Sequential hydrolysis of FAD by ecto-5′ nucleotidase CD73 and alkaline phosphatase is required for uptake of vitamin B(2) into cells

Extracellular hydrolysis of flavin-adenine dinucleotide (FAD) and flavin mononucleotide (FMN) to riboflavin is thought to be important for cellular uptake of vitamin B(2) because FAD and FMN are hydrophilic and do not pass the plasma membrane. However, it is not clear whether FAD and FMN are hydrolyzed by cell surface enzymes for vitamin B(2) uptake. Here, we show that in human cells, FAD, a major form of vitamin B(2) in plasma, is hydrolyzed by CD73 (also called ecto-5′ nucleotidase) to FMN. Then, FMN is hydrolyzed by alkaline phosphatase to riboflavin, which is efficiently imported into cells. We determined that this two-step hydrolysis process is impaired on the surface of glycosylphosphatidylinositol (GPI)-deficient cells due to the lack of these GPI-anchored enzymes. During culture of GPI-deficient cells with FAD or FMN, we found that hydrolysis of these forms of vitamin B(2) was impaired, and intracellular levels of vitamin B(2) were significantly decreased compared with those in GPI-restored cells, leading to decreased formation of vitamin B(2)-dependent pyridoxal 5′-phosphate and mitochondrial dysfunction. Collectively, these results suggest that inefficient uptake of vitamin B(2) might account for mitochondrial dysfunction seen in some cases of inherited GPI deficiency.