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1. (CSDB ID: 46397) | report error |
| ?%a-D-Glcp-(1-3)-+ | -6)-a-D-Glcp-(1- | Show graphically |
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Leuconostoc mesenteroides
(NCBI TaxID 1245,
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gmail.com>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, Lac(4)-Cyt-c, Lac(9)-Cyt-c, Dex(5)(10kD)-Cyt-c, Dex(8)(10kD)-Cyt-c, and Dex(3) (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 Dex(3)(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
apoptosis, chemical glycosylation, drug delivery, pharmaceutical protein, protein formulation, protein stability
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