Chemical glycosylation of cytochrome c improves physical and chemical protein stability

Abstract

Background: Cytochrome c (Cyt c) is an apoptosis-initiating protein when released into the cytoplasm of eukaryotic cells and therefore a possible cancer drug candidate. Although proteins have been increasingly important as pharmaceutical agents, their chemical and physical instability during production, storage, and delivery remains a problem. Chemical glycosylation has been devised as a method to increase protein stability and thus enhance their long-lasting bioavailability.

Results: Three different molecular weight glycans (lactose and two dextrans with 1 kD and 10 kD) were chemically coupled to surface exposed Cyt c lysine (Lys) residues using succinimidyl chemistry via amide bonds. Five neo-glycoconjugates were synthesized, Lac4-Cyt-c, Lac9-Cyt-c, Dex5(10kD)-Cyt-c, Dex8(10kD)-Cyt-c, and Dex3(1kD)-Cyt-c. Subsequently, we investigated glycoconjugate structure, activity, and stability. Circular dichroism (CD) spectra demonstrated that Cyt c glycosylation did not cause significant changes to the secondary structure, while high glycosylation levels caused some minor tertiary structure perturbations. Functionality of the Cyt c glycoconjugates was determined by performing cell-free caspase 3 and caspase 9 induction assays and by measuring the peroxidase-like pseudo enzyme activity. The glycoconjugates showed ≥94% residual enzyme activity and 86 ± 3 to 95 ± 1% relative caspase 3 activation compared to non-modified Cyt c. Caspase 9 activation by the glycoconjugates was with 92 ± 7% to 96 ± 4% within the error the same as the caspase 3 activation. There were no major changes in Cyt c activity upon glycosylation. Incubation of Dex3(1 kD)-Cyt c with mercaptoethanol caused significant loss in the tertiary structure and a drop in caspase 3 and 9 activation to only 24 ± 8% and 26 ± 6%, respectively. This demonstrates that tertiary structure intactness of Cyt c was essential for apoptosis induction. Furthermore, glycosylation protected Cyt c from detrimental effects by some stresses (i.e., elevated temperature and humidity) and from proteolytic degradation. In addition, non-modified Cyt c was more susceptible to denaturation by a water-organic solvent interface than its glycoconjugates, important for the formulation in polymers.

Conclusion: The results demonstrate that chemical glycosylation is a potentially valuable method to increase Cyt c stability during formulation and storage and potentially during its application after administration.

Figure 1

Representation of the chemical glycosylation of Cyt c using monofunctionally activated glycans.

Figure 2

Far UV (A), near UV (B), and heme (C) region CD spectra of 0.6 mg/ml Cyt c and Cyt c glycoconjugates in 100 mM phosphate buffer at pH 7.4 and 20°C.

Figure 3

Degradation of Cyt c and Cyt c glycoconjugates by 4 mg/ml trypsin at 37°C. Each experiment was performed in triplicate, the values averaged, and the error bars are the calculated SD.

Figure 4

Degradation of Cyt c and Cyt c glycoconjugates by 5 mg/ml α-chymotrypsin at 37°C. Each experiment was performed in triplicate, the values averaged, and the error bars are the calculated SD.

Figure 5

The effect of 75% relative humidity on the structure of Cyt c and its glycoconjugates. After incubation, the glycoconjugates were lyophilized and dissolved in 10 mM phosphate buffered saline at pH 7.3. Each experiment was performed in triplicate, the values averaged, and the error bars are the calculated SD.

Figure 6

The effect of incubation at 50°C on the structure of Cyt c. After incubation, the glycoconjugates were lyophilized and dissolved in 10 mM phosphate buffered saline at pH 7.3. Each experiment was performed in triplicate, the values averaged, and the error bars are the calculated SD.

Figure 7

Scanning electron microscopy (SEM) images of lyophilized Dex ₃ (1 kD)-Cyt c after the precipitation via solvent displacement with (A) acetonitrile and (B) acetone as desolvating agent.