Using a ubiquitin ligase as an unfolded protein sensor

Abstract

A significant fraction of all proteins are misfolded and must be degraded. The ubiquitin–proteasome pathway provides an essential protein quality control function necessary for normal cellular homeostasis. Substrate specificity is mediated by proteins called ubiquitin ligases. In the endoplasmic reticulum (ER) a specialized pathway, the endoplasmic reticulum associated degradation (ERAD) pathway provides means to eliminate misfolded proteins from the ER. One marker used by the ER to identify misfolded glycoproteins is the presence of a high-mannose (Man5-8GlcNAc2) glycan. Recently, FBXO2 was shown to bind high mannose glycans and participate in ERAD. Using glycan arrays, immobilized glycoprotein pulldowns, and glycan competition assays we demonstrate that FBXO2 preferentially binds unfolded glycoproteins. Using recombinant, bacterially expressed GST-FBXO2 as an unfolded protein sensor we demonstrate it can be used to monitor increases in misfolded glycoproteins after physiological or pharmaceutical stressors.

Introduction

The maintenance of the cellular proteasome is vital for cellular homeostasis. Protein production represents the final step in the flow of genetic information; hence, proteins reflect the sum of errors not only in transcription, splicing, and translation, but also in protein folding, which occurs in the endoplasmic reticulum (ER). High levels of misfolded proteins can disrupt cellular homeostasis, stress the ER, and dysregulate apoptosis. Indeed, the accumulation of specific misfolded proteins contributes to the pathologic sequela of many diseases, such as Alzheimer’s, Parkinson’s and Huntington’s disease [1], [2], [3], [4], [5]. To date, assessing misfolding has only been possible at the level of individual proteins, and a variety of methods have been tried including native and non-native antibodies, circular dichroism, and fluorescence and infrared spectroscopy. It has not been possible, however, to measure the overall burden of protein misfolding placed upon a cell in a normal vs. a stressful situation.

One means to measures the levels of misfolded proteins within a cell may be to examine their attached sugars. Glycoprotein folding occurs in the ER with the assistance of the co-translational addition of oligosaccharides. This 14 sugar oligosaccharide, Glc3Man9(GlcNAc)2, is transferred en bloc to an asparagine residue on a nascent polypeptide. As these glycosylated proteins assume their tertiary structure, the inner GlcNAcGlcNAc core, also called chitobiose, of the attached glycans is sequestered, leaving only terminal sugars exposed [6], [7], [8]. Correctly folded glycoproteins are exported to the Golgi for further processing; incorrectly folded proteins are retro-translocated and degraded by the ubiquitin proteasome pathway. The cellular signal for retro-translocation is, in part, the exposed glycan core [9]. We thought perhaps that using the glycan attachment as a readout might provide a means for measuring overall levels of misfolded glycoproteins.

We wanted to test if the exposed core sugar groups can be exploited to quantify the levels of misfolded glycoproteins. Exposed glycan cores can be recognized by the lectin like ubiquitin ligase FBXO2. FBXO2 was first shown by Yoshida et al. [10] to bind glycoproteins. Subsequently, this group [11], [12], [13] and ours [14], [15] showed FBXO2 preferentially binds glycoproteins through their glycan moiety and not by recognizing the protein component. We thought perhaps if we could prove that FBXO2 bound only unfolded glycoproteins, and that it bound a large array of unfolded glycoproteins, we could use FBXO2’s ability to bind unfolded protein to determine the levels of unfolded proteins within a cell.