Wang Lin, Gamez Alejandra, Sarkissian Christineh N, Straub Mary, Patch Marianne G, Han Gye Won, Striepeke Steve, Fitzpatrick Paul, Scriver Charles R, Stevens Raymond C
Department of Molecular Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA.
Mol Genet Metab. 2005 Sep-Oct;86(1-2):134-40. doi: 10.1016/j.ymgme.2005.05.012. Epub 2005 Jul 11.
Structure-based protein engineering coupled with chemical modifications (e.g., pegylation) is a powerful combination to significantly improve the development of proteins as therapeutic agents. As a test case, phenylalanine ammonia-lyase (PAL, EC 4.3.1.5) was selected for enzyme replacement therapy in phenylketonuria [C.R. Scriver, S. Kaufman, Hyperphenylalaninemia:phenylalanine Hydroxylase Deficiency. The Metabolic and Molecular Bases of Inherited Disease, McGraw-Hill, New York, 2001, Chapter 77], an inherited metabolic disorder (OMIM 261600) causing mental retardation due to deficiency of the enzyme l-phenylalanine hydroxylase (EC 1.14.16.1). Previous in vivo studies of recombinant PAL demonstrated a lowering of blood l-phenylalanine levels; yet, the metabolic effect was not sustained due to protein degradation and immunogenicity [C.N. Sarkissian, Z. Shao, F. Blain, R. Peevers, H. Su, R. Heft, T.M. Chang, C.R. Scriver, A different approach to treatment of phenylketonuria:phenylalanine degradation with recombinant phenylalanine ammonia lyase, Proc. Natl. Acad. Sci. USA 96 (1999) 2339; J.A. Hoskins, G. Jack, H.E. Wade, R.J. Peiris, E.C. Wright, D.J. Starr, J. Stern, Enzymatic control of phenylalanine intake in phenylketonuria, Lancet 1 (1980) 392; C.M. Ambrus, S. Anthone, C. Horvath, K. Kalghatgi, A.S. Lele, G. Eapen, J.L. Ambrus, A.J. Ryan, P. Li, Extracorporeal enzyme reactors for depletion of phenylalanine in phenylketonuria, Ann. Intern. Med. 106 (1987) 531]. Here, we report the 1.6A three-dimensional structure of Rhodosporidium toruloides PAL, structure-based molecular engineering, pegylation of PAL, as well as in vitro and in vivo PKU mouse model studies on pegylated PAL formulations. Our results show that pegylation of R. toruloides PAL leads to promising therapeutic efficacy after subcutaneous injection by enhancing the in vivo activity, lowering plasma phenylalanine, and leading to reduced immunogenicity. The three-dimensional structure of PAL provides a basis for understanding the properties of pegylated forms of PAL and strategies for structure-based re-engineering of PAL for PKU treatment.
基于结构的蛋白质工程与化学修饰（如聚乙二醇化）相结合，是显著改善蛋白质作为治疗剂开发的有力组合。作为一个测试案例，选择苯丙氨酸解氨酶（PAL，EC 4.3.1.5）用于苯丙酮尿症的酶替代疗法[C.R.斯克里弗，S.考夫曼，高苯丙氨酸血症：苯丙氨酸羟化酶缺乏症。《遗传性疾病的代谢和分子基础》，麦格劳-希尔出版社，纽约，2001年，第77章]，这是一种遗传性代谢紊乱疾病（OMIM 261600），由于缺乏L-苯丙氨酸羟化酶（EC 1.14.16.1）导致智力发育迟缓。先前对重组PAL的体内研究表明，血液中L-苯丙氨酸水平有所降低；然而，由于蛋白质降解和免疫原性，代谢效应未能持续[C.N.萨基西安，Z.邵，F.布莱恩，R.皮弗斯，H.苏，R.赫夫特，T.M.张，C.R.斯克里弗，治疗苯丙酮尿症的另一种方法：用重组苯丙氨酸解氨酶降解苯丙氨酸，《美国国家科学院院刊》96（1999）2339；J.A.霍斯金斯，G.杰克，H.E.韦德，R.J.佩里斯，E.C.赖特，D.J.斯塔尔，J.斯特恩，苯丙酮尿症中苯丙氨酸摄入的酶促控制，《柳叶刀》1（1980）392；C.M.安布鲁斯，S.安东尼，C.霍瓦特，K.卡尔加蒂，A.S.莱勒，G.伊彭，J.L.安布鲁斯，A.J.瑞安，P.李，用于降低苯丙酮尿症患者苯丙氨酸水平的体外酶反应器，《内科学年鉴》106（1987）531]。在此，我们报告了红酵母PAL的1.6埃三维结构、基于结构的分子工程、PAL的聚乙二醇化，以及对聚乙二醇化PAL制剂的体外和体内苯丙酮尿症小鼠模型研究。我们的结果表明，红酵母PAL的聚乙二醇化通过增强体内活性、降低血浆苯丙氨酸水平并降低免疫原性，在皮下注射后产生了有前景的治疗效果。PAL的三维结构为理解聚乙二醇化形式的PAL的性质以及基于结构对PAL进行重新工程设计以治疗苯丙酮尿症的策略提供了基础。
