Functional Analysis of the GPI Transamidase Complex by Screening for Amino Acid Mutations in Each Subunit
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-transamida...
| Main Authors: | , , , , , , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
MDPI AG
2021-09-01
|
| Series: | Molecules |
| Subjects: | |
| Online Access: | https://www.mdpi.com/1420-3049/26/18/5462 |
| _version_ | 1797517984095272960 |
|---|---|
| author | Si-Si Liu Fei Jin Yi-Shi Liu Yoshiko Murakami Yukihiko Sugita Takayuki Kato Xiao-Dong Gao Taroh Kinoshita Motoyuki Hattori Morihisa Fujita |
| author_facet | Si-Si Liu Fei Jin Yi-Shi Liu Yoshiko Murakami Yukihiko Sugita Takayuki Kato Xiao-Dong Gao Taroh Kinoshita Motoyuki Hattori Morihisa Fujita |
| author_sort | Si-Si Liu |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-03-10T07:23:48Z |
| format | Article |
| id | doaj.art-1243bbe3a95b4aab90632b9f330bf8b5 |
| institution | Directory Open Access Journal |
| issn | 1420-3049 |
| language | English |
| last_indexed | 2024-03-10T07:23:48Z |
| publishDate | 2021-09-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Molecules |
| spelling | doaj.art-1243bbe3a95b4aab90632b9f330bf8b52023-11-22T14:23:31ZengMDPI AGMolecules1420-30492021-09-012618546210.3390/molecules26185462Functional Analysis of the GPI Transamidase Complex by Screening for Amino Acid Mutations in Each SubunitSi-Si Liu0Fei Jin1Yi-Shi Liu2Yoshiko Murakami3Yukihiko Sugita4Takayuki Kato5Xiao-Dong Gao6Taroh Kinoshita7Motoyuki Hattori8Morihisa Fujita9Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi 214122, ChinaState 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, ChinaKey Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi 214122, ChinaResearch Institute for Microbial Diseases, Osaka University, Suita 565-0871, Osaka, JapanInstitute for Protein Research, Osaka University, Suita 565-0871, Osaka, JapanInstitute for Protein Research, Osaka University, Suita 565-0871, Osaka, JapanKey Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi 214122, ChinaResearch Institute for Microbial Diseases, Osaka University, Suita 565-0871, Osaka, JapanState 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, ChinaKey Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi 214122, ChinaGlycosylphosphatidylinositol (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.https://www.mdpi.com/1420-3049/26/18/5462glyco-diosgeninGPI-anchored proteinsGPI-transamidaseprotein purificationsingle particle |
| spellingShingle | Si-Si Liu Fei Jin Yi-Shi Liu Yoshiko Murakami Yukihiko Sugita Takayuki Kato Xiao-Dong Gao Taroh Kinoshita Motoyuki Hattori Morihisa Fujita Functional Analysis of the GPI Transamidase Complex by Screening for Amino Acid Mutations in Each Subunit Molecules glyco-diosgenin GPI-anchored proteins GPI-transamidase protein purification single particle |
| title | Functional Analysis of the GPI Transamidase Complex by Screening for Amino Acid Mutations in Each Subunit |
| title_full | Functional Analysis of the GPI Transamidase Complex by Screening for Amino Acid Mutations in Each Subunit |
| title_fullStr | Functional Analysis of the GPI Transamidase Complex by Screening for Amino Acid Mutations in Each Subunit |
| title_full_unstemmed | Functional Analysis of the GPI Transamidase Complex by Screening for Amino Acid Mutations in Each Subunit |
| title_short | Functional Analysis of the GPI Transamidase Complex by Screening for Amino Acid Mutations in Each Subunit |
| title_sort | functional analysis of the gpi transamidase complex by screening for amino acid mutations in each subunit |
| topic | glyco-diosgenin GPI-anchored proteins GPI-transamidase protein purification single particle |
| url | https://www.mdpi.com/1420-3049/26/18/5462 |
| work_keys_str_mv | AT sisiliu functionalanalysisofthegpitransamidasecomplexbyscreeningforaminoacidmutationsineachsubunit AT feijin functionalanalysisofthegpitransamidasecomplexbyscreeningforaminoacidmutationsineachsubunit AT yishiliu functionalanalysisofthegpitransamidasecomplexbyscreeningforaminoacidmutationsineachsubunit AT yoshikomurakami functionalanalysisofthegpitransamidasecomplexbyscreeningforaminoacidmutationsineachsubunit AT yukihikosugita functionalanalysisofthegpitransamidasecomplexbyscreeningforaminoacidmutationsineachsubunit AT takayukikato functionalanalysisofthegpitransamidasecomplexbyscreeningforaminoacidmutationsineachsubunit AT xiaodonggao functionalanalysisofthegpitransamidasecomplexbyscreeningforaminoacidmutationsineachsubunit AT tarohkinoshita functionalanalysisofthegpitransamidasecomplexbyscreeningforaminoacidmutationsineachsubunit AT motoyukihattori functionalanalysisofthegpitransamidasecomplexbyscreeningforaminoacidmutationsineachsubunit AT morihisafujita functionalanalysisofthegpitransamidasecomplexbyscreeningforaminoacidmutationsineachsubunit |