Novel Full-length CDNAs Differentially Expressed During

我们克隆了一种新的造血粒细胞集落刺激因子(G-CSF)诱导的嗅球蛋白相关糖蛋白，命名为HGC-1(人G-CSF刺激的克隆-1)。结合改良的两相液体培养系统，采用mRNA差异显示技术。分别用促红细胞生成素、粒细胞集落刺激因子和血小板生成素诱导分离红系、髓系和巨核系的早期前体细胞。从浓缩的细胞中提取的RNA进行差异显示分析，以确定谱系特异性表达基因。人GC-1(HGC-1)是一种新的嗅觉调节素样糖蛋白，在粒细胞分化过程中表达。为了进一步研究GC-1的功能，我们克隆了人GC-1的小鼠同源物的cDNA，并研究了GC-1基因在小鼠胚胎发育过程中和在成年组织中的表达模式。获得了与小鼠Gc-1(MGC-1)相应的基因组和cDNA克隆。MGC-1蛋白与HGC-1蛋白在氨基酸水平上有93%的同源性。对MGC-1的蛋白质序列分析表明，MGC-1属于与嗅觉相关的糖蛋白家族，包括嗅觉相关糖蛋白、TIGR、NOELIN-2和LatroPhilin-1。应用荧光原位杂交技术将MGC-1基因定位于染色体14D3。像其他具有组织限制性表达模式的类似分子的基因一样，MGC-1在小肠和肾脏中高水平表达，在胃、胸腺、脾中中等水平表达，在脑、心脏、肝脏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MGC-1在小鼠胚胎中的表达，发现MGC-1直到E15天才有表达。MGC-1在小肠和胃中均有表达。有趣的是，MGC-1信号在肠道中的分界非常明显，MGC-1特异性地表达于绒毛衬里的肠细胞之间，而不是在固有层或肌层中。MGC-1在胃中也有类似的表达模式，尽管表达较弱。原位杂交显示MGC-1在小肠隐窝表达较强。HGC-1基因已瞬时稳定地转入293细胞。在转染的293细胞中，48h后在培养上清液中检测到HGC-1。在Western印迹中，HGC-1在非还原凝胶中以多聚体形式存在，加入10 mM DTT后，HGC-1被还原为单体。HGC-1在核周和细胞表面也有表达，提示HGC-1是一种分泌型糖蛋白。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。进一步分析，我们发现MGC-1在Pro-B和Pre-B细胞中高水平表达，在成熟B细胞和T细胞中低水平表达，共同定位于小肠的这一区域。粒细胞集落刺激因子作用于髓系祖细胞32D细胞7d后，可诱导MGC-1的表达。综上所述，这些结果提示MGC-1在粒细胞分化中起重要作用，很可能在粘膜免疫中起重要作用。为了进一步了解涉及GC-1效应的潜在途径，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在小肠隐窝表达较强。HGC-1基因已瞬时稳定地转入293细胞。在转染的293细胞中，48h后在培养上清液中检测到HGC-1。在Western印迹中，HGC-1在非还原凝胶中以多聚体形式存在，加入10 mM DTT后，HGC-1被还原为单体。HGC-1在核周和细胞表面也有表达，提示HGC-1是一种分泌型糖蛋白。这与HGC-1仅在具有分泌能力的组织中广泛表达的组织表达结果一致。根据HGC-1是一种糖蛋白并与细胞黏附相关的数据，已有实验筛选出一些可能与HGC-1相互作用的凝集素和细胞黏附蛋白。琼脂糖凝集素下拉实验显示，HGC-1与RCA1强结合，与ConA、WGA弱结合，与DSA、PNA、SNA无结合。纯化的HGC-1可增强NIH3T3和293T/17细胞的铺展和贴壁。转染HGC-1表达载体的293T/17细胞培养上清液中HGC-1含量增加，细胞铺展和贴壁能力增强。免疫共沉淀法显示瞬时转染HGC-1基因的293细胞中HGC-1与钙粘附素相关。HGC-1与钙粘蛋白的相互作用依赖于C端的分子内切结构域，但不需要HGC-1中5个保守的半胱氨酸残基。然而，83、85、246和437位的半胱氨酸残基是HGC-1分泌所必需的，而半胱氨酸226是形成HGC-1多聚体的关键。HGC-1与钙粘附素的潜在联系可能解释了HGC-1参与细胞黏附的原因。尤其是E-钙粘蛋白是上皮性癌症侵袭和生长的广泛抑制因子，其功能的消除是许多肿瘤获得侵袭性表型的关键一步。进一步分析HGC-1和钙粘附素家族黏附蛋白家族的相互作用可能有助于阐明这个假定的肿瘤支持基因的作用。

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.