Monoclonal antibodies recognizing epitopes of calretinins: dependence on Ca2+-binding status and differences in antigen accessibility in colon cancer cells.
Monoclonal antibodies are very helpful tools to investigate the localization and sometimes even the function of specific proteins in cells and tissues. By generating monoclonal antibodies against calretinin-22k (CR-22k), a C-terminally truncated isoform of calretinin (CR) as a result of alternative splicing of the CR mRNA, we envisaged that screening multiple monoclonal antibodies would allow the identification of CR-22k as well as CR. Both proteins share the first 178 amino acids, but have different C-termini. All three antibodies 10C10, 6B3 and 2H4 recognize recombinant CR-22k and the specificity to also recognize CR was demonstrated in brain extracts of different species and human tumour cells, which express CR. All monoclonal antibodies did not crossreact with the closely related protein calbindin D-28k. Antibody binding was depending on the Ca2+-binding status of both forms of calretinin. Generally, the Ca2+-bound form was better recognized than the Ca2+-free form. Carboxy- and amino-terminally truncated CR proteins were expressed in E. coli in order to characterize the epitopes recognized by the three antibodies. Additionally, tryptic and cyanogen bromide fragments were produced to further narrow down the sequences recognized by the three antibodies. 10C10 recognizes an epitope consisting of the linker region between EF-hand domains I and II and the N-terminal part of EF-hand II, while the others (6B3, 2H4) bind to a region including the linker between EF-hand domains III and IV. These antibodies are valuable tools to further investigate the distribution and eventually the specific function of these two proteins in the nervous tissue and under pathological conditions, e.g. in colon tumours and mesotheliomas.
Zimmermann
L
L
Institute of Histology and General Embryology, University of Fribourg, Switzerland.
Schwaller
B
B
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D015153:Blotting, Western; D064032:Calbindin 2; D064026:Calbindins; D002118:Calcium / Q000276:immunology / Q000378:metabolism*; D003110:Colonic Neoplasms / Q000276:immunology* / Q000378:metabolism; D003488:Cyanogen Bromide; D018604:Epitope Mapping; D000939:Epitopes / Q000276:immunology*; D006801:Humans; D007150:Immunohistochemistry; D051379:Mice; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000276:immunology / Q000378:metabolism; D051381:Rats; D064030:S100 Calcium Binding Protein G / Q000276:immunology* / Q000378:metabolism; D017385:Sequence Homology; D014357:Trypsin; D014407:Tumor Cells, Cultured
D000911:Antibodies, Monoclonal; C576764:CALB2 protein, human; C576511:Calb2 protein, mouse; C576512:Calb2 protein, rat; D064032:Calbindin 2; D064026:Calbindins; D000939:Epitopes; D010446:Peptide Fragments; D064030:S100 Calcium Binding Protein G; D014357:Trypsin; D003488:Cyanogen Bromide; D002118:Calcium