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Analysis of expression and function of myosins in hematological cells

血液细胞中肌球蛋白的表达和功能分析

基本信息

批准号：
12671004
负责人：
HIGASHIHARA Masaaki
金额：
$ 2.43万
依托单位：
Kitasato University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2000
资助国家：
日本
起止时间：
2000 至 2001
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-12671004/
关键词：
myosin myosin heavy chain myosin light chain isoform cloning platelet lymphocyte unconventional myosin 血液細胞リン酸化

项目摘要

Our projects provided three conclusions:1) Human platelets express mRNAs which encode MHC-A exclusively, further suggesting that MHC-A is endogenously synthesed in peripheral blood platelets as well as bone marrow megakarypcytes.2) In human lymphocytes, MHC-A is expressed avandantely as compared with unconventional myosin. Further we found that MHC-A had two isoforms; 6.0kbp and 7.5kbp. RT-PCR and northern turning showed several unconventional myosins such as I-c, brush border-I, I-β, V, VIIA, IXA, IXB, XV to less extent in lymphocytes.3) The 20 kDa regulatory myosin light chain (MLC-2) plays an important role in regulating contractile activity in both nonmuscle and smooth muscles. Since MLC genes of hematopoietic cells have yet to be characterized, we cloned the full-length cDNAs of MLC-2 and 17 kDa essential myosin light chain (MLC-3) from Meg-01, a human megakaryoblastic leukemia cell line. Both MLC-2 and MLC-3 gene are transcribed ubiquitously in various hematopoietic cells. The MLC-2 open reading frame of 516 nucleotides encoding a protein of 172 residues was detected in cloned cDNA of 967 nucleotides. The Ca2+- binding domain and five phosphorylation sites were highly conserved. The derived amino acid sequence has a 97.1%, 95.9% and 92.4% homology with that of rat aortic smooth muscle, Xenopus oocytes, and chicken gizzard smooth muscle, respectively. The MLC-3 open reading frame of 453 nucleotides encoding a protein of 151 residues was detected in cloned cDNA of 742 nucleotide. The MLC-3 protein is 99.3% identical to that of human fibroblasts. These results suggest that hematopoietic myosin light chain proteins are identical to those of other nonmuscle cells and smooth muscle, thus differing from skeletal and cardiac muscles. MLCs of smooth muscle and nonmuscle cells, including hematopoietic cells, are probably encoded by the same gene.

我们的项目得出了三个结论：1)人血小板表达MHC-A的mRNAs，这进一步表明MHC-A在外周血血小板和骨髓巨核细胞中内源性合成；2)在人的淋巴细胞中，MHC-A的表达高于非常规肌球蛋白。进一步我们发现MHC-A有两种亚型：6.0kbp和7.5kbp。RT-PCRand Northern Turning显示非常规肌球蛋白I-C、Brush Edge-I、I-β、V、VIIA、IXA、IXB、XV在淋巴细胞中的表达程度较低。3)20 kDa调节性肌球蛋白轻链在调节非肌肉和平滑肌的收缩活动中起重要作用。由于造血细胞的MLC基因尚未确定，我们从人巨核白血病细胞系Meg-01中克隆了MLC-2和17 kDa基本肌球蛋白轻链(MLC-3)的全长cDNA。MLC-2和MLC-3基因在多种造血细胞中广泛转录。在克隆的967个核苷酸的cDNA中检测到516个核苷酸的MLC-2开放阅读框，编码172个残基的蛋白质。钙结合区和5个磷酸化位点高度保守。该氨基酸序列与大鼠主动脉平滑肌、非洲爪哇卵母细胞和鸡肌肌的同源性分别为97.1%、95.9%和92.4%。在克隆的742个核苷酸的cDNA中检测到453个核苷酸的MLC-3开放阅读框，编码151个残基的蛋白质。MLC-3蛋白与人成纤维细胞的同源性为99.3%。这些结果表明，造血肌球蛋白轻链蛋白与其他非肌肉细胞和平滑肌的轻链蛋白相同，因此不同于骨骼肌和心肌。包括造血细胞在内的平滑肌和非肌肉细胞的MLCs可能由相同的基因编码。