Exploring Oligosaccharide Synthesis in Human Mammary Gland Epithelial Cells

 描述(申请人提供)：人类乳木糖(HMO)是人类乳汁中含量第三丰富的成分。虽然越来越多的证据表明，它们对母乳喂养的婴儿有利，但人们对HMO如何在母亲的乳房腺体中合成知之甚少。到目前为止，阐明HMO生物合成的关键障碍之一是缺乏合适的模型来合成类似于人类母乳中发现的低聚糖。我们现在提供的初步结果使我们处于克服这一关键障碍的独特地位。我们提出了一个基于乳腺上皮细胞的模型，乳腺上皮细胞来自于从人类乳汁中分离出来的祖细胞。当暴露于催乳素时，这些细胞合成和分泌包含HMO的所有结构特征的寡糖，包括聚乳糖胺的伸长和分支以及HMO中已知的所有连接中的岩藻糖化和唾液酸化。结果具有高度的重复性。细胞培养上清液和相应牛奶样本中的寡糖组成相匹配，并反映了不同母乳捐赠者之间的人际差异。细胞培养上清液中低聚糖的组成变化是通过糖基转移酶特异的小干扰RNA来响应细胞的操纵。到目前为止，这是第一个也是目前唯一一个可用于探索HMO生物合成的模型。我们的应用建议使用这个新的强大的工具来确定当乳腺上皮细胞暴露于催乳素时哪些糖化相关基因有差异表达(特定目标1)，并确定哪些酶有助于HMO的延伸和分支、岩藻糖化和唾液酸化(特定目标2)。拟议的实验和预期的结果具有很高的潜力，可以显著提高我们对HMO如何在人类乳腺中合成的认识和理解。更好地了解HMO在自然界中是如何合成的，将有助于促进体外酶和化学-酶合成HMO。此外，从拟议的研究中获得的知识可能会为生物工程一个基于细胞的系统奠定基础，该系统包含合成低聚糖所需的糖基化机制，而不是一种，而是一种不同的低聚糖的混合物，这些低聚糖类似于天然存在于母乳中的低聚糖的复杂组成。在这些体外和体内系统中产生的HMO可用于体外分析和动物模型的研究，用于临床干预研究，并最终用于补充配方奶粉，为所有婴儿提供HMO的好处。

 DESCRIPTION (provided by applicant): Human Milk ligosaccharides (HMO) are the third most abundant component of human milk. While accumulating evidence suggests that they benefit the breast-fed infant, little is known about how HMO are synthesized in the mother's mammy gland. Up until now, one of the critical barriers in elucidating HMO biosynthesis has been the lack of suitable models that synthesize oligosaccharides that resemble those found in human milk. We now provide preliminary results that place us in a unique position to overcome this critical barrier. We present a model based on mammary gland epithelial cells that were derived from progenitor cells isolated from human milk. Upon exposure to prolactin, these cells synthesize and secrete oligosaccharides that contain all structural features of HMO, including polylactosamine elongation and branching as well as fucosylation and sialylation in all linkages known to occur in HMO. Results are highly reproducible. Oligosaccharide composition in the cell culture supernatant and in the respective milk sample match and reflect interpersonal variations between different milk donors. Oligosaccharide composition in the cell culture media changes in response to cell manipulation with glycosyltransferase-specific small interfering RNA. To date, this is the first and currently only model available to explore HMO biosynthesis. Our application proposes to use this new powerful tool to determine which glycosylation-related genes are differentially expressed when mammary gland epithelial cells are exposed to prolactin (Specific Aim 1) and to identify which enzymes contribute to HMO elongation and branching, fucosylation and sialylation (Specific Aim 2). The proposed experiments and anticipated results have a high potential to significantly advance our knowledge and understanding on how HMO are synthesized in the human mammary gland. A better understanding of how HMO are synthesized in nature will help facilitate in vitro enzymatic and chemo-enzymatic HMO synthesis. In addition, the knowledge gained from the proposed studies may lay the groundwork to bioengineer a cell-based system that contains the glycosylation machinery required to synthesize not one, but a mix of a variety of different oligosaccharides that resemble the complex compositions of oligosaccharides naturally occurring in human milk. HMO produced in these in vitro and in vivo systems can be used for research in in vitro assays and animal models, for clinical intervention studies, and eventually to supplement formula to provide all infants with the benefits of HMO.