Production and Characterization of Peptide Antibodies to the C-Terminal of Frameshifted Calreticulin Associated with Myeloproliferative Diseases

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.

Keywords: calreticulin; epitope mapping; frameshift mutations; myeloproliferative neoplasms; peptide antibodies.

Figure 1

Schematic presentation of calreticulin (CRT) and location of C-terminal frameshift substitutions. The different regions are coloured according to Boelt et al. [13], and the C-terminal part from residue 361 of native CRT, CRTfs L367, and CRTfs K385 are highlighted (bottom). Acidic residues are coloured blue while basic residues are coloured red. The KDEL sequence of native CRT is underlined.

Figure 2

Reactivity of mouse bleeds to peptides used for immunisation tested in enzyme-linked immunosorbent assay. Fourth collection of mouse bleeds were tested for antibody reactivity to peptides 1–3: (a) Four mice (1a–d) were immunised with peptide 1 (CRRMMRTKMRMRRMRRTRRKMRRKMSPARPRTSCREACLQGWTEA), and the collected samples were tested for reactivity to peptides 1–3. (b) Four mice (2a–d) were immunised with peptide 2 (CREACLQGWTEA), and bleeds were tested for reactivity to peptides 1–3. (c) Four mice (3a–d) were immunised with peptide 3 (CLQGWTEA), and bleeds were tested for reactivity to peptides 1–3. Absorbances were corrected for background reactivity by subtracting reactivity from non-coated wells.

Figure 3

Titration of SSI-HYBs analysed by enzyme-linked immunosorbent assay. A two-fold dilution series ranging from 1:10–1:10240 was tested.

Figure 4

Reactivity of SSI-HYB clones to full-length frameshifted calreticulin (CRTfs) K385 and CRT wild-type (wt) analysed by enzyme-linked immunosorbent assay. Absorbances were corrected for background reactivity by subtracting reactivity from non-coated wells.

Figure 5

Reactivity of SSI-HYB clones to N- and C-terminally truncated frameshifted calreticulin peptides by modified enzyme-linked immunosorbent assay: (a) Reactivity of clones to N-terminally truncated resin-bound peptides. (b) Reactivity of clones to C-terminally truncated resin-bound peptides. Absorbances were corrected for background reactivity by subtracting reactivity from non-coated wells.

Figure 6

Reactivity of SSI-HYBs to Ala-substituted peptides analysed by modified enzyme-linked immunosorbent assay. The control peptide CREACLQGWTEA was used for generation of substituted peptides where Cys¹ and Cys⁵ were substituted with Ala, and a single peptide where both Cys residues were substituted with Ala. Absorbances were corrected for background reactivity by subtracting reactivity from non-coated wells.

Figure 7

Structural epitope analysis. (a) The three-dimensional structure of the CREACLQGWTE sequence obtained from PEP FOLD SERVER. The peptide folds into an α-helix structure in which protruding amino acid residues are seen. On the right side of the structure, the side-chain residues of Trp⁹, Cys¹, and Cys⁵ protrude. On the left side of the structure, the side-chain residues of Arg², Glu³, Leu⁶, Gln⁷, and the terminal Thr¹⁰ protrude. (b) The three-dimensional structure of the CREACLQGWTE peptide. Yellow balls = sulphur atoms, red balls = oxygen atoms, grey balls = carbon atoms, white balls = hydrogen atoms, and blue balls = nitrogen atoms. (c) Helical wheel representation of the CREACLQGWTE peptide. The residues in the epitope are presented using one-letter codes. The image was generated using the NetWheels application (http://lbqp.unb.br/NetWheels, accessed on 14 May 2022). The following colours represent amino acid functions: red, polar/basic; blue, polar/acid; green, polar/uncharged; yellow, nonpolar.

Figure 8

Immunostaining of SSI-HYB 385-06 reactivity to frameshifted calreticulin (CRTfs) and wild-type calreticulin (CRTwt). (a) Coomassie Brilliant Blue staining of recombinant CRT proteins. (b) Western blotting of SSI-HYB 385-06 reactivity to CRTfs L367 and K385 and CRTwt. (c) Western blotting of membrane from figure (b) developed with a commercial monoclonal antibody (CRT FMC 75 mAb), recognizing amino acids 34–41 (TSRWIESK) in the N-terminal of CRT. Lane 1: non-reduced CRTfs K385, Lane 2: non-reduced CRTfs L367, Lane 3: non-reduced recombinant CRT, Lane 4: reduced CRTfs K385, Lane 5: reduced CRTfs L367, Lane 6: reduced recombinant CRT.

Figure 9

Reactivity of SSI-HYB 385-06 to frameshifted calreticulin (CRTfs) in MARIMO cells analysed by Western blotting. (a) MARIMO wells were treated with methanol (−brefeldin A (BFA)) or with BFA, whereafter cells were lysed and examined by Western blotting under reduced conditions. The monoclonal antibody FMC-75 to calreticulin (CRT) was used to detect the total intracellular CRT level, as this antibody interacts with an epitope in the N-terminal, thus detecting wildtype and mutated CRT [37]. CRTfs was stained using SSI-HYB 385-06. Actin was used as loading control (b). Quantification of CRT and CRTfs levels based on Western blot in (a).

Figure 10

Intracellular leukocyte staining of frameshifted calreticulin (CRTfs) by flow cytometry. (a) Negative isotype control staining using a mouse monoclonal antibody of irrelevant specificity (β-galactosidase). (b) Staining using SSI-HYB 385-06 recognizing CRTfs. Colours represent: red, unstained cells; blue, non-specific FITC staining; orange, specific CRT staining.