Perturbation of neuronal cobalamin transport by lysosomal enzyme inhibition.

Hua Zhao,Hongyun Li,Brett Garner,Kalani Ruberu

doi:10.1042/bsr20130130

Abstract

Cbl (cobalamin) utilization as an enzyme cofactor is dependent on its efficient transit through lysosomes to the cytosol and mitochondria. We have previously proposed that pathophysiological perturbations in lysosomal function may inhibit intracellular Cbl transport with consequences for down-stream metabolic pathways. In the current study, we used both HT1080 fibroblasts and SH-SY5Y neurons to assess the impact that protease inhibitors, chloroquine and leupeptin (N-acetyl-L-leucyl-L-leucyl-L-argininal), have on the distribution of [57Co]Cbl in lysosomes, mitochondria and cytosol. Under standard cell culture conditions the distribution of [57Co]Cbl in both neurons and fibroblasts was ~5% in lysosomes, 14% in mitochondria and 81% in cytosol. Treatment of cells with either 25 μM chloroquine or 40 μM leupeptin for 48 h significantly increased the lysosomal [57Co]Cbl levels, by 4-fold in fibroblasts and 10-fold in neurons, and this was associated with reduced cytosolic and mitochondrial [57Co]Cbl concentrations. Based on Western blotting of LAMP2 in fractions recovered from an OptiPrep density gradient, lysosomal Cbl trapping was associated with an expansion of the lysosomal compartment and an increase in a subpopulation of lysosomes with increased size and density. Moreover, the decreased mitochondrial Cbl that was associated with lysosomal Cbl trapping was correlated with decreased incorporation of [14C] propionate into cellular proteins/macromolecules, indicating an inhibition of Cbl-dependent Mm-CoA (methylmalonyl-coenzyme A) mutase activity. These results add support to the idea that lysosomal dysfunction may significantly impact upon Cbl transport and utilization.

Highlights

Vitamin B12 [Cbl] is required for erythrocyte formation and DNA synthesis, and plays a critical role in the maintenance of neuron function [1,2]
Previous data indicated that treatment of fibroblasts with chloroquine (25 μM for 21 h) resulted in a dramatic increase in the amount of [57Co]Cbl that eluted from a Sephacryl S-200 column in the same position as TC, consistent with a retention of Cbl in lysosomes [6]
We have shown that inhibition of lysosomal proteolysis using both pH-dependent and -independent approaches leads to lysosomal Cbl accumulation

Summary

Introduction

Vitamin B12 [Cbl (cobalamin)] is required for erythrocyte formation and DNA synthesis, and plays a critical role in the maintenance of neuron function [1,2]. The cellular uptake of the TC–Cbl complex is mediated by the TCR (transcobalamin receptor) on the cell surface. The endocytosed complex is delivered to the lysosome where the Cbl is released subsequent to TC proteolysis, whereas the TCR is recycled back to the cell surface [3]. Once Cbl is transported out of the lysosome, it is converted to either MeCbl (methyl cobalamin) in the cytosol or AdoCbl (adenosyl cobalamin) in the mitochondria. MeCbl is used to transform Hcy (homocysteine) to methionine (Met) via cytosolic MS (methionine synthase), whereas AdoCbl is required for the conversion of Mm-CoA (methylmalonyl-coenzyme A) to Succ-CoA (succinyl-coenzyme A) via MMCM (mitochondrial methylmalonyl-coenzyme A mutase). Extracellular propionic acid is efficiently taken up into cells, converted to propionyl-CoA, converted to Mm-CoA (via the sequential actions Mm-CoA racemase and MMCM), the incorporation of [14C]-propionic acid into cellular macromolecules such as proteins [that are precipitated by TCA (trichloroacetic acid) in vitro] is an established clinical and basic research tool for evaluating the Cbl-dependent activity of MMCM [4,5]

Methods

Results

Conclusion