title: Molecular basis of antibody binding to mucin glycopeptides in lung cancer.
abstract: Glycopeptides bearing Tn epitopes are emerging targets for cancer diagnosis and immunotherapy. In this study, we analyzed membrane proteins containing O-glycosylated tandem repeat (TR) sequences in lung cancer patients of different types and stages, using gene microarray data in public domain. The expression of Tn and glycopeptide epitopes on the surface of lung cancer cell lines were studied by monoclonal IgG antibodies 14A, 16A, and B72.3. The binding of mAbs to synthetic glycopeptides were studied by surface plasmon resonance. Nine mucin mRNAs were found to be expressed in lung cancer patients but at similar level to healthy individuals. At protein level, a glycopeptide epitope on cancer cell surface is preferably recognized by mAb 16A, as compared to peptide-alone (14A) or sugar-alone epitopes (B72.3). 14A and 16A favor clustered TR containing more than three TR sequences, with 10-fold lower Kd than two consecutive TR. B72.3 preferrably recognized clustered sialyl-Tn displayed on MUC1 but not other O-glycoproteins, with 100-fold stronger binding when MUC1 is transfected as a sugar carrier, while the total sugar epitopes remain unchanged. These findings indicate that clusters of both TR backbones and sugars are essential for mAb binding to mucin glycopeptides. Three rules of antibody binding to mucin glycopeptides at molecular level are presented here: first, the peptide backbone of a glycopeptide is preferentially recognized by B cells through mutations in complementarity determining regions (CDRs) of B cell receptor, and the sugar-binding specificity is acquired through mutations in frame work of heavy chain; secondly, consecutive tandem repeats (TR) of peptides and glycopeptides are preferentially recognized by B cells, which favor clustered TR containing more than three TR sequences; thirdly, certain sugar-specific B cells recognize and accommodate clustered Tn and sialyl-Tn displayed on the surface of a mucin but not other membrane proteins.
authors.affiliation: Shanghai Pulmonary Hospital Affiliated with Tongji University School of Medicine, Shanghai 200092, P.R. China.
authors.forename: Jin
authors.initials: J
authors.lastname: Qu
authors.affiliation: CAS Key Laboratory of Receptor Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, and iHuman Institute, Shanghai Tech University, Shanghai 201203, P.R. China.
authors.forename: Hongtao
authors.initials: H
authors.lastname: Yu
authors.affiliation: Tianjin Cancer Hospital, Tianjin 300060, P.R. China.
authors.forename: Fenge
authors.initials: F
authors.lastname: Li
authors.affiliation: Shenzhen Hospital of Chinese Medicine, Shenzhen, Guangdong 518033, P.R. China.
authors.forename: Chunlei
authors.initials: C
authors.lastname: Zhang
authors.affiliation: Biochemical Institute, Medical Faculty, Christian-Albrechts-University of Kiel, 24105 Kiel, Germany.
authors.forename: Ahmad
authors.initials: A
authors.lastname: Trad
authors.affiliation: Department of Chemistry, California State University, Fresno, CA 93740, USA.
authors.forename: Cory
authors.initials: C
authors.lastname: Brooks
authors.affiliation: Northwestern University Feinberg School of Medicine, Chicago, IL 60611, USA.
authors.forename: Bin
authors.initials: B
authors.lastname: Zhang
authors.affiliation: Department of Epigenetics and Molecular Carcinogenesis, The University of Texas MD Anderson Cancer Center, Smithville, TX 78957, USA.
authors.forename: Ting
authors.initials: T
authors.lastname: Gong
authors.affiliation: Tianjin Cancer Hospital, Tianjin 300060, P.R. China.
authors.forename: Zhi
authors.initials: Z
authors.lastname: Guo
authors.affiliation: Laboratory of Cellular and Molecular Tumor Immunology, Jiangsu Key Laboratory of Infection and Immunity, Institutes of Biology and Medical Sciences, Soochow University, Suzhou, Jiangsu 215123, P.R. China.
authors.forename: Yunsen
authors.initials: Y
authors.lastname: Li
authors.affiliation: Memorial Sloan-Kettering Cancer Center, New York, NY 10065, USA.
authors.forename: Govind
authors.initials: G
authors.lastname: Ragupathi
authors.affiliation: Mayo Clinic, Jacksonville, FL 32224, USA.
authors.forename: Yanyan
authors.initials: Y
authors.lastname: Lou
authors.affiliation: Division of Cancer Medicine, The University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA.
authors.forename: Patrick
authors.initials: P
authors.lastname: Hwu
authors.affiliation: CAS Key Laboratory of Receptor Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, and iHuman Institute, Shanghai Tech University, Shanghai 201203, P.R. China.
authors.forename: Wei
authors.initials: W
authors.lastname: Huang
authors.affiliation: Shanghai Pulmonary Hospital Affiliated with Tongji University School of Medicine, Shanghai 200092, P.R. China.
authors.forename: Dapeng
authors.initials: D
authors.lastname: Zhou
mesh_terms: D000911:Antibodies, Monoclonal / Q000276:immunology / Q000378:metabolism*; D015295:Antigens, Tumor-Associated, Carbohydrate / Q000276:immunology / Q000378:metabolism; D001402:B-Lymphocytes / Q000276:immunology / Q000378:metabolism; D045744:Cell Line, Tumor; D000939:Epitopes / Q000276:immunology; D006020:Glycopeptides / Q000276:immunology / Q000378:metabolism*; D006031:Glycosylation; D006801:Humans; D007074:Immunoglobulin G / Q000276:immunology / Q000378:metabolism; D007167:Immunotherapy / Q000379:methods; D008175:Lung Neoplasms / Q000276:immunology / Q000378:metabolism*; D018396:Mucin-1 / Q000276:immunology / Q000378:metabolism*; D011485:Protein Binding / Q000276:immunology*; D012333:RNA, Messenger / Q000276:immunology
chemical_list: D000911:Antibodies, Monoclonal; D015295:Antigens, Tumor-Associated, Carbohydrate; D000939:Epitopes; D006020:Glycopeptides; D007074:Immunoglobulin G; D018396:Mucin-1; D012333:RNA, Messenger; C055491:sialosyl-Tn antigen