Biochemical and Proteomic Studies of Human Pyridoxal 5′-Phosphate-Binding Protein (PLPBP)

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.