Molecular cloning and functional expression of a cDNA encoding glycosylation-inhibiting factor.
Molecular cloning and functional expression of a cDNA encoding glycosylation-inhibiting factor.
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DOI：
10.1073/pnas.90.21.10056
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发表时间：
1993-11
影响因子：
11.1
通讯作者：
T. Mikayama;T. Nakano;H. Gomi;Yukimitsu Nakagawa;Yun-Cai Liu;M. Sato;A. Iwamatsu;Y. Ishii;W. Weiser;K. Ishizaka
中科院分区：
文献类型：
--
作者：
T. Mikayama;T. Nakano;H. Gomi;Yukimitsu Nakagawa;Yun-Cai Liu;M. Sato;A. Iwamatsu;Y. Ishii;W. Weiser;K. Ishizaka
通过使用基于来自小鼠T细胞杂交瘤的糖基化抑制因子(GIF)的部分氨基酸序列的探针，分离了编码小鼠GIF的全长cDNA。通过使用小鼠GIF cDNA作为探针，从产生GIF的人T细胞杂交瘤的cDNA文库中分离编码人GIF的cDNA克隆。 cDNA 编码一个由 115 个氨基酸组成的推定 12.5-kDa 肽。 Northern 印迹分析显示了一个 0.6 kb 的转录本。针对大肠杆菌衍生的重组 13-kDa 肽结合杂交瘤衍生的 GIF 的多克隆兔抗体。尽管该肽不含信号肽序列，但将cDNA转染至COS-1细胞中导致13-kDa肽的分泌，但该肽的生物活性明显低于杂交瘤来源的GIF。然而，编码由降钙素前体的N端前区和人GIF组成的融合蛋白的嵌合cDNA的表达以及与弗林蛋白酶cDNA的共转染以允许融合蛋白的细胞内裂解导致13-kDa肽的分泌，其生物活性与杂交瘤来源的GIF相当。转染的 COS-1 上清液中的 13-kDa 肽和 GIF 生物活性均与针对杂交瘤来源的人 GIF 的单克隆抗体结合。这些结果表明 13-kDa 肽代表重组 GIF，但肽的翻译后修饰对于生物活性的产生很重要。 GIF cDNA与编码巨噬细胞迁移抑制因子的cDNA具有高度同源性。但重组GIF未能抑制人单核细胞迁移，且重组人巨噬细胞迁移抑制因子不具有GIF生物活性。
By using probes based on partial amino acid sequence of glycosylation-inhibiting factor (GIF) from a mouse T-cell hybridoma, a full-length cDNA encoding mouse GIF was isolated. A cDNA clone encoding human GIF was isolated from cDNA libraries of a GIF-producing human T-cell hybridoma by using mouse GIF cDNA as a probe. The cDNAs encode a putative 12.5-kDa peptide of 115 amino acids. Northern blot analysis demonstrated a single, 0.6-kb transcript. Polyclonal rabbit antibodies against the Escherichia coli-derived recombinant 13-kDa peptide bound hybridoma-derived GIF. Although the peptide did not contain a signal peptide sequence, transfection of the cDNA into COS-1 cells resulted in secretion of 13-kDa peptide, but the peptide had substantially less bioactivity than the hybridoma-derived GIF. However, expression of a chimeric cDNA encoding a fusion protein consisting of the N-terminal pro region of calcitonin precursor and human GIF and cotransfection with furin cDNA to allow intracellular cleavage of the fusion protein resulted in secretion of 13-kDa peptide that was comparable to hybridoma-derived GIF in its bioactivity. Both the 13-kDa peptide and GIF bioactivity in the transfected COS-1 supernatant bound to a monoclonal antibody against hybridoma-derived human GIF. These results indicate that the 13-kDa peptide represents recombinant GIF, but posttranslational modification of the peptide is important for generation of the bioactivity. The GIF cDNA had high homology with the cDNA encoding macrophage migration inhibitory factor. However, the recombinant GIF failed to inhibit migration of human monocytes, and recombinant human macrophage migration inhibitory factor did not have GIF bioactivity.