Functional Analysis of the GPI Transamidase Complex by Screening for Amino Acid Mutations in Each Subunit
Si-Si Liu,
Fei Jin,
Yi-Shi Liu,
Yoshiko Murakami,
Yukihiko Sugita,
Takayuki Kato,
Xiao-Dong Gao,
Taroh Kinoshita,
Motoyuki Hattori,
Morihisa Fujita
Affiliations
Si-Si Liu
Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi 214122, China
Fei Jin
State Key Laboratory of Genetic Engineering, Collaborative Innovation Center of Genetics and Development, Shanghai Key Laboratory of Bioactive Small Molecules, Department of Physiology and Neurobiology, School of Life Sciences, Fudan University, Shanghai 200438, China
Yi-Shi Liu
Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi 214122, China
Yoshiko Murakami
Research Institute for Microbial Diseases, Osaka University, Suita 565-0871, Osaka, Japan
Yukihiko Sugita
Institute for Protein Research, Osaka University, Suita 565-0871, Osaka, Japan
Takayuki Kato
Institute for Protein Research, Osaka University, Suita 565-0871, Osaka, Japan
Xiao-Dong Gao
Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi 214122, China
Taroh Kinoshita
Research Institute for Microbial Diseases, Osaka University, Suita 565-0871, Osaka, Japan
Motoyuki Hattori
State Key Laboratory of Genetic Engineering, Collaborative Innovation Center of Genetics and Development, Shanghai Key Laboratory of Bioactive Small Molecules, Department of Physiology and Neurobiology, School of Life Sciences, Fudan University, Shanghai 200438, China
Morihisa Fujita
Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi 214122, China
Glycosylphosphatidylinositol (GPI) anchor modification is a posttranslational modification of proteins that has been conserved in eukaryotes. The biosynthesis and transfer of GPI to proteins are carried out in the endoplasmic reticulum. Attachment of GPI to proteins is mediated by the GPI-transamidase (GPI-TA) complex, which recognizes and cleaves the C-terminal GPI attachment signal of precursor proteins. Then, GPI is transferred to the newly exposed C-terminus of the proteins. GPI-TA consists of five subunits: PIGK, GPAA1, PIGT, PIGS, and PIGU, and the absence of any subunit leads to the loss of activity. Here, we analyzed functionally important residues of the five subunits of GPI-TA by comparing conserved sequences among homologous proteins. In addition, we optimized the purification method for analyzing the structure of GPI-TA. Using purified GPI-TA, preliminary single particle images were obtained. Our results provide guidance for the structural and functional analysis of GPI-TA.
