Kuroishi Nobuko, Nagata Sayaka, Akashi Emiko, Ashizuka Shinya, Kato Johji, Yamasaki Motoo, Kitamura Kazuo
Circulatory and Body Fluid Regulation, University of Miyazaki, 5200 Kihara, Kiyotake, Miyazaki 889-1692, Japan.
Frontier Science Research Center, Faculty of Medicine, University of Miyazaki, 5200 Kihara, Kiyotake, Miyazaki 889-1692, Japan.
J Biochem. 2021 Dec 4;170(4):445-451. doi: 10.1093/jb/mvab057.
Adrenomedullin is a biologically active peptide with multiple functions. Here, we have developed a novel human serum albumin-adrenomedullin (HSA-AM) conjugate, which was synthesized by the covalent attachment of a maleimide derivative of adrenomedullin to the 34th cysteine residue of HSA via a linker. Denaturing gel electrophoresis and western blotting for HSA-AM yielded a single band with adrenomedullin immunoreactivity at the position corresponding to a molecular weight (MW) of 73 kDa. Following gel-filtration chromatography, the purified HSA-AM showed a single main peak corresponding with an MW of 73 kDa, indicating that HSA-AM is a monomer. Both adrenomedullin and HSA-AM stimulated the intracellular accumulation of cyclic AMP (cAMP) in HEK-293 cells stably expressing the adrenomedullin 1 receptor. The pEC50 values for adrenomedullin and HSA-AM were 8.660 and 7.208 (equivalent to 2.19 and 61.9 nM as EC50), respectively. The bioavailability of HSA-AM compared with that of adrenomedullin was much improved after subcutaneous administration in the rat, which was probably due to the superior resistance of HSA-AM towards endogenous proteases and its reduced clearance from the blood. HSA-AM may be a promising drug candidate for clinical application.
肾上腺髓质素是一种具有多种功能的生物活性肽。在此，我们开发了一种新型的人血清白蛋白 - 肾上腺髓质素（HSA - AM）偶联物，它是通过肾上腺髓质素的马来酰亚胺衍生物经连接子与HSA的第34位半胱氨酸残基共价连接而合成的。对HSA - AM进行变性凝胶电泳和蛋白质印迹分析，在对应于分子量（MW）为73 kDa的位置产生了一条具有肾上腺髓质素免疫反应性的单一条带。经过凝胶过滤色谱法后，纯化的HSA - AM显示出一个对应MW为73 kDa的单一主峰，表明HSA - AM是一种单体。肾上腺髓质素和HSA - AM均刺激稳定表达肾上腺髓质素1受体的HEK - 293细胞中细胞内环磷酸腺苷（cAMP）的积累。肾上腺髓质素和HSA - AM的pEC50值分别为8.660和7.208（相当于EC50分别为2.19和61.9 nM）。在大鼠皮下给药后，与肾上腺髓质素相比，HSA - AM的生物利用度有了很大提高，这可能是由于HSA - AM对内源性蛋白酶具有更强的抗性且其从血液中的清除率降低。HSA - AM可能是一种有前景的临床应用候选药物。
