State Key Laboratory of Molecular Biology, Shanghai Institute of Biochemistry, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China
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Abstract |
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Keywords: alanine-scanning mutagenesis/dimer-forming surface/insulin/monomer
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Introduction |
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Up to now, there have been two major approaches to obtain monomeric insulin. Monomeric insulin, [B28Lys,B29Pro]insulin, was obtained by inverting the B28B29 sequence of insulin B chain as corresponding to the sequence of IGF-I (Howey et al., 1994). The design of this monomeric insulin was based on the fact that IGF-I is a monomer (DiMarchi et al., 1992
). The second approach is to introduce residues with a charged or large side chain into the dimer-forming surface so as to interfere with the formation of the dimer from the monomer, such as monomeric [B9Asp,B27Glu]human insulin (Kang et al., 1991
), [B16His]human insulin (Kuarshelm and Ludvigsen, 1995
) and [B28Glu]human insulin (Mudaliar et al., 1999
).
There are seven residues buried in thedimer, A21Asn, B12Val, B16Tyr, B24Phe, B25Phe, B26Tyr and B27Thr, among which B12Val, B16Tyr, B24Phe, B25Phe and B26Tyr form a hydrophobic surface that is responsible for the formation of insulin dimer (Peking Insulin Structure Research Group, 1974; Baker et al., 1988
). Kristensen et al. (1997) carried out extensive alanine-scanning mutagenesis of insulin and obtained very interesting results on the insulin receptor binding site and the effects of different residues on insulin biosynthesis and folding. They found that A19Tyr, B11Leu and B13Glu are essential for receptor binding, especially the A19 residue, and they proposed an insulin receptor binding site which consists of B6Leu, B8Gly, B23Gly, B24Phe, A2Ile, A3Val and A19Tyr. However, they only obtained seven purified analogs and did not measure their association properties, although this is important for the clinical use of insulin.
To elucidate the role of the residues buried in the dimer on the association properties of insulin and to screen some monomeric insulins, we mutated the seven residues by alanine-scanning mutagenesis. Here we report four new monomeric insulins. Two of them, [B16Ala]human insulin and [B26Ala]human insulin, retain high in vitro and in vivo biological activities and could be developed as the fast-acting insulin required in clinical practice.
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Materials and methods |
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The Escherichia coli strains used were TG1 and RZ1032 (dut, ung). Yeast Saccharomyces cerevisiae XV7006B (leu2, ura3, pep4) and helper phage R408 were a gift from Michael Smith, University of British Columbia, Vancouver, Canada. Plasmid pPGK/PIP was constructed in our laboratory for secretory expression of porcine insulin precursor (PIP) in yeast (Zhang et al., 1996). Standard cloning protocols of Sambrook et al. (1989) were used.
Construction of mutated plasmid
The single alanine mutations at the positions of B12, B16, B26 and A21 were achieved by means of site-directed mutagenesis on pPGK/PIP using a gapped duplex DNA approach to obtain pPGK/[B12Ala]PIP, pPGK/[B16Ala]PIP, pPGK/[B26Ala]PIP and pPGK/[A21Ala]PIP according to the procedures of Sambrook et al. (1989); the PCR method was used to obtain PIP gene fragments possessing alanine mutations at the positions of B24, B25 and B27. Then the mutant PIP gene fragments were subcloned into the plasmid pPGK/PIP by a second PCR to obtain pPGK/[B24Ala]PIP, pPGK/[B25Ala]PIP and pPGK/[27Ala]PIP. The above seven single alanine mutant PIP genes were confirmed to be correct by DNA sequencing.
Secretory expression and purification of the single alanine mutant PIPs
The single yeast clone transformed with pPGK/PIP containing a single alanine mutation was picked and cultured in a fermenter (16 l) for 3 days (Zhang et al., 1996). The concentration of PIP mutant in the culture medium was determined using radioimmunoassay (RIA) kits (from Shanghai Institute of Biological Products). The single alanine PIP mutant was precipitated from the culture medium with trichloroacetic acid (TCA), followed by purification by gel filtration using a Sephadex G-50 column. The product was further purified using a DEAE-CL-6B Sepharose column or reversed-phase HPLC. The seven single alanine mutant PIPs were homogeneous in native pH 8.3 PAGE (Zhang et al., 1996
).
Conversion of single alanine mutant PIPs into corresponding human insulin mutants
Purified single alanine mutant PIPs were converted into the corresponding single alanine human insulin mutants by means of transpeptidation in the presence of trypsin and an excess amount of L-threonine O-tert-butyl ether tert-butyl ester [Thr(But)-OBut] according to the procedures of Zhang et al (1996).
Bioassays of the seven single alanine human insulin mutants
Receptor binding assay was performed using human placental membrane (Feng et al., 1982).
A semi-quantitative mouse convulsion assay was used to measure the in vivo biological activity. For each dosage, five ICR mice (fasted, weighing 1820 g, purchased from SIPPR/BK) were injected and put in a 35°C chamber and their responses were observed.
Determination of self-association of the single alanine human insulin mutants
The self-association of the seven single alanine human insulin mutants was determined by gel filtration using FPLC (Brems et al., 1992): column, Superdex 75 (HR 10/30); buffer, phosphate-buffered saline, pH 7.4; flow-rate, 0.4 ml/min; concentration, 1.20 mg/ml (2x104 M); room temperature; detection at 280 nm. Recombinant human insulin was used as a positive self-association contrast and [B28Lys,B29Pro]insulin as a negative self-association contrast (Howey et al., 1994
).
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Results |
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Yeast clones transformed with pPGK/[A21Ala]PIP, pPGK/[B12Ala]PIP, pPGK/[B16Ala]PIP, pPGK/[B24Ala]PIP, pPGK/[B25Ala]PIP, pPGK/[B26Ala]PIP and pPGK/[B27Ala]PIP were cultured in a fermenter. The fermentation broth was decanted and centrifuged to remove the cells. Products in the supernatant were precipitated with TCA and then purified by gel filtration using a Sephadex G-50 column followed by a DEAE-CL-6B Sepharose ion-exchange column or reversed-phase HPLC. The purified seven single alanine mutant PIPs were homogeneous as judged by native pH 8.3 PAGE (Figure 1).
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The receptor binding activities of the seven single alanine human insulin mutants with insulin receptor on human placental membrane are shown in Figure 3. The relative receptor binding activities of the seven mutants of human insulin were obtained by comparison of the dosages used for 50% inhibition of [125I] insulin bound with the receptor and are listed in Table II
. From Table II
, we can see that the substitution of the four residues B12Val, B16Tyr, B24Phe and B25Phe with alanine causes a marked decrease in their receptor binding activity, among which B12Val and B24Phe are crucial in the receptor binding site. [A21Ala]insulin, [B27Ala]insulin and [B26Ala]insulin retained more than 50% of their receptor binding activity.
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The in vivo biological activities of the seven human insulin mutants are shown in Table III.
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The dimer formation assay was carried out by FPLC with a Superdex 75 (HR 10/30) column using [B28Lys,B29Pro]human insulin and recombinant human insulin for contrast of the monomer and dimer of insulin at high concentration. The FPLC profiles of recombinant human insulin, [B28Lys,B29Pro]human insulin and the seven single alanine human insulin mutants are shown in Figure 4. The retention times and shapes of the peaks as the identification parameters for the monomer and dimer of insulin are listed in Table IV
, which shows that the [B12Ala]human insulin, [B16Ala]human insulin, [B24Ala]human insulin and [B26Ala]human insulin are monomeric insulin. The results demonstrate that B12Val, B16Tyr, B24Phe and B26Tyr play a key role in the formation of insulin dimer, among which [B16Ala]insulin and [B26Ala]insulin are monomeric insulin with high biological activity.
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Discussion |
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It has been proposed that B12Val, B16Tyr, B24Phe, B25Phe and B26Tyr form a hydrophobic surface which is responsible for the binding of insulin with its receptor based on the crystal structure (Peking Insulin Structure Research Group, 1974; Baker et al., 1988
). This proposition was subsequently extended and developed by many laboratories (Insulin Research Group, Academia Sinica, 1974
; Pullen et al., 1976
; Hodgkin, 1977
; Feng and Zhang, 1991
; Hua et al., 1991
; Liang et al., 1992
; Hu et al., 1993
; Schaffer, 1994
). Recently, Kristensen et al (1997) carried out extensive alanine-scanning mutagenesis of insulin. They obtained 21 new analogs and seven HPLC-purified analogs, and together with 11 single-alanine analogs from the literature they analyzed the role of each side chain in receptor binding and biosynthesis and folding. Through total alanine-scanning mutagenesis, they acquired an overview of the insulin receptor binding region. On the surface of insulin, the conservative residues, including B6Leu, B8Gly, B23Gly, B24Phe, A2Ile, A3Val and A19Tyr, form a patch binding with its receptor. B23Gly, B24Phe and A19Tyr are most likely to interact directly with insulin receptor. A2Ile and A3Val also important for receptor binding although they are buried beneath the C-terminus of the B-chain in the crystal structure. The important role of B6Leu and B8Gly is connected with the structure rather than direct interaction with the receptor. However, they did not obtain the alanine analog of the B12 residue which is absolutely conservative in evolution because of its low expression level. In this study we obtained purified B12Ala insulin mutant and determined its receptor binding activity. Our results demonstrate that B12Val is also crucial in the receptor binding site.
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Notes |
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Acknowledgments |
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References |
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Received February 4, 2000; revised August 30, 2000; accepted September 8, 2000.