Mapping Protein Glycosylation by High-Resolution Single Molecule Imaging

当我们想到细胞功能的主要参与者蛋白质时，我们倾向于想象圆形或细长的结构，它们在全身移动，并通过与其他生物分子相互作用来运作。然而，从根本上说，这是正确的，一个蛋白质可以有大量的结构，每一个结构都以不同的方式与其他蛋白质或生物分子相互作用，并微调蛋白质的功能或它在细胞中的移动位置，甚至它在体内存在的时间。这种结构的多样性来源于蛋白质的化学修饰，最常见和最复杂的类型被称为糖基化，这是一种复杂的、非模板驱动的过程，它将称为聚糖的复杂糖添加到蛋白质的特定位点上。大多数人类蛋白质都被聚糖修饰过，而在向蛋白质中添加聚糖的机制中出现的小故障会导致一种罕见但严重的疾病，即先天性糖基化疾病，几乎所有患者都在成年前死亡。无法将蛋白质糖基化对所有动物都是致命的，包括最低级的生命形式，如酵母。关键的是，在几乎所有的人类疾病状态中都观察到糖基化的变化，包括癌症、糖尿病和衰老。糖基化在生物制药工业中越来越重要，因为大多数基于蛋白质的药物，如单克隆抗体，都是糖基化的，而聚糖可以显著影响其安全性和有效性。此外，病毒利用糖基化将其与宿主结合所需的表面蛋白隐藏在所谓的“聚糖屏蔽”下，因为这些蛋白质“尖峰”是免疫检测和抗体靶向的主要焦点。最后一点，COVID-19疫苗携带编码病毒刺突的遗传信息，这是一种含有66个聚糖的糖蛋白，旨在模仿SARS-CoV-2表面上发现的糖蛋白。糖蛋白在人类健康和疾病进展中普遍存在，但令人惊讶的是，我们对聚糖如何控制蛋白质功能知之甚少。这是因为我们缺乏能够处理糖蛋白复杂性的工具。更具体地说，糖蛋白可以有许多糖基化位点，每个位点都可以被数百种不同的支链聚糖结构中的一种占据，因此可能的组合是巨大的。由于这种结构的复杂性，我们实际上只完全了解少数糖蛋白的结构。弗朗西斯·克里克（Francis Crick）是DNA结构的发现者之一，他曾说过：“如果你想了解功能，就研究结构”——这句话对今天的糖蛋白和近70年前的DNA一样适用。因此，生物学和医学研究领域的许多研究人员需要新的工具来研究糖蛋白结构，以更好地了解和治疗疾病。我们的研究项目旨在通过创建一种全新的方法，通过在单分子水平上对给定糖蛋白（称为糖肽）的成像切片来表征糖蛋白，从而解决结构生物学中的这一问题。然后，我们可以利用这些信息来绘制蛋白质上特定位置的聚糖结构，并精确地描述这两个部分的原子细节。为了使我们的新方法成为可能，我们将推进用于拍摄这些单分子图像的分子“探针”——类似于将黑胶蚀刻版画转换成唱机上的音乐的手写笔。为了证明我们的新方法的潜力，我们将描述几种基于糖蛋白的药物以及一种主要的HIV疫苗候选糖蛋白，这将为了解抗体如何与HIV表面的聚糖相互作用提供急需的见解。这是理解基于结构的疫苗（包括用于COVID-19的疫苗）的功能和功效的关键问题。

When we think about proteins, the major players of cell function, we tend to visualise round or elongated structures that move throughout the body and operate by interacting with other biomolecules. Fundamentally, this is true, however, a single protein can have an enormous number of structures, each of which interacts with other proteins or biomolecules in different ways and fine-tunes how a protein functions or where it moves in the cell or even how long it lasts in the body. This structural diversity is derived from chemical modifications to the protein and the most common and complex type is called glycosylation - an intricate, non-template driven process that adds complex sugars, termed glycans, to specific sites on a protein. The majority of human proteins are modified by glycans and minor faults in the machinery that adds glycans to proteins leads to a rare but severe condition in humans called Congenital Disorders of Glycosylation, with nearly all those afflicted dying before adulthood. The inability to glycosylate proteins is lethal in all animals including the lowest forms of life, such as yeast. Critically, changes in glycosylation are observed in nearly all human disease states including cancer, diabetes as well as aging. Glycosylation is increasingly important in the biopharmaceutical industry, as most protein-based drugs, like monoclonal antibodies, are glycosylated and glycans can significantly influence their safety and efficacy. Moreover, viruses use glycosylation to hide their surface proteins, which are required for host binding, under so-called "glycan shields" as these protein "spikes" are the principle focus for immune detection and antibody targeting. As a final point, COVID-19 vaccines carry the genetic message to encode the viral spike - a glycoprotein with 66 glycans that has been designed to mimic those found on the surface of SARS-CoV-2. Glycoproteins are prevalent and central in human health and disease progression but we know surprisingly little of how glycans control the function of proteins. This is because we lack the tools capable of dealing with the complexity of glycoproteins. More specifically, a glycoprotein can have many glycosylation sites, each of which can be occupied by one of hundreds of various branched glycan structures - the possible combinations is therefore enormous. Because of this structural complexity we really only fully know the structure of a handful of glycoproteins. Francis Crick, who co-discovered the structure of DNA, once said, "If you want to understand function, study structure" - a statement that is as revenant for glycoproteins today as it was for DNA nearly 70 years ago. Therefore, researchers across many areas of biological and medical research need new tools to study glycoprotein structure to better understand and treat disease. Our research project aims to solve this problem in structural biology by creating a completely new way to characterise glycoproteins by imaging sections of a given glycoprotein (termed a glycopeptide) at the single molecule level. We can then use this information to map what glycan structures are at specific sites on a protein with exact atomic detail of both parts. To make our new method possible, we will advance the molecular "probes" used for taking these single-molecule images - akin to a stylus that translates vinyl etchings into music on a record player. To demonstrate the potential of our new method, we will characterise several glycoprotein-based drugs as well as a lead HIV vaccine candidate glycoprotein, which will add much needed insight into how antibodies interact with glycans on the surface of HIV. This is a key issue for understanding the function and efficacy of structure-based vaccines, including those used for COVID-19.