Novel Full-length CDNAs Differentially Expressed During

新型全长 cDNA 在过程中差异表达

• 批准号: 7151522
• 负责人: GRIFFIN P. RODGERS
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF DIABETES AND DIGESTIVE AND KIDNEY DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7151522
• 关键词: cadherins; cell adhesion molecules; cell differentiation; cell population study; colony stimulating factor; complementary DNA; differential display technique; erythropoietin; fluorescent in situ hybridization; gene expression; genetic mapping; glycosylation; hematopoiesis; hematopoietic stem cells; human tissue; in situ hybridization; laboratory mouse; lectin; messenger RNA; myeloid stem cell; protein protein interaction; thrombopoietic factor; tissue /cell culture; western blottings

We have cloned a novel hematopoietic granulocyte colony-stimulating factor (G-CSF)-induced olfactomedin-related glycoprotein, termed hGC-1 (human G-CSF-stimulated clone-1). mRNA differential display was used in conjunction with a modified two-phase liquid culture system. Cultures were enriched for early precursors of erythroid, myeloid, and megakaryocytic lineages, which were isolated after induction with erythropoietin, G-CSF, and thrombopoietin, respectively. RNA from the enriched cells was subjected to differential display analysis to identify lineage-specific expressed genes. Human GC-1 (hGC-1) is a novel olfactomedin-like glycoprotein that is expressed during granulocytic differentiation. To further characterize the function of GC-1, we have cloned the cDNA for the mouse homologue of human GC-1 and investigated the pattern of GC-1 gene expression in the mouse during embryonic development and in adult tissues. Genomic and cDNA clones corresponding to mouse GC-1 (mGC-1) were isolated. The mGC-1 protein shares 93% homology with hGC-1 at the amino acid level. The protein sequence of mGC-1 indicates that it belongs to the olfactomedin-related glycoprotein family, which includes olfactomedin, TIGR, NOELIN-2 and latrophilin-1. Fluorescence in situ hybridization (FISH) was used to map the mGC-1 gene locus to chromosome 14D3. Like other olfactomedin-like genes with tissue-restricted patterns of expression, mGC-1 is expressed at high levels in the small intestine and kidney, at moderate levels in the stomach, thymus, spleen, and no expression was detected in brain, heart, liver or lung. These results are consistent with the distribution of hGC-1 in human tissues. Analysis of mGC-1 expression by in situ hybridization in mouse embryos showed that mGC-1 is not expressed until day E15. mGC-1 expression was detected in the small intestine and stomach. Interestingly, a very clear demarcation of the mGC-1 signal was evident in the intestine, where mGC-1 was specifically expressed between the enterocytes lining the villi, but not in the lamina propria or in the muscularis layers. There was a similar, albeit weaker, pattern of mGC-1 expression in stomach. In situ hybridization showed mGC-1 is strongly expressed in the crypts of small intestine. hGC-1 cDNA has been transfected into 293 cells transiently and stably. In transfected 293 cells, hGC-1 was detected in the culture medium after 48h of transfection. In the Western blot, hGC-1 showed a multimer form in non-reducing gel, which was reduced to monomer after adding 10mM DTT. hGC-1 was also detected in the perinuclear region and cell surface.These data suggest hGC-1 is a secreted glycoprotein. This is consistent with tissue expression result that hGC-1 is only abundantly expressed in tissues which has secretion capability. On further analysis, we find high level expression of mGC-1 in pro-B and pre-B cells and low-level expression in mature B and T-cells that co-localizes in this region of the small intestine. When the myeloid progenitor 32D cell line was exposed to G-CSF for 7 days, mGC-1 expression was induced. Taken together, these results suggest that mGC-1 play an important role in granulocytic differentiation, and quite likely in mucousal immunity. To gain further insight into the potential pathways involved in GC-1 effects we performed further analysis in the 293 cell line. In situ hybridization showed mGC-1 is strongly expressed in the crypts of small intestine. hGC-1 cDNA has been transfected into 293 cells transiently and stably. In transfected 293 cells, hGC-1 was detected in the culture medium after 48h of transfection. In the Western blot, hGC-1 showed a multimer form in non-reducing gel, which was reduced to monomer after adding 10mM DTT. hGC-1 was also detected in the perinuclear region and cell surface.These data suggest hGC-1 is a secreted glycoprotein. This is consistent with tissue expression result that hGC-1 is only abuuundantly expressed in tissues which has secretion capability. Based on the data that hGC-1 is a glycoprotein and associated with cell adhesion, experiments have been performed to screen some lectins and cell adhesion proteins which might interact with hGC-1. Agarose bound lectin pull down assay shows hGC-1 strongly bound RCAI, weakly with ConA and WGA and no binding was detected with DSA, PNA and SNA. The purified hGC-1 enhances NIH3T3 and 293T/17 cell spread and attachment. hGC-1 enriched cell culture supernatants of 293T/17 cells transfected with hGC-1 expression vector also enhance cell spread and cell attachment. Co-immunoprecipitation demonstrated a association of hGC-1 with cadherin in 293 cells transiently transfected with hGC-1 cDNA. The interaction of hGC-1 with cadherin depends on the C-terminal olfactomedin domain, but does not require the five well conserved cysteine residues in hGC-1. However, cysteine residue at 83, 85, 246 and 437 is essential for hGC-1 secretion and cysteine 226 is critical for hGC-1 multimer formation. The potential association of hGC-1 with cadherin might explain the involvement of hGC-1 with cell adhesion. E-cadherin in particular, serves as a widely acting suppressor of invasion and growth of epithelial cancers, and its functional elimination represents a key step in the acquisition of the invasive phenotype for many tumors. Further analysis of the interaction of hGC-1 and the cadherin family of adhesion proteins may help clarify the role of this putative tumor suppessor gene.

我们克隆了一种新的造血粒细胞集落刺激因子（G-CSF）诱导的嗅素相关糖蛋白，命名为hGC-1（人G-CSF刺激克隆-1）。mRNA差异显示与改进的两相液体培养系统结合使用。培养丰富红细胞、髓细胞和巨核细胞谱系的早期前体，分别在促红细胞生成素、G-CSF和血小板生成素诱导后分离。来自富集细胞的RNA进行差异显示分析，以鉴定谱系特异性表达基因。人GC-1 （hGC-1）是一种在粒细胞分化过程中表达的新型嗅糖蛋白样糖蛋白。为了进一步表征GC-1的功能，我们克隆了人类GC-1的小鼠同源基因cDNA，并研究了GC-1基因在小鼠胚胎发育和成年组织中的表达模式。分离出小鼠GC-1 （mGC-1）的基因组克隆和cDNA克隆。mGC-1蛋白在氨基酸水平上与hGC-1具有93%的同源性。mGC-1的蛋白序列表明其属于olfactomedin相关糖蛋白家族，该家族包括olfactomedin、TIGR、NOELIN-2和latrophilin-1。采用荧光原位杂交（FISH）技术将mGC-1基因定位到染色体14D3上。与其他具有组织限制性表达模式的olfactomedin样基因一样，mGC-1在小肠和肾脏中高水平表达，在胃、胸腺、脾脏中中等水平表达，在脑、心、肝和肺中未检测到表达。这些结果与hGC-1在人体组织中的分布一致。通过原位杂交分析小鼠胚胎中mGC-1的表达，发现mGC-1直到E15天才表达。在小肠和胃中检测到mGC-1表达。有趣的是，在肠道中，mGC-1信号的界限非常明显，其中mGC-1在绒毛衬里的肠细胞之间特异性表达，而不在固有层或肌层中表达。胃中也有类似的mGC-1表达模式，尽管较弱。原位杂交显示mGC-1在小肠隐窝中强烈表达。hGC-1 cDNA瞬时稳定转染293细胞。转染293细胞48h后，培养液中检测到hGC-1。Western blot结果显示，hGC-1在非还原性凝胶中呈多聚体形态，加入10mM DTT后还原为单体。在核周区和细胞表面也检测到hGC-1。这些数据表明hGC-1是一种分泌糖蛋白。这与hGC-1仅在具有分泌能力的组织中大量表达的组织表达结果一致。通过进一步分析，我们发现mGC-1在前B细胞和前B细胞中高水平表达，而在小肠这一区域共定位的成熟B细胞和t细胞中低水平表达。当髓系祖细胞32D暴露于G-CSF 7 d时，诱导mGC-1表达。综上所述，这些结果表明mGC-1在粒细胞分化中起重要作用，并且很可能在粘膜免疫中起重要作用。为了进一步了解GC-1作用的潜在途径，我们在293细胞系中进行了进一步的分析。原位杂交显示mGC-1在小肠隐窝中强烈表达。hGC-1 cDNA瞬时稳定转染293细胞。转染293细胞48h后，培养液中检测到hGC-1。Western blot结果显示，hGC-1在非还原性凝胶中呈多聚体形态，加入10mM DTT后还原为单体。在核周区和细胞表面也检测到hGC-1。这些数据表明hGC-1是一种分泌糖蛋白。这与hGC-1仅在具有分泌能力的组织中大量表达的组织表达结果一致。基于hGC-1是一种与细胞粘附相关的糖蛋白，我们进行了筛选可能与hGC-1相互作用的凝集素和细胞粘附蛋白的实验。琼脂糖结合凝集素拉下实验显示，hGC-1与RCAI结合较强，与ConA和WGA结合较弱，与DSA、PNA和SNA无结合。纯化后的hGC-1增强了NIH3T3和293T/17细胞的扩散和附着。转染hGC-1表达载体的293T/17细胞培养上清液中富集hGC-1后，细胞扩散和细胞附着增强。在瞬时转染hGC-1 cDNA的293细胞中，共免疫沉淀证实了hGC-1与钙粘蛋白的关联。hGC-1与钙粘蛋白的相互作用依赖于c端olfactomedin结构域，而不需要hGC-1中5个保守的半胱氨酸残基。然而，83、85、246和437处的半胱氨酸残基对hGC-1的分泌至关重要，而226处的半胱氨酸残基对hGC-1多聚体的形成至关重要。hGC-1与钙粘蛋白的潜在关联可能解释了hGC-1参与细胞粘附的原因。特别是e -钙粘蛋白，作为一种广泛作用的上皮性癌症侵袭和生长抑制因子，其功能消除是许多肿瘤获得侵袭性表型的关键步骤。进一步分析hGC-1与粘附蛋白钙粘蛋白家族的相互作用可能有助于阐明这一假定的肿瘤抑制基因的作用。