Research Article Received: 5 July 2014
Revised: 10 December 2014
Accepted: 7 January 2015
Published online in Wiley Online Library: 24 February 2015
(wileyonlinelibrary.com) DOI 10.1002/psc.2756
Microwave-assisted solid-phase peptide synthesis of neurosecretory protein GL composed of 80 amino acid residues Keiko Masuda, Haruka Ooyama, Kenshiro Shikano, Kunihiro Kondo, Megumi Furumitsu, Eiko Iwakoshi-Ukena and Kazuyoshi Ukena* We recently identified a novel cDNA encoding a small secretory protein of 80 amino acid residues, termed neurosecretory protein GL (NPGL), from the chicken hypothalamus. Homologs of NPGL have been reported to be present in mammals, such as human and rat. NPGL is amidated at its C-terminus, contains an intramolecular disulfide bond, and is hydrophobic in nature. In this study, we have optimized the synthesis of the entire 80-amino acid peptide sequence of rat NPGL by microwave-assisted solid-phase peptide synthesis. NPGL was obtained with a 10% yield when the coupling reactions were performed using 1-[Bis(dimethylamino) methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium-3-oxid hexafluorophosphate (HATU) at 50 °C for 5 min, and Fmoc deprotections were performed using 40% piperidine containing 0.1 M HOBt. Furthermore, the disulfide bond of NPGL was formed with 20% yield with the use of glutathione-containing redox buffer and 50% acetonitrile. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd. Keywords: small protein; NPGL; microwave; solid-phase peptide synthesis; disulfide bond formation
Introduction
454
Solid-phase peptide synthesis (SPPS) is a well-established and utilized method for convenient and rapid preparation of various peptides [1]. Although SPPS is typically used to synthesize peptides of approximately 30 amino acids in length, the peptide yield gradually decreases with repeated condensation reactions. As a result, synthesis of longer peptides by SPPS is difficult. Furthermore, interand intramolecular aggregations result in inaccessibility of the reagents to the reactive terminal of an elongating peptide. These problems can be partially addressed with the use of more reactive coupling reagents, double coupling, excessive amount of reagents, and low-loading or highly distensible solid supports. Alternatively, the introduction of pseudoproline dipeptides [2] or O-acyl residues [3] into the peptide backbone can help disrupt the β-sheet structures that are considered to be the source of intermolecular aggregation during chain elongation. Microwave irradiation is reported to have several advantages over conventional SPPS, including a reduction in the reaction time, improved reaction efficiency, and a suppression of inter- and intramolecular aggregations [4]. Since microwave irradiation has been utilized in SPPS [5], several microwave-assisted automatic peptide synthesizers have been developed and are in use commercially. Furthermore, microwaveassisted SPPS has been used for the synthesis of various so-called ‘difficult peptides’, including β-amyloid 1-42 [6], the C-terminus of the MuLV CTL epitope [7], amylin [8], and human exon 1 huntingtin [9]. In some cases, additional modifications, such as disulfide bond formation, are necessary for the synthesis of a functional peptide. Recently, we identified a novel cDNA of a gene that is highly expressed in the chicken hypothalamic infundibulum [10]. The precursor protein derived from this novel cDNA includes one putative small secretory protein flanked at the N-terminus by a signal
J. Pept. Sci. 2015; 21: 454–460
peptide and at the C-terminus by a glycine amidation signal and a dibasic amino acid cleavage site. As the predicted C-terminal amino acids of the small protein were Gly-Leu-NH2, the small protein has been named neurosecretory protein GL (NPGL). NPGL also exists in mammals, such as human and rat, and the homology between chicken and human/rat NPGL is 85% (Figure 1A). In our attempt to generate chicken NPGL by SPPS, after overcoming the first hurdle of insufficient peptide synthesis, we successfully synthesized a long peptide containing the sequence of rat NPGL in a preliminary experiment. We had previously reported that chronic subcutaneous administration of rat NPGL increases the body weight gain without affecting the food intake in chickens [10]. The deduced structure of rat NPGL is shown in (Figure 1B). Rat NPGL comprises of 80 amino acid residues with an amidated C-terminus. Furthermore, NPGL contains two highly conserved cysteine residues, suggesting the presence of an intramolecular disulfide bond. In our preliminary experiment, we first attempted to synthesize rat NPGL by conventional SPPS and yielded negligible amount due to the length and hydrophobicity of NPGL. Despite using pseudoproline dipeptides, the yield of NPGL was
Revised: 10 December 2014
Accepted: 7 January 2015
Published online in Wiley Online Library: 24 February 2015
(wileyonlinelibrary.com) DOI 10.1002/psc.2756
Microwave-assisted solid-phase peptide synthesis of neurosecretory protein GL composed of 80 amino acid residues Keiko Masuda, Haruka Ooyama, Kenshiro Shikano, Kunihiro Kondo, Megumi Furumitsu, Eiko Iwakoshi-Ukena and Kazuyoshi Ukena* We recently identified a novel cDNA encoding a small secretory protein of 80 amino acid residues, termed neurosecretory protein GL (NPGL), from the chicken hypothalamus. Homologs of NPGL have been reported to be present in mammals, such as human and rat. NPGL is amidated at its C-terminus, contains an intramolecular disulfide bond, and is hydrophobic in nature. In this study, we have optimized the synthesis of the entire 80-amino acid peptide sequence of rat NPGL by microwave-assisted solid-phase peptide synthesis. NPGL was obtained with a 10% yield when the coupling reactions were performed using 1-[Bis(dimethylamino) methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium-3-oxid hexafluorophosphate (HATU) at 50 °C for 5 min, and Fmoc deprotections were performed using 40% piperidine containing 0.1 M HOBt. Furthermore, the disulfide bond of NPGL was formed with 20% yield with the use of glutathione-containing redox buffer and 50% acetonitrile. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd. Keywords: small protein; NPGL; microwave; solid-phase peptide synthesis; disulfide bond formation
Introduction
454
Solid-phase peptide synthesis (SPPS) is a well-established and utilized method for convenient and rapid preparation of various peptides [1]. Although SPPS is typically used to synthesize peptides of approximately 30 amino acids in length, the peptide yield gradually decreases with repeated condensation reactions. As a result, synthesis of longer peptides by SPPS is difficult. Furthermore, interand intramolecular aggregations result in inaccessibility of the reagents to the reactive terminal of an elongating peptide. These problems can be partially addressed with the use of more reactive coupling reagents, double coupling, excessive amount of reagents, and low-loading or highly distensible solid supports. Alternatively, the introduction of pseudoproline dipeptides [2] or O-acyl residues [3] into the peptide backbone can help disrupt the β-sheet structures that are considered to be the source of intermolecular aggregation during chain elongation. Microwave irradiation is reported to have several advantages over conventional SPPS, including a reduction in the reaction time, improved reaction efficiency, and a suppression of inter- and intramolecular aggregations [4]. Since microwave irradiation has been utilized in SPPS [5], several microwave-assisted automatic peptide synthesizers have been developed and are in use commercially. Furthermore, microwaveassisted SPPS has been used for the synthesis of various so-called ‘difficult peptides’, including β-amyloid 1-42 [6], the C-terminus of the MuLV CTL epitope [7], amylin [8], and human exon 1 huntingtin [9]. In some cases, additional modifications, such as disulfide bond formation, are necessary for the synthesis of a functional peptide. Recently, we identified a novel cDNA of a gene that is highly expressed in the chicken hypothalamic infundibulum [10]. The precursor protein derived from this novel cDNA includes one putative small secretory protein flanked at the N-terminus by a signal
J. Pept. Sci. 2015; 21: 454–460
peptide and at the C-terminus by a glycine amidation signal and a dibasic amino acid cleavage site. As the predicted C-terminal amino acids of the small protein were Gly-Leu-NH2, the small protein has been named neurosecretory protein GL (NPGL). NPGL also exists in mammals, such as human and rat, and the homology between chicken and human/rat NPGL is 85% (Figure 1A). In our attempt to generate chicken NPGL by SPPS, after overcoming the first hurdle of insufficient peptide synthesis, we successfully synthesized a long peptide containing the sequence of rat NPGL in a preliminary experiment. We had previously reported that chronic subcutaneous administration of rat NPGL increases the body weight gain without affecting the food intake in chickens [10]. The deduced structure of rat NPGL is shown in (Figure 1B). Rat NPGL comprises of 80 amino acid residues with an amidated C-terminus. Furthermore, NPGL contains two highly conserved cysteine residues, suggesting the presence of an intramolecular disulfide bond. In our preliminary experiment, we first attempted to synthesize rat NPGL by conventional SPPS and yielded negligible amount due to the length and hydrophobicity of NPGL. Despite using pseudoproline dipeptides, the yield of NPGL was
