Functional analysis of glycosylation in Etanercept: Effects over potency and stability.

Etanercept is a biotechnological product that has a complex glycosylation profile. To elucidate Etanercept glycosylation effect over biological activity and stability, we deglycosylated sequentially this molecule. Sequential deglycosylation was performed to understand which glycans are critical for Etanercept folding and activity. Extended study showed that gross glycosylation differences, affect thermal stability, hydrodynamic radius, pI, CDC, ADCC, protection against oxidation and charge surface exposition with any effect (within biological assay dispersion) over TNFα neutralization, indicating which glycoforms have a critical effect over Etanercept ADCC, CDC and stability. In this regard, complete remotion of sialic acids have a predominant importance over pI, ADCC, CDC and surface charge while N and O glycosylation over thermal stability, hydrophobicity, aggregation and protection against oxidation. Our research suggest that gross differences in the glycosylation profile are relevant for the stability and biological main activities of Etanercept, and that significant differences that affect the activities related to this fusion protein could be detected with proper analytical methods and stability studies.

Ribonuclease PH interacts with an acidic ribonuclease E site through a basic 80-amino acid domain.

In this work, we characterize the domains for the in vivo interaction between ribonuclease E (RNase E) and ribonuclease PH (RNase PH). We initially explored the interaction using pull-down assays with full wild-type proteins expressed from a chromosomal monocopy gene. Once the interaction was confirmed, we narrowed down the sites of interaction in each enzyme to an acidic 16-amino acid region in the carboxy-terminal domain of RNase E and a basic 80-amino acid region in RNase PH including an α3 helix. Our results suggest two novel functional domains of interaction between ribonucleases.