Imaging the glycosylation state of cell surface glycoproteins by two-photon fluorescence lifetime imaging microscopy.


A cell surface protein s glycosylation state can profoundly influence its biological capabilities and can report on the physiological state of the underlying cell. Thus, visualization of particular protein glycoforms is an important though challenging goal. Most protein-directed imaging methods (e.g. green fluorescent protein (GFP) tags, fluorescent antibodies) are not sensitive to the glycosylation state of a protein. Our research group previously developed a method for imaging certain glycan structures on native glycoproteins by metabolic labeling with functionalized sugars. However, this glycan-targeted imaging method cannot reveal the identity of proteins to which the labeled glycans are attached. Imaging of a specific protein glycoform will require integration of the identifies of both the protein and the glycan. Other groups have recently made strides towards this goal. Sçderberg and co-workers used proximity ligation to detect a specific glycoform of the tumor marker MUC2. More recently, Haga et al. used azido sugar labeling of GFPtagged proteins to image cell surface glycoproteins by Fçrster resonance energy transfer (FRET) fluorescence microscopy. Due to their reliance on GFP-tagged proteins, however, this method cannot image endogenous glycoproteins or proteins that are not amenable to fluorescent protein fusion. Even so, there are some limitations to a traditional FRETbased technique. The distance between the donor and acceptor fluorophores in a FRET experiment is related to the efficiency of energy transfer and typically precludes the use of two large macromolecules, such as immunoglobulin G (IgG; > 10 nm). Another compounding factor for imaging of specific protein glycoforms is the discrepancy between protein copy number and glycan abundance. The difference in abundance between common types of glycans and a specific protein can be orders of magnitude on the cell surface. This large difference in relative number can complicate analyses in imaging applications. For example, in a typical FRET-based experiment, the donor fluorophore is excited, and emission from the acceptor fluorophore is monitored. In the case of high acceptor fluorophore concentration, acceptor bleedthrough can occur causing a false positive FRET signal (see Figure 1 in the Supporting Information). Herein, we present a new approach to image endogenous protein glycoforms using a combination of azido sugar labeling and two-photon fluorescence lifetime imaging microscopy (FLIM). We rely on a small (< 7 nm) targeting moiety, an antigen-binding fragment (Fab), to introduce the donor fluorophore and locate the protein component. We applied our previously developed glycan labeling strategy to introduce the acceptor fluorophore. In this scheme, cells were first incubated with an azido sugar, peracetylated N-azidoacetylmannosamine (Ac4ManNAz), which is processed by the cellular machinery and incorporated into glycoproteins as azido sialic acid (SiaNAz). Subsequent bioorthogonal reaction with a cyclooctyne–fluorophore conjugate delivers the acceptor fluorophore within a minimal distance (Figure 1). A common method for circumventing acceptor bleedthrough is to focus on the donor fluorophore s emission in a FRET experiment. Energy transfer between the donor and acceptor fluorophore results in two major changes to the donor s physical properties. The first is reduction in emission from the donor. Imaging this photon reduction in a population of cells requires normalization by photobleaching of the acceptor to reveal the maximum amount of donor emission, a difficult and tedious task when the field of view contains numerous cells. The other change for the donor fluorophore upon energy transfer is a decrease in fluorescence lifetime. This time-dependent property is advantageous since no further experimentation or sample manipulation is necessary. We sought to utilize the decrease in fluorescence lifetime of the donor fluorophore associated with FRET to monitor the sialylation state of a given glycoprotein through two-photon FLIM. Overexpression of the integrin aVb3 subtype is observed in a variety of cancers and is often correlated with invasiveness due to its pro-angiogenic function. Integrin aVb3 possesses [*] B. Belardi, Prof. A. de la Zerda, D. R. Spiciarich, Prof. C. R. Bertozzi Departments of Chemistry and Molecular and Cell Biology Howard Hughes Medical Institute, University of California Berkeley, CA 94720 (USA) E-mail:

DOI: 10.1002/anie.201307512
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@article{Belardi2013ImagingTG, title={Imaging the glycosylation state of cell surface glycoproteins by two-photon fluorescence lifetime imaging microscopy.}, author={Brian Belardi and Adam de la Zerda and David R Spiciarich and Sophia L. Maund and Donna M . Peehl and Carolyn R Bertozzi}, journal={Angewandte Chemie}, year={2013}, volume={52 52}, pages={14045-9} }