Biochemical features of the adhesion G protein-coupled receptor CD97 related to its auto-proteolysis and HeLa cell attachment activities
文献类型：期刊论文
| 作者 | Yang, Li-yun1,2; Liu, Xiao-fang1; Yang, Yang1; Yang, Lin-lin1; Liu, Kai-wen2; Tang, Yu-bo1; Zhang, Min1; Tan, Min-jia1 ; Cheng, Shan-mei1; Xu, Ye-chun1
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| 刊名 | ACTA PHARMACOLOGICA SINICA
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| 出版日期 | 2017-01 |
| 卷号 | 38期号:1页码:56-68 |
| ISSN号 | 1671-4083 |
| 关键词 | CD97 adhesion GPCR auto-proteolysis HeLa cell attachment N-glycosylation |
| DOI | 10.1038/aps.2016.89 |
| 文献子类 | Article |
| 英文摘要 | CD97 belongs to the adhesion GPCR family characterized by a long ECD linked to the 7TM via a GPCR proteolytic site (GPS) and plays important roles in modulating cell migration and invasion. CD97 (EGF1-5) is a splicing variant of CD97 that recognizes a specific ligand chondroitin sulfate on cell membranes and the extracellular matrix. The aim of this study was to elucidate the extracellular molecular basis of the CD97 EGF1-5 isoform in protein expression, auto-proteolysis and cell adhesion, including epidermal growth factor (EGF)like domain, GPCR autoproteolysis-inducing (GAIN) domain, as well as GPS mutagenesis and N-glycosylation. Both wild-type (WT) CD97-ECD and its truncated, GPS mutated, PNGase F-deglycosylated, and N-glycosylation site mutated forms were expressed and purified. The auto-proteolysis of the proteins was analyzed with Western blotting and SDS-PAGE. Small angle X-ray scattering (SAXS) and molecular modeling, were used to determine a structural profile of the properly expressed receptor. Potential N-glycosylation sites were identified using MS and were modulated with PNGase F digestion and glyco-site mutations. A flow cytometry-based HeLa cell attachment assay was used for all aforementioned CD97 variants to elucidate the molecular basis of CD97-HeLa interactions. A unique concentration-dependent GPS auto-proteolysis was observed in CD97 EGF1-5 isoform with the highest concentration (4 mg/mL) per sample was self-cleaved much faster than the lower concentration (0.1 mg/mL), supporting an intermolecular mechanism of auto-proteolysis that is distinct to the reported intramolecular mechanism for other CD97 isoforms. N-glycosylation affected the auto-proteolysis of CD97 EGF1-5 isoform in a similar way as the other previously reported CD97 isoforms. SAXS data for WT and deglycosylated CD97ECD revealed a spatula-like shape with GAIN and EGF domains constituting the body and handle, respectively. Structural modeling indicated a potential interaction between the GAIN and EGF5 domains accounting for the absence of expression of the GAIN domain itself, although EGF5-GAIN was expressed similarly in the wild-type protein. For HeLa cell adhesion, the GAIN truncated forms showed dramatically reduced binding affinity. The PNGase F-deglycosylated and GPS mutated forms also exhibited reduced HeLa attachment compared with WT CD97. However, neither N-glycosylation mutagenesis nor auto-proteolysis inhibition caused by N-glycosylation mutagenesis affected CD97-HeLa cell interactions. A comparison of the HeLa binding affinities of PNGase F-digested, GPS-mutated and N-glycosylation-mutated CD97 samples revealed diverse findings, suggesting that the functions of CD97 ECD were complex, and various technologies for function validation should be utilized to avoid single-approach bias when investigating N-glycosylation and auto-proteolysis of CD97. A unique mechanism of concentration-dependent auto-proteolysis of the CD97 EGF1-5 isoform was characterized, suggesting an intermolecular mechanism that is distinct from that of other previously reported CD97 isoforms. The EGF5 and GAIN domains are likely associated with each other as CD97 expression and SAXS data revealed a potential interaction between the two domains. Finally, the GAIN and EGF domains are also important for CD97-HeLa adhesion, whereas N-glycosylation of the CD97 GAIN domain and GPS auto-proteolysis are not required for HeLa cell attachment. |
| WOS关键词 | ACTIVATION ANTIGEN CD97 ; EGF-TM7 RECEPTOR ; LIGAND CD55 ; CHONDROITIN SULFATE ; ENDOTHELIAL-CELLS ; GPS MOTIF ; EXPRESSION ; EMR2 ; AUTOPROTEOLYSIS ; CLEAVAGE |
| 资助项目 | National Natural Science Foundation of China[21372238] ; National Natural Science Foundation of China[21572244] ; State Education Ministry grant for Returned Overseas Chinese Scholars[X-0801-15-020] |
| WOS研究方向 | Chemistry ; Pharmacology & Pharmacy |
| 语种 | 英语 |
| CSCD记录号 | CSCD:5906817 |
| 出版者 | ACTA PHARMACOLOGICA SINICA |
| WOS记录号 | WOS:000393540900005 |
| 源URL | [http://119.78.100.183/handle/2S10ELR8/275740]  |
| 专题 | 生物技术药物研发中心（筹） 药物安全性评价中心 |
| 通讯作者 | Song, Gao-jie; Huang, Wei |
| 作者单位 | 1.Chinese Acad Sci, Shanghai Inst Mat Med, CAS Ctr Excellence Mol Cell Sci, CAS Key Lab Receptor Res, Shanghai 201203, Peoples R China; 2.Shanghai Tech Univ, iHuman Inst, Shanghai 201210, Peoples R China |
| 推荐引用方式 GB/T 7714 | Yang, Li-yun,Liu, Xiao-fang,Yang, Yang,et al. Biochemical features of the adhesion G protein-coupled receptor CD97 related to its auto-proteolysis and HeLa cell attachment activities[J]. ACTA PHARMACOLOGICA SINICA,2017,38(1):56-68. |
| APA | Yang, Li-yun.,Liu, Xiao-fang.,Yang, Yang.,Yang, Lin-lin.,Liu, Kai-wen.,...&Huang, Wei.(2017).Biochemical features of the adhesion G protein-coupled receptor CD97 related to its auto-proteolysis and HeLa cell attachment activities.ACTA PHARMACOLOGICA SINICA,38(1),56-68. |
| MLA | Yang, Li-yun,et al."Biochemical features of the adhesion G protein-coupled receptor CD97 related to its auto-proteolysis and HeLa cell attachment activities".ACTA PHARMACOLOGICA SINICA 38.1(2017):56-68. |
入库方式： OAI收割
来源：上海药物研究所
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