Exploring Oligosaccharide Synthesis in Human Mammary Gland Epithelial Cells

 描述（由申请方提供）：人乳中的利戈替尼（HMO）是人乳中含量第三高的成分。虽然越来越多的证据表明它们对母乳喂养的婴儿有益，但人们对HMO如何在母亲的乳腺中合成知之甚少。到目前为止，阐明HMO生物合成的关键障碍之一是缺乏合成类似于人乳中发现的寡糖的合适模型。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。我们提出了一个模型的基础上，乳腺上皮细胞来源于从人乳中分离的祖细胞。暴露于催乳素后，这些细胞合成并分泌含有HMO所有结构特征的寡糖，包括聚乳糖胺延长和分支以及已知在HMO中发生的所有键中的岩藻糖基化和唾液酸化。结果具有高度重现性。细胞培养上清液和相应乳汁样品中的寡糖组成匹配并反映不同乳汁供体之间的人际差异。细胞培养基中的寡糖组成响应于用糖基转移酶特异性小干扰RNA进行的细胞操作而变化。迄今为止，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。我们的申请建议使用这种新的强大的工具，以确定哪些糖基化相关基因的差异表达时，乳腺上皮细胞暴露于催乳素（具体目标1），并确定哪些酶有助于HMO延伸和分支，岩藻糖基化和唾液酸化（具体目标2）。拟议的实验和预期的结果具有很大的潜力，显着推进我们的知识和理解如何在人类乳腺合成HMO。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促和化学酶促HMO合成。此外，从拟议的研究中获得的知识可能为生物工程基于细胞的系统奠定基础，该系统包含合成所需的糖基化机制，而不是一种，而是多种不同寡糖的混合物，类似于母乳中天然存在的寡糖的复杂组成。在这些体外和体内系统中产生的HMO可用于体外试验和动物模型的研究，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的益处。

 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.