Synthesis of a glycosylated angiotensin-related peptide, Big angiotensin-25 — ASN Events
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Synthesis of a glycosylated angiotensin-related peptide, Big angiotensin-25 (#37)

Hajime Hibino 1 , Sayaka Nagata 2 , Kinta Hatakeyama 3 , Maki Asami 2 , Mariko Tokashiki 2 , Kenji Kuwasako 4 , Johji Kato 4 , Yujiro Asada 3 , Kazuo Kitamura 2 , Yuji Nishiuchi 1 5
  1. SAITO Research Center, Peptide Inst. Inc., Ibaraki-Shi, Osaka, Japan
  2. Circulatory and Body Fluid Regulation, Faculty of Medicine, University of Miyazaki, Miyazaki-Shi, Miyazaki, Japan
  3. Pathology, Faculty of Medicine, University of Miyazaki, Miyazaki-shi, Miyazaki, Japan
  4. Frontier Science Research Center, University of Miyazaki, Miyazaki-Shi, Miyazaki, Japan
  5. Graduate School of Science, Osaka University, Toyonaka-Shi, Osaka, Japan

The renin-angiotensin system plays key roles in the regulation of blood pressure and electrolyte, and body fluid dynamics. Recently, big angiotensin-25 (Bang-25), which consists of the angiotensinogen (Aogen) sequence (1-25) having the N-linked complex-type nonasaccharide (Asn14) and cystine structure (Cys18), has been isolated from human urine by Kitamura et al. In this study, we have synthesized Bang-25 not only to confirm its reported primary structure including the N-linked oligosaccharide but also to use it for elucidating the physiological function. Introduction of a complex-type nonasaccharide into Asn14 of Bang-25 was performed by the following procedures: 1) a solid-phase synthesis using glycosylated Fmoc-Asn as a building block, and 2) a transglycosylation reaction of [Asn(GlcNAc)14]-Bang-25 using the mutant of Endo-M1 . In the course of chain assembly by the conventional SPPS, however, Fmoc-His(Trt) was never coupled with the amino group of glycosylated Asn14 due to the steric hindrance. In contrast, the microwave-assisted SPPS allowed a complete His incorporation without its racemization when Fmoc-His(MBom) was used2 . As for the chemo-enzymatic synthesis, the transglycosylation reaction could be facilitated by improving the solubility of [Asn(GlcNAc)14]-Bang-25 in which the O-acyl isopeptide structure and the disulfide linkage, Cys18-Cys(Arg)5-NH2, were introduced. The synthetic Bang-25 was confirmed to be identical to the natural product using RP-HPLC and MALDI-TOF MS analyses. Using the synthetic peptide, it was found that Bang-25 was abundantly expressed in a number of human tissues and that renin processed Bang-25 into angiotensin I (Ang I), but its production from Bang-25 was much slower than that from Aogen. By contrast, chymase rapidly digested Bang-25 to produce Ang II while it did not act on Aogen. Although the physiological function of Bang-25 remains unclear, our findings suggest that Bang-25 is processed from Aogen and may represent an alternative, renin-independent path for Ang II synthesis in tissue.

  1. Umekawa, M. et. al., J. Biol. Chem., 2010, 285, 511-521.
  2. Hibino, H. et al., J. Pep. Sci., 2012, 18, 763-769.