Protein-lipid interactions and the influence of cellular lipid environments on glycosylation processes

在受体分子中加入碳水化合物残基是一种普遍存在的蛋白质修饰，在生命的所有领域都是保守的。糖基化事件的范围从单糖的添加到高度复杂的碳水化合物链连接到蛋白质和脂质受体。不同长度、组成和排列的糖链的大量可能组合导致了人类糖链的无与伦比的复杂性，这是由大约200种不同底物的糖基转移酶带来的。这种巨大的结构多样性反映在可受糖基化影响的广泛功能上，包括蛋白质折叠、运输/定位和蛋白质活性调节。糖基化缺陷，如发生在先天性糖基化障碍（CDGs）中的糖基化缺陷，预计也会对膜结构域的脂质组成产生直接影响，例如，通过扰乱参与脂质合成和信号传导的酶的定位。除了直接影响脂质合成外，异常糖基化还可以间接影响脂质和脂质信号传导。在这两种情况下，脂质稳态的改变可能会导致这些多系统疾病的病理生理。在这里，我们的目的是研究脂质在调节糖基化酶的组装，活性和特异性中的作用。我们将重点关注磷酸甘露糖合成酶，这是n -糖基化以及C-和o -甘露糖基化的关键酶。使用体外和细胞方法，我们将研究膜脂对酶的结构和功能的贡献。此外，我们将与研究小组的其他成员合作，使用不同的真核模型系统，从人类患者成纤维细胞到小鼠和鱼类模型，研究cgd是否以及如何影响细胞内脂质稳态。采用定量纳米质谱法，我们将研究CDG中脂质途径的改变，以确定低糖基化对细胞脂质稳态的影响。综上所述，我们的目的是阐明脂质和糖基化酶之间的相互作用，并确定糖基化缺陷如何影响细胞和机体脂质稳态。

The addition of carbohydrate residues to acceptor molecules is a ubiquitous modification of proteins conserved across all domains of life. Glycosylation events range from the addition of monosaccharides to the attachment of highly complex carbohydrate chains to protein and lipid acceptors. The high number of possible combinations of different lengths, compositions and arrangements of the glycan chains results in an unmatched complexity of the human glycome, which is brought about by ~200 glycosyltransferases with different substrates. This enormous structural diversity is reflected in the broad range of functions that can be affected by glycosylation, including protein folding, trafficking/localisation and regulation of protein activity. Glycosylation defects such as those that occur in congenital disorders of glycosylation (CDGs) would also be expected to have a direct impact on the lipid composition of membrane domains, e.g. by perturbing the localisation of enzymes involved in lipid synthesis and signalling. In addition to its direct effects on lipid synthesis, aberrant glycosylation can also indirectly affect lipids and lipid signalling. In both cases, altered lipid homeostasis is likely to contribute to the pathophysiology of these multisystemic disorders. Here we aim to investigate the roles of lipids in modulating the assembly, activity and specificity of glycosylation enzymes. We will focus on dolichol phosphate mannose synthase, a key enzyme in N-glycosylation as well as C- and O-mannosylation. Using both in vitro and cellular approaches we will investigate contributions of membrane lipids to the structure and function of the enzyme. In addition, in collaboration with the other members of the Research Unit, we will study whether and how CGDs affect intracellular lipid homeostasis, using different eukaryotic model systems, ranging from human patient fibroblasts to mouse and fish models. Employing quantitative nano-mass spectrometry, we will study lipid pathway alterations in CDG to determine the consequences of hypoglycosylation on cellular lipid homeostasis. Taken together, we aim to elucidate the interplay between lipids and glycosylation enzymes and to determine how glycosylation defects can effect cellular and organismal lipid homeostasis.