Biochemical and proteomic studies of human pyridoxal 5′-phosphate-binding protein (plpbp)

Abstract

The pyridoxal 5′-phosphate-binding protein (PLPBP) is an evolutionarily conserved protein linked to pyridoxal 5′-phosphate-binding. Although mutations in PLPBP were shown to cause vitamin B6-dependent epilepsy, its cellular role and function remain elusive. We here report a detailed biochemical investigation of human PLPBP and its epilepsy-causing mutants by evaluating stability, cofactor binding, and oligomerization. In this context, chemical cross-linking combined with mass spectrometry unraveled an unexpected dimeric assembly of PLPBP. Furthermore, the interaction network of PLPBP was elucidated by chemical cross-linking paired with co-immunoprecipitation. A mass spectrometric analysis in a PLPBP knockout cell line resulted in distinct proteomic changes compared to wild type cells, including upregulation of several cytoskeleton-and cell division-Associated proteins. Finally, transfection experiments with vitamin B6-dependent epilepsy-causing PLPBP variants indicate a potential role of PLPBP in cell division as well as proper muscle function. Taken together, our studies on the structure and cellular role of human PLPBP enable a better understanding of the physiological and pathological mechanism of this important protein.