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Modeling Glyco-Collagen Conjugates Using Host-Guest Strategy to Alter Phenotypic Cell Migration and In Vivo Wound Healing Sivakoti Sangabathuni, Raghavendra Vasudeva Murthy, Madhuri Gade, Harikrishna Bavireddi, Suraj Toraskar, Mahesh V. Sonar, Krishna N. Ganesh, and Raghavendra Kikkeri ACS Nano, Just Accepted Manuscript • DOI: 10.1021/acsnano.7b01789 • Publication Date (Web): 27 Oct 2017 Downloaded from http://pubs.acs.org on October 29, 2017
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Modeling
Glyco-Collagen
Conjugates
Using
Host-Guest Strategy to Alter Phenotypic Cell Migration and In Vivo Wound Healing Sivakoti Sangabathuni,†,# Raghavendra Vasudeva Murthy,†,# Madhuri Gade,† Harikrishna Bavireddi,† Suraj Toraskar,† Mahesh V. Sonar,† Krishna N. Ganesh,*,† Raghavendra Kikkeri*,† †
Indian Institute of Science Education and Research, Dr. Homi Bhabha Road, Pune-411008,
India. #
Equal contribution
E-mail:
[email protected];
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ABSTRACT The constructs and study of combinatorial libraries of structurally defined homologs extracellular matrix (ECM) glycopeptides can significantly accelerate the identification of cell surface markers involved in a variety of physiological and pathological processes. Herein, we present a simple and reliable host-guest approach to design high-throughput glyco-collagen library to modulate the primary and secondary cell lines migration process. 4-Amidoadamantyl-substituted collagen peptides and β-cyclodextrin (β-CD) appended with mono or disaccharides were used to construct self-assembled glyco-collagen conjugates (GCC) which were found to be thermally stable, with a triple helix structures and nanoneedles-like morphologies that altered cell migration processes. We also investigated the glycopeptides mechanisms of action, which included interactions with integrins and cell signalling kinases. Finally, we report murine wound models to demonstrate the real-time application of GCC. As a result of our observations, we claim that the host-guest model of ECM glycopeptides offers an effective tool to expedite identification of specific glycopeptides to manipulate cell morphogenesis, cell differentiation metastatic processes and its biomedical applications. KEYWORDS: collagen, cyclodextrin, carbohydrates, self-assembly, wound healing, mouse model.
Protein glycosylation is a post-translational process responsible for more than 50% of protein modifications in nature. Attaching glycans to proteins results in a dramatic increase in the bioavailability, stability and solubility of proteins.1-5 The major roadblock in applying glycopeptides or glycoproteins to delineate the biology of disease pathways is the limited availability of affordable and accessible synthetic platforms useful for deciphering the
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biochemical basis of glycoprotein interactions. Recently, signature-based profiling of glycopeptides has provided a powerful strategy for mimicking the protein structure and understand the functions of post-translational modifications.6-10 Similar synthetic approaches were recently used to prepare tumour-associated MUC-1 conjugates, collagen,11 coiled-coils and anti-freezing glycopeptides for studying structure-function relationships of native proteins.12-14 Synthesis of glycopeptides is usually carried out by incorporating glycosylated amino acid residues in the peptide skeleton following a step-wise process15 or via convergent synthesis, in which functionalization is performed in the final step.14,16,17 Host-guest interaction between sugar functionalized β-cyclodextrin (β-CD) and adamantyl scaffolds has recently been used for the convergent assembly of multivalent glycodendrimers.18,19 In comparison to the step-wise process, host-guest strategy dictates high-throughput processes which yield dynamic combinatorial libraries in a short span. Based on such advantages, we used 4-adamantyl-capped peptides and sugar-appended β-cyclodextrin and created a versatile combinatorial library of glycopeptides. We have combined solid phase peptide synthesis, high-throughput phenotypiccells migration assay, molecular biology and murine wound healing model to alter specific glycopeptides mediated cell migration. Briefly, we designed a library of glyco-collagen conjugates (GCC), one of the most abundant fibrous structural proteins in the extracellular matrix that is extensively involved in cell migration process.20-24 We modified the collagen peptide (CP) with 4-adamantyl derivatives to induce host-guest interactions with β-cyclodextrin derivatives, in a way that the triple helicity of the collagen remained intact. We have used these molecules for performing high-throughput cell migration assays using primary and secondary cell lines. Moreover, we have conducted mechanistic studies to identify specific combinations that can evidently be involved in murine wound healing processes. Our convergent method of
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screening cell migration is highly scalable and allows identifying different targets. It may represent a platform to be used in drug and inhibitors discovery. RESULTS AND DISCUSSIONS Building glyco-collagen library: Previous studies on functionalization of collagen have revealed that the middle site (Y-position) of proline-hydroxyproline-glycine triad is ideal for bioconjugations without hampering the triple helix structure of collagen.14 In order to monitor the biological responses mediated by glyco-collagens, we synthesized two control CPs (C-1 and C2) and adamantyl-CP (C-3) using a solid phase peptide synthesizer following standard protocols (Figures 1, S1 and S2). The CPs were purified by reverse phase semi-preparative HPLC (Figure S4 and Table S1) and characterized by MALDI-TOF data. The binding events between specific glycans and their complementary receptors occur in a multivalent and cooperative manner.25,26 Hence, β-cyclodextrin (β-CD) conjugates of common mono/disaccharide derivatives, found in mammalian cell surfaces, were synthesized (G-2 to G-7) to mimic the collagen sugar microenvironment (Figures 1).27-29 Circular dichroism (CD) spectra and the corresponding thermal unfolding curves of peptides C-1 to C-3 and their β-CD complexes were used to verify the triple-helix formation of the CPs (Figure S7 and Table S2, S3). The morphological structures of peptides C-1, C-2 and C-3 were confirmed using Scanning Electron Microscopy (SEM). As seen from SEM images, peptides C-1, C-2 self-assembled into nano-needle structures whereas, C-3 displayed flat nano-needles with an average size of 800 nm × 120 nm (Figure S8ii). Collagen peptide C-3 spontaneously formed a non-covalently conjugated complex by mixing it with stoichiometric amounts (1:1) of β-CD derivatives (G-1 to G-7) and formed a combinatorial library of GCC (Figure S5a). ESI-MS (soft ionization) data for C-3/G-1 reveals a
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m/z of 862 [M+2K+2H]+/4 (Figure S5b) corresponding to host-guest complexes of GCC. The peaks at m/z 768 and 1135, corresponding to individual C-3 and G-1 respectively, stem from partial dissociation of C-3/G-1 complex due to collision of the analyte ions with neutral gas molecules during the ionization process. Similarly, mass spectral data of C-3/G-4 (m/z 1242; [M+K+3H]+/4), C-3/G-5 (m/z 1213; [M+K+3H]+/4), C-3/G-10 (m/z 1525; [M+K+3H]+/4) and C3/G-6 (m/z 1289; [M+K]+/4) confirmed the existence of supramolecular assemblies of respective GCCs and β-CD derivatives (Figure S5c-i). 1H-NOESY NMR also supported the anchoring of βCD on the adamantyl moiety and formation of a supramolecular assembly. Overlapping of ProHyp of C-3 and β-CD peaks in the range 4.0 to 3.2 ppm prevented the assignment of β-CD peaks required for identifying changes due to C-3/G-1 complex formation. However, the adamantyl Ha-Hc peaks of C-3 revealed strong NOE interactions with the protons in β-CD region (Figure S6), confirming the formation of supramolecular complex between adamantane and β-CD, not with the phenyl group. The morphology of the complexes was confirmed by SEM studies (Figure S8ii-iii and Table S5). GCC C-3/G-1 formed bi-fringe needle-like morphologies, with slightly different topology of the host-guest complexes as compared to its native form. Similar morphology was observed for C-3/G-2 complexes. To address the carbohydrate-protein interaction of GCC, their binding affinity to mannose-specific ConA and galactose-specific-PNA was examined. Inhibition of binding studies of these lectins with mannose-BSA and galactoseBSA, in the presence of GCC and the IC50 value were evaluated. As expected, C-3/G-4 exhibited strong inhibition as compared to other GCC combinations, while PNA lectin showed strong binding preference to C-3/G-3 glycopeptide. The results clearly indicated that glycopeptides display strong preferences toward specific lectins (Figure S9 and Table S6).
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Figure 1. Molecular structure of collagen peptides (C-1 to C-3) and sugar-capped β-cyclodextrin derivatives (G-1 to G-7). Cell migration assays using a library of combinatorial GCC. We have carried out cell migration assays using the synthetic GCC. Primary fibroblast cells, isolated from murine tissues (ear and skin)30 and secondary fibroblast cell lines, such as NIH-3T3 and BJ cells were used in our studies. A variety of human cancer cells, including cervical epithelial cancer (HeLa), breast cancer (MDA-MB-231) and endothelial cells (hUVEC) were also used in our cell migration studies. The choice of the specific cell lines was based on the fact that fibroblasts and endothelial cells are directly involved in collagen mediated wound healing processes31 and cells, isolated from primary murine tissues, often mimic in vivo responses. Confirming the specific glycocollagen molecules responsibility for cancer cells motility has implications in basic and translational research for targeting invasion and metastatic processes.32
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Cell migration assays were carried out using standard literature protocols.33 Phenotypic cells were seeded in 24-well plates, allowed to form a monolayer by maintaining at 37 °C in a CO2 incubator (Figure 2i), each monolayer was scratched with a 1000 µl sterile tip generating wounds. Seven different combination of GCCs (200 µM) were added and the wells were assayed. Commercially available Collagen-II (200 µM), carbohydrates (G-1 to G-7) and bare CPs (C-1 to C-3) were used as controls. The bright field images were recorded every single hour until all specific combinations of GCCs resulted in 100 % wound healing as compared to untreated cells. We hypothesized that the disparity in cell surface sugar receptors on different cells types influence the rate of the wound healing process. The hierarchical clustering results were used to trace particular combinations of GCCs responsible for specific cell type wound healing and to elucidate the mechanism(s) of these processes (Figure 2ii). For consistency, results were collected in duplicates and performed in triplicates. The hierarchical clustering analysis (HCA) responses of HeLa cell migration indicated the presence of several distinct clusters. Although many combinations of the sugar-peptide complexes revealed increased cell migration during the wound healing process as compared to the individual components, particularly C-3 GCC addition induced stronger cell migration responses as compared to that of the C-1, C-2 GCCs. Among the sugars, lactose conjugated to peptide (C-3/G-7) showed 90-100 % wound healing after 6 hours, maltose (C-3/G-6) - 80-90 % and fucose (C-3/G-5) - 70-80 %. HeLa cells were shown to express several types of sugar receptors, among them lactose receptors are considered as ‘Trojan horse’ (Figures 2ii and S10iv).34 Comparative studies of wound healing behavior of two-fibroblast cell lines from different origins (human and mouse), i.e., NIH-3T3 and BJ, were carried out (Figures 2ii and S10ii-iii).
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Both cell lines exhibited different sugar based hierarchical clusters, indicating differences in cell surface sugar receptors. It has been shown that fibroblasts in general express growth factors, which selectively bind to heparin and glucosamine sugar moieties,35 while NIH-3T3 cells do not contain galactose specific asialoglycoprotein receptors.36 Thus, even though both cells are of the same phenotype, they may display disparity in cell migration in the presence of glycopeptides conjugations. With regard to the NIH-3T3 cell line, the combination of C-3/G-4 exhibited 90100 % cell migration in comparison to other GCCs. In contrast C-3/G-3 and C-3/G-2 were effective in wound healing of BJ cells after 10 h (Figure S10ii). The breast cancer cell lines MDA-MB-231 exhibited superior wound healing after 6 hours when galactose (C-3/G-2), glucose (C-3/G-3) GCCs were administered (Figure S10v).37 Based on these results, we hypothesize that the origin of HCA of specific cell line is associated with the interaction between specific glyco-collagen peptides and its receptors on the cell surfaces. The experiments with the primary cell lines isolated from different organs of murine (ear and skin) also demonstrated that C-3 complex with β-cyclodextrins modified with galactose (G2) exhibited 90-100 % wound healing in both primary cell lines (Figures 2ii-iii and S10 vi-vii) while, glucose (G-3) and lactose (G-7) displayed a variable cell migration. Endothelial hUVEC cells displayed 90-100 % wound healing effects with complexes of galactose (C-3/G-2), complexes with fucose (C-3/G-5) and lactose (C-3/G-7) exhibited 70-80 % wound healing (Figure S10i).38 All these values were found to be superior to control collagen-II, suggesting that specific sugar-peptide combinations fine-tune the microenvironment essential for cell migration as compared to the full-length collagen peptide.
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Figure 2. (i) Schematic representation of the synthesis of a library of glyco-collagen peptides using host-guest methods and its effect in cell migration (ii) Hierarchical clustering analysis (HCA) of cell migration assay with different GCC combinations after 6 h (HeLa, MDA-MB231), 10 h for (BJ, hUVEC, NIH-3T3), and 10 h for skin and ear cells extracted from a mouse; Conc. of GCC (200 µM), Conc. C-1 to C-3 (200 µM) and Conc. G-1 to G-7 (200 µM). The
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origin of HCA of a specific cell line is associated with the interaction between specific glycocollagen peptides and their receptors on cell surfaces (iii) Bright field images of progressing wound healing of skin cell lines after 10 h; (a) C-3; (b) C-3/G-1; (c) C-3/G-2; (d) C-3/G-3; (e) C-3/G-4; (f) C-3/G-5; (g) C-3/G-6; (h) C-3/G-7. In order to elucidate the mechanism of wound healing, we performed fluorescent imaging experiments with 5-carboxyfluorescein conjugated C-3 peptide (C-3-F) with four distinct combinations of sugars (G-1, G-2, G-5, G-7) (Figure S2 and S8(i)). The data from confocal images with two secondary cell lines (HeLa and BJ) and primary cell line isolated from skin, expressing different rates of migration with C-3-F GCCs, were generated (Figure 3i). After incubation for 6 hours of C-3-F and different combinations of GCC, fluorescence was observed on the cell surfaces and inside the cells. Among them, C-3-F and C-3-F/G-1 did not bind to all the three cell lines, while BJ cells express C-3-F/G-2, C-3-F/G-7 glycopeptides on the cell surfaces in a more pronounced manner as compared to other glycopeptides. These findings suggest that C-3-F GCC derivatives bind to the cell adhesive molecules through specific sugarpeptide combination. Thus, the sugar and collagen sequence of glycopeptides seem to be crucial factors in cell uptake and specific interactions. We hypothesize that the glycopeptides bind to cell surface receptors, such as integrins to regulate cell migration processes while, weak fluorescence intensity indicates less impact of GCCs on cell migration. Similar trend was also observed in the skin primary cell line and HeLa cells. To support this hypothesis, the level of integrins on the cell surfaces were examined in the presence of specific GCC combinations. Integrins are major class of cell adhesion molecules, mediating cell-cell and cellextracellular matrix interactions.39 X-ray crystal structure analysis has shown that the triple helical structure of collagen and the presence of 4-hydroxyproline are essential for the binding of integrins.40-42 Integrins (α1β1) are collagen receptors, involved in cell migration and metastatic
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processes found in fibroblast, endothelial and epithelial cells.43 All collagens are expected to bind specific integrin receptors and up-regulate the expression levels, causing the production of matrix metalloproteinases (MMPs). Using multiple antibodies for FACS analysis, we distinguished the level of integrin expression in the presence of specific GCC in primary (skin) and secondary fibroblast cells (BJ cells) (Figure 3ii and Table S8). In case of BJ cells, C-3/G-5 complex was found to have a small effect on integrin expression (31.7 %) whereas, C-3/G-2 expressed high levels (65.8 %) of integrin compared to C-3/G-7 combination (41.3 %). In contrast, C-3/G-2 expressed 7.4 % of integrins in HeLa and 60.4 % in primary skin fibroblast cells, demonstrating that the cell surface binding of GCC results in up regulation of integrins, which may influence MMPs production and promote wound healing process. To examine the mechanism by which integrin induces cell migrations, the ERK/FAK pathways were investigated with BJ and skin fibroblast cells.44 Elevated expression levels of these proteins are considered as a general marker of cell migration. To test, if the GCCs inducing integrin elevate the ERK and FAK, protein extracts of GCC (C-3/G-2 and C-3/G-7) treated cells at 6 h were analyzed for the expression level of ERK and FAK. Western blot analysis of FAK and ERK showed that BJ cell treated with C-3/G-2 expressed high level of ERK and FAK compared to C-3/G-7. In contrast, skin fibroblast cells express strong ERK and FAK in presence of both C-3/G-2 and C-3/G-7 conjugates (Figure 3iii). Overall, these results suggest that the cell adhesion molecules (integrins) and sugar receptors synergistically work together to influence cell migration.
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Figure 3. (i) Fluorescence images of different cells BJ, Hela, and skin incubated for 6 h with (a) C-3-F; (b) C-3-F/G-1; (c) C-3-F/G-2, (d) C-3-F/G-5 and (f) C-3-F/G-7 respectively (green fluorescent correspond to C-3- F, blue fluorescent corresponds to DAPI) (ii) Flow cytometry of integrin (α1β1) expression in BJ cell line after treated with (a) untreated, (b) C-3/G-2, (c) C-3/G5 and (d) C-3/G-7; integrin expression in HeLa cell line after treatment with (e) untreated, (f) C3/G-2, (g) C-3/G-5 and (h) C-3/G-7; integrin expression in skin cell line (i) untreated, (j) C-3/G2, (k) C-3/G-5 and (l) C-3/G-7 (iii) The expressions of FAK, pFAK, ERK, pERK, α-tublin in (a) BJ cell lines and (b) skin cell lines (35 µg whole-cell lysate of proteins) with (1) control; (2) C-3/G-2 and (3) C-3/G-7 were analysed by western blotting. Protein loading was assessed by probing the blots with anti- α-tublin. Based on the in vitro screening experiments, we explored the wound healing effect of C3/G-7 in wild-type mice model.45 Determination of wound healing rate and histology of the wound is a useful method for studying the real time application of these GCC.46 Previously, hydrogel was encapsulated with fibroblast, collagen and growth factor molecules showed wound healing properties in mice model.47-49 Hence, it is expected that self-assembled GCC could also be a promising platform for in vivo wound healing. In order to study this phenomenon, a 5 mm excisional wound was created on the dorsal surface of the mice and C-3/G-7, collagen-II were applied on top of the wound and the decrease in the wound area was monitored for 14 days (Figure 4ii). As shown in Figures 4, the wound area of C-3/G-7 - treated mice was significantly reduced as compared to that of collagen-II - treated and control mice at post wounding days 3, 5 and 8 respectively. Complete wound closure was observed in C-3/G-7 on day ~11 whereas natural collagen and control showed similar wound healing on days ~12 and ~14 respectively (Figures 4i and S11). To further confirm the progress of the wound healing, histomorphometric analysis was performed at the wound biopsies on day 8 of post wounding. Histology images of mouse showed substantial growth of epidermis layer in C-3/G-7 treated mouse as compared to control and natural collagen-treated mice, suggesting the faster wound healing rate with C-3/G-7
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peptide (Figure 4iii). In summary, our study shows that the use of the supramolecular strategy represents a valid step in identifying specific biomarkers for physiological and pathological processes. (i)
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Figure 4. (i) Change in total wound sizes with respect to time from day 0 to day 14 in mice (ii) Respective pictures of wound taken from mice by treatment of collagen II (Col-II) and C-3/G-7 at different days (iii) Histological analysis of wounds after treatment with haematoxylin and eosin stain in presence of (a) Normal (without wound); (b) Control; (c) collagen II; (d) C-3/G-7 after 8 days (arrow indicates the wound healing process, scale bar = 200 µm, n = 6, Data shown as mean ± SD, Two way ANOVA post-test for significance *p
