Selection of novel peptide mimics of the GD2 ganglioside from a constrained phage-displayed peptide library.
Aberrant glycosylation is a universal feature of cancer cells. There are quantitative and qualitative changes in expression of gangliosides observed in tumors of a neuroectodermal origin such as neuroblastoma, melanoma and astrocytoma. The presence of large amounts of GD2 ganglioside on neuroblastoma cells, as compared to normal cells, opens the possibilities to use the tumor-associated carbohydrate antigen in diagnosis and immunotherapeutic approaches. In the quest for immunogens potentially capable of eliciting anti-GD2 ganglioside immune responses, we performed affinity purification of phage-displayed peptides from the LX-8 library (12-mer containing disulphide bridge). The library was screened with the biotinylated anti-GD2 ganglioside 14G2a mAb monoclonal antibody. Our goal was to isolate and characterize peptide mimics of GD2 ganglioside. Numerous individual phage clones that bound 14G2a mAb were identified with the application of immunoblotting technique in the phage pools yielded from the pannings. The phage-borne peptides were tested for their anti-GD2 ganglioside antibody binding ability using ELISA. Among these clones five different phage-displayed peptide sequences were identified. Moreover, we showed that the secondary structure of the peptides, stabilized by the disulfide bridging between cysteine residues at positions 2 and 11, was crucial for the binding of the peptides to 14G2a mAb. In a separate set of experiments, we observed a competition of the peptides, expressed on phages as well as in their synthetic form, with the nominal antigen GD2 ganglioside expressed on IMR-32 neuroblastoma cells for binding to 14G2a mAb. Based on the obtained results we concluded that all of these 5 peptides were mimics of the GD2 ganglioside.
Horwacik
Irena
I
Faculty of Biochemistry, Biophysics and Biotechnology, The Jagiellonian University, Krakow, Poland. irena@awe.mol.uj.edu.pl
Czaplicki
Dominik
D
Talarek
Katarzyna
K
Kowalczyk
Aleksandra
A
Bolesta
Elzbieta
E
Kozbor
Danuta
D
Rokita
Hanna
H
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000276:immunology; D001667:Binding, Competitive; D045744:Cell Line, Tumor; D002999:Clone Cells; D004220:Disulfides; D004797:Enzyme-Linked Immunosorbent Assay; D005732:Gangliosides / Q000737:chemistry* / Q000276:immunology; D006801:Humans; D015151:Immunoblotting; D018716:Molecular Mimicry; D008969:Molecular Sequence Data; D009447:Neuroblastoma / Q000473:pathology; D019151:Peptide Library; D010455:Peptides / Q000737:chemistry / Q000302:isolation & purification*; D011485:Protein Binding; D020539:Sequence Analysis, Protein
D000911:Antibodies, Monoclonal; D004220:Disulfides; D005732:Gangliosides; D019151:Peptide Library; D010455:Peptides; C019403:ganglioside, GD2