Special Issue Article Received: 9 December 2014
Revised: 19 December 2014
Accepted: 21 December 2014
Published online in Wiley Online Library
(wileyonlinelibrary.com) DOI 10.1002/psc.2744
Three-chain insulin analogs demonstrate the importance of insulin secondary structure to bioactivity‡ Fangzhou Wu,a Joseph R. Chabenne,b Vasily M. Gelfanov,a John P. Mayera and Richard D. DiMarchia* This report describes the chemical synthesis and biological characterization of novel three-chain insulin analogs with a destabilized secondary structure. The analogs, obtained by chemical synthesis via a single-chain precursor and selective enzymatic digestion, were used to investigate the role of the highly conserved ‘insulin fold’. Biological characterization through in vitro biochemical signaling showed extremely low activity at each insulin receptor when compared with native insulin. We conclude that the ‘insulin fold’ is a structural foundation that supports insulin biological action. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd. Keywords: insulin; single-chain insulin analogs; enzyme-assisted synthesis; insulin receptor interaction
Introduction Insulin is a peptide hormone secreted by pancreatic β cells that is essential to the regulation of glucose metabolism [1]. It is a member of a peptide superfamily, which also includes the insulin-like growth factors, relaxin, and insulin-like peptides [2]. The primary structure of insulin was determined by Sanger and colleagues in 1955, and its higher-order structure was subsequently revealed by crystallography and NMR spectroscopy [3–6]. Insulin consists of two peptide chains (A and B) with a total of 51 amino acids. The A chain possesses 21 amino acids and the B chain a total of 30. The two chains are tethered together by two inter-chain disulfide bonds (CysA7–CysB7 and CysA20–CysB19), whereas the A chain contains an additional intra-chain disulfide bond (CysA6–CysA11) (Figure 1). Insulin is stored in the pancreas as a zinc-complexed hexamer, which slowly dissociates to its active monomeric form which binds and activates insulin receptors on the plasma membrane of insulintarget tissues [7]. The insulin peptide family possesses a unique higher-order structure, termed the ‘insulin fold’ [8,9]. It is a highly conserved motif throughout the superfamily [10–13]. Its salient features include (i) three invariant disulfide bonds, (ii) an interior aliphatic hydrophobic core constituted by numerous conserved residues, (iii) a central Bchain α helix, and (iv) two α helices located at each end of the A chain oriented by an intra-chain disulfide bond [10] (Figure 1). Despite the sizable knowledge regarding the insulin structure, the question of how this important hormone engages its receptor and transmits its signal remains a source of continual study [14]. Structural analysis of non-native analogs supports the notion that insulin adopts a distinct conformation at its receptor relative to its crystal structure, with increasing clarity emerging regarding its active conformation [15,16]. A number of residues within the insulin fold are highly conserved throughout the insulin superfamily and appear to support receptor recognition [10,13,17,18].
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In this study, we systematically interrogated the conserved secondary structure and its relative importance in insulin receptormediated bioactivity. We designed and synthesized three novel, three-chain insulin analogs that lack a specific peptide bond at different sites in the insulin fold. We hypothesized that the absence of a single bond would destabilize the structure because the hormone is stabilized by the main chain hydrogen bonding between covalently linked amino acid residues [19]. The three novel insulin analogs were obtained by enzymatic cleavage of a synthetically derived single-chain insulin precursor [20]. We characterized each insulin analog by in vitro insulin receptor analysis and CD spectroscopy. The three sites of modification are located in the A chain Cterminal α helix, the A chain intra-chain loop, and the B chain central α helix (Figure 1).
* Correspondence to: Richard DiMarchi, Department of Chemistry, Indiana University, Bloomington, IN, 47405, USA. E-mail: [email protected] ‡
Invited Article for the Anniversary Issue 2015 of Journal of Peptide Science.
a Department of Chemistry, Indiana University, Bloomington, IN, 47405, USA b AIT Bioscience, Indianapolis, IN, 46278, USA Abbreviations: 6-CI-HOBt, 6-chloro-1-hydroxybenzotriazole; Boc, tertbutyloxycarbonyl; BSA, bovine serum albumin; CD, circular dichroism; DIC, N,N′-diisopropylcarbodiimide; DMF, N,N′-dimethylformamide; ELISA, enzyme-linked immunosorbent assay; Fmoc, 9-fluorenylmethyloxycarbonyl; HEK, human embryonic kidney; IRA, insulin receptor A; IRB, insulin receptor B; LC–MS, liquid chromatography–mass spectrometry; MALDI-TOF-MS, matrix-assisted laser desorption/ionization-time of flight-mass spectrometry; NMP, N-methyl-2pyrrolidinone; NMR, nuclear magnetic resonance; OD, optical density; OtBu, 5tert-butyl ester; Pbf, 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl; PBS, phosphate buffered saline; RP-HPLC, reversed phase-high pressure liquid chromatography; tBu, tert-butyl; TFA, trifluoroacetic acid; TIS, triisopropylsilane; TMB, 3,3′,5,5′-tetramethylbezidine; Trt, trityl.
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WU ET AL. Tyr(tBu). The Fmoc protecting group was removed by treatment with 20% piperidine in NMP, the peptide coupling was initiated by utilizing a tenfold excess of amino acid derivative with DIC/6CI-HOBt in NMP as activation reagents. Peptides were constructed on either Rink amide (ChemMatrix) resin with the C-terminal asparagine introduced by coupling Fmoc-Asp-OtBu [22] or by use of preloaded Fmoc-Asn(Trt)-Wang resin. Completed peptidyl-resins were washed and briefly dried under vacuum then treated with a TFA cleavage cocktail (20-ml solution for 0.1-mmol resin) containing TFA/H2O/TIS/anisole/2-mercaptoethanol (90/2.5/2.5/2.5/2.5) for 2 h to release peptide from the solid support. Cleaved peptides were precipitated and washed with cold diethyl ether, the ether phase was decanted and crude peptidyl-pellet dried under vacuum for 1 h prior to refolding. Single-chain Insulin Analogs Refolding and Purification
Figure 1. Schematic representation and three-dimensional structure of insulin (PDB file: 9INS). The insulin A chain is shown in green, and the B chain is shown in cyan. The A, B, C indicate the secondary structure elements interrogated in this study.
Materials and Methods Materials Reagents, including endoproteinase Lys-C and trypsin, were acquired from Sigma-Aldrich (St. Louis, MO, USA) except otherwise specified. NMP and acetonitrile were products from Avantor Performance materials Inc. (Center Valley, PA, USA). 6-CI-HOBt, Wang resin (0.51 mmol/g) and Fmoc-protected amino acids were purchased from AAPPTec (Louisville, KY, USA). TFA and H-Rink Amide-ChemMatrix Resin were obtained from Oakwood Chemicals (West Columbia, SC, USA) and PCAS BioMatrix Inc. (Quebec, Canada), respectively. Piperidine was supplied by Alfa Aesar (Ward Hill, MA, USA). Amino acid cartridges for the ABI peptide synthesizer were ordered from Midwest Biotech (Fishers, IN, USA). Water was produced from a GenPure UV/UF xCAD plus water purification system (Thermo Scientific, Waltham, MA, USA). Analytical RP-HPLC was performed on a Beckman Gold Instrument utilizing Phenomenex Kinetex C8 2.6 μ 100 A (75 × 60 mm) column, with 1 ml/min flow rate and a gradient of 10–80% acetonitrile in water containing 0.1% TFA at 10 min unless otherwise noted. LC–MS analysis was conducted on Agilent 1260 infinity LC–MS system coupled with Agilent 6120 Quadrupole mass spectrometer, using the identical column and solvent conditions as described earlier. MALDI-TOF-MS was carried out on a Bruker Daltonics Autoflex MALDI-TOF mass spectrometer using CHCA matrix.
Peptide Synthesis Single-chain insulin analogs were synthesized by standard Fmocbased solid phase peptide protocol on an Applied Biosystem 433A (Applied Biosytem, Foster city, CA, USA) or Symphony peptide synthesizer (Protein Technologies Inc., Tucson, AZ, USA) [21]. The following side chain protecting groups were utilized for individual amino acid residues: Arg(Pbf), Asp(OtBu), Asn(Trt), Cys(Trt), Gln (Trt), Glu(OtBu), His(Trt), Lys(Boc), Orn(Boc), Ser(tBu), Thr(tBu), and
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Approximately 120 mg of crude linear peptide (~0.1 mM) was dissolved in 200 ml of 20-mM glycine buffer containing 2-mM Lcysteine hydrochloride (~70 mg). The pH of the solution was adjusted to ~10.5 with sodium hydroxide, and the reaction mixture was stirred, open to air at 4 °C. The progress of the folding was monitored by analytical RP-HPLC (column: described in the Materials Section) using an ammonium bicarbonate/acetonitrile solvent system. The folding reaction, which was typically completed in 24– 72 h, was acidified to pH 8.0 with concentrated hydrochloric acid, and the reaction mixture was loaded onto a preparative C8 Luna column (AXIA packed, 250 × 21.2 mm) for purification. Elution was achieved by using a linear gradient of acetonitrile in 25-mM ammonium bicarbonate, pH 8.0. The desired fractions were identified by analytical RP-HPLC (column: described in the Materials section, eluent: 25-mM ammonium bicarbonate in aqueous acetonitrile, pH 8.0) and MALDI-TOF-mass spectrometry and lyophilized to yield a white fluffy powder. Enzymatic Digestion of Single-chain Insulin Analogs For the Lys-C cleavage of single-chain insulin analogs, purified peptides (2–5 mg) were dissolved in 1 ml, 25-mM ammonium bicarbonate buffer (pH = 8.0) combined with 10 μl of Lys-C stock solution (3 UN/100 μl H2O). The reaction was incubated at 37 °C, and the progress of the enzymatic digestion was monitored by analytical HPLC and MALDI-TOF mass spectrometry. Trypsin digestion of single-chain insulin analogs was initiated by dissolving the purified peptide (~5 mg) in 1 ml, 25-mM ammonium bicarbonate buffer (pH = 8.0) with addition of 10–20 μl trypsin stock solution (1 mg/ml). The reaction was incubated at 37 °C for at least 8 h, and the progress of digestion was monitored by analytical LC–MS. Upon completion, the reaction mixture was diluted tenfold with 20% aqueous acetonitrile containing 0.1% TFA, and the digestion product was purified by preparative HPLC using a linear gradient of acetonitrile in 0.1% TFA(aq) on a C8-reverse phase column (AXIA packed, 250 × 21.2 mm). The desired fractions were identified by analytical HPLC and MALDI-TOF-mass spectrometry and lyophilized. Determination of Biological Activity of Insulin Analogs To measure receptor phosphorylation of the insulin A or B receptor, transfected HEK293 cells were plated in poly-lysine-coated 96 well tissue culture plates (Costar #3596, Cambridge, MA, USA) and cultured in Dulbecco’s modified Eagle medium (DMEM)
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THREE-CHAIN INSULIN ANALOGS supplemented with 100 IU/ml penicillin, 100 mg/ml streptomycin, 10-mM HEPES, and 0.25% bovine growth serum (HyClone SH30541, Logan, UT, USA) for 16–20 h at 37 °C, 5% CO2, and 90% humidity. Serial dilutions of insulin or test analogs were prepared in DMEM supplemented with 0.5% bovine serum albumin (Roche Applied Science #100350, Indianapolis, IN, USA) and added to the wells with adhered cells. After a 15-min incubation at 37 °C in humidified atmosphere with 5% CO2, the cells were fixed with 5% paraformaldehyde for 20 min at room temperature, washed twice with phosphate buffered saline pH 7.4, and blocked with 2% bovine serum albumin in PBS for 1 h. The plate was then washed three times and filled with horseradish peroxidaseconjugated antibody against phosphotyrosine (Upstate biotechnology #16-105, Temecula, CA, USA) reconstituted in PBS with 2% bovine serum albumin per manufacturer’s recommendation. After 3 h of incubation at room temperature, the plate was washed four times, and 0.1 ml of TMB single solution substrate (Invitrogen, #00-2023, Carlsbad, CA, USA) was added to each well. Color development was stopped 5 min later by adding 0.05-ml 1N HCl. Absorbance at 450 nm was measured on Titertek Multiscan MCC340 (ThermoFisher, Pittsburgh, PA, USA). Absorbance versus peptide concentration dose–response curves were plotted, and EC50 values were determined by using Origin software (OriginLab, Northampton, MA, USA). Circular Dichroism Purified peptides were dissolved in 10-mM aqueous potassium phosphate buffer (pH 7.0) at a concentration of ~25 μM. The samples were analyzed by CD spectroscopy using a JASCO J-715 spectropolarimeter at wavelength of 300–190 nm, and data were collected through ten scans in a 1-mm path-length cuvette at a scan speed of 100 nm/min. The data were normalized by subtracting the background signal and appropriately smoothed.
The millidegree values were converted to mean residue ellipticity by the equation: mean residue ellipticity = millidegrees/(pathlength in millimeters × the molar concentration of protein × the number of residues) [23].
Results Synthesis of Peptides 1–4 and Enzymatic Digestion Chemical synthesis of the analogs utilized a single-chain insulin precursor, in which the B29 lysine was directly connected to the A chain N-terminus. A lysine-linked poly-glutamate extension at the N-terminus of the B chain facilitated solubility of the peptide in alkaline-folding buffer (Table 1) [20,24]. This single-chain insulin precursor ensured the correct disulfide bond formation and yielded the two-chain desB30 insulin upon treatment with Lys-C (Figure 2) [24]. Peptide 1 was successfully prepared by conventional Fmoc-based solid phase peptide synthesis on a preloaded Fmoc-Asn(Trt)-Wang resin utilizing an automated peptide synthesizer. The completed peptidyl-resin was treated with a TFA cleavage cocktail to deprotect and release peptide from solid support, and the peptide was directly dissolved into the folding buffer without further purification or characterization [24]. Correct folding of the peptide was confirmed by an earlier eluting peak in the analytical RP-HPLC profile resulting from reduced hydrophobicity of the correctly folded peptide. The protein was purified by RP-HPLC to remove misfolded isomers, multimers, and other impurities. Correctly folded peptide 1 was subsequently digested by enzyme Lys-C to generate the two-chain desB30 insulin with high efficiency and selectivity, as indicated by a single peak in analytical HPLC and the desired mass after digestion, without other detectable impurities (Figure 3). The in vitro insulin receptor activity of two-chain peptide 1 was tested in an insulin receptor-based
Figure 2. Synthetic strategy utilized on control peptide 1 to generate two-chain desB30 insulin. The insulin A chain is shown in green, and the B chain is shown in cyan, whereas the N-terminal solubility tag is shown in black.
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WU ET AL.
Figure 3. Analytical RP-HPLC and MALDI-TOF mass spectrum (single-chain control peptide 1 before digestion, MS calculated: 6537.3 Da, found: 6537.6 Da; single-chain control peptide 1 after digestion, MS calculated: 5724.5 Da, found: 5725.5 Da.) of single-chain control peptide 1 before and after Lys-C digestion.
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Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.
J. Pept. Sci. 2015
THREE-CHAIN INSULIN ANALOGS
Insulin Rec-A
Insulin Rec-B
0.8
0.6 0.7 0.6
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0.4
0.3
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1
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1E-3
0.01
0.1
1
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Peptide concentration(nM)
Figure 4. Insulin receptor specific phosphorylation of the two-chain control peptide 1 at insulin receptor A and insulin receptor B. Table 1. Amino acid sequence of single-chain insulin precursor‡
a
The lysine and ornithine residues in the sequence are highlighted in red.
Table 2. Insulin receptor in vitro bioactivity‡ Peptide number Insulin 1 8 5 9 6 10 7
Insulin receptor A (% native)
Insulin receptor B (% of native)
100 126.67 ± 1.53 43.67 ± 1.53 1.10 ± 0.14 35.67 ± 15.01 1.17 ± 0.15 37.33 ± 11.68 0.97 ± 0.59
100 116.67 ± 18.23 50.10 ± 6.26 1.07 ± 0.85 63.00 ± 24.76 2.04 ± 0.71 43.67 ± 12.89 1.10 ± 0.95
a
Percent standard value is from the comparison of peptide concentration where half-maximal phosphorylation is occurring between the insulin analog tested and native insulin, after trypsin digestion. Each standard value was obtained by the average of at least three independent experiments, with the standard deviation also reported.
phosphorylation assay. In this assay, the two-chain form of peptide 1 stimulated a full agonist response at both insulin receptor isoforms, with slightly enhanced potency relative to native insulin (Figure 4, Table 2). The in vitro receptor activity confirms the formation of correctly disulfide-paired desB30 insulin analog as improperly disulfide-paired analogs, and single-chain peptides are of much reduced potency. On the basis of this strategy, we designed peptides 2–4 (Table 1), which were analogous to peptide 1 but for an additional lysine mutation within a specific structural element of interest. The lysine substitution in peptide 2 occurs at A9Ser, peptide 3 at A14Tyr, and
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peptide 4 at B17Leu. These additional peptides (2–4) were synthesized using the same procedure as utilized with peptide 1 and subsequently treated with Lys-C. Treatment with Lys-C should simultaneously remove the N-terminal solubility extension and transform the single-chain molecule to a three-chain analog without loss of disulfide bond integrity through cleavage at B29 and the additional inserted lysine. The folding efficiency of peptides 3 and 4 was comparable with peptide 1, whereas peptide 2 resulted in a much lower yield. Apparently, the A9 lysine mutation imposed a limitation on folding efficiency. Mass spectrometry analysis of the digestion product revealed a mass
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WU ET AL.
9
29
30
9
29
30
Figure 5. Trypsin cleavage toward single-chain insulin precursors leads to (a) A Orn, B Arg des-B two-chain insulin and (b) A Lys, B Arg des-B threechain insulin.
Insulin Rec-A 0.50
Insulin Peptide9: two-chain
0.7 0.6
0.40
OD450
OD450
0.45
Insulin Rec-B
I
0.35
0.5 0.4
0.30 0.3
0.25 0.20 1E-3
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Peptide concentrtaion(nM) 17
17
Figure 6. Insulin receptor specific phosphorylation of peptide 10 (two-chain B Orn mutant) and peptide 7 (three-chain B Lys mutant) at insulin receptor A and insulin receptor B.
that was 18 Da less than the mass of the expected three-chain analogs. This indicated that one of the three lysine residues in the single-chain synthetic precursor was inert to Lys-C enzymatic treatment. This was further confirmed by sulfitolysis, and in each analog, the single uncleaved site proved to be the lysine residue within the targeted secondary structural site. A series of more aggressive enzymatic digestion protocols were assessed but proved unsuccessful. These results indicated that a new peptide construct was required to obtain the desired three-chain insulin analogs. It also demonstrated the structural rigidity of each of the unique sites, as high-order structure can limit proteolysis in a manner that was not observed at the two less-structured sites in the precursor. Alternative Route to Three-chain Insulin Analogs We designed a new peptide strategy to afford the three-chain insulin analogs that employed the higher efficiency of trypsin cleavage, relative to Lys-C. In peptides 5–7, the B29 lysine and the lysine at the Nterminal pre-sequence were replaced by arginine. The B22 arginine was substituted with histidine to prevent tryptic digestion, whereas lysine residues within the desired secondary structural elements
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were conserved. Peptides 8–10 originated from peptides 5–7, with the single lysine in the sequence replaced by an ornithine residue (Table 1). Ornithine retains the positively charged side chain of lysine but is resistant to trypsin cleavage. As a result, the correctly folded peptides 8–10 would be converted to two-chain insulin analogs by trypsin digestion and bear a single ornithine substitution. These peptides would be compared with the analogous three-chain insulin analogs with a lysine at the site of additional cleavage. The precursors to these six, single-chain insulin analogs were synthesized, folded, purified, and subsequently digested with trypsin. Treatment of peptides 5–7 with trypsin produced the desired three-chain insulin analogs as determined by LC–MS, whereas peptides 8–10 provide the two-chain insulin counterparts (Figure 5). The crude HPLC profiles following trypsin digestion were less straightforward than the previous Lys-C digestions, which necessitated additional purification by RP-HPLC to obtain a pure product to be used in subsequent biological characterization and structural determination. In Vitro Analyses of Two-chain and Three-chain Insulin Analogs All the insulin analogs were tested for their ability to stimulate insulin receptor-mediated phosphorylation, at the A and B isoforms.
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J. Pept. Sci. 2015
THREE-CHAIN INSULIN ANALOGS
(a)
(b)
(c)
Figure 7. Comparative CD spectrum between two-chain and three-chain insulin analogs. (A) Comparative CD spectrum between two-chain peptide 9 and three-chain peptide 6. (b) Comparative CD spectrum between two-chain peptide 10 and three-chain peptide 7. (c) Comparative CD spectrum between twochain peptide 8 and three-chain peptide 5.
The results are reported in Table 2 [25]. Each of the two-chain A9 and A14 ornithine mutants induced a full agonism at both insulin receptors with approximately half the potency relative to native hormone. The three-chain insulin analogs displayed significantly reduced activities at both insulin receptors with a consistent reduction in potency of approximately a hundredfold in each analog. When comparing the two-chain and the three-chain insulins, there is no meaningful difference in receptor isoform activity, but in each instance, the former is nearly 50 times more potent than the latter (Figure 6, Table 2). Circular Dichroism Spectroscopy Each of the insulin analogs was studied by CD in the wavelength range between 190 and 300 nm. The CD spectrum of the A14 Orn, two-chain analog exhibited negative bands at 208 and 222 nm and a positive band at 193 nm, indicative of a well-defined α-helical structure. Conversely, the analysis of the A14 Lys, three-chain analog displayed reduced negative bands at 208 and 222 nm, with negative ellipticity near 193 nm. This result is consistent with destabilization of the α-helical content as a result of a single peptide bond cleavage at A14 [Figure 7(a)]. Similar results were obtained in the comparative analysis of the B17 Orn two-chain and the B17 Lys three-chain analogs, where the single additional cleavage at B17 destabilized the conformation through reduced α-helical content [Figure 7(b)]. The A9 Orn two-chain and A9 Lys three-chain insulin analogs displayed a similar CD spectrum in the 200–300-nm range, especially the negative bands at 222 and 208 nm. However, the three-chain insulin analog showed much reduced ellipticity than its two-chain counterpart, indicative of a less-ordered overall structure resulting from peptide bond cleavage within the intra-chain A loop [Figure 7(c)].
Discussion Chemical synthesis of insulin through a single-chain precursor is a recently developed methodology and was utilized to generate a set of two-chain and three-chain insulin analogs. The use of a single-chain insulin precursor facilitates correct disulfide bond formation, simplifies purification, and affords an excellent yield of the desired product, as demonstrated by the successful synthesis of peptide 1 [20,24,26,27]. The incorporation of additional lysines at unique sites enabled the synthesis of three-chain insulin analogs, an accomplishment never before realized. This approach takes advantage of the inherent capability of the native sequence to
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efficiently fold into the multiple disulfide structure and subsequently to site-specifically digest the molecule to the desired three-chain form. The alternative approach of synthetically building the three-chain analogs as a direct target, without application of enzyme action is envisioned to be more troublesome than the synthesis of a native two-chain insulin, which is more complex than preparation through a single-chain precursor. Unexpectedly, the lysine residues inserted in each of the three secondary structural sites proved completely resistant to Lys-C digestion. We attributed this phenomenon to steric factors imposed by the ordered peptide backbone, which hindered Lys-C proteolysis and rendered it less efficient in reaction relative to the two other lysine sites in the single-chain precursor. Trypsin proved of higher enzymatic activity and enabled lysine cleavage at the desired site, which is consistent with literature reports [28]. The use of trypsin required a change in synthetic strategy that employed ornithine as a lysine structural mimetic resistant to proteolysis. The results confirmed the successful application of trypsin in treatment of peptides 5–7 to produce the three-chain insulin synthetic target analogs. The two-chain, ornithine-substituted insulin analogs proved to be high potency, balanced insulin agonists at both receptor isoforms. This was anticipated because the mutations were purposefully selected to minimize change in receptor activity in the two-chain form. The two-chain A9 ornithine mutants induced modest reduction in activity relative to native insulin, and the bioactivity of A14 and B17 two-chain, ornithine mutants was generally consistent with alanine mutations previously reported [18]. In stark contrast, each of the corresponding three-chain lysine substituted, insulin analogs exhibited extremely low bioactivities at both insulin receptors. Quantitatively, the analogs showed more than an order of magnitude decrease in potency relative to their two-chain, ornithine counterparts (Table 2). We attributed the extremely low potency to the destabilization in the secondary structure at each of the three points where peptide bond cleavage was introduced. To confirm this conclusion, CD-based structural analysis was performed and demonstrated a sizable reduction in helical content. Recent biophysical studies have demonstrated that insulin is a dynamic macromolecule capable of movement within a variable degree and that such mobility is a vital element to bioactivity [29]. In the present study, we report that lessened secondary structure through site-specific peptide bond hydrolysis has a deleterious effect on insulin receptor signaling. We interpret these results to imply that the interior inter-chain hydrophobic core supported by multiple helical structures within the insulin fold is relatively rigid and essential
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WU ET AL. for receptor interactions at surface residues within this region. This presumably is the basis for the high degree of conservation of multiple amino acids within the insulin core. Among such amino acid residues believed to be involved in receptor recognition are A12Ser, A13Leu, A17Glu, and A19Tyr located within the A chain C-terminal helical segment and B10His, B12Val, B13Glu, B16Tyr, B17Leu on the B chain central helical segment, with A8Thr within the A intra-chain loop.
9 10 11 12 13
Conclusion In summary, we have utilized a unique chemical approach that entailed the use of enzymatic cleavage to generate a set of unique, three-chain insulin analogs. The conceptual approach can be applied to other peptides and proteins to interrogate the importance of secondary structure. These three-chain insulin analogs displayed uniformly low bioactivity, and their destabilized structures were confirmed by preliminary structural analysis. These results provide direct experimental evidence of the indispensable role of the classical insulin fold to support biochemical signaling.
14 15 16 17 18
Acknowledgements We thank Mr. Jay Levy and Mr. Dave Smiley for their assistance in peptide synthesis, purification, and characterization. We also thank Drs. Alex Zaykov and Bin Yang for their thoughtful discussions throughout the project. We appreciate the help from Ms. Angela Hansen and Dr. Jonathan Karty in the Indiana University mass spectrometry facility for the peptide characterization. Partial funding in support of this research was received from Calibrium.
20 21 22 23
References
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2Zn pig insulin crystals at 1.5 Å resolution. Philos. Trans. R. Soc. Lond. B Biol. Sci. 1988; 319(1195): 369–456. Kline AD, Justice RM, Jr. Complete sequence-specific 1H NMR assignments for human insulin. Biochemistry 1990; 29(12): 2906–2913. Murray-Rust J, McLeod AN, Blundell TL, Wood SP. Structure and evolution of insulins: implication for receptor binding. Bioessays 1992; 14(5): 325–331. Rinderknecht E, Humbel RE. The amino acid sequence of human insulinlike growth factor I and its structural homology with proinsulin. J. Biol. Chem. 1978; 253(8): 2769–2776. Bedarkar S, Turnell WG, Blundell TL, Schwabe C. Relaxin has conformational homology with insulin. Nature 1977; 270(5636): 449–451. Mayer JP, Zhang F, DiMarchi RD. Insulin structure and function. Biopolymers 2007; 88(5): 687–713. De Meyts P, Whittaker J. Structural biology of insulin and IGF1 receptors: implications for drug design. Nat. Rev. Drug Discov. 2002; 1(10): 769–783. Hua QX, Shoelson SE, Kochoyan M, Weiss MA. Receptor binding redefined by a strutural switch in a mutant human insulin. Nature 1991; 354(6350): 238–241. Ward CW, Lawrence MC. Ligand-induced activation of the insulin receptor: a multi-step process involving structural changes in both the ligand and the receptor. Bioessays 2009; 31(4): 422–434. Pullen RA, Lindsay DG, Wood SP, Tickle IJ, Blundell TL, Wollmer A, Krail G, Brandenburg D, Zahn H, Gliemann J, Gammeltoft S. Receptor-binding region of insulin. Nature 1976; 259(5542): 369–373. Kristensen C, Kjeldsen T, Wiberg FC, Schäffer L, Hach M, Havelund S, Bass J, Steiner DF, Andersen AS. Alanine scanning mutagenesis of insulin. J. Biol. Chem. 1997; 272(20): 12978–12983. Pauling L, Corey RB, Branson HR. The structure of proteins: two hydrogen-bonded helical configurations of the polypeptide chain. Proc. Natl. Acad. Sci. U. S. A. 1951; 37(4): 205–211. Zaykov AN, Mayer JP, Gelfanov VM, DiMarchi RD. Chemical synthesis of insulin analogs through a novel precursor. ACS Chem. Biol. 2014; 9(3): 683–691. Chan WC, White PD. Fmoc Solid Phase Peptide Synthesis: a Practical Approach, Oxford University Press: Oxford, 2000. Garcia-Ramos Y, Paradis-Bas M, Tulla-Puche J, Albericio F. ChemMatrix ((R)) for complex peptides and combinatorial chemistry. J. Pept. Sci. 2010; 16(12): 675–678. Greenfield NJ. Using circular dichroism spectra to estimate protein secondary structure. Nat. Protoc. 2006; 1(6): 2876–2890. Tofteng AP, Jensen KJ, Schäffer L, Hoeg-Jensen T. Total synthesis of desB30 insulin analogues by biomimetic folding of single-chain precursors. Chembiochem 2008; 9(18): 2989–2996. Belfiore A, Frasca F, Pandini G, Sciacca L, Vigneri R. Insulin receptor isoforms and insulin receptor/insulin-like growth factor receptor hybrids in physiology and disease. Endocr. Rev. 2009; 30(6): 586–623. Avital-Shmilovici M, Mandal K, Gates ZP, Phillips NB, Weiss MA, Kent SB. Fully convergent chemical synthesis of ester insulin: determination of the high resolution X-ray structure by racemic protein crystallography. J. Am. Chem. Soc. 2013; 135(8): 3173–3185. Sohma Y, Kent SB. Biomimetic synthesis of lispro insulin via a chemically synthesized “mini-proinsulin” prepared by oxime-forming ligation. J. Am. Chem. Soc. 2009; 131(44): 16313–16318. Rawlings ND, Barrett AJ. Families of serine peptidases. Methods Enzymol. 1994; 244: 19–61. Chothia C, Lesk AM, Dodson GG, Hodgkin DC. Transmission of conformational change in insulin. Nature 1983; 302(5908): 500–505.
Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.
J. Pept. Sci. 2015
Revised: 19 December 2014
Accepted: 21 December 2014
Published online in Wiley Online Library
(wileyonlinelibrary.com) DOI 10.1002/psc.2744
Three-chain insulin analogs demonstrate the importance of insulin secondary structure to bioactivity‡ Fangzhou Wu,a Joseph R. Chabenne,b Vasily M. Gelfanov,a John P. Mayera and Richard D. DiMarchia* This report describes the chemical synthesis and biological characterization of novel three-chain insulin analogs with a destabilized secondary structure. The analogs, obtained by chemical synthesis via a single-chain precursor and selective enzymatic digestion, were used to investigate the role of the highly conserved ‘insulin fold’. Biological characterization through in vitro biochemical signaling showed extremely low activity at each insulin receptor when compared with native insulin. We conclude that the ‘insulin fold’ is a structural foundation that supports insulin biological action. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd. Keywords: insulin; single-chain insulin analogs; enzyme-assisted synthesis; insulin receptor interaction
Introduction Insulin is a peptide hormone secreted by pancreatic β cells that is essential to the regulation of glucose metabolism [1]. It is a member of a peptide superfamily, which also includes the insulin-like growth factors, relaxin, and insulin-like peptides [2]. The primary structure of insulin was determined by Sanger and colleagues in 1955, and its higher-order structure was subsequently revealed by crystallography and NMR spectroscopy [3–6]. Insulin consists of two peptide chains (A and B) with a total of 51 amino acids. The A chain possesses 21 amino acids and the B chain a total of 30. The two chains are tethered together by two inter-chain disulfide bonds (CysA7–CysB7 and CysA20–CysB19), whereas the A chain contains an additional intra-chain disulfide bond (CysA6–CysA11) (Figure 1). Insulin is stored in the pancreas as a zinc-complexed hexamer, which slowly dissociates to its active monomeric form which binds and activates insulin receptors on the plasma membrane of insulintarget tissues [7]. The insulin peptide family possesses a unique higher-order structure, termed the ‘insulin fold’ [8,9]. It is a highly conserved motif throughout the superfamily [10–13]. Its salient features include (i) three invariant disulfide bonds, (ii) an interior aliphatic hydrophobic core constituted by numerous conserved residues, (iii) a central Bchain α helix, and (iv) two α helices located at each end of the A chain oriented by an intra-chain disulfide bond [10] (Figure 1). Despite the sizable knowledge regarding the insulin structure, the question of how this important hormone engages its receptor and transmits its signal remains a source of continual study [14]. Structural analysis of non-native analogs supports the notion that insulin adopts a distinct conformation at its receptor relative to its crystal structure, with increasing clarity emerging regarding its active conformation [15,16]. A number of residues within the insulin fold are highly conserved throughout the insulin superfamily and appear to support receptor recognition [10,13,17,18].
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In this study, we systematically interrogated the conserved secondary structure and its relative importance in insulin receptormediated bioactivity. We designed and synthesized three novel, three-chain insulin analogs that lack a specific peptide bond at different sites in the insulin fold. We hypothesized that the absence of a single bond would destabilize the structure because the hormone is stabilized by the main chain hydrogen bonding between covalently linked amino acid residues [19]. The three novel insulin analogs were obtained by enzymatic cleavage of a synthetically derived single-chain insulin precursor [20]. We characterized each insulin analog by in vitro insulin receptor analysis and CD spectroscopy. The three sites of modification are located in the A chain Cterminal α helix, the A chain intra-chain loop, and the B chain central α helix (Figure 1).
* Correspondence to: Richard DiMarchi, Department of Chemistry, Indiana University, Bloomington, IN, 47405, USA. E-mail: [email protected] ‡
Invited Article for the Anniversary Issue 2015 of Journal of Peptide Science.
a Department of Chemistry, Indiana University, Bloomington, IN, 47405, USA b AIT Bioscience, Indianapolis, IN, 46278, USA Abbreviations: 6-CI-HOBt, 6-chloro-1-hydroxybenzotriazole; Boc, tertbutyloxycarbonyl; BSA, bovine serum albumin; CD, circular dichroism; DIC, N,N′-diisopropylcarbodiimide; DMF, N,N′-dimethylformamide; ELISA, enzyme-linked immunosorbent assay; Fmoc, 9-fluorenylmethyloxycarbonyl; HEK, human embryonic kidney; IRA, insulin receptor A; IRB, insulin receptor B; LC–MS, liquid chromatography–mass spectrometry; MALDI-TOF-MS, matrix-assisted laser desorption/ionization-time of flight-mass spectrometry; NMP, N-methyl-2pyrrolidinone; NMR, nuclear magnetic resonance; OD, optical density; OtBu, 5tert-butyl ester; Pbf, 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl; PBS, phosphate buffered saline; RP-HPLC, reversed phase-high pressure liquid chromatography; tBu, tert-butyl; TFA, trifluoroacetic acid; TIS, triisopropylsilane; TMB, 3,3′,5,5′-tetramethylbezidine; Trt, trityl.
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WU ET AL. Tyr(tBu). The Fmoc protecting group was removed by treatment with 20% piperidine in NMP, the peptide coupling was initiated by utilizing a tenfold excess of amino acid derivative with DIC/6CI-HOBt in NMP as activation reagents. Peptides were constructed on either Rink amide (ChemMatrix) resin with the C-terminal asparagine introduced by coupling Fmoc-Asp-OtBu [22] or by use of preloaded Fmoc-Asn(Trt)-Wang resin. Completed peptidyl-resins were washed and briefly dried under vacuum then treated with a TFA cleavage cocktail (20-ml solution for 0.1-mmol resin) containing TFA/H2O/TIS/anisole/2-mercaptoethanol (90/2.5/2.5/2.5/2.5) for 2 h to release peptide from the solid support. Cleaved peptides were precipitated and washed with cold diethyl ether, the ether phase was decanted and crude peptidyl-pellet dried under vacuum for 1 h prior to refolding. Single-chain Insulin Analogs Refolding and Purification
Figure 1. Schematic representation and three-dimensional structure of insulin (PDB file: 9INS). The insulin A chain is shown in green, and the B chain is shown in cyan. The A, B, C indicate the secondary structure elements interrogated in this study.
Materials and Methods Materials Reagents, including endoproteinase Lys-C and trypsin, were acquired from Sigma-Aldrich (St. Louis, MO, USA) except otherwise specified. NMP and acetonitrile were products from Avantor Performance materials Inc. (Center Valley, PA, USA). 6-CI-HOBt, Wang resin (0.51 mmol/g) and Fmoc-protected amino acids were purchased from AAPPTec (Louisville, KY, USA). TFA and H-Rink Amide-ChemMatrix Resin were obtained from Oakwood Chemicals (West Columbia, SC, USA) and PCAS BioMatrix Inc. (Quebec, Canada), respectively. Piperidine was supplied by Alfa Aesar (Ward Hill, MA, USA). Amino acid cartridges for the ABI peptide synthesizer were ordered from Midwest Biotech (Fishers, IN, USA). Water was produced from a GenPure UV/UF xCAD plus water purification system (Thermo Scientific, Waltham, MA, USA). Analytical RP-HPLC was performed on a Beckman Gold Instrument utilizing Phenomenex Kinetex C8 2.6 μ 100 A (75 × 60 mm) column, with 1 ml/min flow rate and a gradient of 10–80% acetonitrile in water containing 0.1% TFA at 10 min unless otherwise noted. LC–MS analysis was conducted on Agilent 1260 infinity LC–MS system coupled with Agilent 6120 Quadrupole mass spectrometer, using the identical column and solvent conditions as described earlier. MALDI-TOF-MS was carried out on a Bruker Daltonics Autoflex MALDI-TOF mass spectrometer using CHCA matrix.
Peptide Synthesis Single-chain insulin analogs were synthesized by standard Fmocbased solid phase peptide protocol on an Applied Biosystem 433A (Applied Biosytem, Foster city, CA, USA) or Symphony peptide synthesizer (Protein Technologies Inc., Tucson, AZ, USA) [21]. The following side chain protecting groups were utilized for individual amino acid residues: Arg(Pbf), Asp(OtBu), Asn(Trt), Cys(Trt), Gln (Trt), Glu(OtBu), His(Trt), Lys(Boc), Orn(Boc), Ser(tBu), Thr(tBu), and
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Approximately 120 mg of crude linear peptide (~0.1 mM) was dissolved in 200 ml of 20-mM glycine buffer containing 2-mM Lcysteine hydrochloride (~70 mg). The pH of the solution was adjusted to ~10.5 with sodium hydroxide, and the reaction mixture was stirred, open to air at 4 °C. The progress of the folding was monitored by analytical RP-HPLC (column: described in the Materials Section) using an ammonium bicarbonate/acetonitrile solvent system. The folding reaction, which was typically completed in 24– 72 h, was acidified to pH 8.0 with concentrated hydrochloric acid, and the reaction mixture was loaded onto a preparative C8 Luna column (AXIA packed, 250 × 21.2 mm) for purification. Elution was achieved by using a linear gradient of acetonitrile in 25-mM ammonium bicarbonate, pH 8.0. The desired fractions were identified by analytical RP-HPLC (column: described in the Materials section, eluent: 25-mM ammonium bicarbonate in aqueous acetonitrile, pH 8.0) and MALDI-TOF-mass spectrometry and lyophilized to yield a white fluffy powder. Enzymatic Digestion of Single-chain Insulin Analogs For the Lys-C cleavage of single-chain insulin analogs, purified peptides (2–5 mg) were dissolved in 1 ml, 25-mM ammonium bicarbonate buffer (pH = 8.0) combined with 10 μl of Lys-C stock solution (3 UN/100 μl H2O). The reaction was incubated at 37 °C, and the progress of the enzymatic digestion was monitored by analytical HPLC and MALDI-TOF mass spectrometry. Trypsin digestion of single-chain insulin analogs was initiated by dissolving the purified peptide (~5 mg) in 1 ml, 25-mM ammonium bicarbonate buffer (pH = 8.0) with addition of 10–20 μl trypsin stock solution (1 mg/ml). The reaction was incubated at 37 °C for at least 8 h, and the progress of digestion was monitored by analytical LC–MS. Upon completion, the reaction mixture was diluted tenfold with 20% aqueous acetonitrile containing 0.1% TFA, and the digestion product was purified by preparative HPLC using a linear gradient of acetonitrile in 0.1% TFA(aq) on a C8-reverse phase column (AXIA packed, 250 × 21.2 mm). The desired fractions were identified by analytical HPLC and MALDI-TOF-mass spectrometry and lyophilized. Determination of Biological Activity of Insulin Analogs To measure receptor phosphorylation of the insulin A or B receptor, transfected HEK293 cells were plated in poly-lysine-coated 96 well tissue culture plates (Costar #3596, Cambridge, MA, USA) and cultured in Dulbecco’s modified Eagle medium (DMEM)
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THREE-CHAIN INSULIN ANALOGS supplemented with 100 IU/ml penicillin, 100 mg/ml streptomycin, 10-mM HEPES, and 0.25% bovine growth serum (HyClone SH30541, Logan, UT, USA) for 16–20 h at 37 °C, 5% CO2, and 90% humidity. Serial dilutions of insulin or test analogs were prepared in DMEM supplemented with 0.5% bovine serum albumin (Roche Applied Science #100350, Indianapolis, IN, USA) and added to the wells with adhered cells. After a 15-min incubation at 37 °C in humidified atmosphere with 5% CO2, the cells were fixed with 5% paraformaldehyde for 20 min at room temperature, washed twice with phosphate buffered saline pH 7.4, and blocked with 2% bovine serum albumin in PBS for 1 h. The plate was then washed three times and filled with horseradish peroxidaseconjugated antibody against phosphotyrosine (Upstate biotechnology #16-105, Temecula, CA, USA) reconstituted in PBS with 2% bovine serum albumin per manufacturer’s recommendation. After 3 h of incubation at room temperature, the plate was washed four times, and 0.1 ml of TMB single solution substrate (Invitrogen, #00-2023, Carlsbad, CA, USA) was added to each well. Color development was stopped 5 min later by adding 0.05-ml 1N HCl. Absorbance at 450 nm was measured on Titertek Multiscan MCC340 (ThermoFisher, Pittsburgh, PA, USA). Absorbance versus peptide concentration dose–response curves were plotted, and EC50 values were determined by using Origin software (OriginLab, Northampton, MA, USA). Circular Dichroism Purified peptides were dissolved in 10-mM aqueous potassium phosphate buffer (pH 7.0) at a concentration of ~25 μM. The samples were analyzed by CD spectroscopy using a JASCO J-715 spectropolarimeter at wavelength of 300–190 nm, and data were collected through ten scans in a 1-mm path-length cuvette at a scan speed of 100 nm/min. The data were normalized by subtracting the background signal and appropriately smoothed.
The millidegree values were converted to mean residue ellipticity by the equation: mean residue ellipticity = millidegrees/(pathlength in millimeters × the molar concentration of protein × the number of residues) [23].
Results Synthesis of Peptides 1–4 and Enzymatic Digestion Chemical synthesis of the analogs utilized a single-chain insulin precursor, in which the B29 lysine was directly connected to the A chain N-terminus. A lysine-linked poly-glutamate extension at the N-terminus of the B chain facilitated solubility of the peptide in alkaline-folding buffer (Table 1) [20,24]. This single-chain insulin precursor ensured the correct disulfide bond formation and yielded the two-chain desB30 insulin upon treatment with Lys-C (Figure 2) [24]. Peptide 1 was successfully prepared by conventional Fmoc-based solid phase peptide synthesis on a preloaded Fmoc-Asn(Trt)-Wang resin utilizing an automated peptide synthesizer. The completed peptidyl-resin was treated with a TFA cleavage cocktail to deprotect and release peptide from solid support, and the peptide was directly dissolved into the folding buffer without further purification or characterization [24]. Correct folding of the peptide was confirmed by an earlier eluting peak in the analytical RP-HPLC profile resulting from reduced hydrophobicity of the correctly folded peptide. The protein was purified by RP-HPLC to remove misfolded isomers, multimers, and other impurities. Correctly folded peptide 1 was subsequently digested by enzyme Lys-C to generate the two-chain desB30 insulin with high efficiency and selectivity, as indicated by a single peak in analytical HPLC and the desired mass after digestion, without other detectable impurities (Figure 3). The in vitro insulin receptor activity of two-chain peptide 1 was tested in an insulin receptor-based
Figure 2. Synthetic strategy utilized on control peptide 1 to generate two-chain desB30 insulin. The insulin A chain is shown in green, and the B chain is shown in cyan, whereas the N-terminal solubility tag is shown in black.
J. Pept. Sci. 2015
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WU ET AL.
Figure 3. Analytical RP-HPLC and MALDI-TOF mass spectrum (single-chain control peptide 1 before digestion, MS calculated: 6537.3 Da, found: 6537.6 Da; single-chain control peptide 1 after digestion, MS calculated: 5724.5 Da, found: 5725.5 Da.) of single-chain control peptide 1 before and after Lys-C digestion.
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Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.
J. Pept. Sci. 2015
THREE-CHAIN INSULIN ANALOGS
Insulin Rec-A
Insulin Rec-B
0.8
0.6 0.7 0.6
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OD450
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0.4
0.3
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0.1
1
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1E-3
0.01
0.1
1
10
100
1000
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Peptide concentration(nM)
Figure 4. Insulin receptor specific phosphorylation of the two-chain control peptide 1 at insulin receptor A and insulin receptor B. Table 1. Amino acid sequence of single-chain insulin precursor‡
a
The lysine and ornithine residues in the sequence are highlighted in red.
Table 2. Insulin receptor in vitro bioactivity‡ Peptide number Insulin 1 8 5 9 6 10 7
Insulin receptor A (% native)
Insulin receptor B (% of native)
100 126.67 ± 1.53 43.67 ± 1.53 1.10 ± 0.14 35.67 ± 15.01 1.17 ± 0.15 37.33 ± 11.68 0.97 ± 0.59
100 116.67 ± 18.23 50.10 ± 6.26 1.07 ± 0.85 63.00 ± 24.76 2.04 ± 0.71 43.67 ± 12.89 1.10 ± 0.95
a
Percent standard value is from the comparison of peptide concentration where half-maximal phosphorylation is occurring between the insulin analog tested and native insulin, after trypsin digestion. Each standard value was obtained by the average of at least three independent experiments, with the standard deviation also reported.
phosphorylation assay. In this assay, the two-chain form of peptide 1 stimulated a full agonist response at both insulin receptor isoforms, with slightly enhanced potency relative to native insulin (Figure 4, Table 2). The in vitro receptor activity confirms the formation of correctly disulfide-paired desB30 insulin analog as improperly disulfide-paired analogs, and single-chain peptides are of much reduced potency. On the basis of this strategy, we designed peptides 2–4 (Table 1), which were analogous to peptide 1 but for an additional lysine mutation within a specific structural element of interest. The lysine substitution in peptide 2 occurs at A9Ser, peptide 3 at A14Tyr, and
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peptide 4 at B17Leu. These additional peptides (2–4) were synthesized using the same procedure as utilized with peptide 1 and subsequently treated with Lys-C. Treatment with Lys-C should simultaneously remove the N-terminal solubility extension and transform the single-chain molecule to a three-chain analog without loss of disulfide bond integrity through cleavage at B29 and the additional inserted lysine. The folding efficiency of peptides 3 and 4 was comparable with peptide 1, whereas peptide 2 resulted in a much lower yield. Apparently, the A9 lysine mutation imposed a limitation on folding efficiency. Mass spectrometry analysis of the digestion product revealed a mass
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WU ET AL.
9
29
30
9
29
30
Figure 5. Trypsin cleavage toward single-chain insulin precursors leads to (a) A Orn, B Arg des-B two-chain insulin and (b) A Lys, B Arg des-B threechain insulin.
Insulin Rec-A 0.50
Insulin Peptide9: two-chain
0.7 0.6
0.40
OD450
OD450
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Insulin Rec-B
I
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0.5 0.4
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0.25 0.20 1E-3
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0.01
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Peptide concentrtaion(nM) 17
17
Figure 6. Insulin receptor specific phosphorylation of peptide 10 (two-chain B Orn mutant) and peptide 7 (three-chain B Lys mutant) at insulin receptor A and insulin receptor B.
that was 18 Da less than the mass of the expected three-chain analogs. This indicated that one of the three lysine residues in the single-chain synthetic precursor was inert to Lys-C enzymatic treatment. This was further confirmed by sulfitolysis, and in each analog, the single uncleaved site proved to be the lysine residue within the targeted secondary structural site. A series of more aggressive enzymatic digestion protocols were assessed but proved unsuccessful. These results indicated that a new peptide construct was required to obtain the desired three-chain insulin analogs. It also demonstrated the structural rigidity of each of the unique sites, as high-order structure can limit proteolysis in a manner that was not observed at the two less-structured sites in the precursor. Alternative Route to Three-chain Insulin Analogs We designed a new peptide strategy to afford the three-chain insulin analogs that employed the higher efficiency of trypsin cleavage, relative to Lys-C. In peptides 5–7, the B29 lysine and the lysine at the Nterminal pre-sequence were replaced by arginine. The B22 arginine was substituted with histidine to prevent tryptic digestion, whereas lysine residues within the desired secondary structural elements
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were conserved. Peptides 8–10 originated from peptides 5–7, with the single lysine in the sequence replaced by an ornithine residue (Table 1). Ornithine retains the positively charged side chain of lysine but is resistant to trypsin cleavage. As a result, the correctly folded peptides 8–10 would be converted to two-chain insulin analogs by trypsin digestion and bear a single ornithine substitution. These peptides would be compared with the analogous three-chain insulin analogs with a lysine at the site of additional cleavage. The precursors to these six, single-chain insulin analogs were synthesized, folded, purified, and subsequently digested with trypsin. Treatment of peptides 5–7 with trypsin produced the desired three-chain insulin analogs as determined by LC–MS, whereas peptides 8–10 provide the two-chain insulin counterparts (Figure 5). The crude HPLC profiles following trypsin digestion were less straightforward than the previous Lys-C digestions, which necessitated additional purification by RP-HPLC to obtain a pure product to be used in subsequent biological characterization and structural determination. In Vitro Analyses of Two-chain and Three-chain Insulin Analogs All the insulin analogs were tested for their ability to stimulate insulin receptor-mediated phosphorylation, at the A and B isoforms.
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J. Pept. Sci. 2015
THREE-CHAIN INSULIN ANALOGS
(a)
(b)
(c)
Figure 7. Comparative CD spectrum between two-chain and three-chain insulin analogs. (A) Comparative CD spectrum between two-chain peptide 9 and three-chain peptide 6. (b) Comparative CD spectrum between two-chain peptide 10 and three-chain peptide 7. (c) Comparative CD spectrum between twochain peptide 8 and three-chain peptide 5.
The results are reported in Table 2 [25]. Each of the two-chain A9 and A14 ornithine mutants induced a full agonism at both insulin receptors with approximately half the potency relative to native hormone. The three-chain insulin analogs displayed significantly reduced activities at both insulin receptors with a consistent reduction in potency of approximately a hundredfold in each analog. When comparing the two-chain and the three-chain insulins, there is no meaningful difference in receptor isoform activity, but in each instance, the former is nearly 50 times more potent than the latter (Figure 6, Table 2). Circular Dichroism Spectroscopy Each of the insulin analogs was studied by CD in the wavelength range between 190 and 300 nm. The CD spectrum of the A14 Orn, two-chain analog exhibited negative bands at 208 and 222 nm and a positive band at 193 nm, indicative of a well-defined α-helical structure. Conversely, the analysis of the A14 Lys, three-chain analog displayed reduced negative bands at 208 and 222 nm, with negative ellipticity near 193 nm. This result is consistent with destabilization of the α-helical content as a result of a single peptide bond cleavage at A14 [Figure 7(a)]. Similar results were obtained in the comparative analysis of the B17 Orn two-chain and the B17 Lys three-chain analogs, where the single additional cleavage at B17 destabilized the conformation through reduced α-helical content [Figure 7(b)]. The A9 Orn two-chain and A9 Lys three-chain insulin analogs displayed a similar CD spectrum in the 200–300-nm range, especially the negative bands at 222 and 208 nm. However, the three-chain insulin analog showed much reduced ellipticity than its two-chain counterpart, indicative of a less-ordered overall structure resulting from peptide bond cleavage within the intra-chain A loop [Figure 7(c)].
Discussion Chemical synthesis of insulin through a single-chain precursor is a recently developed methodology and was utilized to generate a set of two-chain and three-chain insulin analogs. The use of a single-chain insulin precursor facilitates correct disulfide bond formation, simplifies purification, and affords an excellent yield of the desired product, as demonstrated by the successful synthesis of peptide 1 [20,24,26,27]. The incorporation of additional lysines at unique sites enabled the synthesis of three-chain insulin analogs, an accomplishment never before realized. This approach takes advantage of the inherent capability of the native sequence to
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efficiently fold into the multiple disulfide structure and subsequently to site-specifically digest the molecule to the desired three-chain form. The alternative approach of synthetically building the three-chain analogs as a direct target, without application of enzyme action is envisioned to be more troublesome than the synthesis of a native two-chain insulin, which is more complex than preparation through a single-chain precursor. Unexpectedly, the lysine residues inserted in each of the three secondary structural sites proved completely resistant to Lys-C digestion. We attributed this phenomenon to steric factors imposed by the ordered peptide backbone, which hindered Lys-C proteolysis and rendered it less efficient in reaction relative to the two other lysine sites in the single-chain precursor. Trypsin proved of higher enzymatic activity and enabled lysine cleavage at the desired site, which is consistent with literature reports [28]. The use of trypsin required a change in synthetic strategy that employed ornithine as a lysine structural mimetic resistant to proteolysis. The results confirmed the successful application of trypsin in treatment of peptides 5–7 to produce the three-chain insulin synthetic target analogs. The two-chain, ornithine-substituted insulin analogs proved to be high potency, balanced insulin agonists at both receptor isoforms. This was anticipated because the mutations were purposefully selected to minimize change in receptor activity in the two-chain form. The two-chain A9 ornithine mutants induced modest reduction in activity relative to native insulin, and the bioactivity of A14 and B17 two-chain, ornithine mutants was generally consistent with alanine mutations previously reported [18]. In stark contrast, each of the corresponding three-chain lysine substituted, insulin analogs exhibited extremely low bioactivities at both insulin receptors. Quantitatively, the analogs showed more than an order of magnitude decrease in potency relative to their two-chain, ornithine counterparts (Table 2). We attributed the extremely low potency to the destabilization in the secondary structure at each of the three points where peptide bond cleavage was introduced. To confirm this conclusion, CD-based structural analysis was performed and demonstrated a sizable reduction in helical content. Recent biophysical studies have demonstrated that insulin is a dynamic macromolecule capable of movement within a variable degree and that such mobility is a vital element to bioactivity [29]. In the present study, we report that lessened secondary structure through site-specific peptide bond hydrolysis has a deleterious effect on insulin receptor signaling. We interpret these results to imply that the interior inter-chain hydrophobic core supported by multiple helical structures within the insulin fold is relatively rigid and essential
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WU ET AL. for receptor interactions at surface residues within this region. This presumably is the basis for the high degree of conservation of multiple amino acids within the insulin core. Among such amino acid residues believed to be involved in receptor recognition are A12Ser, A13Leu, A17Glu, and A19Tyr located within the A chain C-terminal helical segment and B10His, B12Val, B13Glu, B16Tyr, B17Leu on the B chain central helical segment, with A8Thr within the A intra-chain loop.
9 10 11 12 13
Conclusion In summary, we have utilized a unique chemical approach that entailed the use of enzymatic cleavage to generate a set of unique, three-chain insulin analogs. The conceptual approach can be applied to other peptides and proteins to interrogate the importance of secondary structure. These three-chain insulin analogs displayed uniformly low bioactivity, and their destabilized structures were confirmed by preliminary structural analysis. These results provide direct experimental evidence of the indispensable role of the classical insulin fold to support biochemical signaling.
14 15 16 17 18
Acknowledgements We thank Mr. Jay Levy and Mr. Dave Smiley for their assistance in peptide synthesis, purification, and characterization. We also thank Drs. Alex Zaykov and Bin Yang for their thoughtful discussions throughout the project. We appreciate the help from Ms. Angela Hansen and Dr. Jonathan Karty in the Indiana University mass spectrometry facility for the peptide characterization. Partial funding in support of this research was received from Calibrium.
20 21 22 23
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