Characterization of Glycoproteoforms of Integrins α2 and β1 in Megakaryocytes in the Occurrence of JAK2V617F Mutation-Induced Primary Myelofibrosis.
Characterization of Glycoproteoforms of Integrins α2 and β1 in Megakaryocytes in the Occurrence of JAK2V617F Mutation-Induced Primary Myelofibrosis.
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DOI：
10.1016/j.mcpro.2022.100213
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发表时间：
2022-04
期刊：
影响因子：
--
通讯作者：
Ravid K
中科院分区：
文献类型：
--
作者：
Gaye MM;Ward CM;Piasecki AJ;Stahl VL;Karagianni A;Costello CE;Ravid K
原发性骨髓纤维化（PMF）是一种容易发生白血病转化的肿瘤，目前治疗方法有限。在诊断为 PMF 的个体中，最常见的突变是激活 Janus 激酶 2 (JAK2) 酶的 JAK2V617F 体细胞点突变。我们早期关于 JAK2V617F 巨核细胞 (MK) 中 β1 整合素过度活跃和 α2β1 复合物粘附活性增强的报告使我们检验了新的假设，即这种突变通过糖基化的变化导致翻译后修饰。样品来源于从 Vav1-hJAK2V617F 和 WT 小鼠获得的 MK 的免疫沉淀。通过 SDS-PAGE 分离免疫沉淀组分，并在自下而上的糖蛋白组学工作流程中使用 LC-MS/MS 技术进行分析。在免疫沉淀中，观察到与整联蛋白 β1 的 12 个潜在 N-糖基化位点中的 11 个相对应的糖肽型以及与整联蛋白 α2 的所有 9 个潜在糖基化位点相对应的糖肽型。比较了 WT 和 JAK2V617F 表型的糖肽型的两种整联蛋白。观察到的总体趋势是 PMF MK 中的 JAK2V617F 突变导致 β1 糖基化发生变化；在大多数情况下，它会导致糖肽状整合面积的增加。我们还观察到，在突变的 MK 中，整合素 α2 糖基化的变化比整合素 β1 糖基化的变化更大，这一发现表明整合素 α2 糖基化的改变也可能影响激活。此外，与整合素 α2 和 β1 共免疫沉淀的细胞骨架相关蛋白的鉴定证明了本研究中使用的方法的潜力，可以在肽水平上深入了解 MK 中整合素激活的后果。我们发现的广泛而详细的糖基化模式为未来与 JAK2V617F 突变细胞相比对照细胞中每个位点的功能研究提供了基础。数据可通过 ProteomeXchange 获得，标识符为 PXD030550。 β1、α2 整合素从 WT 和 JAK2V617F 小鼠巨核细胞的骨髓中分离出来。糖肽形式的特征在于整合素 β1 上 12 个潜在 N 连接位点中的 11 个。糖肽形式的特征在于整合素 α2 上所有九个潜在的 N 连接位点。 JAK2V617F 巨核细胞的两种蛋白在多个位点都有糖基化变化。从 WT 和骨髓纤维化 (JAK2V617F) 小鼠巨核细胞的骨髓中分离出的整合素 β1 和 α2 的多个位点记录了糖基化的变化。整合素 β1 的 12 个潜在 N-糖基化位点中的 11 个和整合素 α2 上的所有 9 个潜在糖基化位点的糖肽型均得到了表征。
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 modification via changes in glycosylation. Samples were derived from immunoprecipitation of MKs obtained from Vav1-hJAK2V617F and WT 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. Data are available via ProteomeXchange with identifier PXD030550. β1, α2 Integrins are isolated from bone marrow of WT and JAK2V617F mouse megakaryocytes. Glycopeptiforms are characterized at 11 of 12 potential N-linked sites on integrin β1. Glycopeptiforms are characterized at all nine potential N-linked sites on integrin α2. JAK2V617F megakaryocytes had glycosylation changes in both proteins at multiple sites. Changes in glycosylation were documented at multiple sites in integrins β1 and α2 isolated from bone marrow of WT and myelofibrotic (JAK2V617F) mouse megakaryocytes. Glycopeptiforms at 11 out of the 12 potential N-glycosylation sites of integrin β1 and at all nine potential glycosylation sites on integrin α2 were characterized.
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影响因子：
4.8
作者：
Eliades, Alexia;Papadantonakis, Nikolaos;Ravid, Katya
通讯作者：
Ravid, Katya
影响因子：
3.5
作者：
Jacobs PP;Sackstein R
通讯作者：
Sackstein R
影响因子：
64.8
作者：
Jumper J;Evans R;Pritzel A;Green T;Figurnov M;Ronneberger O;Tunyasuvunakool K;Bates R;Žídek A;Potapenko A;Bridgland A;Meyer C;Kohl SAA;Ballard AJ;Cowie A;Romera-Paredes B;Nikolov S;Jain R;Adler J;Back T;Petersen S;Reiman D;Clancy E;Zielinski M;Steinegger M;Pacholska M;Berghammer T;Bodenstein S;Silver D;Vinyals O;Senior AW;Kavukcuoglu K;Kohli P;Hassabis D
通讯作者：
Hassabis D
影响因子：
14.9
作者：
Deutsch EW;Csordas A;Sun Z;Jarnuczak A;Perez-Riverol Y;Ternent T;Campbell DS;Bernal-Llinares M;Okuda S;Kawano S;Moritz RL;Carver JJ;Wang M;Ishihama Y;Bandeira N;Hermjakob H;Vizcaíno JA
通讯作者：
Vizcaíno JA
影响因子：
4.4
作者：
Danzer, Carsten;Eckhardt, Katrin;Krek, Wilhelm
通讯作者：
Krek, Wilhelm