Effects of exogenous B-cell immunoglobulin binding protein (BIP) and BIP protein inducer X (BIX) on myelination

Abstract

B-cell immunoglobulin binding protein (BiP) is an essential endoplasmic reticulum (ER) chaperone that belongs to the Hsp70 family, normally found in the ER lumen. However, BiP also has other extracellular and intracellular functions. As it is unclear whether peripheral BiP always has a signal and/or ER retention sequence and the possibility that different variants of BiP could have differing biochemical impacts, we produced and biochemically characterised four variants of BiP (see Chapter 3). The variants differed depending on the presence or the absence of signal and ER retention peptides. BiP variants were purified using nickel affinity chromatography, and protein size and quality were assessed using SDS/PAGE gels. In addition, we characterised the nucleotide-binding properties of variants in the absence and the presence of divalent cations Mg2+, Mn2+, and Ca2+. Interestingly, in the absence of cations, ADP has a higher binding affinity for BiP than ATP. The presence of divalent cations results in a decrease of the Kd values of both ADP and ATP, indicating higher affinities of both nucleotides for BiP. ATPase assays were carried out to study the enzyme activity of these variants and to characterise kinetic parameters. Variants with the signal sequence had higher specific activities than those without. Both Mg2+ and Mn2+ efficiently stimulated the ATPase activity of these variants at low micromolar concentrations, whereas calcium failed to stimulate BiP ATPase. Our novel findings indicate the potential functionality of the BiP signal sequence, and also reveal the effect of physiological concentrations of cations on the nucleotide-binding properties and enzyme activities of all variants. Studies have shown that BiP is essential for the survival of oligodendrocytes and is upregulated during developmental myelination. We hypothesized that the exogenous addition of BiP would enhance myelination in multiple sclerosis (Chapter 4). To address this, preliminary experiments to study the uptake of BiP by cells were conducted by adding FITC-labelled BiP to B104 neuroblastoma cells and spinal cord myelinating cultures. These experiments confirmed that BiP was taken up by cells in a dose- and time-dependent manner. In addition, the dose-dependent effects of unlabelled BiP were assessed and a dose of 20 μg/ml was selected for all other experiments. BiP was found to have no effect on myelination but increased the microglia number 2.5 times. This effect could be possibly due to BiP acting on cells directly, however heat inactivation experiments of BiP should be conducted to differentiate the effects of the protein from the endotoxins. Our results suggest that BiP could be detrimental as well. In an alternative approach (described in Chapter 5), we induced the expression of BiP using a chemical inducer of BiP (BIX). As the use of BIX in spinal cord myelinating cultures is not established before, we performed dose-dependent effects using BIX and 5 μg/ml of dose was used for further experiments. Surprisingly BIX had a detrimental effect on the microglia, oligodendrocytes, and myelin. We performed RNA sequencing to study the differentially expressed genes and used the Reactome pathway browser to identify the pathways possibly involved in the BIX effects observed. The genes related to oligodendrocytes and microglia were found to be downregulated, confirming the observed toxicity of BIX towards these cell populations. Astrocytes and neurons related genes were also down-regulated but they seem to be resistant compared to oligodendrocytes and microglia. Additionally, the top 25 most upregulated and downregulated pathways suggested that BIX interferes with the cell cycle, cell division, metabolism, and apoptosis. BIX exerted similar effects when added to organotypic slice cultures, suggesting that the effects of BIX are reproducible in a different model of myelination. Neither BiP nor BIX proved to be therapeutic in our culture models. The application of BIX as a therapeutic should be pursued with caution. Our results highlight the need for further investigation of BiP’s therapeutic potential within the context of myelinating disorders.