Vitamin B12 is a precursor of methylcobalamin, which is a cofactor in many metabolic processes, is an important nutrient for the formation of red blood cells and the maintenance of a healthy nervous system, and has been implicated in cancer development. Additionally, within ecologically relevant microbial communities it is necessary to support life for many microbes. The development of an activity-based B12 probe is useful to explore the biochemical interactions of vitamin B12 by live cell imaging and LC-MS based proteomics. We have developed a click chemistry compatible activity-based vitamin B12 probe (B12-ABP) in which the ribose-5'-hydroxy group of vitamin B12 (cyanocobalamin) is modified to contain a terminal alkyne and a diazirine photo-crosslinker. Uptake of vitamin B12 (cyanocobalamin) is facilitated by the cobalamin-binder gastric intrinsic factor (IF), which recognizes a 460 kDa receptor, cubilin, and is transported across the cell bound to another vitamin B12-binding protein, transcobalamin II (TC II). The plasma TC II�vitamin B12 complex is taken up by cells requiring B12 via the TC II receptor (TC II-R), a 47 kDa protein that is expressed as a functional dimer in cellular plasma membranes. Binding of the B12-ABP with intrinsic factor (IF) and transcobalamin (II) was validated in intestinal epithelial (CaCo2) and lung epithelial (A549) cells.  CaCo2 cells can express both cubilin and transcobalamin proteins, whereas A549 cells express both cubilin and transcobalamin in presence of gastric intrinsic factor only.  Uptake and transport of B12-ABP in CaCo2 and A549 cells was confirmed using flow cytometry, western blotting, RT-PCR, live cell imaging and proteomics.  We also evaluated the performance of B12-ABP in a complex microbial community, wherein we identified the spatio-temporal uptake of B12 by the various microbial community members.

Development and application of an activity-based vitamin B<sub>12</sub> probe for protein profiling in living cells and microbial communities

248th National Meeting (2014)

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Development and application of an activity-based vitamin B12 probe for protein profiling in living cells and microbial communities

Development and application of an activity-based vitamin B₁₂ probe for protein profiling in living cells and microbial communities