N-Glycosylation influences the structure and self-association abilities of recombinant nucleolin.

Nucleolin is a major nucleolar protein involved in fundamental processes of ribosome biogenesis, regulation of cell proliferation and growth. Nucleolin is known to shuttle between nucleus, cytoplasm and cell surface. We have previously found that nucleolin undergoes complex N- and O-glycosylations in extra-nuclear isoforms. We found that surface nucleolin is exclusively glycosylated and that N-glycosylation is required for its expression on the cells. Interestingly, the two N-glycans are located in the RNA-binding domains (RBDs) which participate in the self-association properties of nucleolin. We hypothesized that the occupancy of RBDs by N-glycans plays a role in these self-association properties. Here, owing to the inability to quantitatively produce full-size nucleolin, we expressed four N-glycosylation nucleolin variants lacking the N-terminal acidic domain in a baculovirus/insect cell system. As assessed by heptafluorobutyrate derivatization and mass spectrometry, this strategy allowed the production of proteins bearing or not paucimannosidic-type glycans on either one or two of the potential N-glycosylation sites. Their structure was investigated by circular dichroism and fluorimetry, and their ability to self-interact was analyzed by electrophoresis and surface plasmon resonance. Our results demonstrate that all nucleolin-derived variants are able to self-interact and that N-glycosylation on both RBD1 and RBD3, or RBD3 alone, but not RBD1 alone, modifies the structure of the N-terminally truncated nucleolin and enhances its self-association properties. In contrast, N-glycosylation does not modify interaction with lactoferrin, a ligand of cell surface nucleolin. Our results suggest that the occupancy of the N-glycosylation sites may contribute to expression and functions of surface nucleolin.