ser]-insulin and DKP-[A7, B7Ser]-insulin were also studied[11, 12].
The data revealed that the [A7-B7] inter-chain disulfide bond is crucial for
maintaining the native conformation and biological activity of insulin, and two
inter-chain disulfide bonds are important for efficient folding/secretion of
insulin precursor in yeast[11]. Yet, the detailed role of [A7-B7]
disulfide bond in the structure and folding of insulin precursor has to be
fully elucidated.
In this paper, [A7, B7Ser]-human proinsulin
([A7, B7Ser]-HPI) was prepared by replacing cysteine residues with serine at A7
and B7 positions in the HPI, and its physico-chemical properties, biological
activities as well as the in vitro
refolding behaviors were studied.
1 Materials and Methods
1. 1 Materials
Escherichia
coli strain DH5α was used as host
cell. The expression vector pBV220 was a gift from Dr. HOU Yun-De of the
Institute of Virology, Chinese Academic of Preventive Medicine. Plasmid pJG103
containing human proinsulin gene was previously constructed in our laboratory
(derived from pBV220)[13]. PCR primers were synthesized by Sangon. T4
DNA ligase, Taq DNA polymerase, EcoRI, BamHI were purchased from Promega. Trypsin
with an activity of 10 400 u/mg was a Sigma product. DTT was from BBI.
5,5’-dithio-bis-(2-nitrobenzonic acid) (DTNB) and BSA were from SABC. Other
chemicals of analytical or chromatographic grade were all
local products. 125I-insulin radioimmunoassay kit was kindly
provided by Navy Radioimmunoassay Technique Center (Beijing, China).
1. 2 Construction of mutant gene and
preparation of protein
The [A7, B7Ser]-HPI gene was obtained by site-directed mutagenesis using PCR with
pJG103 as the template. After cleavage with EcoRI
and BamHI, the mutant gene was
inserted into pBV220 and the recombinant expression plasmid was transformed E. coli strain DH5α. The positive colony was identified by restriction mapping
analysis and further confirmed by DNA sequencing. Both HPI and [A7, B7Ser]-HPI
were expressed and proteins purified following procedure described previously[13].
The crude protein was then proceeded a final purification by reversed-phase fast protein liquid chromatography
(RP-FPLC) on a ResourceTM (3 mL) column and then analyzed by 15%
native PAGE. The purified proteins with about 95% purity were lyophilized and
stored at –20 °C for later
testing.
1. 3 In
vitro refolding assay of fully reduced proteins
1 mg HPI or [A7, B7Ser]-HPI was dissolved in
1 mL 0.1 mol/L Tris-HCl buffer (pH 8.0), containing 8 mol/L urea, 1 mmol/L EDTA
and 20-fold (in terms of thiol group) excess of DTT. After incubation for 2 h
at 37 °C, the reaction mixture was dialyzed against 2000 volumes of refolding
buffer (pH 10.8), containing 0.05 mol/L glycine-NaOH, at 4 °C for 24 h to
remove reducing agent and denaturant. 10 μL of refolding product was then
analyzed by both 15% native PAGE and 15% non-reductive SDS-PAGE. After staining
the gel with Coomassie brilliant blue, the proinsulin bands were quantified by
densitometry using software Glyko Bandscan (Glyko, Inc., USA).
1. 4 Analysis of tryptic digestion
2 mg HPI or [A7, B7Ser]-HPI was dissolved in
1mL 0.05 mol/L Tris-HCl buffer (pH 7.2). Trypsin was dissolved in 1 mmol/L HCl
to different concentrations. 2 μL trypsin was then added to 20 μL HPI or [A7,
B7Ser]-HPI at defined enzyme /substrate (W/W)
ratios. After incubation at 37 °C for 0.5 h, the reaction was stopped by
addition of equal volume of 2× native PAGE loading buffer. 10 μL of reaction
mixture was analyzed by 15% native PAGE. The corresponding des-B30Thr-insulin
bands were quantified by software Glyko Bandscan.
1. 5 Analysis of thiol groups oxidation
2 mg HPI or [A7, B7Ser]-HPI was dissolved in
1 mL of 0.05 mol/L Tris-HCl buffer (pH 8.0), containing 8 mol/L urea, 1 mmol/L
EDTA, and 20-fold (in terms of thiol group) excess of DTT. After 2 h incubation
at 37 °C, the pH of the reaction mixture was adjusted to 2~3 and then the
reaction mixture was loaded onto a desalting FPLC HiTrapTM column (5
mL). The column was eluted with 0.1% TFA to remove DTT in excess. The reduced
protein fraction was then collected. 20 μL sample was mixed with 0.5 mL DTNB
solution to determine numbers of thiol group at 0 h. The rest sample was mixed
with 1/10 volume 0.5 mol/L glycine-NaOH (pH 10.8) and incubated at 4 °C for
refolding. The numbers of thiol group at various time periods during refolding
were determined with DTNB according to the Ellman’s method[14].
1. 6 Analysis of receptor and antibody
binding activities
Crude insulin receptor
preparation was obtained from human placenta for insulin receptor binding
assay[15] and immuno assay was performed as described in the manual
of 125I-insulin radio immunoassay kits. Two measurements were
calculated as mean±SD.
1. 7 Circular dichroism (CD) studies
The HPI or [A7, B7Ser]-HPI was dissolved in
double distilled water (pH 7.0). Protein concentration was determined by UV
absorbency, and adjusted to 0.2. CD spectra were recorded by a Jasco-715
circular dichroism spectropolarimeter at 25 °C and from 190 nm to 240 nm. The
cell path length was 1 mm. The relative secondary structure contents were
calculated according to the method of Chen et al.[16].
2 Results
2. 1 Comparison of in vitro refolding of HPI and [A7, B7Ser]-HPI
HPI and [A7, B7Ser]-HPI were fully reduced by
DTT and then dialyzed against the refolding buffer (pH 10. 8) for renaturation.
In native PAGE gel, HPI and [A7, B7Ser]-HPI purified by RP-FPLC are shown to be
a major single band with similar mobility rate (lanes 1 and 2 in Fig.1). Both
crude refolded products show many bands on the gel (lanes 3 and 4 in Fig.1).
The relative percentage of the band of the correctly refolded product was
quantified by densitometry. The refolding yield for [A7, B7Ser]-HPI is 88%,
almost equals the yield for HPI of 82%. The same result can be seen by
non-reductive SDS-PAGE analysis (Fig.2). Most refolded products are in
monomeric form, and only small amounts are dimers and polymers (lanes 3 and 4
in Figs.1 and 2).
Fig.1
15% native PAGE analysis of HPI and [A7, B7Ser]-HPI before and after the
in vitro refolding
1, 2, HPI and [A7, B7Ser]-HPI purified by RP-FPLC, respectively; 3, 4, crude
in vitro refolding products of HPI
and [A7, B7Ser]-HPI, respectively.
2. 2 Sensitivity of HPI and [A7, B7Ser]-HPI
to tryptic digestion
Both HPI and [A7, B7Ser]-HPI were digested
with trypsin, and analyzed by native PAGE (Fig. 3). Des-B30Thr-insulin as well
as some intermediates during tryptic digestion of HPI can be observed [15].
Des-B30Thr-insulin shows similar mobility rate on native PAGE gel to that of
porcine insulin [Fig. 3(A)]. The tryptic peptide mapping pattern of [A7,
B7Ser]-HPI is similar to that of HPI as both proteins share the same tryptic
cleaving sites. The mobility rate of des-B30Thr-[A7, B7Ser]-insulin is a little
slower than that of porcine insulin [Fig. 3(B)]. With the increase of trypsin
concentration, the acquired des-B30Thr-[A7, B7Ser]-insulin was partially
further converted into des-octapeptide-[A7, B7Ser]-insulin at the
enzyme/substrate ratio of 1/400. The insulin analogue band disappeared
completely at the enzyme/substrate ratio of 1/25. Meanwhile, HPI could only be
digested to des-B30Thr-insulin even at the enzyme/substrate ratio of 1/25. This
suggests that [A7, B7Ser]-HPI is much more sensitive to tryptic digestion than
HPI. The yields of des-B30Thr-insulin or des-B30Thr-[A7, B7Ser]-insulin at
different enzyme/substrate ratios were quantified and plotted in Figure 3(C).
Fig.2 15% non-reductive SDS-PAGE analysis of
crude in vitro refolding products
of HPI and [A7, B7Ser]-HPI
M,
molecular weight marker; 1, crude refolded HPI; 2, crude refolded [A7,
B7Ser]-HPI.
Fig.3 Tryptic digestion assay of HPI and [A7,
B7Ser]-HPI by 15% native PAGE analyses in (A) and (B), respectively
(A) 1, HPI. 2 to 7, digestion products at enzyme/substrate (W/W) ratios of 1/600, 1/400, 1/200,
1/100, 1/50, 1/25, respectively; 8, porcine insulin. (B) 1, [A7, B7Ser]-HPI; 2
to 8, digestion products at enzyme/substrate (W/W) ratios of 1/1600, 1/800, 1/400, 1/200, 1/100, 1/50, 1/25,
respectively; 9, porcine insulin. The lanes for des-B30Thr-insulin and
des-B30Thr-[A7, B7Ser]-insulin were indicated in (A) and (B), respectively.
(C), a plot indicating the digestic production rate at different E/S ratios. ●, des-B30Thr-insulin; ○, des-B30Thr-[A7, B7Ser]-insulin.
2. 3 In
vitro oxidation of the thiol groups of reduced proteins
The time courses of the in vitro oxidation of thiol groups of reduced HPI and [A7,
B7Ser]-HPI are shown in Figure 4. In the first half hour, the oxidation rate of
reduced [A7, B7Ser]-HPI was very fast, similar to that of HPI. After one hour,
although oxidation of reduced HPI was continuously at a relatively high speed,
the oxidation rate of reduced [A7, B7Ser]-HPI decreased
significantly.
Fig. 4
Time course of oxidation of reduced HPI (●) and [A7, B7Ser]-HPI (○)
2.
4 Antibody and receptor binding
assays
The results of antibody and
receptor binding assays of HPI and [A7, B7Ser]-HPI are shown in Figure 5(A) and
5(B), respectively. Compared the dosages used for 50% inhibition of 125I-insulin
binding between HPI and [A7, B7Ser]-HPI, the antibody
binding activity of [A7, B7Ser]-HPI remains at 20.6%. However, the receptor
binding activity of [A7, B7Ser]-HPI is only 1.6% of that of HPI.
Fig. 5 Biological activity
assays of HPI (●) and [A7, B7Ser]-HPI (○)
(A)
antibody binding activity assay; (B) receptor binding activity assay. Data are
shown as mean±SD of two
measurements.
2. 5 Comparison of CD spectra
There are significant differences between the
CD spectra of HPI and [A7, B7Ser]-HPI (Fig. 6). The relative secondary
structure contents were calculated. The α-helix of [A7, B7Ser]-HPI is about
24%, much lower than that of HPI, which is 39%.
Fig. 6
CD spectra of HPI and [A7, B7Ser]-HPI
The
block line represents HPI, and the dotted line represents [A7, B7Ser]-HPI.
3 Discussion
The three disulfide bonds are absolutely
conserved in evolution of insulin family members. Deletion of the intra-A chain
disulfide bond resulted in an unfolding of the N-terminal α-helix of insulin A
chain (A1-A8) and great decrease of biological activity[15,17,18].
It was shown that the deletion of [A7-B7] inter-chain
disulfide bond yielded even more serious
effect on the structure and the biological activity of insulin[11,12].
In our study, significant changes exist in CD spectrum between [A7,
B7Ser]-HPI and HPI, and the α-helix content in the mutant is much lower than in the
native molecule. This agrees well with the results reported previously[11,12].
The tryptic digestion shows that [A7, B7Ser]-HPI is much more sensitive to
proteolytic action than HPI, which indicates that the removal of the [A7-B7]
disulfide bond results in a great structural change in proinsulin molecule.
This also agrees well with the CD data. The receptor binding activity of [A7,
B7Ser]-HPI is much lower than that of HPI, which is also similar to the data
reported previously[11,12].
However, its immunoactivity is partially retained. This indicates that the
conformation for antibody binding sites may have not been changed greatly,
although great changes have taken place in its receptor binding site as elucidated[12].
Guo et al.[11] found that removal of the
[A7-B7] disulfide bond resulted in a great decrease in the yield of recombinant
insulin precursor secreted from yeast, and the mutant
without the inter-chain disulfide bond [A20-B19] even failed to secrete from
the yeast cell. Based on these observations, they concluded that the two
inter-chain disulfide bonds were important for efficient folding/secretion in vivo, and they also inferred that the
[A20-B19] disulfide bond formed earlier than [A7-B7]. While in our study, the in vitro refolding yield of [A7,
B7Ser]-HPI is even a little higher than that of the wild type, indicating that
the deletion of [A7-B7] disulfide bond has little effect on the refolding yield
of proinsulin. With two less thiol groups than reduced HPI, the reduced [A7,
B7Ser]-HPI may have less possibility to form dimers or polymers during
refolding. It was suggested that there is a complex quality control system in
the in vivo secretory pathway, and
only proteins that pass a stringent selection can be secreted at last[19].
Based on our study, the decrease of the α-helix content and the increase of
sensitivity to tryptic digestion indicate a much looser conformation of [A7,
B7Ser]-HPI than the wild type. Even if the [A7, B7Ser]-insulin precursor might
have folded correctly in yeast cell, its structure is unstable and easier to be
attacked by protease in vivo, so that
the secretion yield of [A7, B7Ser]-insulin precursor decreased greatly[11].
The time course of oxidation of
thiol groups in reduced protein may provide some
information to illustrate the folding pathway. In our earlier work, the
differences in the time courses of oxidation of reduced insulin A chain,
reduced A and B chains, and reduced proinsulin between the [A6-A11] disulfide
bond deleted mutant and the wild type were studied. The intra-A chain disulfide
bond forms first in the folding pathway of insulin precursor was
proposed[9].
This was also verified by the investigation of the oxidation of recombinant
porcine insulin precursor as a major folding pathway[10]. Herein we
compared the time course of oxidation of reduced [A7, B7Ser]-HPI with that of
HPI and the final refolding yields of both proteins. The result indicates that
more than two thiol groups in the reduced [A7, B7Ser]-HPI disappeared in the
first half hour and the speed of oxidation was fast. This suggests that,
similar to HPI, the [A6-A11] intra-A chain disulfide bond in [A7, B7Ser]-HPI
has formed first. The oxidation rate of [A7, B7Ser]-HPI was then slowed down, indicating
a possibly slow formation of the [A20-B19] inter-chain disulfide bond, which
means that deletion of the [A7-B7] inter-chain
disulfide bond does have influence on the
formation of [A20-B19] disulfide bond. Even though, as reduced [A7, B7Ser]-HPI
has two less thiol groups as compared with reduced HPI, the former protein
gives an equal, if not higher, yield to the latter (Figs. 1 and 2). In another study of our laboratory on
[A20-B19] disulfide bond deleted HPI, it was demonstrated that the deletion of
[A20-B19] disulfide bond did not affect the oxidation of the other four thiol
groups significantly, but the refolding yield was decreased to about 45% as
compared to 88% for [A7, B7Ser]-HPI at similar
conditions (unpublished data). This indicates that formation of the [A20-B19]
disulfide bond is more important than that of [A7-B7] disulfide bond in the
proinsulin folding pathway and the former disulfide bond is very likely to form
earlier than the latter. Based on all the above results, we propose a very possible proinsulin folding pathway: During the
folding, the intra-A chain disulfide bond forms first and very fast, which can
stabilize partially folded A chain secondary structure[9]. The
C-peptide may serve as an intra-molecular chaperone-like function to prevent B
chain from aggregation and help A chain with partially native like structure to
pair with B chain to form the two inter-chain disulfide bonds[20].
The formation of the [A20-B19] disulfide bond is more crucial than [A7-B7]
disulfide bond for the correct folding of proinsulin and forms very likely
earlier than the latter.
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