Gold Nanoparticles Conjugated with Glycopeptides for Lectin Detection Protein & Peptide Letters, 2018, Vol. 25, No. 1 85

lactose-binding lectins primarily expressed on hepatocytes and associated with a clearance of glycoproteins that carry galactose units at the termini of their glycan chains [5]. Spe-cific expression of ASGPR on liver cancer cells is useful for receptor-mediated drug delivery [8], however, detail physio-logical properties of ASGPR are still unclear. The selective detection and imaging of galactose-binding proteins includ-ing ASGPR are required to find out the complex biological processes mediated by them. Recently, inorganic nanomaterials such as metal nanopar-ticles [10-12] and nano-surface [13-16] have attracted much attention in protein detection. Gold nanoparticle (GNP) is one of attractive materials for lectin detection, because GNP shows red color due to a characteristic surface plasmon ab-sorption around 520 nm and its absorption changes by the formation of aggregates with lectins that have multiple bind-ing sites to sugars [12, 17-22]. Generally, the interaction between lectins and monosaccharides is weak [1], however, the arrangement of many saccharide units on the nanoparti-cles enhances the binding affinity to lectins due to the multi-valent interaction called the cluster glycoside effect [23, 24]. In many cases, monosaccharide derivatives easy to synthe-size have been used for the conjugation with GNPs. We, previously, demonstrated that sugar-modified pep-tides (glycopeptides) were useful ligands for the detection and characterization of lectins compared to the sugar unit alone [25, 26]. A synthetic α-helix peptide library with a monosaccharide was prepared by a systematic substitution of amino acids around the Lys having a monosaccharide unit in its side chain [25]. In addition, a semi-synthetic β-loop gly-copeptide library was constructed by the combination of a phage display technique and a selective chemical modifica-tion reaction [26]. A mannose unit was selectively intro-duced into the side chain of the Cys arranged at the center of randomized loop region. In these glycopeptide libraries, the sugar unit and surrounding amino acids cooperatively worked for high binding affinity to target lectins. Further-more, mannose-modified peptides were combined with GNPs to produce probes for the selective detection of conca-navalin A (ConA), a mannose-binding lectin [27]. Glycopep-tide-conjugated GNPs could detect ConA as a color change from red to violet in naked eye with high binding affinity. Therefore, the combination of GNPs and glycopeptides is useful approach for the lectin detection with a diverse affin-ity and selectivity. To expand our glycopeptide-GNP conju-gates for the detection of other lectins, in this study, we have developed gold nanoparticles conjugated with galactose-modified peptides for the detection of galactose-binding lect-ins and imaging of a galactose binding protein on cell sur-face.

2. MATERIALS AND METHODS

All chemicals and solvents were of reagent or HPLC grade and were used without further purification. Ricinus Communis Agglutinin I (RCA120), lactose and α-methyl mannose were purchased from Sigma-Aldrich. RP-HPLC was performed on the Hitachi L7000 system using a COS-MOSIL 5C18-ARII (φ10×250 mm) column for purification with a linear gradient of acetonitrile/0.1% trifluoroacetic acid (TFA) at a flow rate of 3.0 mL/min. Electrospray ionization

mass spectrometry (ESI-MS) was conducted on a Shimadzu LCMS-2010. Transmission electron microscopic (TEM) observation was performed on a Hitachi H-7500 electron microscope (Hitachi, Tokyo, Japan). Dynamic light scatter-ing (DLS) was measured with Zetasizer Nano ZS90. Galactose-modified peptides were synthesized by the solid-supported peptide synthesis method (Supplementary Material). Briefly, all peptide were synthesized on a TentaGel S RAM resin (Hipep) by the Fmoc solid-phase method. The allyl (Al) was used as a side chain protection group of Glu to introduce of 2’-aminoethyl-O-β-D-galactopyranoside. After stepwise elongation of amino acids, the side chain protection group of Glu(OAl) was selectively removed by using Pd(PPh3)4 and 2’-aminoethyl-O-β-D-galactopyranoside was introduced into the side chain of Glu on the resin. After cleavage and side chain deprotection of peptides, galactose-modified peptides were purified by re-versed phase HPLC and identified by ESI-MS. Detail procedure for preparation of galactose-modified peptides (GP/GNPs) was described in Supplementary Mate-rial Section. Briefly, GNP with 22 nm size was prepared by reduction of HAuCl4 using trisodium citrate. GNPs were coated with LA-PEG-Mal and LA-PEG by the selective re-action between Au atom and a lipoic acid (LA) unit (PEG: polyethylene glycol). After removal of excess amount of LA-PEG-Mal and LA-PEG by centrifugation, galactose-modified peptides were conjugated with the coated GNPs via a thioether linkage. Absorption spectra were recorded at 25°C on a Shimadzu UV-2550 spectrometer using a quartz cell with 1.0 cm path length. The stock solution of RCA120 was prepared with HEPES buffer (10 mM pH 7.0 containing 150 mM NaCl, 1.0 mM CaCl2, 1.0 mM MnCl2). The RCA120 solution was added to the 200 µL of GP/GNP solution (0.5 nM, HEPES buffer). The final concentration of RCA120 was varied from 0.5 nM to 1000 nM. After 12 h incubation at 4°C, absorption spectra were measured. The effective concentration in aggregation of GP/GNPs (EC50) was estimated based on the absorbance change at 525 nm using GraphPad Prism. The RCA120 solution was added to the 200 µL of GP/GNP solution containing 60 mM α-methyl mannose or 60 mM lactose (Lac) (0.5 nM, HEPES buffer). The final concentration of RCA120 was fixed at 100 nM. After 12 h incubation at 4°C, absorption spectra were measured. HepG2 and MCF7 cells were placed on 22×22 mm cover slip in 6 well cell culture plates (2×105 cells / well) and cul-tured overnight at 37ºC under 5% CO2 condition. After washing twice with PBS, 1 mL of GP/GNPs (0.2 nM) in 10 mM HEPES buffer were added in each well and incubated for 18 h at 37ºC under 5% CO2 condition. After incubation, cells were washed twice with PBS and fixed with 4% para-formaldehyde solution for 15 min. The cover slips were coated with 90% glycerol and sealed to slide glass. Dark-field images based on elastic light scattering were taken us-ing a Nikon microscope (TieU) with an immersion dark field condenser. For the inhibition between HepG2 cells and WF(Gal)/GNP, Lac or Me-Man (100 mM) were co-incubated with WF(Gal)/GNPs, and dark-field images of cells were obtained in the same manner described above.

86 Protein & Peptide Letters, 2018, Vol. 25, No. 1 Tsutsumi et al.

3. RESULTS AND DISCUSSION

RCA120, a galactose-binding lectin, was selected as a model lectin. Galactose-modified peptides with various amino acids (hydrophobic, hydrophilic and anionic residues) around a galactose unit were designed based on the previous study (Figure 1a) [27]. Cationic residues were not used, be-cause the cationic glycopeptide formed insoluble aggregation during the conjugation with citric acid-coated GNP [27, 28]. 2’-Aminoethyl-O-β-D-galactopyranoside (Gal) was intro-duced into the side chain of Glu as a galactose unit. Cys resi-due was arranged at the C-terminus of peptides via a (aeea)2 linker as a conjugation unit with GNP. Although structure of these glycopeptides is random coil (data not shown), multi-ple modification of glycopeptides on GNP can contribute the strong binding affinity by the multivalent effect [27]. GNP was coated with PEG to maintain well-dispersed condition after conjugation with glycopeptides. GNPs conjugated with GP/GNPs were prepared in a manner shown in Figure 1b. At first, GNP was coated with LA-PEG and LA-PEG-Mal. Then, GPs were introduced into PEG-coated GNPs via a thioether linkage produced by the reaction between a Mal group and Cys at C-terminus of the peptides. The optimal content of LA-PEG-Mal for both stable dispersion of GP/GNPs and binding to RCA120 was determined as 20% (detail is described in Supplementary Material Section). In the titration experiment of RCA120, GP/GNPs showed a decrease of absorbance according to the addition of RCA120 (Figure 2a and Figure S3). On the other hand, there is no significant spectral change of LA-PEG/GNP that does not have a galactose unit in the presence of RCA120 (Figure 2b). These results suggest that the aggregation of GP/GNPs is induced through the selective binding to RCA120. The absor-bance changes of GP/GNPs at 525 nm were plotted against

the concentration of RCA120 (Figure 3) and titration curves were obtained to evaluate the effective concentration in ag-gregation of GP/GNPs (EC50, Table 1). The EC50 values of AA(Gal)/GNP, WF(Gal)/GNP, TS(Gal)/GNP and ED(Gal)/ GNP were estimated as 66.2 nM, 43.2 nM, 38.6 nM and 104.4 nM, respectively. TS(Gal)/GNP showed the lowest EC50 value among GP/GNPs. RCA120 has several binding sites for the galactose, and there are hydrophilic amino acids (Thr24, Glu26, Gln35, Asn42 and Asp44) around one of galactose binding sites (Figure S4a). This result indicates that the hydrogen bonds between these amino acids and Thr/Ser residues of TS(Gal) contribute to the efficient aggre-gation of TS(Gal)/GNP. Since RCA120 also has hydrophobic amino acids (Trp93 and Tyr125) around the another galac-tose binding site (Figure S4b), hydrophobic interaction be-tween these amino acids and Trp/Phe residues of WF(Gal) might work well for the aggregation of WF(Gal)/GNP. ED(Gal)/GNP showed the highest EC50 value, due to that it might be caused by electrostatic repulsion between anionic amino acids (Glu26 and Asp44) and Glu/Asp of ED(Gal). Table 1. EC50 values of GP/GNPs aggregation by RCA120.

EC50 / nM EC50 / nM

([BSA] = 10 µM)

AA(Gal)/GNP 66.2 58.9

WF(Gal)/GNP 43.2 51.4

TS(Gal)/GNP 38.6 52.3

ED(Gal)/GNP 104.4 n. d.a a: not determined

Figure 1. (a) Sequences of galactose-modified peptides (GPs). (b) Schematic illustration of the preparation of gold nanoparticles (GNPs) conjugated with GPs.

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Gold Nanoparticles Conjugated with Glycopeptides for Lectin Detection Protein & Peptide Letters, 2018, Vol. 25, No. 1 87

Figure 3. The titration plot of absorbance change of GP/GNPs with RCA120 at 525 nm. Diamond: AA(Gal)/GNP, square: WF(Gal)/ GNP, triangle: TS(Gal)/GNP, cross mark: ED(Gal)/GNP.

The absorbance of GP/GNPs also decreased according to the increase of RCA120 concentration in the presence of excess BSA (10 µM) (Figure S5). The EC50 values of AA(Gal)/ GNP, WF(Gal)/GNP and TS(Gal)/GNP were estimated as 58.9 nM, 51.4 nM and 52.3 nM, respectively. These EC50 values are similar to those in the absence of BSA. These re-sults suggest that AA(Gal)/GNP, WF(Gal)/GNP and TS(Gal)/GNP can selectively interact with RCA120. On the other hand, the EC50 value of ED(Gal)/GNP was not deter-mined because the absorbance change of ED(Gal)/GNP did not reach to the maximum compared to other GP/GNPs. ED(Gal)/GNP might interact with BSA. Next, inhibition experiments in the aggregation of WF(Gal)/GNP with RCA120 were performed (Figure 4). The absorbance of WF(Gal)/GNP decreased by the aggregation with RCA120. In the presence of excess lactose (Lac) (60 mM), there was no significant absorbance change of

WF(Gal)/GNP according to the addition of RCA120. On the contrary, in the presence of excess α-methyl mannose (Me-Man) (60 mM), the absorbance of WF(Gal)/GNP decreased by the addition of RCA120 and the absorption spectrum was similar to that of aggregated WF(Gal)/GNP with RCA120. These results clearly showed that Lac inhibits the WF(Gal)/ GNP binding with RCA120, but Me-Man does not, and that WF(Gal)/GNP selectively interacts with RCA120 and forms the aggregate. Finally, a galactose binding protein on the surface of HepG2 cells was visualized by using GP/GNPs as optical probes, because GNP was available for optical imaging due to its strong light scattering property [29, 30]. Scattering light derived from GNP was observed on the surface of HepG2 cells incubated with GNPs conjugated with GPs (Figure S6). WF(Gal)/GNP showed the strongest scattering light on the cell surface among GP/GNPs (Figure S6 and Figure 5a). ASGPR is a candidate of galactose binding pro-teins on the cell surface, because it is reported that liver can-cer cell lines including HepG2 cells highly express ASGPR [31]. It is indicated that ASGPR of rat has a Trp residue near the galactose binding site of its carbohydrate recognition domain by previous reports [32, 33], and ASGPR on human cells has a similar sequence to that of rat cells [34]. Hydro-phobic/aromatic interaction between this Trp residue of pro-tein and Trp/Phe residues of WF(Gal) might effectively work for the imaging of ASGPR on the surface of HepG2 cells. There was no significant scattering light on the surface of HepG2 cells incubated with LA-PEG/GNP or WF(Gal)/GNP in the presence of excess Lac (Figure 5b and 5c). On the other hand, light scattering image similar to WF(Gal)/GNP was obtained on HepG2 cells incubated with WF(Gal)/GNP in the presence of excess Me-Man (Figure 5d). These results suggest that WF(Gal)/ GNP selectively visualizes ASGPR on HepG2 cell surface. The background scattering light in Figure 5a and 5d might come from secretory galactose bind-ing proteins, because the background scattering light de-crease in the presence of excess Lac (Figure 5c). In the case of MCF7 cells on which the expression level of a galactose binding lectin is low, LA-PEG/GNP and all GP/GNPs did not show any scattering light on the cell surface (Figure S7).

Figure 2. (a) Absorption spectra of WF(Gal)/GNP upon the addition of RCA120: [WF(Gal) /GNPs] = 0.5 nM, [RCA120] = 0, 0.5, 1.0, 2.0, 5.0, 10, 20, 50, 100, 200, 500 and 1000 nM in 10 mM HEPES buffer (pH 7.0 containing 150 mM NaCl, 1.0 mM CaCl2, 1.0 mM MnCl2). (b) Absorption spectra of LA-PEG/GNP (0.5 nM) in the absence and presence of RCA120 (100 nM) in 10 mM HEPES buffer.

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88 Protein & Peptide Letters, 2018, Vol. 25, No. 1 Tsutsumi et al.

This result also supports that WF(Gal)/GNP selectively bind to the galactose binding protein on the surface of HepG2 cells.

CONCLUSION

We have developed gold nanoparticles with galactose-modified peptides (GP/GNPs) as organic-inorganic hybrid probes for lectin detection and imaging. GP/GNPs can detect RCA120 by the selective binding and the aggregation forma-

tion. The aggregation ability of GP/GNPs for RCA120 de-pends on the amino acid residues surrounding the galactose unit. In particular, the EC50 value of TS(Gal)GNP in aggre-gation with RCA120 is 38.6 nM, that is the most effective among GP/GNPs, in which the hydrophilic side chains of Thr/Ser residues intensifies the binding of the galactose unit to RCA120. It is demonstrated that the interaction between GP/GNPs and RCA120 is selective by the titration experiment in the presence of BSA and the inhibition experiment using excess lactose. Furthermore, a galactose binding protein on

Figure 4. Inhibition of the aggregation of WF(Gal)/GNP induced by RCA120 with excess Lac, but not with excess Me-Man. (a) Absorption spectra of WF(Gal)/GNP, [WF(Gal)/GNP] = 0.5 nM, [RCA120] = 100 nM, [Me-Man or Lac] = 60 mM, in 10 mM HEPES buffer (pH 7.0 containing 150 mM NaCl, 1.0 mM CaCl2, 1.0 mM MnCl2). (b) The absorbance changes of WF(Gal)/GNP in the presence of RCA120, RCA120 and Lac, RCA120 and Me-Man.

Figure 5. Dark-field light scattering images of HepG2 cells incubated with (a) WF(Gal)/GNP, (b) LA-PEG/GNP, (c) WF(Gal)/GNP and Lac, and (d) WF(Gal)/GNP and Me-Man. Scale bar is 50 µm.

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Gold Nanoparticles Conjugated with Glycopeptides for Lectin Detection Protein & Peptide Letters, 2018, Vol. 25, No. 1 89

the surface of HepG2 cells is successfully visualized using WF(Gal)/GNP as an optical probe. It is demonstrated that the binding of WF(Gal)/GNP to the surface of HepG2 cells is also selective by the inhibition experiment using lactose and imaging experiment of MCF7 cells. Thus, GNPs conjugated with glycopeptides will be useful probes for the selective detection and imaging of lectins.

CONSENT FOR PUBLICATION

Not applicable.

CONFLICT OF INTEREST

The authors declare no conflict of interest, financial or otherwise.

ACKNOWLEDGEMENT

This work was supported in part by JSPS KAKENHI. The authors acknowledge Prof. Hideya Yuasa, Tokyo Insti-tute of Technology, for the guidance of synthesis of 2’-Aminoethyl-O- β-D-galactopyranoside.

SUPPLEMENTARY MATERIAL

Supplementary material is available on the publisher's website along with the published article.

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