An N-glycosylation Modifiable Self-Assembling Polypeptide Tag for Formation of Protein Hydrogels

Introduction:: Hydrogels generated from self-assembling peptides are attractive as they can be tuned by varying the amino acid sequence, can be designed to respond to stimuli, and are generally biocompatible with cells and tissues. Here, we have designed a novel recombinant polypeptide tag consisting of a repeating
Actinobacillus plueropneumoniae

N-glycosyltransferase (ApNGT) consensus sequence. This polypeptide tag was then fused onto the N-terminus of superfolder green fluorescent protein for fluorometric detection (N-glycotag-sfGFP). Repetitive motifs containing asparagine and tryptophan in the N-glycotag sequence provide intermolecular non-covalent interactions that promote self-assembly of the fusion protein in a temperature dependent manner. N-glycotag-sfGFP (N-GT-sfGFP) could be efficiently enzymatically glycosylated with ApNGT and UDP-Glucose (Glu), respectively, to make Glu-N-GT-sfGFP, further varying the self-assembly state.

Materials and Methods:: N-GT-sfGFP and ApNGT were generated using recombinant bacterial expression. Briefly, pET-21d(+) plasmids encoding the target proteins were transformed into Origami™ B(DE3) competent cells, expressed via isopropyl β-D-1-thiogalactopyranoside induction, and purified via immobilized metal affinity chromatography. N-GT-sfGFP was glycosylated using 25 molar excess of nucleotide sugar in the presence of ApNGT. Glycosylation was confirmed via MALDI-TOF mass spectrometry. For turbidity measurements, absorbance of N-GT-sfGFP and Glucose modified N-GT-sfGFP were measured at 600 nm and 620 nm from 4°C to 55°C. To examine the effects of hydrogen bonding on N-GT-sfGFP self-assembly, sodium thiocyanate was used to block intermolecular hydrogen bonding and absorbance measured at 600 nm. N-GT-sfGFP gels were formed at 1 mM concentration and extruded via syringe into gel molds. A rheometer was used to determine the strain for the linear viscoelastic region (LVER) of the gel. Frequency sweeps at constant LVER strain were performed at 4°C, 25°C, 37°C, and 55°C to determine the storage modulus (G’) and loss modulus (G”). Shear-thinning and recovery was determined by measuring viscosity at alternating shear rates of 0.5 Hz and 100 Hz.

Results, Conclusions, and Discussions:: N-glycotag-sfGFP was efficiently enzymatically glycosylated as evidenced by a mass shift corresponding to the installation of carbohydrates on the glycotag domain. At lower (300 μM-500 μM) concentrations, N-glycotag-sfGFP underwent a sol-gel transition at 4°C, and began flowing when heated to 50°C. N-glycotag-sfGFP at 1 mM concentration formed a weak viscoelastic gel at all measured frequencies and temperatures, as determined by the ratio of G” to G’. Frequency sweeps at increasing temperatures revealed that the storage modulus registered a four-fold decrease when heated from 4°C to 55°C. N-GT-sfGFP flows under high shear rate as displayed by a decrease in viscosity, and showed partial recovery of viscosity when alternated back to low shear rate, supporting injectability of the gel. Sodium thiocyanate resulted in a cloud point depression of N-GT-sfGFP, suggesting that the driving force of N-GT-sfGFP phase behavior is hydrogen bonding. Phase change behavior was completely abrogated when glycosylated, as Glucose-N-GT-sfGFP did not display a sol-gel transition and was miscible from 4°C - 55°C. These data demonstrate the ability of glycosylation to influence phase behavior. Future work will involve further rheological analysis on varying concentrations of N-GT-sfGFP gel, using glycosylation to tune the self-assembly of N-GT-sfGFP, and replacing the sfGFP with enzyme for immobilization of active enzyme into a protein gel.

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References (Optional): : (1) Kightlinger, W., Lin, L., Rosztoczy, M. et al., Nat Chem Biol, 2018, 14, 627-635.