Université Lille Nord de France, Lille, France.
FEBS J. 2011 Jul;278(14):2552-64. doi: 10.1111/j.1742-4658.2011.08180.x. Epub 2011 Jun 3.
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.
核仁素是一种主要的核仁蛋白，参与核糖体生物发生、细胞增殖和生长的基本过程。核仁素有核质穿梭功能。我们之前发现核仁素在核外异构体中经历复杂的 N-和 O-糖基化。我们发现表面核仁素仅被糖基化，并且 N-糖基化是其在细胞表面表达所必需的。有趣的是，两个 N-聚糖位于 RNA 结合结构域（RBD）中，这些结构域参与核仁素的自我缔合特性。我们假设 RBD 被 N-聚糖占据在这些自我缔合特性中起作用。在这里，由于无法定量产生全长核仁素，我们在杆状病毒/昆虫细胞系统中表达了四个缺乏 N 端酸性结构域的 N-糖基化核仁素变体。如七氟丁酸衍生化和质谱评估所示，该策略允许在一个或两个潜在 N-糖基化位点上产生具有或不具有低聚糖型聚糖的蛋白质。通过圆二色性和荧光法研究了它们的结构，并通过电泳和表面等离子体共振分析了它们相互作用的能力。我们的结果表明，所有核仁素衍生变体都能够自我相互作用，并且 RBD1 和 RBD3 上的 N-糖基化或仅 RBD3 上的 N-糖基化修饰了 N 端截断核仁素的结构并增强了其自我缔合特性。相反，N-糖基化不会修饰与乳铁蛋白（核仁素表面的配体）的相互作用。我们的结果表明，N-糖基化位点的占据可能有助于表面核仁素的表达和功能。
