Deciphering the biophysical basis by which Glycosaminoglycans CONtrol growth factor signaling during development: a biomimetic approach (GlyCON)

近年来，越来越多的证据表明，组织中生长因子（GF）的活性和生物利用度是由细胞外基质决定的，特别是由蛋白聚糖的硫酸乙酰肝素（HS）链决定的，其与GF的不同碱性结构域相互作用。然而，HS结构（N-和O-硫酸化的序列）和GF的结合位点（碱性氨基酸的序列）如何影响分子水平上的相互作用在很大程度上是未知的。此外，硫酸软骨素（CS），一种与HS结构相似的糖胺聚糖（GAG）也与许多GF结合，尽管亲和力降低。该财团获得的初步证据表明，CS水平增加的小鼠突变体的软骨细胞与缺陷的HS结构指向CS的补偿功能，至少在软骨细胞。然而，尚未系统地研究组织中GAG的组成如何影响GF活性。通过GlyCON，我们建立了一个跨学科的项目，将材料科学和发育生物学联系起来。三个国际团队紧密合作，以获得对HS和CS控制活动，演示和分发GF的机制的基本见解。基于合作伙伴的科学专业知识，我们将使用胚胎内软骨骨化过程作为模型，研究决定HS和CS与骨形态发生蛋白（BMP）和印度刺猬（Ihh）相互作用的分子机制。在我们的多学科方法中，我们将破译GAG结构如何控制这些GF的生物活性以及GAG组成的改变如何影响发育过程，组织稳态和HS相关疾病的基本原理，如体内多发性骨软骨瘤（MO）。具体来说，我们将创建一个具有不同硫酸化模式的HS寡糖库，并将其在仿生平台上进行模拟。这些平台将被开发用于HS-BMP和HS-Ihh相互作用的结合亲和力和化学计量的生物物理学研究，以及HS结构如何决定GF对共培养细胞的生物活性的后续生物学研究。HS结构的变化对组织稳态的影响将通过对HS合成酶中携带特定缺陷的小鼠突变体的体内分析来研究。在第二部分中，我们将分析GF与CS的生物物理相互作用，并确定两种GAG的相对组成如何影响它们的生物学特性。（HS和CS）影响GF的生物活性，在体外使用在仿生平台上培养的细胞和在体内在小鼠突变体的原代细胞中进行。我们的最终目的是在GAG-GF相互作用的分子基础上产生详细的知识，从而为将来研究其他GF铺平道路，组织和罕见的GAG相关疾病，并最终用于新的治疗方法。

During recent years, increasing evidence has been accumulated that the activity and bioavailability of growth factors (GFs) in the tissue is determined by the extracellular matrix, in particular by the heparan sulfate (HS) chains of proteoglycans, which interact with distinct basic domains of the GFs. Nevertheless, it is largely unknown how the HS structure (the sequence of N- and O- sulfation) and the binding sites of the GFs (the sequence of basic amino acids) affect the interaction at the molecular level. Moreover, chondroitin sulfate (CS), a glycosaminoglycan (GAG) with structural similarities to that of HS also binds to many GFs, although with reduced affinity. Preliminary evidence gained by the consortium indicates that CS levels are increased in chondrocytes of mouse mutants with a defective HS structure pointing to a compensatory function of CS at least in chondrocytes. How the composition of GAGs in a tissue affects GF activity has however not been systematically investigated. With GlyCON, we have established a multidisciplinary project bridging material science and developmental biology. Three international teams interact in a tight collaboration to gain basic insight into the mechanism by which HS and CS control the activity, presentation and distribution GFs. Based on the scientific expertise of the partners, we will use the process of embryonic endochondral ossification as a model to investigate the molecular mechanisms that determine the interaction of HS and CS with bone morphogenetic proteins (BMP) and Indian hedgehog (Ihh). In our multi-disciplinary approach, we will decipher basic principles of how the GAG structure controls the bioactivity of these GFs and how alterations in the GAG composition affect developmental processes, tissue homeostasis and HS related diseases, like multiple osteochondromas (MO) in vivo.Specifically, we will create a library of HS oligosaccharides exhibiting distinct sulfation patterns and immobilize these on biomimetic platforms. These platforms will be developed for biophysical studies of the binding affinity and stoichiometry of the HS-BMP and HS-Ihh interactions and for subsequent biological investigation of how the HS structure determines the bioactivity of the GF towards co-cultured cells. The impact of changes in the HS structure on tissue homeostasis will be investigated by the in vivo analyses of mouse mutants carrying specific defects in HS synthesizing enzymes. In the second part, we will anlayze the biophysical interaction of GF with CS and determine how the relative composition of both GAGs (HS and CS) affects the bioactivity of GFs in vitro using cells cultured on biomimetic platforms and in vivo in primary cells of mouse mutants.Our final aim is to generate detailed knowledge on the molecular basis of the GAG–GF interaction, thereby paving the way for future studies of other GF, tissues and rare GAGs associated diseases and ultimately for new therapeutic approaches.