Pancreatic-type ribonucleases (ptRNases) are a class of highly conserved secretory proteins that accomplish diverse biological functions through the cleavage of RNA and are tightly regulated by a cytosolic ribonuclease inhibitor (RI) protein. ptRNases that are capable of evading RI can readily degrade cellular RNA, triggering apoptosis, and are being pursued as cancer chemotherapeutic agents. Though homologous to the well-characterized bovine enzyme RNase A, human RNase 1 contains three sites for N-linked glycosylation (Asn34, Asn76, and Asn88) that are recognized by an endogenous oligosaccharyltransferase. Despite their ubiquitous expression, the characterization of different RNase 1 glycoforms has been hindered by difficulties in producing substantial quantities of homogenously glycosylated protein. By using an engineered yeast strain, we have produced RNase 1 glycoforms with exquisitely controlled patterns of glycosylation consisting of a human-like core heptasaccharide (Man5GlcNAc2) at each glycosylation site. We have determined that glycosylation of RNase 1 at any position attenuates enzymatic activity but improves overall protein stability. Inspired by the interplay between protein and carbohydrate we sought to employ a previously identified dextran carrier to deliver therapeutic payloads, including RNase 1. We have prepared several different protein-dextran conjugates through site-selectively installed triazole linkers and have observed enhanced toxicity in cancer cell lines. Given our interests in the cytosolic uptake of clinically-relevant biomolecules, we have developed an exogenous nitroreductase-probe system to monitor the endosomal escape of RNase 1. Using this optimized system, we are also measuring differences in the overall rate of endocytosis across different cell lines.

Human ribonuclease 1: Endogenous glycosylation, cellular internalization, and cytotoxic capability

256th National Meeting (2018)

The official archive and repository for all ACS Meetings & Expositions abstracts and select posters or presentations.