Visualizing a protein's sugars.

Abstract

As the Human Genome Project was declared complete in the early 21th century, it was obvious that the human genome contains fewer genes than previously predicted [1]. Soon scientists realized that the biodiversity and complexity observed in humans cannot be simply explained by numbers of genes nor by the size of the human genome. Many other factors such as epigenetics and ‘postprocessing’ chemical processes (i.e. post-transcriptional regulation of RNA and post-translational modifications or PTM of proteins) beyond the central dogma exist and contribute significantly to complexity of humans. Among various PTMs, glycosylation—adding sugar molecules or glycans onto the protein backbones— is one of the most ubiquitous and complicated forms. More than half of the proteome consists of glycoproteins and most cell-surfaceproteinsareglycosylated.The glycans are essential for mediating the dynamics and function of the proteins that they are attached to. Many proteins share similar or even same glycan structures. Furthermore, the reactions of attaching glycans to proteins, under the control of an array of metabolic enzymes, usually occur after protein synthesis. Therefore, glycans are traditionally labeled and imaged on the entire proteome [2]. The identity of the individual protein could not be differentiated. However, to elucidate biological effects of glycosylation on a protein of interest, it is highly desirable to visualize glycans in a protein-specific manner by using fluorescence microscopy. This challenge has not been solved in the field for a long time until recently [3]. The research group of Dr. Xing Chen at Peking University reported a method for protein-specific imaging of glycans [3], representing an important breakthrough in chemical glycobiology. Dr. Chen's strategy utilizes a fluorescence imaging technique based on Forster resonance energy transfer (FRET). For FRET to happen, two fluorophores, a donor–acceptor pair, need to be in close proximity (<10 nm). In a FRET imaging experiment, a light is used to excite the donor and the energy is transferred to the acceptor, which emits a fluorescent signal for detection and imaging. Chen and co-workers installed a FRET acceptor to glycans attached to various proteins by taking advantage of the previously developed metabolic glycan labeling technique [2]. Simultaneously, they used a genetically encoded tag [4] to attach a FRET donor to the protein of interest. Since the tag is genetically encoded, the donor only resides on the specific protein of interest. Due to the distance constrain for FRET, only acceptor bound to the glycans on the same protein can be excited through intramolecular FRET, whereas the excess acceptors attached to other proteins will not respond. Therefore, the FRET effect serves to selectively image glycans of the same protein labeled with donor (Fig. 1). Using this technique, Chen and co-workers studied functional roles of sialylated glycans in β2 integrin activation. β2 integrins are adherent receptors expressed on the surface of leukocytes, and their activation is crucial in mediating leukocyte trafficking. They imaged the sialylated glycans on αXβ2 integrin and revealed that sialylation is important for activation; it would be almost impossible for such a study without the protein-specific imaging technique. The team also demonstrated generic applicability of their method to various cell-surface glycoproteins. Figure 1 The FRET-based methodology for protein-specific imaging of cell-surface glycans. Revised from [3] (Copyright 2014, American Chemical Society). Glycobiology has long been impeded by the lack of imaging tools. The metabolic glycan labeling coupled with bioorthogonal chemistry, pioneered by the Bertozzi's group at University of California, Berkeley, has revolutionized molecular imaging of glycome in living cells [2]. The challenge, however, remained for specifically imaging glycans on an individual protein of interest. Chen's work provides a smart solution to this challenge. One beauty of their work is that the methodology builds on the metabolic glycan labeling technique and site-specific labeling of proteins. There are many other protein labeling methods available that could be applied as well [5]. Therefore, this methodology should be readily adopted by the community to study many important glycoproteins.