人β-半乳糖苷酶R299L突变体的获得与活性研究<sup>*</sup>

doi:10.13523/j.cb.2203055

中国生物工程杂志

2022, Vol. 42

Issue (9): 39-49 DOI: 10.13523/j.cb.2203055

技术与方法

人β-半乳糖苷酶R299L突变体的获得与活性研究^*

章嫣^1,²,马文豪²,赵天意²,吴小兵^2,^**(

),盛望¹,杨怡姝^1,^**(

)

1.北京工业大学生命科学与生物工程学院北京 100124
2.北京锦篮基因科技有限公司北京 100023

Acquisition and Activity Exploration of Human β-Galactosidase R299L Mutant

ZHANG Yan^1,²,MA Wen-hao²,ZHAO Tian-yi²,WU Xiao-bing^2,^**(

),SHENG Wang¹,YANG Yi-shu^1,^**(

)

1. Beijing University of Technology, Beijing 100124, China
2. Beijing GeneCradle Pharmaceutical Co., Ltd., Beijing 100023, China

全文: PDF(4685 KB) HTML

摘要：

目的: GM1神经节苷脂贮积症是一种由半乳糖苷酶beta 1(galactosidase beta 1, GLB1)基因突变引起的β-半乳糖苷酶(β-galactosidase,β-gal)活性降低导致的严重的溶酶体贮积病。该病以进行性、致命性神经退行性病变为特征,目前尚无有效的治疗手段,AAV载体介导的基因治疗被认为是最有希望的治疗方法。通过基因定点突变获得具有较高β-gal活性的GLB1突变体,以期用于后续AAV介导的基因治疗。方法: 对人类和其他6种脊椎动物GLB1基因进行多序列比对分析,筛选出部分氨基酸位点进行定点突变,采用携带突变位点的重组质粒和AAV9载体转染或感染HEK-293细胞,比较突变体与未突变体的活性差异。对GM1模型鼠注射携带coGLB1-R299L的rAAV9病毒,探究该突变体的体内活性表达。结果: 从15个突变体中筛选出coGLB1-R299L突变体,经质粒转染导入细胞后,其β-gal活性比具有野生型氨基酸序列的coGLB1增加了30%~40%。AAV体外感染实验中,rAAV9-coGLB1-R299L组的β-gal活性较未感染的细胞对照组提升了约2.2倍。体内结果显示,rAAV9-coGLB1-R299L在模型鼠体内广泛表达,心脏、肝脏、脾脏、肺、脑组织中β-gal活性显著提升。结论: 获得了具有更高β-gal活性的突变体coGLB1-R299L,初步探究了rAAV9-coGLB1-R299L的体外表达效果和模型鼠体内β-半乳糖苷酶的表达与分布,为该突变体应用于AAV介导的GM1神经节苷脂病治疗奠定基础。

关键词： GM1神经节苷脂贮积症; 半乳糖苷酶beta 1基因; β-半乳糖苷酶; 腺相关病毒载体; 定点突变

Abstract:

Objective: GM1 gangliosidosis is a devastating lysosomal storage disease featured by progressive and fatal neurodegeneration, caused by a mutation in the galactosidase beta 1 (GLB1) gene that reduces the activity of human beta-galactosidase. Currently, no effective therapy exists for the affected individuals, and AAV gene therapy is viewed as the most promising method. The object of this research is to obtain a mutant of GLB1 with higher β-gal activity through genetic modification, so as to be used in subsequent AAV-mediated gene therapy. Methods: The sequence of galactosidase beta 1 gene from human and other six vertebrates was analyzed by multiple sequence alignment, and some amino acid sites were selected for modification. HEK-293 cells were transfected or infected with either the recombinant plasmid or rAAV9 carrying the mutant site, and the activity of the mutant was compared with that of the control. The rAAV9 virus carrying coGLB1-R299L was injected into GM1 mice to explore the expression of the mutant in vivo. Results: The coGLB1-R299L mutant was screened from 15 mutants and then transfected into HEK-293 cells. It displayed higher β-gal activity with a 30%~40% increase compared with that of the wild-type amino acid sequence. The β-gal activity of rAAV9-coGLB1-R299L group was 2.2 times higher than that of the cell control group in the AAV infection experiment in vitro. The results in vivo showed that rAAV9-coGLB1-R299L was widely expressed in the GM1 mice, and the β-gal enzyme activities in the heart, liver, spleen, lung and brain tissue were significantly increased. Conclusion: A mutant coGLB1-R299L with higher β-gal activity is obtained, and the in vitro expression and distribution of enzyme activity of rAAV9-coGLB1-R299L in model mice was preliminarily explored, laying a foundation for the application of this mutant in the treatment of AAV-mediated GM1 gangliosidosis.

Key words: GM1 gangliosidosis Galactosidase beta 1 gene β-galactosidase Adeno-associated virus vector Site directed mutation

收稿日期: 2022-03-24 出版日期: 2022-10-10

ZTFLH:

Q819

基金资助: * 北京市自然科学基金(7202002)

通讯作者: 吴小兵,杨怡姝 E-mail: wuxiaobing@bj;yishu-y@bjut.edu.cn

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引用本文:

章嫣,马文豪,赵天意,吴小兵,盛望,杨怡姝. 人β-半乳糖苷酶R299L突变体的获得与活性研究^*[J]. 中国生物工程杂志, 2022, 42(9): 39-49.

ZHANG Yan,MA Wen-hao,ZHAO Tian-yi,WU Xiao-bing,SHENG Wang,YANG Yi-shu. Acquisition and Activity Exploration of Human β-Galactosidase R299L Mutant. China Biotechnology, 2022, 42(9): 39-49.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.2203055 或 https://manu60.magtech.com.cn/biotech/CN/Y2022/V42/I9/39

表1 突变体构建引物

图1 GLB1突变体的设计与构建

图2 GLB1突变体的β-gal活性

图3 X-gal 底物染色法原位检测细胞中β-gal活性

图4 四种299位氨基酸突变体酶活性比较

图5 AAV9介导coGLB1-R299L在体外表达β-gal

图6 AAV9介导coGLB1-R299L在模型鼠体内表达β-gal

[1]	Caciotti A, Garman S C, Rivera-Colón Y, et al. GM1 gangliosidosis and Morquio B disease: an update on genetic alterations and clinical findings. Biochimica et Biophysica Acta (BBA) - Molecular Basis of Disease, 2011, 1812(7): 782-790. doi: 10.1016/j.bbadis.2011.03.018
[2]	Nicoli E R, Annunziata I, D’Azzo A, et al. GM1 gangliosidosis-A mini-review. Frontiers in Genetics, 2021, 12: 734878. doi: 10.3389/fgene.2021.734878
[3]	Magistretti P J, Geisler F H, Schneider J S, et al. Gangliosides: treatment avenues in neurodegenerative disease. Frontiers in Neurology, 2019, 10: 859. doi: 10.3389/fneur.2019.00859 pmid: 31447771
[4]	Brunetti-Pierri N, Scaglia F. GM1 gangliosidosis: review of clinical, molecular, and therapeutic aspects. Molecular Genetics and Metabolism, 2008, 94(4): 391-396. doi: 10.1016/j.ymgme.2008.04.012 pmid: 18524657
[5]	Hofer D, Paul K, Fantur K, et al. GM1 gangliosidosis and Morquio B disease: expression analysis of missense mutations affecting the catalytic site of acid β-galactosidase. Human Mutation, 2009, 30(8): 1214-1221. doi: 10.1002/humu.21031
[6]	Pattali R, Mou Y C, Li X J. AAV9 Vector: a novel modality in gene therapy for spinal muscular atrophy. Gene Therapy, 2019, 26(7-8): 287-295. doi: 10.1038/s41434-019-0085-4 pmid: 31243392
[7]	Verdera H C, Kuranda K, Mingozzi F. AAV vector immunogenicity in humans: a long journey to successful gene transfer. Molecular Therapy, 2020, 28(3): 723-746. doi: S1525-0016(20)30003-4 pmid: 31972133
[8]	Lee E J, Guenther C M, Suh J. Adeno-associated virus (AAV) vectors: rational design strategies for capsid engineering. Current Opinion in Biomedical Engineering, 2018, 7: 58-63. doi: 10.1016/j.cobme.2018.09.004 pmid: 31106283
[9]	Liu S C, Ma W H, Feng Y Y, et al. AAV9-coGLB1 improves lysosomal storage and rescues central nervous system inflammation in a mutant mouse model of GM1 gangliosidosis. Current Gene Therapy, 2022, 22(4): 352-365. doi: 10.2174/1566523222666220304092732 pmid: 35249485
[10]	Liu S C, Feng Y Y, Huang Y L, et al. A GM1 gangliosidosis mutant mouse model exhibits activated microglia and disturbed autophagy. Experimental Biology and Medicine (Maywood, N J), 2021, 246(11): 1330-1341.
[11]	张梦瑶, 杨峰, 刘开东, 等. 利用同源重组技术构建DKK1、BMP4真核表达载体及共转染成纤维细胞表达的研究. 华北农学报, 2018, 33(5): 117-124. doi: 10.7668/hbnxb.2018.05.017
	Zhang M Y, Yang F, Liu K D, et al. Construction of eukaryotic expression vector of DKK1, BMP4 gene by homologous recombination and its expression in fibroblasts. Acta Agriculturae Boreali-Sinica, 2018, 33(5): 117-124. doi: 10.7668/hbnxb.2018.05.017
[12]	Guo P, El-Gohary Y, Prasadan K, et al. Rapid and simplified purification of recombinant adeno-associated virus. Journal of Virological Methods, 2012, 183(2): 139-146. doi: 10.1016/j.jviromet.2012.04.004 pmid: 22561982
[13]	吴小兵, 董小岩, 伍志坚, 等. 一种快速高效分离和纯化重组腺病毒伴随病毒载体的方法. 科学通报, 2000, 45(19): 2071-2075.
	Wu X B, Dong X Y, Wu Z J, et al. A method of rapidly and efficiently isolating and purifying recombinant adeno-associated viral vector. Chinese Science Bulletin, 2000, 45(19): 2071-2075.
[14]	彭建强, 彭忞, 谭淑萍, 等. 一种高效快速浓缩腺相关病毒载体的方法. 病毒学报, 2005, 21(1): 11-14.
	Peng J Q, Peng M, Tan S P, et al. A method of efficiently and rapidly condensing adeno-associated viral vector. Chinese Journal of Virology, 2005, 21(1): 11-14.
[15]	Kimura M, Aviv A. Measurement of telomere DNA content by dot blot analysis. Nucleic Acids Research, 2011, 39(12): e84. doi: 10.1093/nar/gkr235
[16]	Lee B Y, Han J, Im J S, et al. Senescence-associated beta-galactosidase is lysosomal beta-galactosidase. Aging Cell, 2006, 5(2): 187-195. doi: 10.1111/j.1474-9726.2006.00199.x pmid: 16626397
[17]	Cadaoas J, Hu H M, Boyle G, et al. Galactosialidosis: preclinical enzyme replacement therapy in a mouse model of the disease, a proof of concept. Molecular Therapy - Methods & Clinical Development, 2021, 20: 191-203.
[18]	Kwapiszewski R, Szczudlowska J, Kwapiszewska K, et al. Determination of acid β-galactosidase activity: methodology and perspectives. Indian Journal of Clinical Biochemistry, 2014, 29(1): 57-62. doi: 10.1007/s12291-013-0318-z pmid: 24478550
[19]	Baek R C, Broekman M L D, Leroy S G, et al. AAV-mediated gene delivery in adult GM1-gangliosidosis mice corrects lysosomal storage in CNS and improves survival. PLoS One, 2010, 5(10): e13468. doi: 10.1371/journal.pone.0013468
[20]	Kasperzyk J L, d’Azzo A, Platt F M, et al. Substrate reduction reduces gangliosides in postnatal cerebrum-brainstem and cerebellum in GM 1 gangliosidosis mice. Journal of Lipid Research, 2005, 46(4): 744-751. doi: 10.1194/jlr.M400411-JLR200 pmid: 15687347
[21]	Suzuki Y. Chemical chaperone therapy for GM1-gangliosidosis. Cellular and Molecular Life Sciences: CMLS, 2008, 65(3): 351-353. doi: 10.1007/s00018-008-7470-2
[22]	Condori J, Acosta W, Ayala J, et al. Enzyme replacement for GM1-gangliosidosis: uptake, lysosomal activation, and cellular disease correction using a novel β-galactosidase: RTB lectin fusion. Molecular Genetics and Metabolism, 2016, 117(2): 199-209. doi: 10.1016/j.ymgme.2015.12.002 pmid: 26766614
[23]	Sawada T, Tanaka A, Higaki K, et al. Intracerebral cell transplantation therapy for murine GM1 gangliosidosis. Brain and Development, 2009, 31(10): 717-724. doi: 10.1016/j.braindev.2008.11.004 pmid: 19118961
[24]	Chen J C, Luu A R, Wise N, et al. Intracerebroventricular enzyme replacement therapy with β-galactosidase reverses brain pathologies due to GM 1 gangliosidosis in mice. Journal of Biological Chemistry, 2020, 295(39): 13532-13555. doi: 10.1074/jbc.RA119.009811
[25]	Ward N J, Buckley S M K, Waddington S N, et al. Codon optimization of human factor Ⅷ cDNAs leads to high-level expression. Blood, 2011, 117(3): 798-807. doi: 10.1182/blood-2010-05-282707
[26]	Juven-Gershon T, Cheng S S, Kadonaga J T. Rational design of a super core promoter that enhances gene expression. Nature Methods, 2006, 3(11): 917-922. pmid: 17124735
[27]	Ferizi M, Aneja M K, Balmayor E R, et al. Human cellular CYBA UTR sequences increase mRNA translation without affecting the half-life of recombinant RNA transcripts. Scientific Reports, 2016, 6: 39149. doi: 10.1038/srep39149 pmid: 27974853
[28]	Jalkanen A L, Coleman S J, Wilusz J. Determinants and implications of mRNA poly(A) tail size - does this protein make my tail look big? Seminars in Cell & Developmental Biology, 2014, 34: 24-32.
[29]	Chang J L, Jin J P, Lollar P, et al. Changing residue 338 in human factor IX from arginine to alanine causes an increase in catalytic activity. Journal of Biological Chemistry, 1998, 273(20): 12089-12094. doi: 10.1074/jbc.273.20.12089 pmid: 9575152
[30]	Wu W M, Xiao L, Wu X, et al. Factor IX alteration p.Arg338Gln (FIX Shanghai) potentiates FIX clotting activity and causes thrombosis. Haematologica, 2021, 106(1): 264-268. doi: 10.3324/haematol.2019.216713 pmid: 32079698

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