Definition of Imaging Glycosylation - Report Example

Imaging glycosylation Introduction The biological capabilities and physiological of a cell are determined by the surface protein’s glycosylation. However, it is critical to undertake cell visualization as it central to cell biological capabilities and physiological states. The protein-directed imaging such as Green Fluorescent Protein remains unreactive method to the glycosylation prompting our research team to design a metabolic functionalized sugar labelling approach to image particular structures on local glycoproteins. Regrettably, this devised approach is limited to the revelation of protein identity being labeled glycan. However, effective imaging must integrate the identifiers of the glycan and protein. Therefore, the difficulty of visualizing specific protein glycoforms has also prompted Soderberg alongside its co-workers to apply different approaches such as proximity ligation in detecting particular glycoforms particularly, the tumor marker, MUC2. Body The recent studies conducted by Haga et al. applied azido sugar labeling of GFP-attached proteins for carrying out cell surface glycoprotein imaging. They used Foster resonance energy transfer (FRET) fluorescence microscopy. However, their dependence on GFP-tagged proteins in imaging led to the study inability to image the endogenous glycoproteins or unamenable to fluorescent protein fusion. However, the traditional FRET-attached techniques have a number of shortcomings. The efficiency of energy transfer is linked to the distance between acceptor and donor fluorophores as constituted in FRET experiments. Thus, such relationships prohibit functionality of two enormous macromolecules like immunoglobulin G (Ig; >nm). Also, the incongruity between glycan profusion and protein copy number is a key compounding aspect for imaging of particular glycoforms. Such discrepancies are so disastrous to the cell surface leading to increased difficulties in the examination of imaging applications. For instance, during the FRET-dependent experiments, the excitement of donor fluorophore is executed after which the investigator observed the acceptor fluorophore’s emissions. Where the investigation involves high acceptor fluorophore accumulation that are the possibility of bleed-through of acceptor fluorophore. If that is the case, a false positive FRET signal occurrence is an inevitability as illustrated below in figure A. Figure 1: a false positive FRET signal Following the shortcomings attached to the FRET-tagged imaging, an alternative is presented for imaging endogenous protein glycoforms. This approach utilizes a merger between azido sugars labelling and two-photon fluorescence lifetime imaging microscopy (FLIM). The present method depends on a small less than 7 nm targeting moiety and an antigen-binding fragment (Fab) that ushers in the donor fluorophore as well as locating the particular component of the protein. The approach borrows from the previous method’s glycan labeling mechanism when introducing the acceptor fluorophore. The researchers kicked off the experiment by first incubating the cells in an azido sugar, peracetylated N-azido-acetylmannosamine. Such incubator is processed by the cellular machinery and subsequently integrated with glycoproteins as azido sialic acid. Within a minimal distance and with succeeding biorthogonal reaction with a cyclooctyne-fluorophore conjugate produces the acceptor fluorophore as illustrated in figure A. The method uses the popular approach that circumvents acceptor bleed-through that concentrates on the emission of the donor fluorophore in a FRET experiment. Two primary alterations in the physical properties of the donor occur as energy transmit between the acceptor fluorophore and the donor. One of the alterations is the emission reduction from the donor. The photobleaching normalization of the acceptor facilitates the photon reduction in population cells to reveal the maximum volume of donor emission. Such a process is tedious and difficult task should the field of view has many cells. The second alteration in the donor fluorophore following energy transmission is a decline in fluorescence lifespan. Here, the problems of sample manipulation and experimentation are eliminated due to the advantageous utilization of time-dependent property. Moreover, the approach examines the decline in the donor’s fluorophore fluorescence lifetime linked to FRET in monitoring the sialylation state of a particular glycoprotein via two-photon FLIM. The commonly experienced overexpression of the integrin subtype in a range of cancers and is found to correlate with the invasiveness as a result of the pro-angiogenic function. The integrin has nine potential N-glycosylation alongside four reported sites on the alpha subcategory. Also, integrin has two reported as well as four potential sites on the beta subcategory. The recent studies on this topic point out that the integrin angiogenic and migratory functions can drastically change be changed by the glycosylation state. Particularly, two independent reports uncovered that integrin’s sialylation was a key requirement for the proliferation and regeneration as well as migration during the healing of wound assays. Accordingly the study conducted by Panjwani alongside its co-workers found an inevitable linkage between the glycosylation state and the VEGF- and bFGF-mediated angiogenesis. Indeed, the linkage results from the interaction with galectin-3. The study was inspired by the significance of glycosylation state of the integrin and subsequently pursued such an integrin to showcase a proof-of-concept glycoprotein to image its individual sialylation condition. Here, the investigators initially sought to determine the possibility of endogenous integrin being sialylated in the U87MG cell line of glioblastoma. This followed the known facts that attach glioblastoma cell line to the expression of heterodimer complex at greater heights. The immunoprecipitation of the integrin from U87MG lysate took place. Subsequent assessment of the presence of sialic acid on either subunit was carried through the application of lectin blotting. The distinguishing of alpha 2, 3- from alpha 2, 6-linked sialosides was made possible by blotting with Maackia amanuensis agglutinin also known as MAA as well as Sambucus nigra lectins, SNA in that order. The observation of MAA binding to either of integrin subunits followed the immuno-precipitation. Here, MAA only bound on alpha 2, 3-linked residues hence their sialylation. This is further dramatically explained in figure B below. Figure 2 A: MAA binding on a2, 3-linked The study’s FRET mechanism is predicted on SiaNAz integration into the N-glycans the integrin as reflected in figure 1 above. This was justified through the supplementation of cells using Ac4ManNAc or Ac4ManNAz for a period of three days. Subsequently, cell incubation with a blotin-conjugated phosphine took place. Such a biorthogonal investigation is appropriate for the functionality of azido. Further, the probe subjected cell lysates to immunoprecipitation using an anti-integrin antibody. Here, the detection of SiaNAz was noted through western blotting. Also, there was an observation of Azide-driven labelling for the beta and alpha integrin chains as shown in the figure 2 B above. Such observation points towards the presence of SiaNAz in either the heterodimer subunits. A further confirmation of azide-specific signal occurred due to the presence of SiaNAz remains. The digestion of the lysates with sialidase resulted from the observation of a declined signal on each integrin as revealed in the figure 2 B above. On the other hand, the investigators further shifted gear and focused on the dual labelling approach to select the sufficient FRET pair. The primary objective of the dual strategy was to eliminate the cellular autofluorescence following the known facts of the occurrence of firm Flavin- and riboflavin-linkage to autofluorescence. Therefore, the emphasis here was with respect to fluorophores’ donor emitting over 600 nm. Accordingly, the investigators ultimately settled on fluorophore combination of Alexa Fluor 594 (594) as well as Alexa Flour 647 (647). The decision was informed by the maximum emission and the Foster pair radius of Alexa Flour 594’s that were 617 and 8.5 nm respectively. Subsequently, the commencement of the donor Fab fragment conjugate preparation was based on the treatment of the monoclonal antibody against integrin (LM609 clone) with the peptides. The peptides were central to cleaving the complete length IgG between the Fc and Fab sections and the unreacted IgG following the Protein A resin incubation. Accordingly, the reaction between Alexa Flour 594 N-hydroxysuccinimidyl ester and the lysine followed. Such a reaction furnished the Fab-594 conjugate. The synthesis of the acceptor cyclooctyne from the commercially available reagents was performed using the DIBAC amine alongside the Alexa Fluor 647 N-hydroxysuccinimidyl ester. The ability to construct the donor (Fab-594) easily with the acceptor (DIBAC-647) that targets at moieties justifies the versatility of the dual method and its subsequent applicability to further glycoprotein targets. The observation of particular cellular labelling of membrane-driven integrin possible of inhibition by the availability of anti-integrin IgG followed the U87MG cells treatment with Fab-594. Further, the researchers determined the DIBAC-647’s faithfulness in the biorthogonal investigation for azides. At this point, cells were treated for a period of three days with both Ac4ManNAc and Ac4ManNAz. The treatment was proceeded by incubation of the cells with DIBAC-647. The alteration in the cell surface labelling was solely noted amongst those cells fed with Ac4ManNAz as illustrated in the figure 2 C and D above. After such double confirmation, the investigators shifted their tune to monitor the FRET through a two-photon FLIM strategy. Here, donor’s fluorophore was excited through near-IR/IR femtosecond pulses alongside examining the time-driven fluorescence deterioration in the nanosecond intervention. The energy transfer is central in this experiment as it helps in the depletion of excited donor’s state should the acceptor fluorophore be in proximity to the fluorophore of the donor. The depletion culminates into a declined of the donor’s molecule fluorescence lifespan. The researchers noted that Fab-594 showcased an in vitro features lifetime value of 3.09 ns. Such time value was reached at by fitting the fluorescence emission for the decline of Fab-594 to an individual exponential as revealed in figure 3 below. Figure 3: Determination of Fab-594 Lifetime The FLIM takes an individual pixel of an image monitoring of fluorescence emission based on some set time criterion. Subsequently, the data gathered from the emission are fitted to determine the lifetime values that are ultimately showcased as heat-map images. Such images were extracted through incubation of U87MG cells for three days in Ac4ManNAz or Ac4ManNAc. Accordingly, sequential DIBAC–647 and Fab–594 cell labeling occurred. The Ac4ManNAc-treated cells showed a mean lifetime value of 2.91 ns as reflected in figure 3 A. Moreover, such cells surface revealed a relatively fix lifetime value showcased by the deep blue color in figure 3 A. On contrary Ac4ManNAz-traeated cells had a mean lifetime value declining to 2.60 as illustrated in figure 3 B above. Such an observation was in line with the FRET’s cell-surface occurring between DIBAC and Fab-594. Thus, there was a more heterogeneous in figure 3 B relative to A. These far flung heterogeneous findings is showcased by the green-yellow categories in figure 3 B above. Jointly, such data, as presented in figure 3, demonstrate the sialylatedness of the cell surface integrin on U87MG cells. In addition, the examination of the lifetime was showcased by histogram construction illustrating the reactions with both Ac4ManNAc and Ac4ManNAz cell populations as depicted in figure 3 C above. Also, the unimodal (red) to multimodal (blue) distribution was noted with respect to the decline in the lifetime readings for the cells solely labelled with Fab-594 donor against those treated with both the DIBAC-647 acceptor alongside the Fab-594 donor. Such alterations stand to showcase that differences in the magnitude of sialylation on particular integrin molecules. Moreover, as depicted in figure four A below, it was discovered that cells that had no Fab-594 treatment but DIBAC-647 acceptor-labelled demonstrated minute signals. Figure 4: minimal Signals of Cell lacking Fab-594 Further, the investigators shifted attention to the evaluation of exact instigator or origin of the observed FRET. This, probe focused on the analysis of possible chances of SiaNAz residues on the integrin individually and related Sialic acid-adjusted glycolipids or glycoproteins. The investigators distinguished between sialylated integrin-driven FRET from background FRET. This followed their reasoning that acceptor moiety was never bound to their focused integrin and hence adding an acceptor fluorophore to the surface of a cell while simultaneously warranting the unbounding acceptor moiety. They initially incubated the cells in dipalmitoyl phosphatidylethanolamine-647. The DPPE-647 integrate the acceptor with a cell membranes uniformity. This was proceeded with Fab-594 labelling. The outcomes were that there was no alteration in the value of the lifetime. The surface of the cells showcased a fixed hue as those cells possessing the Fab-594 fragments solely as demonstrated in figure 4 B, D above. Subsequently, the investigators concluded that there was no relationship between sialic acid on nearby proteins and lipids and the levels of the signals of the FRET resulting from integrin complexes. Further, the investigators probed the possibility of the noted FRET signal being a sialylation stated-driven circumstance. Here, the investigators treated Ac4ManNAz-fed U87MG cells with a sialidase. This treatment helped in cleaving SiaNAz residues from the surface of the cell glycoconjugates. The investigators then treated cells with the DIBAC–647 and then gestated with the Fab– 594. The investigators realized that the processes of sialidase treatment eliminated the depression in the process and indication that the SiaNAz filtrates on integrin aVb3 were crucial in the observation of FRET. An exceptional and most crucial aspect of this platform is that it can articulately analyze the glycosylation state of the endogenous glycoproteins as well as image cavernous in a specimen. However, this is largely attributed to the two-photon approach of excitation common in this type of analysis. Similarly, the investigators were able to show that the human prostate adenocarcinoma tissue portions could be metabolically integrated azido sugars into their glycoprotein tissues. According to the investigators, the prostate cancer cells occasionally undergoes an upregulation in the integrin aVb3 and avails an opportunity to apply the imaging procedure to human tissue slices. The precision-cut adenocarcinoma material slices of grade 3-4 that were consequent of the 8 mm core in the fundamental prostatectomy cultured in either Ac4ManNAz or Ac4ManNAc. The investigators first ascertained that the aVb3 immune occasioned from the tissue lysate, and like the aVb3 plagiarized from the U87MG lysate, it was recognized by MAA and was integrated with the SiaNAz deposits into its glycan tissues. Correspondingly, the investigators found out that the use of fluorescence microscopy would label the prostate tissues in the aVb3- and azide-reliant processes as indicated in figure five below. Figure 5: Fluorescence Microscopy Labelling of Prostate Tissue Next, the investigators applied the developed FLIM FRET technique to the tissue slices by first discoloring with DIBAC–647 and later with the Fab–594. It was ascertained that the blue color observed displayed the features of the Fab–594 t valuation treated with Ac4ManNAc. Notably, the Ac4ManNAc tissues displayed a depression in the t valuation that could be retracted once treated with the appropriate sialidase that was unswerving with the FRET and the sialylated integrin aVb3 entrenched within the material slices. Conclusion In essence, the investigators were tasked with developing a FILM FRET based technique used to envisage the glycosylation state of particular glycoprotein components. Arguably, they were able to develop successfully the FILM FRET based method that could overcome intrinsic confines of the definitive FRET imaging that could be destabilized by bleed-through. The technique has enabled proper imaging of discrete proteins glycoforms in the slices of the tissues. In conclusion, the method has contributed immensely to the growth of the toolkit used to characterize the cell superficial glycomes with molecular precisions. The investigators recommend that this technique be applied in future to image the cognate cell superficial ligands for the concealed lectins and the analytical purposes to monitor the glycosylation eminence of precise glycoproteins. Work Cited Brian B. Adam de la Z., David R. S, Sophia L. M, Donna M. P, & Carolyn R. B. Imaging the Glycosylation State of Cell Surface Glycoproteins: By Two-Photon Fluorescence Lifetime Imaging Microscopy. Angewandte. 2013. Print. J. D. Marth, P. K. Grewal, Nat. Rev. Immunol. 2008, 8, 874 – 887. K. Ohtsubo, S. Takamatsu, M. T. Minowa, A. Yoshida, M.Takeuchi, J. D. Marth, Cell 2005, 123, 1307 – 1321. Read More