Proteomics

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Glycoproteoforms of Integrins α2 and β1 in Megakaryocytes in the Occurrence of Primary Myelofibrosis


ABSTRACT: Primary myelofibrosis (PMF) is a myeloproliferative neoplasm prone to leukemic transformation, for which limited treatment is available. Amongst individuals diagnosed with PMF, the most prevalent mutation is the JAK2V617F somatic point mutation that activates the Janus kinase 2 (JAK2) enzyme. Our earlier reports on hyperactivity of β1 integrin and enhanced adhesion activity of the α2β1 complex in JAK2V617F megakaryocytes (MKs) led us to examine the new hypothesis that this mutation leads to post-translational modification via changes in glycosylation. Samples were derived from immunoprecipitation of MKs obtained from Vav1-hJAK2V617F and wild type mice. Immunoprecipitated fractions were separated by SDS-PAGE and analyzed using LC-MS/MS techniques in a bottom-up glycoproteomics workflow. In the immunoprecipitate, glycopeptiforms corresponding to 11 out of the 12 potential N-glycosylation sites of integrin β1, and to all nine potential glycosylation sites of integrin α2, were observed. Glycopeptiforms were compared across WT and JAK2V617F phenotypes for both integrins. The overall trend observed is that JAK2V617F mutation in PMF MKs leads to changes in β1 glycosylation; in most cases, it results in an increase in the integrated area of glycopeptiforms. We also observed that, in mutated MKs, changes in integrin α2 glycosylation were more substantial than those observed for integrin β1 glycosylation, a finding that suggests that altered integrin α2 glycosylation may also affect activation. Additionally, the identification of proteins associated to the cytoskeleton that were co-immunoprecipitated with integrins α2 and β1 demonstrated the potential of the methodology employed in this study to provide some insight, at the peptide level, into the consequences of integrin activation in MKs. The extensive and detailed glycosylation patterns we uncovered provide a basis for future functional studies of each site in control cells as compared to JAK2V617F mutated cells.

INSTRUMENT(S): Orbitrap Fusion Lumos, Q Exactive HF

ORGANISM(S): Mesocricetus Auratus (golden Hamster) Mus Musculus (mouse)

TISSUE(S): Blood Cell, Ovary Cell Line, Bone Marrow

DISEASE(S): Myelofibrosis

SUBMITTER: MAISSA GAYE  

LAB HEAD: Catherine E. Costello

PROVIDER: PXD030550 | Pride | 2022-02-22

REPOSITORIES: Pride

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Publications

Characterization of Glycoproteoforms of Integrins α2 and β1 in Megakaryocytes in the Occurrence of JAK2V617F Mutation-Induced Primary Myelofibrosis.

Gaye Maissa M MM   Ward Christina M CM   Piasecki Andrew J AJ   Stahl Vanessa L VL   Karagianni Aikaterini A   Costello Catherine E CE   Ravid Katya K  

Molecular & cellular proteomics : MCP 20220217 4


Primary myelofibrosis (PMF) is a neoplasm prone to leukemic transformation, for which limited treatment is available. Among individuals diagnosed with PMF, the most prevalent mutation is the JAK2V617F somatic point mutation that activates the Janus kinase 2 (JAK2) enzyme. Our earlier reports on hyperactivity of β1 integrin and enhanced adhesion activity of the α2β1 complex in JAK2V617F megakaryocytes (MKs) led us to examine the new hypothesis that this mutation leads to posttranslational modific  ...[more]

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