An in situ Dynamic Continuum of Supramolecular Phosphoglycopeptides Enables Formation of 3D Cell Spheroids

Article abstract of DOI:10.1002/anie.201710269

Higher-order assemblies of proteins, with a structural and dynamic continuum, is an important concept in biology, but these insights have yet to be applied in designing biomaterials. Dynamic assemblies of supramolecular phosphoglycopeptides (sPGPs) transf

Full text of DOI:10.1002/anie.201710269

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Accepted Article
Title: An in-situ Dynamic Continuum of Supramolecular
Phosphoglycopeptides Enables Formation of 3D Cell Spheroids
Authors: Huaimin Wang, Junfeng Shi, Zhaoqianqi Feng, Rong Zhou,
Shiyu Wang, Avital A. Rodal, and Bing Xu
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To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.201710269
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An in-situ Dynamic Continuum of Supramolecular
Phosphoglycopeptides Enables Formation of 3D Cell Spheroids
Huaimin Wang,[a] Junfeng Shi,[a] Zhaoqianqi Feng,[a] Rong Zhou,[a] Shiyu Wang,[b] Avital A. Rodal,[b]
and Bing Xu*[a]
Abstract: Higher-order assemblies of proteins, with a structural and
dynamic continuum, is a new paradigm in biology, but these insights
have yet to be applied in designing biomaterials. Dynamic
assemblies of supramolecular phosphoglycopeptides (sPGPs)
transform a 2D cell sheet to 3D cell spheroids. A ligand-receptor
interaction between a glycopeptide and a phosphopeptide produces
sPGPs that form nanoparticles, which transform to nanofibrils upon
partially enzymatic dephosphorylation. The assemblies form
dynamically and hierarchically in situ on the cell surface, and interact
with extracellular matrix molecules and effectively abolish contact
inhibition of locomotion (CIL) of the cells. Integrating molecular
recognition, catalysis, and assembly, these active assemblies act as
a dynamic continuum to disrupt CIL, thus illustrating a new kind of
biomaterials for regulating cell behavior.
cellular milieu exhibit emergent properties.[11] To verify the roles
of saccharides and dephosphorylation, we intentionally excluded
known cell adhesion sequences in the phosphopeptides or the
glycopeptides. We used the ligand-receptor interaction[12] (Fig.
1B) between a phosphopeptide (1P) and a glycopeptide (2) to
form a sPGP (1P:2), which self-assembled to form oligomers
(e.g., (1P:2)6, Fig. 1C) and existed as nanoparticles with
diameters of 8±2 nm (Fig. 1D). Adding alkaline phosphatase
[13]
(ALP)
to the solution of 1P:2 turned the nanoparticles into
nanofibrils with diameters of 8±2 nm (Fig. 1D). Liquid
chromatography–mass spectrometry (LC-MS) revealed that the
assemblies of 1P:2 were partially dephosphorylated (Fig. 1E).
Circular dichroism (CD) spectra (Fig. S1) of enzymatic
dephosphorylation of 1P exhibited a positive peak of 192 nm
and a negative peak around 212 nm, suggesting a β-sheet
arrangement. Mixture of 1P with
2 yields the assemblies
Playing multifarious roles in cell organization, extracellular matrix
adopting the conformation that similar with 2. Being partially
dephosphorylated by ALP, 1P:2 yields the assemblies slightly
increasing the signal of CD spectra, evidenced by two negative
peaks at 200 nm and 212 nm. These results confirm that the
assemblies of 1P:2 are dynamic (i.e., reversible binding between
(
ECM) interacting with cells in 3D environment differ
[
1]
fundamentally from in 2D. The elucidation of cell adhesion
molecules has inspired attaching cell adhesion sequences (e.g.,
Arg-Gly-Asp (RGD)) to solid surfaces, polymeric materials, or
supramolecular materials[4] for 2D or 3D cell culture.[5] However,
two important post-translation features of ECM, glycosylation
and phosphorylation, are largely overlooked. Every living cell is
[2]
[3]
1P (or 1) with 2) and reactive (i.e., as substrates of
phosphatases), thus generating a dynamic continuum of the
assemblies consisting of peptide, phosphopeptide, and
glycopeptide (Fig. 1A). Such dynamic assemblies are
particularly important for functionally mimicking ECM because
the ECM components themselves are oligomeric, multifunctional,
and dynamic.
[6]
covered with complex array of glycans; many proteins that
[7]
associate with cell-matrix adhesion are phosphorylated or
dephosphorylated dynamically. Therefore, biomaterials acting as
an artificial microenvironment for guiding cells must mimic the
[
8]
functions of glycoproteins and phosphoproteins.
But the
[9]
synthesis of glycans remains a daunting challenge, and the
controlled protein phosphorylation is hardly an easy task. Thus,
it is imperative to develop a facile approach for generating next
generation biomaterials that mimic the functions of extracellular
glycoproteins and phosphoproteins.
To sidestep the difficulty of synthesizing glycoproteins and
phosphoproteins, we chose to generate higher-order assemblies
of supramolecular phosphoglycopeptides (sPGPs, Fig. 1A) as
their functional mimics because (i) higher-order assemblies of
proteins, with a structural and dynamic continuum, are emerging
as a new paradigm in biology to understand cellular functions,[10]
but these insights have yet to be applied in designing the next
generation biomaterials; (ii) assemblies of small molecules in the
[
a]
Dr. H. Wang, Dr. J. Shi, Ms. Z. Feng, Dr. R. Zhou and Prof. Dr. B.
Xu
Department of chemistry
Brandeis University
415 South St, Waltham, MA 02454, USA
Fax: (+) 1-781-736-2516
E-mail: bxu@brandeis.edu
Ms. S. Wang, and Prof. A. Rodal
Department of Biology
[
b]
Brandeis University
Figure 1. (A) Illustration of sPGPs, formed by non-covalent interaction, as a
dynamic continuum in cell milieu. (B) Ligand-receptor interactions (green dash
lines, dissociation constant (Kd) is 7.1 M)) between vancomycin (Van, 2) and
the D-Ala-D-Ala containing phosphopeptides (Nap-FFpYGGaa, 1P) result in a
415 South St, Waltham, MA 02454, USA
Supporting information for this article is given via a link at the end of
the document.
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2
sPGP (1P:2). (C) Enzymatic conversion of a dimer of the sPGP ((1P:2) , stick
model) to a dimer of supramolecular glycopeptide ((1:2) , CPK model)), and
2
We
introduced
a
nitrobenzofurazan
(NBD)-capped
phosphopeptide (NBD-1P) because NBD fluoresces intensely
when forming assemblies.
the CPK model of a hexamer of 1P:2. The dimer and hexamer are derived
[
19]
from the known crystal structure.[
14]
Adding NBD-1P:2 into the HS-5
(D) Transmission electron microscopic
(
TEM) images of 1P:2 (300 µM) before (left) and after (right) being treated by
cell culture induced the cell spheroids after 24 h (Fig. S5). Live
cell imaging (video S3) showed that the fluorescence increased
gradually, indicating that more assemblies of NBD-1:2 formed
upon dephosphorylation (Fig. 2C) over time, as well as
suggesting the formation of intermediate structures. The
fluorescence on the cells (Fig. 2D) and the size of the cell
spheroids increase simultaneously, indicating that assembling of
NBD-1:2, indeed, is an in-situ, dynamic process. Meanwhile, the
HS-5 cells treated by only NBD-1P or by NBD-1:2 (Fig. S6)
exhibit little fluorescence and hardly form spheroids. These
results confirm that the reactive sPGP assemblies act as a
dynamic continuum of nanostructures to induce 3D spheroids.
We synthesized the analogs of 1P and mixed each of them
with 2 for the cell assay. Scrambling the sequence of 1P results
in 3P and 4P, and introducing D-phenylalanine and D-tyrosine
generates 5P. 3P:2, 4P:2, or 5P:2 induced the cell spheroids
while 3P, 4P or 5P alone was ineffective, indicating that the
regiochemistry or stereochemistry of the FFY segment in 1P
hardly affected the functions of the dynamic assemblies.
Replacing the diphenylalanine by dialanine yields 6P, and 6P:2
only induced the HS-5 spheroids slowly (after 48 h incubation).
The acetyl-capped phosphopeptide (7P) was unable to self-
assemble after dephosphorylation (Fig. S8), and (7P:2) failed to
induce the cell spheroids. Using L-Ala-L-Ala to replace D-Ala-D-
Ala produced 8P, which was unable to form sPGP and failed to
induce cell spheroid in the presence of 2. These results confirm
that the self-assembling ability of the sPGPs and the formation
of sPGPs are the prerequisites for inducing cell spheroids.
Moreover, adding D-Ala-D-Ala, which competitively bound to 2 in
the presence of 1P and led to more long nanofibrils (comparing
to without D-Ala-D-Ala), resulted in more pronounced formation
of cell spheroids (Fig. S10). The activities of these analogs, as
the molecular validation, confirm that in-situ, hierarchical, and
dynamic assembling of sPGP enables 2D to 3D cell organization.
a phosphatase (ALP, 1 U/mL. 24 h). Scale bar is 20 nm. (E) Phosphatases
(
(
ALP, 0.2, 0.5, and 1 U/mL) catalyzed conversion of 1P (300 μM) and 1P:2
300 μM) within 48 h.
To test if the designed assemblies functionally mimic ECM, we
used spheroid formation by a fibroblast cell line (HS-5[15]) as a
simple assay (Fig. 2A), for several reasons: (i) Fibroblast
[1]
synthesize 3D ECM slowly (i.e., requiring days) . Thus, if the
cells rapidly switch from a 2D sheet to 3D spheroids, the
assemblies likely act as ECM. (ii) Changing from 2D to 3D, cells
have to remodel ECM so the assemblies must be adaptive and
dynamic. (iii) In vitro assay reduces the cost and the complexity
of in vivo models. (iv) Most importantly, the addition of
phosphoproteins (e.g., FGF2) or glycoproteins (e.g., TGF-β1)
induces clustering of HS-5 cells. Thus, the assemblies of the
sPGPs, if acting as functional mimics of glycoproteins and
phosphoproteins, should induce 3D spheroids of HS-5 cells. Live
imaging confirmed that, after being incubated with 1P:2 for 24 h,
the HS-5 cells migrate and arise from surface to form 3D cell
spheroids (Fig. 2A, video S1). In controls, HS-5 cells, without
any treatment, remained as a 2D cell sheet (Fig. S2), likely due
[
16]
[
17]
to contact inhibition of locomotion (CIL) (video S2).
The 3D
spheroids spread to revert back to the 2D cell sheet after the
removal of 1P:2 (Fig. 2A), indicating that the assemblies
maintain the 3D spheroids. As shown in Fig. 2B, adding 1P or 2
alone into the culture of HS-5 cells failed to lead to 3D spheroids.
Neither the completely dephosphorylated complex (1:2) nor the
complex of 1P with vacomycin aglycon (1P:2Agl) induced cell
spheroids. Co-incubation the cocktail of phosphatase
inhibitors[ with 1P:2 prevented formation of cell spheroids (Fig
S4). These results confirm that the phosphotyrosine on the
peptide (1P), the glycoside on 2, and the in-situ
dephosphorylation reaction are indispensable for inducing 3D
spheroids.
18]
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Figure 2. In-situ, dynamic assembling of sPGPs transforms a cell sheet to cell spheroids. (A) The illustration of forming 3D spheroids from a 2D cell sheet upon
the addition of sPGP and the reversibility of the process. (B) HS-5 cells only form 2D sheets when being co-cultured with phosphopeptide (1P), glycopeptide (2),
1:2 or 1P:2Agl (vancomycin aglycon), respectively, for 48 h. Scale bars (in A, B, and C) are 50 μm. (C) Fluorescence changes during the formation of cell
spheroids induced by NBD-1P:2. (D) Change of fluorescence on HS-5 cells treated with NBD-1P:2 within 24 h at λex = 488 nm (three repeats). (E) Structures of
the analogs of 1P. (F) Summary of the states of the HS-5 cells co-cultured with different analogs of 1P without or with 2. All molecular concentrations are 300 μM.
Since the sPGP assemblies and the cell spheroids formed
simultaneously, we examined whether the assemblies
interacted with the components of ECM or cell adhesion
molecules. Immunofluorescence staining reveals that
fibronectin, laminin, collagen III, and collagen IV partially co-
localize with the assemblies of NBD-1P:2 (Fig. 3A, videos S4
to S7). Mβ-CD, a disruptor of lipid rafts,[20] hardly prevents
formation of the cell spheroids or the co-localization of
fibronectin with the assemblies of NBD-1P:2 (video S8), further
confirming that the interaction between the assemblies and
ECM components (independent from plasma membrane) is
critical for forming cell spheroids. Notably, the morphology of
fibronectin in the cell spheroids is long, fibrous (Figs. 3A and
S11), differing from the short puncta in the control (Fig. S11).
Since ECM dynamics involve adhesion molecules,[21] we also
adhesion, hardly co-localize with the assemblies (Fig. S13,
videos S11 and S12). These results exclude the direct
interactions between the assemblies and integrins.
Furthermore, immunofluorescent staining shows no co-
localization of E-cadherin, N-cadherin, or E-selectin with the
assemblies of NBD-1P:2 (Fig. S13, videos S13 to15)).
Moreover, antibody blocking by antagonistic monoclonal
antibody (mAb) to β integrin, E-cadherin, N-cadherin, or E-
1
selectin is unable to prevent 1P:2 to induce the cell spheroids
(Fig. S14). Western blot (Fig. 3D) indicates that the expression
level of β integrin, E-cadherin, N-cadherin, as well as FGF2 or
1
TGF-β1[16], remains constant. Collectively, these results
confirm that the dynamic assemblies of the sPGPs interact with
ECM components (Fig. 3E) and robustly induce formation of
cell spheroids.
5 1
examined the localization of α and β integrins, which are
major fibronectin receptors, and observed little overlap
between these integrins and the assemblies (Fig. 3B and S13,
v 3 3
videos S9 and S10). Antibodies against α β and β integrins,
receptors for vitronectin and a distinctive type of cell-matrix
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control to achieve 3D cell organization. It not only validates the
dynamic continuum of the assemblies of small molecules as
dynamic biomaterials to control cell behaviors for potential
applications such as tissue engineering and wound healing,
but also provides a simple model for studying collective
migration of cells.
Acknowledgements
This work was partially supported by NIH (CA142746), NSF
(DMR-1420382), and W. M. Keck Foundation. ZF thanks the
Dean’s fellowship. We thank Dr. Torokstorb for sharing HS-5
cells.
Keywords: self-assembly • vancomycin • enzyme • cell spheroid
•
extracellular matrix
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exists at the edge of the spheroids, while little cluster like
fluorescence at the intercellular boundary (Fig. 3C, video S17).
In the control HS-5 cells, the microtubules extend through the
cell bodies (Fig. S15). These results suggest that the dynamic
assemblies of sPGPs on the cell membrane provide additional
forces to affect the actin (and tubulin) polymerization
machinery at the leading edge, which alter migratory behavior
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COMMUNICATION
Huaimin Wang, Junfeng Shi, Zhaoqianqi
Feng, Rong Zhou, Shiyu Wang, Avital A.
Rodal, and Bing Xu*
Page No. – Page No.
An in-situ Dynamic Continuum of
Supramolecular
Phosphoglycopeptides Enables
Formation of 3D Cell Spheroids
Enzymatically formed dynamic biomaterials in cell milieu modulate
intercellular interactions to generate 3D cell spheroids.
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