Production and characterization of peptide antibodies to the C-terminal of frameshifted calreticulin associated with myeloproliferative diseases

Farah Perveen Mughal, Ann Christina Bergmann, Ha Uyen Buu Huynh, Sarah Hyllekvist Jørgensen, Inaam Mansha, Meliha Kesmez, Patrick Mark Schürch, Alexandre Pierre André Theocharides, Paul Robert Hansen, Tina Friis, Morten Orebo Holmström, Evaldas Ciplys, Rimantas Slibinskas, Peter Højrup, Gunnar Houen*, Nicole Hartwig Trier

*Corresponding author for this work

Research output: Contribution to journalJournal articleResearchpeer-review

35 Downloads (Pure)

Abstract

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.

Original languageEnglish
Article number6803
JournalInternational Journal of Molecular Sciences
Volume23
Issue number12
ISSN1661-6596
DOIs
Publication statusPublished - Jun 2022

Keywords

  • calreticulin
  • epitope mapping
  • frameshift mutations
  • myeloproliferative neoplasms
  • peptide antibodies

Fingerprint

Dive into the research topics of 'Production and characterization of peptide antibodies to the C-terminal of frameshifted calreticulin associated with myeloproliferative diseases'. Together they form a unique fingerprint.

Cite this