Biochemical features of the adhesion G protein-coupled receptor CD97 related to its auto-proteolysis and HeLa cell attachment activities

doi:10.1038/aps.2016.89

	Biochemical features of the adhesion G protein-coupled receptor CD97 related to its auto-proteolysis and HeLa cell attachment activities
	Yang, Li-yun 1,2; Liu, Xiao-fang 1; Yang, Yang 1; Yang, Lin-lin 1; Liu, Kai-wen2 ; Tang, Yu-bo 1; Zhang, Min 1; Tan, Min-jia 1; Cheng, Shan-mei 1; Xu, Ye-chun 1; Yang, Huai-yu 1; Liu, Zhi-jie2 ; Song, Gao-jie2 ; Huang, Wei1,2
	2017-01
发表期刊	ACTA PHARMACOLOGICA SINICA
ISSN	1671-4083
卷号	38 期号:1 页码:56-68
发表状态	已发表
DOI	10.1038/aps.2016.89
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.
关键词	CD97 adhesion GPCR auto-proteolysis HeLa cell attachment N-glycosylation
收录类别	SCI
语种	英语
资助项目	State Education Ministry grant for Returned Overseas Chinese Scholars[X-0801-15-020]
WOS研究方向	Chemistry ; Pharmacology & Pharmacy
WOS类目	Chemistry, Multidisciplinary ; Pharmacology & Pharmacy
WOS记录号	WOS:000393540900005
出版者	ACTA PHARMACOLOGICA SINICA
WOS关键词	ACTIVATION ANTIGEN CD97 ; EGF-TM7 RECEPTOR ; LIGAND CD55 ; CHONDROITIN SULFATE ; ENDOTHELIAL-CELLS ; GPS MOTIF ; EXPRESSION ; EMR2 ; AUTOPROTEOLYSIS ; CLEAVAGE
原始文献类型	Article
引用统计
文献类型	期刊论文
条目标识符	https://kms.shanghaitech.edu.cn/handle/2MSLDSTB/2941
专题	生命科学与技术学院_博士生生命科学与技术学院 iHuman研究所 iHuman研究所_PI研究组_刘志杰组 iHuman研究所_PI研究组_程建军组
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
第一作者单位	上海科技大学
通讯作者单位	上海科技大学
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.