Sulfation of glycosaminoglycans depends on the catalytic activity of lithium-inhibited phosphatase BPNT2 in vitro.

Golgi-resident bisphosphate nucleotidase 2 (BPNT2) is a member of a family of magnesium-dependent, lithium-inhibited phosphatases that share a three-dimensional structural motif that directly coordinates metal binding to effect phosphate hydrolysis. BPNT2 catalyzes the breakdown of 3'-phosphoadenosine-5'-phosphate, a by-product of glycosaminoglycan (GAG) sulfation. KO of BPNT2 in mice leads to skeletal abnormalities because of impaired GAG sulfation, especially chondroitin-4-sulfation, which is critical for proper extracellular matrix development. Mutations in BPNT2 have also been found to underlie a chondrodysplastic disorder in humans. The precise mechanism by which the loss of BPNT2 impairs sulfation remains unclear. Here, we used mouse embryonic fibroblasts (MEFs) to test the hypothesis that the catalytic activity of BPNT2 is required for GAG sulfation in vitro. We show that a catalytic-dead Bpnt2 construct (D108A) does not rescue impairments in intracellular or secreted sulfated GAGs, including decreased chondroitin-4-sulfate, present in Bpnt2-KO MEFs. We also demonstrate that missense mutations in Bpnt2 adjacent to the catalytic site, which are known to cause chondrodysplasia in humans, recapitulate defects in overall GAG sulfation and chondroitin-4-sulfation in MEF cultures. We further show that treatment of MEFs with lithium (a common psychotropic medication) inhibits GAG sulfation and that this effect depends on the presence of BPNT2. Taken together, this work demonstrates that the catalytic activity of an enzyme potently inhibited by lithium can modulate GAG sulfation and therefore extracellular matrix composition, revealing new insights into lithium pharmacology.

Bisphosphate nucleotidase 2 (BPNT2), a molecular target of lithium, regulates chondroitin sulfation patterns in the cerebral cortex and hippocampus.

Bisphosphate nucleotidase 2 (BPNT2) is a member of a family of phosphatases that are directly inhibited by lithium, the first-line medication for bipolar disorder. BPNT2 is localized to the Golgi, where it metabolizes the by-products of glycosaminoglycan sulfation reactions. BPNT2-knockout mice exhibit impairments in total-body chondroitin-4-sulfation which lead to abnormal skeletal development (chondrodysplasia). These mice die in the perinatal period, which has previously prevented the investigation of BPNT2 in the adult nervous system. Previous work has demonstrated the importance of chondroitin sulfation in the brain, as chondroitin-4-sulfate is a major component of perineuronal nets (PNNs), a specialized neuronal extracellular matrix which mediates synaptic plasticity and regulates certain behaviors. We hypothesized that the loss of BPNT2 in the nervous system would decrease chondroitin-4-sulfation and PNNs in the brain, which would coincide with behavioral abnormalities. We used Cre-lox breeding to knockout Bpnt2 specifically in the nervous system using Bpnt2 floxed (fl/fl) animals and a Nestin-driven Cre recombinase. These mice are viable into adulthood, and do not display gross physical abnormalities. We identified decreases in total glycosaminoglycan sulfation across selected brain regions, and specifically show decreases in chondroitin-4-sulfation which correspond with increases in chondroitin-6-sulfation. Interestingly, these changes were not correlated with gross alterations in PNNs. We also subjected these mice to a selection of neurobehavioral assessments and did not identify significant behavioral abnormalities. In summary, this work demonstrates that BPNT2, a known target of lithium, is important for glycosaminoglycan sulfation in the brain, suggesting that lithium-mediated inhibition of BPNT2 in the nervous system warrants further investigation.