Characterization of an immunodominant cancer-specific O-glycopeptide epitope in murine podoplanin (OTS8).
Auto-antibodies induced by cancer represent promising sensitive biomarkers and probes to identify immunotherapeutic targets without immunological tolerance. Surprisingly few epitopes for such auto-antibodies have been identified to date. Recently, a cancer-specific syngeneic murine monoclonal antibody 237, developed to a spontaneous murine fibrosarcoma, was shown to be directed to murine podoplanin (OTS8) with truncated Tn O-glycans. Our understanding of such cancer-specific auto-antibodies to truncated glycoforms of glycoproteins is limited. Here we have investigated immunogenicity of a chemoenzymatically produced Tn-glycopeptide derived from the putative murine podoplanin O-glycopeptide epitope. We found that the Tn O-glycopeptide was highly immunogenic in mice and produced a Tn-glycoform specific response with no reactivity against unglycosylated peptides or the O-glycopeptide with extended O-glycan (STn and T glycoforms). The immunodominant epitope was strictly dependent on the peptide sequence, required Tn at a specific single Thr residue (Thr(77)), and antibodies to the epitope were not found in naive mice. We further tested a Tn O-glycopeptide library derived from human podoplanin by microarray analysis and demonstrated that the epitope was not conserved in man. We also tested human cancer sera for potential auto-antibodies to similar epitopes, but did not detect such antibodies to the Tn-library of podoplanin. The reagents and methods developed will be valuable for further studies of the nature and timing of induction of auto-antibodies to distinct O-glycopeptide epitopes induced by cancer. The results demonstrate that truncated O-glycopeptides constitute highly distinct antibody epitopes with great potential as targets for biomarkers and immunotherapeutics.
Steentoft
Catharina
C
Department of Basic Sciences and Environment (IGM)-Bioorganic Chemistry, University of Copenhagen, Faculty of Life Sciences, Thorvaldsensvej 40, DK-1871, Frederiksberg C, Denmark.
Schjoldager
Katrine T
KT
Cló
Emiliano
E
Mandel
Ulla
U
Levery
Steven B
SB
Pedersen
Johannes W
JW
Jensen
Knud
K
Blixt
Ola
O
Clausen
Henrik
H
D000595:Amino Acid Sequence; D000818:Animals; D000918:Antibody Specificity / Q000276:immunology; D004797:Enzyme-Linked Immunosorbent Assay; D006020:Glycopeptides / Q000737:chemistry / Q000276:immunology*; D006031:Glycosylation; D006801:Humans; D016056:Immunodominant Epitopes / Q000737:chemistry* / Q000276:immunology*; D007074:Immunoglobulin G / Q000276:immunology; D008562:Membrane Glycoproteins / Q000737:chemistry* / Q000276:immunology*; D051379:Mice; D008969:Molecular Sequence Data; D009369:Neoplasms / Q000737:chemistry* / Q000276:immunology*; D040081:Protein Array Analysis; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D000098853:Podoplanin
D006020:Glycopeptides; D016056:Immunodominant Epitopes; D007074:Immunoglobulin G; D008562:Membrane Glycoproteins; C490441:Pdpn protein, mouse; D000098853:Podoplanin