Research Paper
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Acta Biochim Biophys Sin
2005,37:673-679 |
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doi:10.1111/j.1745-7270.2005.00093.x |
Mutational Analysis of the
Absolutely Conserved B8Gly: Consequence on Foldability and Activity of Insulin
Zhan-Yun GUO1, Zhou ZHANG1, Xiao-Yuan JIA1, Yue-Hua TANG1,2, and You-Min FENG1,2*
1 Key Laboratory of
Proteomics, Institute of Biochemistry and Cell Biology, Shanghai Institutes for
Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China;
2 Institute of
Biochemistry, Zhejiang Sci-Tech University, Hangzhou 310018, China
Received: March 25,
2005
Accepted: June 24,
2005
*Corresponding
author: Tel, 86-21-54921133; Fax, 86-21-54921011; E-mail,
[email protected]
Abstract B8Gly is absolutely conserved
in insulins during evolution. Moreover, its corresponding position is always occupied
by a Gly residue in other members of insulin superfamily. Previous work showed
that Ala replacement of B8Gly significantly decreased both the activity and the
foldability of insulin. However, the effects of substitution are complicated,
and different replacements sometimes cause significantly different results.
To analyze the effects of B8 replacement by different amino acids, three new
insulin/single-chain insulin mutants with B8Gly replaced by Ser, Thr or Leu
were prepared by protein engineering, and both their foldability and activity
were analyzed. In general, replacement of B8Gly by other amino acids causes
significant detriment to the foldability of single-chain insulin: the
conformations of the three B8 mutants are essentially different from that of
wild-type molecules as revealed by circular dichroism; their disulfide
stabilities in redox buffer are significantly decreased; their in vitro
refolding efficiencies are decreased approximately two folds; the structural
stabilities of the mutants with Ser or Thr substitution are decreased
significantly, while Leu substitution has little effect as measured by
equilibrium guanidine denaturation. As far as biological activity is
concerned, Ser replacement of B8Gly has only a moderate effect: its insulin
receptor-binding activity is 23% of native insulin. But Thr or Leu replacement
produces significant detriment: the receptor-binding potencies of the two
mutants are less than 0.2% of native insulin. The present results suggest that
Gly is likely the only applicable natural amino acid for the B8 position of
insulin where both foldability and activity are concerned.
Key words insulin; foldability;
activity; disulfide; stability
As early as the 1960s,
Anfinsen et al. first demonstrated the 3-D structure of a globular
protein is uniquely determined by its amino acid sequence [1]. Since then
significant advances have been made in the understanding of protein folding
through experimental and theoretical approaches. For small proteins with
two-state folding, topology is a major determinant of the folding rate and
greatly influences the structure of the transition-state ensemble [2-4]. Studies on the disulfide-coupled
folding of some small globular proteins, such as bovine pancreatic trypsin
inhibitor and ribonuclease A, have revealed a sequence of preferred kinetic
intermediates, which define a folding pathway [5-11].
In vivo the protein folding is assisted by the molecular chaperones and
folding enzymes, especially for large proteins [12-14],
some chaperones can even provide the missing steric information for protein
folding [15].
Insulin is a structurally and
functionally well-characterized small globular protein containing A- and
B-chain linked by three disulfides (one intrachain bond, A6-A11; two interchain
bonds, A7-B7 and A20-B19). Its 3-D structure has been well solved by X-ray
crystallography [16,17] and nuclear magnetic resonance (NMR) [18]. In vitro
the separate A- and B-chain of insulin can be recombined efficiently [19], but
in vivo a single-chain polypeptide precursor (preproinsulin) is
synthesized. When B29Lys and A1Gly were linked by a peptide bond directly, the
mini-proinsulin still retained the 3-D structure identical to that of insulin
[20,21]. Our laboratory has constructed a single-chain insulin (PIP) in which
the C-terminal of porcine insulin B-chain and the N-terminal of its A-chain
were linked together by a dipeptide, Ala-Lys. PIP can fold correctly and be
secreted efficiently from transformed yeast cells, and can be converted to
insulin by enzymatic treatment [22]. It can be reasonably presumed that the 3-D
structure of PIP is identical or very similar to that of
insulin/mini-proinsulin.
Although significant advances
have been made in the understanding of protein folding, an understanding of how
the folding information is stored in the amino acid sequence is still elusive.
Through protein engineering, various mutants with one specific residue
replaced by different amino acids can be prepared and analyzed. This makes it
possible to analyze the contribution of individual residues to the foldability
and activity of the whole polypeptide chain. The structure and function of
insulin have been extensively investigated, and in vitro PIP can
spontaneously fold into native structure with a defined folding pathway characterized
by kinetically preferred intermediates [23-25].
Therefore, insulin/PIP provides a well-characterized model for the study of
protein folding as well as structure-function relationships. In insulins B8Gly
is absolutely conserved during evolution, moreover, its corresponding position
is always occupied by a Gly residue in other members of the insulin
superfamily, such as insulin-like growth factor I and II, relaxin, and
bombyxin. B8Gly is located at the conjunction of an invariant a-helix (B9-B19)
and a segment of variable conformation (B1-B8).
The latter segment undergoes the T (extended)®(helical) transition in
insulin hexamer. In R-state the main-chain dihedral angles are those of an L-amino
acid, while in T-state B8Gly lies in the D-region of the Ramachandran
plot. Using combinatorial peptide chemistry, Zhao et al. synthesized B8
L-amino acid and D-amino acid peptide libraries [26]. The B8 L-amino
acid library yielded only a trace amount of insulin, while the D-amino
acid library resulted in native foldability.
Replacement of B8Gly by Ala
significantly decreased the receptor-binding activity of insulin [27-29] and the foldability of PIP [30].
These suggest that B8Gly contributes to both the activity and foldability of
insulin. However, the effects of replacement are complicated, and different substitutions
sometimes cause essentially different results.
To further understand the
contribution of B8Gly to both the activity and foldability of insulin, we
prepared three new insulin/PIP mutants with B8Gly replaced with Ser, Thr or Leu
by means of protein engineering. These three residues were chosen mainly
because they are different with Ala in size or in hydrophilic property. The
effects of the replacements on the foldability and activity of insulin/PIP were
analyzed. The results also suggest that Gly is likely the only applicable
natural amino acid for the B8 position of insulin when both activity and
foldability are considered.
Materials and Methods
Materials
The Escherichia coli
strains DH12S and RZ1032 (dut-, ung-) were used. Saccharomyces cerevisiae XV700-6B (leu2,
ura3, pep4) and helper phage R408 were kindly provided by Michael SMITH (University
of British Columbia, Vancouver, Canada). Plasmid pVT102-U/aMFL-PIP for secretory expression of PIP
was constructed previously [22]. The mutagenesis oligonucleotide primers were
chemically synthesized. The other chemical reagents used were of analytical
grade.
The Vydac reverse-phase C8
column (5 mm, 4.6 mm´250 mm) (Vydac, Hesperia,
USA), Gilson 306 HPLC system (Gilson, Beltline, USA), and Gilson 115 UV
(Gilson) detector were used. In the HPLC analysis, a gradient elution was
used. Solvent A was 0.15% aqueous trifluoroacetic acid (TFA); solvent B was 60%
acetonitrile containing 0.125% TFA. The elution gradient was as follows: 0
min, 0% solvent B; 1 min, 0% solvent B; 5 min, 40% solvent B; 35 min, 80%
solvent B; 36 min, 100% solvent B; 38 min, 100% solvent B; 40 min, 0% solvent
B; 45 min, 0% solvent B.
DNA manipulation
The expression vectors
encoding the B8 mutants were constructed using a gapped duplex DNA approach for
site-directed mutagenesis [31]. The plasmid pVT102-U/aMFL-PIP
was used as the mutagenesis template, and the expected mutations were
confirmed by DNA sequencing.
Expression and purification of
B8 mutants
The expression vectors
encoding the B8 mutants were transformed into S. cerevisiae XV700-6B
(leu2, ura3, pep4), respectively. Then the transformed yeast cells were
cultured in a 16-liter fermenter and the secreted PIP mutants were purified
from the media supernatant as follows.The target protein in the media
supernatant was precipitated by trichloroacetic acid at the final concentration
of 5% (W/V). The pellet containing the target protein was dissolved
with 1 M acetic acid several times, and each time the resulting supernatant was
applied to a Sephadex G-50 column (Pharmacia, Uppsala, Sweden) pre-balanced
with 1 M acetic acid. Then the product separated by gel filtration was
lyophilized and applied to an ion-exchange column (DEAE-Sepharose-CL-6B)
pre-balanced with buffer [50 mM Tris-HCl, 40% (V/V) isopropyl alcohol,
pH 7.8], and eluted with a linear gradient of NaCl solution from 0 M to 0.2 M.
The eluted analog from the ion-exchange column was lyophilized, then dissolved
with 2-3 ml water, acidified to pH
2.0 with TFA, and centrifuged. The pellet containing the mutant was further
purified by C4 reverse-phase HPLC. The purity of these three analogs was
analyzed by native polyacrylamide gel electrophoresis (PAGE), pH 8.3, and
analytical C8 reverse-phase HPLC, respectively.
Circular dichroism analysis and
guanidine titration
The samples were dissolved in
35 mM phosphate buffer (pH 7.0). The protein concentration was determined by UV
absorbance at 276 nm with the extinction coefficient of 1.0 ml∙mg-1∙cm-1, and the final concentration
was adjusted to 0.2 mg/ml. Circular dichroism (CD) measurements were
performed on a Jasco-715 CD spectropolarimeter (Jasco Corp., Tokyo, Japan) at
25 ºC.
The near-UV spectra were scanned from 300 nm to 245 nm with a cell of 1.0 cm
path length; the far-UV spectra were scanned from 250 nm to 190 nm with a cell
of 0.1 cm path length. The data were expressed as molar ellipticity. The
software J-700 for Windows Secondary Structure Estimation (Version 1.10.00;
Jasco Corp.) was used for secondary structural content evaluation from CD
spectra.
For guanidine titration the
samples were dissolved to a final concentration of 0.2 mg/ml in 35 mM phosphate
buffer (pH 7.0) containing different concentrations of guanidine chloride. The
spectra of samples in different concentrations of denaturant were recorded from
230 nm to 220 nm at 25 ºC. The CD signal at 222 nm was used to
monitor the denaturation, and the measured data were fitted with the two-state
model.
Disulfide stability in redox
buffer
The wild-type PIP and the B8
mutants were dissolved in the buffer (0.1 M Tris-HCl, 1 mM EDTA, pH 8.7) containing
different concentrations of reduced glutathione (GSH; Amresco, Solon, USA) and
oxidized glutathione (GSSG; Amresco), respectively. The final protein
concentration was 0.15 mg/ml. The reaction was carried out at 0 ºC
overnight. After incubation, one-fifth volume of freshly prepared 0.5 M sodium
iodoacetate solution was added to modify the free thiol groups. Then the
mixture was analyzed by native PAGE, pH 8.3.
In vitro refolding analysis
The sample was reduced in
buffer containing 0.5 M Gly-HCl, 1 mM EDTA, pH 9.5, 10 mM dithiothreitol (DTT;
Sigma, St. Louis, USA) at 15 ºC for 30 min. The final concentration of
sample is 0.5 mg/ml. After reduction 6 ml
of solution was removed and immediately mixed with 3 ml
sodium iodoacetate solution (0.5 M). The carboxymethylation was carried out at
room temperature for 5 min, then the sample was analyzed by native PAGE, pH
8.3, to examine whether the samples were fully reduced. The fully reduced
sample was 10-fold diluted with the refolding buffer. The refolding was carried
out under the conditions indicated in Table 1 for 2 h. After
incubation, 100 ml of refolding mixture was
acidified to pH 2.0 with TFA, then analyzed by C8 reverse-phase HPLC and
detected at 230 nm. The refolding yield was calculated from the peak area of
the folded sample.
Conversion of single-chain
mutants to double-chain mutants
The purified B8 PIP analogs
were dissolved in the reaction buffer (0.1 M NH4HCO3, pH 8.5) at a final
concentration of 3 mg/ml. Then Lys-C endoproteinase was added to the solution
at a mass ratio of 1:500. The enzymatic cleavage was carried out at 25 ºC
overnight, then [desB30]insulin analogs were purified by C8 reverse-phase
HPLC.
Insulin receptor-binding assay
The receptor-binding assay of
[desB30]insulin mutants with insulin receptor was performed using human
placental membrane as previously described [32]. A total of 250 mg membrane insulin receptor was incubated
with 125I-insulin
(approximately 105 cpm) and 0.4 ml native insulin or sample solution (50 mM
Tris-HCl, 1% BSA, pH 7.5) at 4 ºC overnight. After incubation the unbound 125I-insulin was washed with
ice-cold buffer (50 mM Tris-HCl, 0.1% BSA, pH 7.5) three times and the
radioactivity of the precipitate was measured. The receptor binding
activity of the sample was calculated from the concentration that caused 50%
inhibition of 125I-insulin binding to the receptor.
Results
Expression and purification of
the B8 mutants
Three B8 mutants, [B8Ser]PIP,
[B8Thr]PIP and [B8Leu]PIP, were purified from the fermentation supernatant as
described in "Materials and Methods". After purification, the
purities were analyzed by analytical C8 reverse-phase HPLC and native pH 8.3
PAGE. As shown in Fig. 1, the three PIP analogs showed a single peak on
HPLC and a single band on native PAGE, therefore all of them were homogeneous.
Their molecular weights were analyzed by electrospray mass spectrometry and are
listed in Table 2. All of the measured values were well consistent with
the theoretical values, suggesting the expected mutations had occurred in the
three analogs. As far as the secretion yield was concerned, [B8Leu]PIP was
approximately 3-4 mg obtained from 8 liters of
fermentation supernatant; [B8Ser]PIP and [B8Thr]PIP were approximately 1-2 mg; while the wild-type PIP was
approximately 50 mg from the same volume of fermentation supernatant.
Therefore, mutation of B8Gly had significant detriment on the secretion efficiency
of PIP from transformed yeast cells. Additionally, the secretion efficiency of
[B8Leu]PIP was higher than that of [B8Ser]PIP and [B8Thr]PIP. This difference
was probably caused by their different structural stability: [B8Leu]PIP was
much more stable than the other two B8 mutants as shown by following stability
analysis. This result was confirmed by direct analysis of the secretion
product in the medium supernatant by native PAGE (pH 8.3, silver staining) and
densitometry (data not shown).
Circular dichroism analysis on
secondary structure
The conformational changes of the three B8 mutants were analyzed by CD. As shown in Fig. 2, both near-UV and far-UV spectra of the three mutants were different from that of the wild-type PIP. The estimated a-helix content of [B8Ser]PIP, [B8Thr]PIP, [B8Leu]PIP and wild-type PIP was 33%, 29%, 36% and 46%, respectively. Therefore, replacement of B8Gly by Ser, Thr, or Leu had significant effect on the conformation of PIP. Although the present CD analysis suggests that the secondary/tertiary structures of the three mutants had been disturbed, it is difficult to deduce where the disturbance occurred. High-resolution analyses (crystal or NMR) will be used to analyze the structural disturbance of these mutants in future.
Structural stability analysis
Our previous results showed
that Ala substitution at the B8 position significantly decreased the structural
stability of PIP [30]. Here, the effects of different mutations on the structural
stability of PIP were analyzed using guanidine titration. The denaturation was
monitored by CD, and the signals at 222 nm, a helix-sensitive wavelength, were
used to plot against the concentrations of denaturant, guanidine chloride. The
normalized denaturation curves of wild-type PIP and the three B8 mutants are
shown in Fig. 3, and the calculated thermodynamic parameters, Cmid and DGº,
are listed in Table 3. Both Ser and Thr replacement of B8Gly
significantly decreased the structural stability of PIP; this is similar to the
effect of Ala substitution. Unexpectedly, Leu replacement of B8Gly has little
effect on the structural stability of PIP.
Disulfide stability analysis
The structure of PIP/insulin must
be stabilized by its three disulfides. In turn, the structure affects the
disulfide stability. Here, the effect of B8 mutation on the disulfide stability
of PIP was analyzed. As shown in Fig. 4, on each gel the upper band is the
native species with intact disulfides (N), and the lowest band is the species
whose disulfides are fully reduced (R). For each sample, the disulfides of some
species were reduced after incubation in the redox buffer, and these reduced
species ran faster on the native PAGE after modification of their free thiol
groups with sodium iodoacetate. On each gel, the different lanes represent
different redox potentials (GSH:GSSG) where the sample is incubated. From lane
2 to lane 8, the ratio of GSH:GSSG was increased gradually, therefore more and
more native species were converted into reduced species. The more stable the
disulfides, the higher the ratio of GSH:GSSG needed in order to convert the
same percentage of native species into reduced species. As shown in Fig. 4,
for wild-type PIP, when 50% of native species were converted into reduced
species, the ratio of GSH:GSSG is between 20:1 and 30:1; while for the three
analogs, the ratio is less than 14:1. Therefore, the disulfides of the three
PIP analogs are much more easily reduced than those of the wild-type PIP.
In vitro refolding analysis
In vitro PIP can spontaneously fold
into the native structure with high efficiency. When B8Gly was replaced by
alanine, the refolding efficiency of PIP decreased significantly [30]. Here,
the refolding yields of the three B8 mutants were analyzed under different
refolding conditions, including different pH, different temperature and
different redox potential. As shown in Table 1, the refolding yields of
the three PIP analogs are approximately half that of the wild-type PIP under
different refolding conditions. This implies that B8Gly is critical for
efficient folding of the PIP sequence.
Insulin receptor-binding
activity
By endoproteinase Lys-C
cleavage the three PIP mutants were converted into [desB30]insulin mutants,
and their receptor-binding activities were measured (Fig. 5). The
insulin analog with B8Gly replaced by Ser still retained significant
receptor-binding activity, 23% of native insulin; while Thr and Leu replacement
produced serious detriment to biological activity: their receptor-binding
activities were less than 0.2% of native insulin. Therefore, as far as activity
is concerned, the B8 position is tolerant to Ser substitution, but not tolerant
to Thr or Leu replacement.
Discussion
In the present study, three
new PIP/insulin mutants in which the absolutely conserved B8Gly was replaced by
Ser, Thr or Leu, were prepared and their foldability and activity were analyzed.
Together with previous results [30], it is known that B8Gly is critical for the
foldability of the insulin. Once B8Gly was mutated, the foldability of insulin
decreased significantly; the native structure was disturbed; the in vitro
refolding efficiency was decreased; the disulfides were more easily reduced in
redox buffer; and the structural stability was decreased (except in the instance
of Leu replacement). In terms of biological activity, B8Gly replacement with
Ala, Thr or Leu has serious detriment. According to the insulin-receptor
interaction model [28,33], B8Gly does not directly interact with the insulin
receptor. Therefore, the direct contribution of the B8 position is likely to
be on foldability, and the decrease in biological activity is the result of
local and/or global structural disturbance caused by B8 replacement. The B8
position is located at the conjunction of an invariant a-helix
(B9-B19) and a segment of variable
conformation (B1-B8). Although the R-state
conformation has been observed in crystal, in solution the conformation of
insulin/mini-proinsulin is similar to the T-state of the crystal structure
[17,18,20]. In the T-state, the main-chain dihedral angles of the B8 position
are those of a D-amino acid. In the natural amino acids, only Gly is a
non-chiral residue and can adopt the main-chain dihedral angles of D-amino
acid. Therefore, Gly is likely the only applicable natural amino acid for the
B8 position as far as foldability and activity are concerned.
Some interesting phenomena
were observed in the present study. B8 substitutions usually significantly decrease
the structural stability as measured by guanidine titration. However, it is
tolerant to Leu replacement. Because Leu has a much larger side-chain than
Ala, Ser, or Thr, it is difficult to deduce how the large side-chain of Leu is
accommodated in the mutant. In terms of biological activity, the B8 position
is tolerant to Ser substitution but not to Ala, Ser or Leu replacement. Ser
replacement has serious detriment on foldability, why is it tolerant for
activity? To answer these questions, the high-resolution structures of these
mutants need to be solved.
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