Assembling custom side chains on proteoglycans to interrogate their function in living cells

Proteoglycans (PGs) are composed of a core protein and one or more chains of glycosaminoglycans (GAGs). The highly heterogeneous GAG chains play an irreplaceable role in the functions of PGs. However, the lack of an approach to control the exact structure of GAG chains conjugated to PGs tremendously hinders functional studies of PGs. Herein, by using glypican-3 as a model, we establish an aldehyde tag-based approach to assemble PGs with specific GAG chains on the surface of living cells. We show that the engineered glypican-3 can regulate Wnt and Hedgehog signaling like the wild type. Furthermore, we also present a method for studying the interaction of PGs with their target glycoproteins by combining the assembly of PGs carrying specific GAG chains with metabolic glycan labeling, and most importantly, we obtain evidence of GPC3 directly interacting with Frizzled. In conclusion, this study provides a very useful platform for structural and functional studies of PGs with specific GAG chains.

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October 2018
Data Policy information about availability of data All manuscripts must include a data availability statement. This statement should provide the following information, where applicable: -Accession codes, unique identifiers, or web links for publicly available datasets -A list of figures that have associated raw data -A description of any restrictions on data availability The data that support the findings of this study are available from the corresponding author upon reasonable request.

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Life sciences study design
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Sample size
Sample size was determined based on similar studies in this field.
Data exclusions Data points were excluded when there was a technical mistake during the experimental procedure.

Replication
Experiments were performed in at least three independent biological replicates. All attempts at replication were successful and gave similar results.
Randomization Randomization was not used and relevant to this study, since it did not involved an allocation of an intervention or a trial with human or animal participants.

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Blinding was not used and relevant to this study, since it did not involved an allocation of an intervention or a trial with human or animal participants Reporting for specific materials, systems and methods We require information from authors about some types of materials, experimental systems and methods used in many studies. Here, indicate whether each material, system or method listed is relevant to your study. If you are not sure if a list item applies to your research, read the appropriate section before selecting a response. (RRID:AB_2576208), . An anti-GPC3 mouse monoclonal antibody (αGCN) was prepared in our laboratory (Han et.al Chem Commun. 2017 The axis labels state the marker and fluorochrome used (e.g. CD4-FITC).

Materials
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Sample preparation
The cells were suspended by gentle pipetting; then, they were collected by centrifuging for 5 min at 1200 rpm, blocked with blocking buffer after washing with ice-cold PBS, and then were incubated with FITC-conjugated streptavidin for 1 h at room temperature in the dark. Finally, the cells were washed twice with PBS and analysed.

Instrument Beckman CoulterCytoFLEX
Software CytExpert Cell population abundance The total collected and analyzed cell numbers were 10000 per sample. This number was according the FSC and SSC gate after removing the dead and clumped cells.

Gating strategy
First, gate the FSC and SSC as P1 gate after removing the dead and clumped cells. Second, analyzed all cells fluorescence intensity in P1 gate.
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