title: Biochemical features of the adhesion G protein-coupled receptor CD97 related to its auto-proteolysis and HeLa cell attachment activities.
abstract: 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.
authors.affiliation: CAS Key Laboratory of Receptor Research, CAS Center for Excellence in Molecular Cell Science, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China.|iHuman Institute, Shanghai Tech University, Shanghai 201210, China.
authors.forename: Li-Yun
authors.initials: LY
authors.lastname: Yang
authors.affiliation: CAS Key Laboratory of Receptor Research, CAS Center for Excellence in Molecular Cell Science, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China.
authors.forename: Xiao-Fang
authors.initials: XF
authors.lastname: Liu
authors.affiliation: CAS Key Laboratory of Receptor Research, CAS Center for Excellence in Molecular Cell Science, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China.
authors.forename: Yang
authors.initials: Y
authors.lastname: Yang
authors.affiliation: CAS Key Laboratory of Receptor Research, CAS Center for Excellence in Molecular Cell Science, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China.
authors.forename: Lin-Lin
authors.initials: LL
authors.lastname: Yang
authors.affiliation: iHuman Institute, Shanghai Tech University, Shanghai 201210, China.
authors.forename: Kai-Wen
authors.initials: KW
authors.lastname: Liu
authors.affiliation: CAS Key Laboratory of Receptor Research, CAS Center for Excellence in Molecular Cell Science, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China.
authors.forename: Yu-Bo
authors.initials: YB
authors.lastname: Tang
authors.affiliation: CAS Key Laboratory of Receptor Research, CAS Center for Excellence in Molecular Cell Science, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China.
authors.forename: Min
authors.initials: M
authors.lastname: Zhang
authors.affiliation: CAS Key Laboratory of Receptor Research, CAS Center for Excellence in Molecular Cell Science, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China.
authors.forename: Min-Jia
authors.initials: MJ
authors.lastname: Tan
authors.affiliation: CAS Key Laboratory of Receptor Research, CAS Center for Excellence in Molecular Cell Science, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China.
authors.forename: Shan-Mei
authors.initials: SM
authors.lastname: Cheng
authors.affiliation: CAS Key Laboratory of Receptor Research, CAS Center for Excellence in Molecular Cell Science, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China.
authors.forename: Ye-Chun
authors.initials: YC
authors.lastname: Xu
authors.affiliation: CAS Key Laboratory of Receptor Research, CAS Center for Excellence in Molecular Cell Science, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China.
authors.forename: Huai-Yu
authors.initials: HY
authors.lastname: Yang
authors.affiliation: iHuman Institute, Shanghai Tech University, Shanghai 201210, China.
authors.forename: Zhi-Jie
authors.initials: ZJ
authors.lastname: Liu
authors.affiliation: iHuman Institute, Shanghai Tech University, Shanghai 201210, China.
authors.forename: Gao-Jie
authors.initials: GJ
authors.lastname: Song
authors.affiliation: CAS Key Laboratory of Receptor Research, CAS Center for Excellence in Molecular Cell Science, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China.|iHuman Institute, Shanghai Tech University, Shanghai 201210, China.
authors.forename: Wei
authors.initials: W
authors.lastname: Huang
mesh_terms: D015703:Antigens, CD / Q000235:genetics / Q000378:metabolism*; D002448:Cell Adhesion / Q000502:physiology*; D006031:Glycosylation; D006367:HeLa Cells; D006801:Humans; D008961:Models, Structural; D016296:Mutagenesis; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase / Q000378:metabolism; D020033:Protein Isoforms / Q000378:metabolism; D059748:Proteolysis; D043562:Receptors, G-Protein-Coupled
chemical_list: C095004:ADGRE5 protein, human; D015703:Antigens, CD; D020033:Protein Isoforms; D043562:Receptors, G-Protein-Coupled; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase