Recent Chemical Biology Approaches for Profiling Cell Surface

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Recent Chemical Biology Approaches for Profiling Cell Surface Sialylation Status Joshua Whited, Xiaoqing Zhang, Huan Nie, Dan Wang, Yu Li, and Xue-Long Sun ACS Chem. Biol., Just Accepted Manuscript • DOI: 10.1021/acschembio.8b00456 • Publication Date (Web): 27 Jul 2018 Downloaded from http://pubs.acs.org on August 1, 2018

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ACS Chemical Biology

Recent Chemical Biology Approaches for Profiling Cell Surface Sialylation Status Joshua Whited,1 Xiaoqing Zhang,2 Huan Nie,2 Dan Wang,1 Yu Li2 and Xue-Long Sun1* 1

Department of Chemistry, Chemical and Biomedical Engineering and Center for

Gene Regulation in Health and Disease (GRHD), Cleveland State University, 2121 Euclid Avenue, Cleveland, Ohio, 44115, USA 2

School of Life Science and Technology, Harbin Institute of Technology, 2 Yikuang-jie,

Harbin, Heilongjiang, 5001, China

To be submitted as a Review Paper to ACS Chemical Biology

*Corresponding author. Tel.: +1 216 687 3919; fax: +1 216 687 9298; Email address: [email protected] (X.-L. Sun)

Running title: Profiling cell surface sialylation status

Keywords: Sialic acid (SA), Polysialic acids (PSAs), Metabolic glyco-engineering and labeling,

Bioorthogonal

chemistry,

Click

Chemistry,

Periodate

oxidation

and

aniline-catalyzed oxime ligation (PAL), Boronic acid labeling, MALDI Imaging Mass Spectrometry (MALDI-IMS)

1

ACS Paragon Plus Environment

ACS Chemical Biology 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ABSTRACT: Sialic acids (SAs) often exist as the terminal sugars of glycans of either glycoproteins or glycolipids on the cell surface and thus are directly involved in biological processes, such as cell - cell, cell - ligand, and cell - pathogen interactions. Cell surface SAs expression levels and their linkages are collectively referred as cell surface sialylation status, which represent varying cellular states and contribute to the overall functionality of a cell. Accordingly, systemic and specific profiling of the cell surface sialyation status is critical in deciphering the structures and functions of cell surface glycoconjugates and the molecular mechanisms of their underlying biological processes. In recent decades, several advanced chemical biology approaches have been developed to profile the cell surface sialyation status of both in vitro and in vivo samples, including metabolic labeling, direct chemical modification, and boronic acid coupling approaches. Various investigative technologies have been also explored for their unique competence, including fluorescent imaging, flow cytometry, Raman imaging, magnetic resonance imaging (MRI), and MALDI imaging MS (MALDI-IMS). In particular, investigation of sialylation status of a specific glycoprotein on the cell surface has been explored. This review highlights the recent advancements of chemical biology approaches for profiling cell surface sialyation status. It is expected that this review will provide researchers different choices for both biological and biomedical researches and applications.

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I. INTRODUCTION Sialic acids are a family of 9-carbon monosaccharides containing a carboxylic acid. They are often located at the terminal position of N- and O-linked glycan structures of the cell surface and

glycoconjugates.1

secreted

They

are linked

to

either

galactose

(Gal),

or

N-acetylgalactosamine (GalNAc) units via α2,3- or α2,6-linkage, or to SA via α2,8- or 2,9-linkage (Fig. 1).2 The specific linkage formation is controlled by a specific enzyme during the sialylation process. N-acetylneuraminic acid (Neu5Ac) and N-glycolylneuraminic acid (Neu5Gc) are major SAs. It is known that human beings synthesize Neu5Ac but not Neu5Gc.3 In addition, 2-keto-3-deoxy-D-glycero-D-galacto-nononic acid (KDN), a deaminated neuraminic acid, is identified in humans and other mammals.4 So far, various modifications of 4-, 5-, 7-, 8- and 9-hydroxyls of SAs have been confirmed, which produce more than 50 SA species.5 Given their electronegative features and terminal location on the glycans of cell surface glycoconjugates, SAs are involved in both physiological and pathological processes, such as in cell-cell interactions, cell activation, differentiation, transformation and migration.5,6

R2O

HO HO AcHN

OR 2 8

9

R2O R1 7 5

1 COOH

6

R 2O

O HO

COOH HO OH O O O R'

Neu5Ac-(α2,3)-Gal (or GalNAc)

O 4

OH

2

O

3

R 1 = NHAc (Neu5Ac) NHGc (Neu5Gc) OH (KND) R 2 , = H, or Ac

HO HO AcHN

OH

COOH HO O O HO HO

O

O R' Neu5Ac-(α2,6)-Gal (or GalNHAc)

R HO HO AcHN

OH

COOH O

HO

HO HO AcHN

O

COOH O

O

HO Neu5Ac-(α2,8)-Neu5Ac

HO HO AcHN

OH O HO

COOH O OH HO AcHN

COOH O

O

HO

Neu5Ac-(α2,9)-Neu5Ac

Figure 1. Diverse structures of sialic acids and sialyl linkages. Neu5Ac is used as a representative sialic acid in the figure. Modified from Ref 2.

SAs expression levels and their linkages on the cell surface, collectively known as sialylation status, are often varied at altered cellular status and environments and are associated with cell properties, phenotypes, functionalities, and thus human health and disease.6 Profiling cell surface sialylation status will contribute to understanding the cell phenotype, functionality, and the molecular mechanisms of related biological processes. Lectins7 and antibodies8 are useful tools to profile cell surface SAs expression as they 3



specifically recognize SA derivatives and their underlying linkages. SA-binding proteins and recombinant proteins have also been developed for detecting specific SAs, sialylglycans and their modifications on cells and/or tissues.9 We have summarized recent advancements of these bio-affinity approaches for directly detecting and profiling cell surface sialylation status.10 On the other hand, chemical biology approaches such as metabolic glyco-engineering that allows the introduction of unique functional groups to cell surface SAs have been developed for sensitive detection of cell surface SAs.11 Also, aldehydes can be selectively derived from SAs on the cell surface by chemical modification with diluted sodium periodate, and selectively ligated with hydrazine and amino-oxy probes for profiling cell surface SAs.12 In addition, phenylboronic acid (PBA) selectively conjugates to the glycerol side chain of SAs at physiological pH and thus has been used for selective recognition of cell surface SAs.13 This review summarizes these recent chemical biology approaches for profiling cell surface sialylation status. Specially, metabolic labeling, SA-specific chemical modification, and boronic acid labeling approaches combined with fluorescent imaging, flow cytometry, Raman imaging, MRI and MALDI-IMS are highlighted (Fig. 2).

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Figure 2. Diverse approaches for profiling cell surface sialylation status: Bio-affinity approaches (a. lectin, b. antibody, c. recombinant protein); Chemical biology approaches (d. metabolic glyco-engineering and labeling, e. chemical modification and conjugation, f. boronic acid coupling-based microscope imaging, flow cytometry, Raman scattering imaging and xenon MRI; and g. MALDI-IMS). Neu5Ac is used as a representative sialic acid in the figure. Neu5Ac-R: Neu5Ac derivative.

I. METABOLIC LABELLING APPROACHES TO PROFILE SAS ON THE CELL SURFACE Metabolic labeling is a recently developed chemical biology strategy to study cell surface SAs.11,14-16 In general, a sugar substrate analog carrying a functional group is incorporated into cell surface glycoconjugates via the metabolic pathway, from which a second probe can be introduced by labeling the functional group for analysis and imaging application. Bertozzi et al. first demonstrated that ketone derived ManNAc (ManLev) could go through the sialylation pathway so as to incorporate a ketone group into the cell surface SAs, from which the ketone could be selectively labeled by biotin-hydrazide and detected by fluorescently 5



labeled streptavidin via flow cytometry.11 This metabolic engineering and labeling approach offers a selective and sensitive detection of cell surface SAs and has become an efficient chemical biology approach to investigate the SAs functions in biological systems, from cells to whole animals. So far, four kinds of SA precursors have been developed to generate SA derivatives on the cell surface through the SA biosynthetic pathway, including GlcNAc, ManNAc, SA, and CMP-SA analogs.17 The development of bioorthogonal chemistry empowered wide applications of metabolic cell surface SA engineering, such as detection, imaging, and isolation of sialyl glycoconjugates of living cells, tissues, and organisms.17-21 Various reactive functional groups such as ketone, azide, alkyne, thiol, alkene, and diazirine can be incorporated into SAs on the cell surface, functionalized under physiological conditions, and in a chemically selective way for imaging, isolation and derivatization applications.21 Among them, the azido functional group is mostly often incorporated into cell surface SAs by using N-azidoacetylmannosamine (ManNAz). The incorporated azido group can be labeled with a probe carrying alkyne and subsequently analyzed by fluorescent microscopic imaging. In general, a high concentration of ManNAz is needed because there is no membrane transporter for ManNAz. Later, per-acetylated ManNAz (Ac4ManNAz) was used in order to increase its cellular intake. Acetylated monosaccharide analog has hydrophobic prosperity to increase its penetrance across the cell membranes. The acetate groups can be hydrolyzed by esterases inside the cells, affording the free monosaccharide, which can be ultimately incorporated into SA biosynthesis pathway. Chen et al. used Ac4ManNAz and copper(I)-catalyzed alkyne-azide cycloaddition (CuAAC) to visualize dynamic sialylation in epithelial–mesenchymal transition (EMT) during embryonic development and organ formation (Fig. 3).20 It was found that sialylation underwent a down regulation during EMT and was then reverted and up regulated in the mesenchymal state after EMT (Fig. 3B). CuAAC was first reported by the Sharpless group22 and Meldal group23 independently and has been widely used as a bioorthogonal reaction. 2-(4-((Bis((1-(tert-butyl)-1H-1,2,3-triazol-4-yl)methyl)amino)methyl)-1H-1,2,3-triazol-1-yl)a cetic acid (BTTAA) is often used to enhance the CuAAC reaction efficiency by maintaining the Cu(I) oxidation state of copper sources. In addition, it protects biomolecules from 6


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oxidative damage during the labeling reaction.24,25 In another study, Choi et al. applied metabolic labeling for real-time monitoring the distribution of polysialic acids (PSAs) in living primary hippocampal neurons.26 In this work, Ac4ManNAz was used to metabolically incorporate N-azidoacetyl SA to PSAs in PSA biochemical pathway. With this method, they could examine the temporal and spatial distributions of PSAs in the primary hippocampal tissues. In particular, a continuous synthesis and recycle of PSA during neuronal development was confirmed. It is expected that this tissue-based labelling method can be applied to other tissues as well.

Figure 3. Profiling cell surface SAs by metabolic incorporation of azide into cell surface SAs of HaCaT by using Ac4ManNAz. A. Schematic illustration of metabolically engineering and click labeling of cell surface SAs carrying azide via the BTTAA-assisted CuAAC. B. Analysis of cell surface SAs carrying azide by conjugation with alkyne-biotin and streptavidin-Alexa Fluor 488, and analyzed by flow cytometry. C. Confocal fluorescence microscopy of the labeled cells. The nuclei were counterstained with Hoechst 33342. Scale bars, 20 µm. D. Immunoblot analysis of azide-incorporated cell surface glycoproteins in HaCaT cells.20 Adapted with permission from Ref 20. Copyright 2015 the American Society for Biochemistry and Molecular Biology.

In general, cell surface SA metabolic engineering often incorporates SA analogs into all glycoproteins with SAs and therefore, it is especially challenging to analyze a specific glycoprotein with the labeled SAs. To this goal, a transmembrane Förster fluorescence 7



resonance energy transfer (FRET) approach was developed, in which a fluorescent probe was introduced to the metabolically engineered SA via bioorthogonal labelling as the fluorescence donor molecule, while another fluorescent probe was introduced to the protein of the interest via genetic approach as the fluorescence acceptor molecule on the cell surface.27,28 For example, a green fluorescent protein (GFP) can be incorporated onto the protein of interest via protein expression. Bertozzi and co-workers found that high concentration of acceptor fluorophore could cause a false positive FRET signal due to acceptor bleedthrough.29 They used two‐photon fluorescence lifetime imaging microscopy (FLIM) in combination of azido sugar labeling to visualize the sialylation status of integrin αVβ3, a cell-adhesion protein associated with prostate cancer metastasis.29 In this study, the donor fluorophore was introduced via fragment-antigen binding (Fab) and the acceptor fluorophore was installed by metabolic engineering of cell surface SAs with azide group and subsequent click labeling with fluorescence molecules containing cyclooctyne. After excitation, the changes in fluorescent lifetime of the donor was monitored, elucidating the presence of both the donor and acceptor fluorophores (Fig. 4).29 Therefore, this technique allows for selective imaging of a specific glycoprotein and thus enables clarification of the functional significance of SA on a specific protein of interest. This is an elegant method to profile the sialylation status of a specific glycoprotein and may facilitate identification and monitoring of the biological role of the target glycoprotein in either physiological or pathological pathway.

Figure 4. Imaging the sialylation state of a specific glycoprotein using metabolic labeling and antibody labeling followed by 2-photon fluorescence lifetime imaging microscopy (FLIM). A. The azide functionality was metabolically incorporated in sialylated glycoproteins of U87MG cells with Ac4ManNAc, followed by click labeling with 8


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fluorophore probe (DIBAC–647) as the acceptor fluorophore. Next, the donor fluorophore was introduced to sialylated integrin αVβ3 glycoprotein with fluorophore-labeled Fab fragment (Fab–594). B. Two-photon FLIM imaging of sialylated integrin αVβ3. Scale bar = 50 µm.29 Adapted with permission from Ref 29. Copyright 2013 John Wiley and Sons.

Alkynes are often used for click labeling but also provide a unique Raman scattering image probes as they have a specific Raman scattering pattern within the Raman-silent region of a cell. Chen et al. developed a bioorthogonal stimulated Raman scattering (SRS) imaging method for visualizing cell surface SA containing alkynes in live cells.30 This SRS imaging method is simple to do without labeling after metabolic cell surface SA engineering with alkynes. On the other hand, Witte et al. demonstrated a live cell MRI with xenon Hyper-CEST biosensor that targeted to the metabolically labeled SA by bioorthogonal chemistry (Fig. 5).31 In this approach, azide groups were metabolically incorporated into the cell surface SAs by using Ac4ManNAz, which were labeled with the multimodal biosensor carrying an alkyne bioorthogonal functional group and xenon host (xenon MRI/fluorescence), then, the cells were imaged using Hyper CEST (Fig. 5B). Xenon biosensors have good pharmacokinetic properties. Therefore, this method may be applied to monitor the dynamic sialylation changes during cellular processes in vivo.

9



Figure 5. MRI with xenon hyper-CEST biosensors targeted to metabolically labeled cell-surface SAs using bioorthogonal chemistry. A. Three key steps for imaging cell surface SAs with Ac4ManNAz and xenon Hyper-CEST biosensors. B. Hyper‐CEST MRI of the live cell. Yellow indicates Xenon Hyper‐CEST MRI of the cells. The percentage CEST effect is displayed (pseudocolor) overlaid a reference proton MRI.31 Details please see Ref. 31. Adapted with permission from Ref 31. Copyright 2015 John Wiley and Sons.

Most recently, Bertozzi’s group applied the metabolic SA engineering and click labeling strategy to image human tissues cultured ex vivo.32 Briefly, human prostate tissue slice cultures were incubated with Ac4ManNAz to introduce azide groups into sialoglycoproteins. Then, tissue slices were fixed and labeled with DIBAC-AlexaFluor 647 (DIBAC-647) for imaging. Meanwhile, this metabolic labelling was used for glycoproteomic study. Indeed, the tissue slices were lysed and labeled with an alkyne affinity biotin probe, which was used for purification and enrichment for subsequent glycoproteomics using mass spectrometry. This 10


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work showed multiple application potentials of the metabolic engineering and bioorthogonal labeling strategy for both basic and clinical research and application. The advantages of metabolic engineering and bioorthogonal labeling strategy of cell surface SAs have been well demonstrated by the examples highlighted above. However, there are issues that limit wide application of this strategy. First, not all unnatural monosaccharides show satisfactory metabolic incorporation efficiency. This is likely the result of low cellular intake because there are no membrane transporters for these monosaccharide analogs. To overcome the lower cellular intake problem, the per-acetylation strategy was applied to ManNAc20,26 and other SA analogs.33,34 Yarema and co-workers conducted a systemic metabolic flux analysis for ManNAc analogues.35 Currently, per-acetylated ManNAc analogs are mostly used for metabolic engineering and labeling of cell surface SAs. Another limitation of metabolic engineering and labeling is the lack of cell-type selectivity when applied in vivo. To overcome this limitation, Chen et al. recently applied a targeted drug delivery strategy for selective labeling of sialylglycans in speciﬁc cell types.36 Briefly, they encapsulated the azido sugars within ligand modified liposomes and delivered them to target cells via specific ligand-receptor recognition. Mice were used to demonstrate the ability of this cell-specific strategy to label and visualize tumor associated sialylglycans. They found this strategy could be used to label and visualize both brain and tumor-associated sialylglycans.37,38 Most recently, they performed a mechanistic study on the liposome-assisted strategy and explored its multiplexing capability, with which the sialylglycans in two distinct cell populations in a co-cultured system could be selectively labeled with two distinct chemical reporters.39 This cell-specific strategy can be used to study the cell surface sialylation of a selected cell population and targeted disease in the future.

II. DIRECT CHEMICAL MODIFICATION APPROACHES TO DETERMINE SAS ON THE CELL SURFACE Direct chemical modification is another method for qualitative and quantitative investigation of SAs on the cell surface. Mild periodate oxidation chemistry has been developed to generate 11



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aldehyde groups from the cell surface SAs, which selectively oxidizes the glycerol side chain of SA to afford a 7-aldehyde SA derivative. This aldehyde group provides a selective modification of SAs via covalent linkage, such as oxime ligation. The advantages associated with chemical modification are attractive. Cells do not need to culture with sugar precursors prior to labeling. Biocompatible reaction conditions and efficient ligation are the key factors to achieve a practical detection of cell surface SAs. Periodate was first used to selectively oxidize SAs on the glycoprotein fetuin in 1964 for glycan sequencing and linkage analysis.40 In this research, quantitative conversion of SA residues to 7-carbon analogues was made by periodate oxidation and subsequent triturated borohydride reduction. Later, this technique was widely used for living cell surface SA modification.12,41-43 The combination of periodate oxidation, biotinylation, and biotin-avidin patterning procedures has made this highly selective oxidation approach suitable for almost any cell type.44,45 In this method, native SA residues presented on cell surface were first converted to aldehydes with diluted sodium periodate, which reacted with hydrazide-biotin to produce biotinylated cells. Then, the biotinylated cells were detected by avidin with a fluorochrome label or other modifications. However, the reaction between an aldehyde and aminooxy or hydrazine reagent was processed at only a modest rate even in optimal conditions.46

To

overcome

this

limitation,

Paulson

and

co-workers

developed

aniline-catalyzed oxime ligation which was demonstrated with an aminooxy-biotin tag in highly improved reaction efficiency.12 In this method, the aldehyde group on SAs generated by mild periodate oxidation was used for imine ligation in the presence of aniline, which is called periodate oxidation with aniline-catalyzed ligation (PAL). With the addition of aniline, the imine ligation efficiency was increased about ten-fold measured by flow cytometry with DTAF-streptavidin. The PAL approach made it possible to use other aminoxy or hydrazide reagents to target the aldehyde group on SAs in an efficient manner. Based on PAL, Cario and co-workers used fluorescent nitrobenzoxadiazole dyes to ligate the aldehyde groups of SAs on the cell surface and image the cells directly (Fig. 6).47 The attractive feature of this method was one-step imaging using the carbonyl-reactive chromophores alternative to

12


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avidin-biotin labeling strategies and simple detection of SAs on cell surface. This method was confirmed to be suitable for imaging of SAs both in vitro and in vivo.47

Figure 6. Fluorescent imaging of cells via PAL labelling. HeLa cells were observed for fluorescent images (488 nm ex/515 nm em) and DIC images (from left to right): no periodate and no NBDAO dye; no periodate and NBDAO dye; and periodate with NBDAO dye. Scale bars represent 50 µm.47 Adapted with permission from Ref 47. Copyright 2012 American Chemical Society.

Recently, the PAL was used in combination with GAL (galactose oxidase and aniline-catalyzed oxime ligation) to identify altered sialylation status and glycosylation sites of sialoglycoproteins in different cell models. For example, Ramya et al. used PAL and GAL to biotinylate the glycans of glycoprotein with aminooxy-biotin in high efficiency and cell viability.48 Applying these specific labeling methods to enrich target glycoproteins, they were able to identify a total of 175 unique N-glycosylation sites, which belonged to 108 nonredundant glycoproteins. In another study, McCombs et al. confirmed glycoproteins sialylation changes in response to neuraminidase treatment.49 They identified specific substrates of two pneumococcal neuraminidases in a human cell line that models the blood-brain barrier by using this combined approaches. As highlighted above, PAL paved the way for selective and efficient in situ labeling of SAs on the cell surface, and the next generation of analysis focused on sensitive detection through signal amplification mechanisms. Nanoparticles have been used for various imaging 13



and sensing platforms.50-52 Ju et al. developed a chemiluminescent (CL) imaging approach that used PAL-mediated biotinylation of cell surface SAs and biotinylated gold nanoparticles (AuNPs) carrying a large amount of horseradish peroxidase (HRP).53 HRP acted to trigger the CL emission to produce an amplified signal for CL imaging. Another report involved a similar approach using a single-walled carbon nanohorn (SWNH) for recognition and mannan-conjugated AuNPs for signal amplification.54 In this research, a biotin tag was introduced to SAs with the PAL technique. The SWNH has binding sites for both biotin and AuNPs, and the bound AuNPs could be released with fructose and detected by an electrochemical detector. With this signal amplification mechanism, this method could quantify cells ranged from 5 × 102 to 1 × 106 cells mL-1 with a detection limit of 380 cells mL-1. So far, several approaches have been developed to improve the efficiency of the imine formation reaction, e.g. oxime/hydrazine ligations.55 The aniline catalyzing mechanism makes PAL a sensitive and rapid method for detecting cell surface SAs, as well as monitoring and tracking cell surface sialylation status. The PAL approach possesses potential future applications in uncovering complex carbohydrate-related biological processes and in clinical diagnosis. Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) is a powerful technique for characterizing complex structures of biomolecules, such as proteins, lipids and glycans. Recently, MALDI imaging MS (MALDI-IMS) was developed to characterize the spatial distribution and relative abundance of various biomolecules on the tissue.56,57 It was found that many diseases are associated with aberrant glycosylation. Therefore, profiling disease-associated/tissue-specific glycans have become one of the most attractive research fields.58 MALDI-IMS was used to profile N-glycans of clinical tissue blocks, which could correlate their molecular expression to histopathological changes and offer an alternative approach for biomarker identification.59-65 This technique consists of four steps: (i) tissue section preparation (e. g. sectioning FFPE tissues, deparaffinization, rehydration, and denaturing tissue proteins), (ii) releasing N-glycans from proteins by printing peptide-N-glycosidase F (PNGase F) over the sections, (iii) spray-coating the tissue with matrix, and (iv) analyzing N-glycans by MALDI-IMS.61 Additionally, glycans can be 14


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extracted from the tissue and derivatized for structural analysis by mass spectrometry or HPLC. One limitation of MALDI-IMS for in situ profiling of sialylation is the labile character of SAs that is prone to in-source/post-source decay, therefore affecting the profiling accuracy.66,67 In addition, partial salt formation with the carboxylic acid of SAs further complicates the signals and reduces the sensitivity. Moreover, like other techniques above, direct MALDI-IMS generally cannot distinguish the α2,3- and α2,6-SA linkages, which leads to unrevealed SA functions that relate to their linkages. Holst et al. addressed these SA related issues by using a linkage-specific SA derivatization approach on FFPE tissues for MALDI-IMS detection.68 They used a two-step derivatization of the carboxylic acid of sialylglycans: first with dimethylamine catalyzed with 1-ethyl-3-(3-(dimethylamino)propyl) carbodiimide (EDC) and 1-hydroxybenzotriazole (HOBt), and the second with ammonia. As a result, α2,6-linked SAs form a stable dimethylamide in the first step, which is unchanged in the presence of ammonia in the second step. However, α2,3-linked SAs react with the neighboring galactose to form a lactone in the first step but is hydrolyzed and the carboxylic acid reacts with the added ammonia to form a stable amide in the second step. These derivatizations result in 27.047 Da positive mass shift of α2,6-linked SAs, and 0.984 Da negative mass shift of α2,3-linked SAs (Fig. 7). This workflow showed the increase detection range of N-glycans by preventing SAs dissociation. Therefore, more SAs related biological information would be accessible. This is an elegant approach to image the cell surface SAs in different linkages and thus will provide a useful tool to distinguish cellular status and phenotypes for clinical diagnostic applications in the future.

15



Figure 7. Specific derivatization and MALDI MS imaging of α2,3-linked SAs and α2,6-linked SAs on the tissue.68 Adapted with permission from Ref 68. Copyright 2016 American Chemical Society.

III. BORONIC ACID-BASED APPROACHES TO DETERMINE SAS ON THE CELL SURFACE Boronic acid forms cyclic ester with cis-diol of carbohydrates under aqueous conditions and thus can be used for carbohydrate recognition applications.69 Phenylboronic acid (PBA) shows specific and robust conjugation with SAs in comparison with other sugars. Therefore, PBA derivatives have been explored as a useful molecular targeting platform for selective profiling cell surface SAs of both in vitro and in vivo samples. For example, a potentiometric method using PBA-modified gold electrode was developed to detect cell surface SAs as well as free SAs.70 This method is based on the charge density change of the electrode due to the binding of SA residues. It was successfully applied to differentiate the degree of tumor metastasis through the detection of cell surface SAs.71 Recently, Zhang’s group reported an inductively coupled plasma mass spectrometry (ICP-MS) for the analysis of the surface SAs of cancer cells.72 This method used biotinylated phenylboronic acid (biotin-PBA) to selective label SAs at physiological pH and the signal was enhanced by gold nanoparticles (AuNPs) in ICP-MS. In addition, PBA-functionalized quantum dots (QDs) was developed to track cell surface SAs continually.73 In particular, the high selectivity of PBA and superior photostability of QDs made it possible for tracking of sialylated glycoproteins and their intracellular distribution, trafficking, and diffusion dynamics on the cell membrane as well. Most recently, Liu et al. designed a PBA-conjugated and alkyne-bridged AuNP dimer probe for Raman scattering imaging of cancer cell surface SAs, in which the alkyne exhibited a single vibrational peak in the cellular Raman silent region (between 1800 and 2800 cm-1).74 They called this a background-free surface-enhanced Raman scattering (SERS) imaging technique (Fig. 8). With this technique, they profiled clinically relevant cancer tissues of different periods of differentiation and multiple metastasis degrees. Briefly, breast cancer tissue specimens were fixed on glass slides followed by incubation with PBA-conjugated and alkyne-bridged AuNP dimer probe for profiling SA expression. The appearance of both B-O 16


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and C≡C stretching peaks indicated the SA-specific alkyne-bridged surface-enhanced Raman scattering tags. This Raman imaging technique provides a three-dimensional visualization and distribution of SA expression of a variety of cells. Additionally, this technique is sensitive enough to profile SA expression at a single cell level without destroying the cells themselves.

Figure 8. Raman scattering imaging of SAs expression in cancer cells and tissues using PBA-conjugated and alkyne-bridged AuNP dimer probe.74 Adapted with permission from Ref 74. Copyright 2017 American Chemical Society.

Like periodate oxidation/ligation approach for profiling cell surface SAs, the profiling sensitivity with PBA ligation can also be improved with signal amplification approaches. Ju and colleagues developed a double signal amplification strategy for sensitive profiling cell surface SAs by using BSA modified quantum dots (PBA-QDs) and polysialic acid modified gold nanoparticles (PSA-AuNPs).75 In this work, cell surface SA moieties were first recognized by PBA-QDs, followed by the conjugation of PSA-AuNPs, through which more PBA-QDs were introduced onto cell surface via PSA bindings, providing strong signals for imaging. The signal was produced by cadmic cation release from the captured QDs to activate a strong fluorescence emission of the metal-responsive dye Rhod-5N. Using PBA-QDs probe, Cao et al. examined SA expression on the cell surface at a single cell level combined with a microfluidic platform.76 This platform provided a sensitive and high-throughput tool for SA analysis. In another study, Ji et al. conjugated PBA with low molecular weight polyethylenimine (PEI1.8k) to generate an amphiphilic PBA-grafted 17



PEI1.8k (PEI-PBA) nanovector for profiling cell surface SAs. PEI-PBA nanovector was originally designed for targeted RNA delivery to cancer cells through the recognition of cancer cell surface SA moieties.77 This alternative route to signal amplification involved PBA tagged fluorescent silica nanoparticles (FSNPs), which have ~350 fold increased fluorescent intensity when compared to a single dye molecule. In this approach, FSNPs were prepared through a reverse microemulsion process, and then modified with PBA tags on their surface via an aqueous 'thiol-ene' click reaction. PBA-FSNPs were selective and sensitive in probing SAs expressed on the surface of living cells.78 Also, Li et al. reported fluorescent SA-imprinted nanoparticles that could stain DU 145 cancer cells but not HeLa cells.79 This fluorescent nanoparticles may find applications for cancer cell imaging. In general, PBA derivatives can react with cis-diols of sugars not only from glycoproteins and glycolipids but also from ribose of RNAs in biological samples through reversible boronate ester formation. Therefore, the big concern is the selectivity when PBA derivatives are used for profiling glycans in biological samples. PBA esterification of sugars prefers basic condition. However, PBA derivatives selectively label cell surface SAs through esterification with its glycerol chain, which is further stabilized by B-N interaction from the 5-NAc of SA at pH 7.13,80 As a result, PBA derivatives exhibit selectivity toward SAs over other common saccharides, such as glucose, mannose, galactose, and ribose in biological samples under physiological conditions. Therefore, PBA can serve as a ligand for detection of SAs in complex biological samples. PBA monomers are known to be relatively weak concerning their interactions with SAs at the monomeric level. PBA-containing polymers have been explored by taking the advantage of polyvalent effect in improving the strength of interaction.81-83 Meta-amide substituted PBA is the most used structure as an efficient SA-binder for the above-described applications. Most recently, Matsumoto A. et al. reported heterocyclic BAs, such as 5-boronopicolinic acid, that can bind SAs specifically in much higher binding affinity than PBA.84 In addition, heterocyclic BAs show higher water solubility, which is often an issue limiting the range of medical applications. Therefore, heterocyclic BAs offer an alternative to the mostly used PBA for detecting cell surface SAs with advanced performance in the future. 18


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IV. SUMMARY Cell surface SAs are directly involved in biological processes as they exist as the terminal sugars of glycan structures of both glycoproteins and glycolipids. Cell surface sialylation status, SAs expression levels and their linkages, represents varying cellular states and contribute to the overall functionality of a cell. Profiling cell surface sialyation status facilitates understanding of SA-carrying glycoconjugate functions and the cell’s functionality as well. In the past decades, several advanced approaches have been developed for visualization, identification, and characterization of cell surface SAs. Lectin- and antibody-based affinity methods are conventional approaches used to directly profile cell surface SAs. In addition, chemical biology methods, such as metabolic engineering, biorthogonal labeling, selective chemical modification combined with fluorescence microscopy, flow cytometry, Raman scattering imaging, MRI, and MALDI-MS imaging have been proven as effective approaches for profiling specific sialylation status of cells, tissues, and in animals as well. Profiling sialylation of a specific cell surface glycoprotein has become doable by using FRET technique. Also, profiling cell surface SAs of a specific cell population has been demonstrated by targeted delivery of metabolic engineering agents. Nanoparticles and quantum dot staining show promising results as well, especially for signal amplification.

The

development

of

chemical

biology,

imaging

technology

and

nanotechnology will contribute to a fully understating of cell functionality and the molecular mechanism of a biological process involved. More research will focus on in vivo investigation with less detrimental and damaging effects of the reagents and reaction conditions towards the cells. Eventually, these advanced approaches will be fully used for both basic and applied biomedical researches and applications. Detecting specific cell surface sialylation status patterns will provide a practical tool for clinical diagnosis applications.

KEYWORDS Sialic acid (SA): a group of 9-carbon monosaccharides containing a carboxylic acid. N-Acetylneuraminic acid (Neu5Ac) and N-glycolylneuraminic acid (Neu5Gc) are the major sialic acids. In addition, 2-keto-3-deoxy-D-glycero-D-galacto-nononic acid (KDN) is a minor sialic acid. 19



Polysialic acids (PSAs): A linear polymer of α-2,8 (or 2,9)-linked sialic acid. Sialylation Status: Sialic acids expression levels and their linkages on the cell surface, collectively known as sialylation status, which are often varied at altered cellular status and environments. Metabolic glyco-engineering and labeling: A sugar substrate analog carrying a functional group is incorporated into cell surface glycoconjugates via the metabolic pathway, from which a second probe can be introduced by labeling the functional group for analysis, imaging, and purification applications. Bioorthogonal chemistry: Chemo-selective reactions for labeling biomolecules in living systems without interfering with native biological processes. Click Chemistry: A class of biocompatible reactions commonly used for selectively labeling of biomolecules of choice with specific tags or probes. The classic click reaction is the Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC). Periodate oxidation and aniline-catalyzed oxime ligation (PAL): An aldehyde group on sialic acids is generated by mild periodate oxidation, which is used for imine ligation in the presence of aniline for analysis and imaging of cell surface sialic acid applications. Boronic acid labeling: Boronic acid forms covalent conjugation with carbohydrates via cyclic ester from the cis-diol of the carbohydrates under aqueous conditions and thus can be used for carbohydrate recognition and imaging applications. MALDI Imaging Mass Spectrometry (MALDI-IMS): A mass spectrometry-based imaging using matrix-assisted laser desorption ionization MS technique, which analyses proteins, lipids and glycans in a tissue, which measures the distribution of the biomolecules at one time without destroying the biological sample.

AUTHOR INFORMATION Corresponding Authors *[email protected] (X.-L. Sun) ORCID Xue-Long Sun: 0000-0001-6483-1709 Notes 20


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The authors declare no competing financial interest.

ACKNOWLEDGEMENTS This work was supported by John C. Vitullo’s Pilot and Bridge Program Award from the Center for Gene Regulation in Health and Disease (GRHD) and Faculty Research Development Award at Cleveland State University (XL. Sun). This work was partially supported by grant from The National Natural Science Foundation of China (31771627, H. Nie).

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Legends of Figures Figure 1. Diverse structures of sialic acids and sialyl linkages. Neu5Ac is used as a representative sialic acid in the figure. Modified from Ref 2.

Figure 2. Diverse approaches for profiling cell surface SAs: Bio-Affinity approaches (a. lectin, b. antibody, c. recombinant protein); Chemical Biology approaches (d. metabolic glyco-engineering and labeling, e. chemical modification and conjugation, f. boronic acid coupling-based microscope imaging, flow cytometry, Raman scattering imaging and xenon MRI; and g. MALDI-IMS). Neu5Ac is used as a representative sialic acid in the figure. Neu5Ac-R: Neu5Ac derivative.

Figure 3. Profiling cell surface SAs by metabolic incorporation of azide into cell surface SAs of HaCaT by using Ac4ManNAz. A. Schematic illustration of metabolically engineering and click labeling of cell surface SAs carrying azide via the BTTAA-assisted CuAAC. B. Analysis of cell surface SAs carrying azide by conjugation with alkyne-biotin and streptavidin-Alexa Fluor 488, and analyzed by flow cytometry. C. Confocal fluorescence microscopy of the labeled cells. The nuclei were counterstained with Hoechst 33342. Scale bars, 20 µm. D. Immunoblot analysis of azide-incorporated cell surface glycoproteins in HaCaT cells.20 Adapted with permission from Ref 20. Copyright 2015 the American Society for Biochemistry and Molecular Biology.

Figure 4. Imaging the sialylation state of a specific glycoprotein using metabolic labeling and antibody labeling followed by 2-photon fluorescence lifetime imaging microscopy (FLIM). A. The azide functionality was metabolically incorporated in sialylated 27



glycoproteins of U87MG cells with Ac4ManNAc, followed by click labeling with fluorophore probe (DIBAC–647) as the acceptor fluorophore. Next, the donor fluorophore was introduced to sialylated integrin αVβ3 glycoprotein with fluorophore-labeled Fab fragment (Fab–594). B. Two-photon FLIM imaging of sialylated integrin αVβ3. Scale bar = 50

µm.29 Adapted with permission from Ref 29. Copyright 2013 John Wiley and Sons.

Figure 5. MRI with xenon hyper-CEST biosensors targeted to metabolically labeled cell-surface SAs using bioorthogonal chemistry. A. Three key steps needed to image metabolically labeled SAs on live cells with xenon Hyper-CEST biosensors. B. Hyper‐CEST MRI with the live cell. Yellow indicates Xenon Hyper‐CEST MRI of the cells treated with Ac4ManNAz. The percentage CEST effect is displayed (pseudocolor) overlaid a reference proton MRI.31 Details please see Ref. 31. Adapted with permission from Ref 31. Copyright 2015 John Wiley and Sons. Figure 6. Fluorescent imaging of cells via PAL labelling. HeLa cells were observed for fluorescent images (488 nm ex/515 nm em) and DIC images (from left to right): no periodate and no NBDAO dye; no periodate and NBDAO dye; and periodate with NBDAO dye. Scale bars represent 50 µm.47 Adapted with permission from Ref 47. Copyright 2012 American Chemical Society.

Figure 7. Specific derivatization and MALDI MS imaging of α2,3-linked SAs and α2,6-linked SAs on the tissue.68 Adapted with permission from Ref 68. Copyright 2016 American Chemical Society. Figure 8. Raman scattering imaging of SAs expression in cancer cells and tissues using PBA-conjugated and alkyne-bridged AuNP dimer probe.74 Adapted with permission from Ref 74. Copyright 2017 American Chemical Society.

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Recent Chemical Biology Approaches for Profiling Cell Surface

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