TY - JOUR
T1 - Production and characterization of peptide antibodies to the C-terminal of frameshifted calreticulin associated with myeloproliferative diseases
AU - Mughal, Farah Perveen
AU - Bergmann, Ann Christina
AU - Huynh, Ha Uyen Buu
AU - Jørgensen, Sarah Hyllekvist
AU - Mansha, Inaam
AU - Kesmez, Meliha
AU - Schürch, Patrick Mark
AU - Theocharides, Alexandre Pierre André
AU - Hansen, Paul Robert
AU - Friis, Tina
AU - Holmström, Morten Orebo
AU - Ciplys, Evaldas
AU - Slibinskas, Rimantas
AU - Højrup, Peter
AU - Houen, Gunnar
AU - Trier, Nicole Hartwig
N1 - Publisher Copyright:
© 2022 by the authors. Licensee MDPI, Basel, Switzerland.
PY - 2022/6
Y1 - 2022/6
N2 - Myeloproliferative Neoplasms (MPNs) constitute a group of rare blood cancers that are characterized by mutations in bone marrow stem cells leading to the overproduction of erythrocytes, leukocytes, and thrombocytes. Mutations in calreticulin (CRT) genes may initiate MPNs, causing a novel variable polybasic stretch terminating in a common C-terminal sequence in the frameshifted CRT (CRTfs) proteins. Peptide antibodies to the mutated C-terminal are important reagents for research in the molecular mechanisms of MPNs and for the development of new diagnostic assays and therapies. In this study, eight peptide antibodies targeting the C-terminal of CRTfs were produced and characterised by modified enzyme-linked immunosorbent assays using resin-bound peptides. The antibodies reacted to two epitopes: CREACLQGWTE for SSI-HYB 385-01, 385-02, 385-03, 385-04, 385-07, 385-08, and 385-09 and CLQGWT for SSI-HYB 385-06. For the majority of antibodies, the residues Cys1, Trp9, and Glu11 were essential for reactivity. SSI-HYB 385-06, with the highest affinity, recognised recombinant CRTfs produced in yeast and the MARIMO cell line expressing CRTfs when examined in Western immunoblotting. Moreover, SSI-HYB 385-06 occasionally reacted to CRTfs from MPN patients when analysed by flow cytometry. The characterized antibodies may be used to understand the role of CRTfs in the pathogenesis of MPNs and to design and develop new diagnostic assays and therapeutic targets.
AB - Myeloproliferative Neoplasms (MPNs) constitute a group of rare blood cancers that are characterized by mutations in bone marrow stem cells leading to the overproduction of erythrocytes, leukocytes, and thrombocytes. Mutations in calreticulin (CRT) genes may initiate MPNs, causing a novel variable polybasic stretch terminating in a common C-terminal sequence in the frameshifted CRT (CRTfs) proteins. Peptide antibodies to the mutated C-terminal are important reagents for research in the molecular mechanisms of MPNs and for the development of new diagnostic assays and therapies. In this study, eight peptide antibodies targeting the C-terminal of CRTfs were produced and characterised by modified enzyme-linked immunosorbent assays using resin-bound peptides. The antibodies reacted to two epitopes: CREACLQGWTE for SSI-HYB 385-01, 385-02, 385-03, 385-04, 385-07, 385-08, and 385-09 and CLQGWT for SSI-HYB 385-06. For the majority of antibodies, the residues Cys1, Trp9, and Glu11 were essential for reactivity. SSI-HYB 385-06, with the highest affinity, recognised recombinant CRTfs produced in yeast and the MARIMO cell line expressing CRTfs when examined in Western immunoblotting. Moreover, SSI-HYB 385-06 occasionally reacted to CRTfs from MPN patients when analysed by flow cytometry. The characterized antibodies may be used to understand the role of CRTfs in the pathogenesis of MPNs and to design and develop new diagnostic assays and therapeutic targets.
KW - calreticulin
KW - epitope mapping
KW - frameshift mutations
KW - myeloproliferative neoplasms
KW - peptide antibodies
U2 - 10.3390/ijms23126803
DO - 10.3390/ijms23126803
M3 - Journal article
C2 - 35743246
AN - SCOPUS:85132067469
SN - 1661-6596
VL - 23
JO - International Journal of Molecular Sciences
JF - International Journal of Molecular Sciences
IS - 12
M1 - 6803
ER -