Expression, purification, crystallization and preliminary X-ray crystallographic analysis of Enpp6

Abstract

Enpp (ectonucleotide phosphodiesterase/pyrophosphatase) 6 is a membrane-bound glycoprotein that hydrolyzes choline-containing compounds such as lysophosphatidylcholine and glycerophosphorylcholine, and presumably participates in choline metabolism. The catalytic domain of mouse Enpp6 was expressed in HEK293T cells, purified using the TARGET tag/P20.1-Sepharose system and crystallized. An X-ray diffraction data set was collected to 1.8 Å resolution. The crystal belonged to space group P1, with unit-cell parameters a=63.7, b=68.8, c=69.7 Å, α=60.6, β=87.0, γ=68.1°. Assuming the presence of two protein molecules per asymmetric unit, the solvent content was estimated to be 49.5%.

Ablation of Enpp6 Results in Transient Bone Hypomineralization

“…The specific function of ENPP6 remains unclear however, given its established lysophospholipase C activity (19)(20)(21) . The similarities of the phenotype described here to that of the Phospho1 -/mouse are clear.…”

“…However, specific candidates which may function as part of the MV mechanism have yet to be identified. Ectonucleotide pyrophosphatase/phosphodiesterase 6 (or glycerophosphocholine cholinephosphodiesterase; ENPP6) is a member of the nucleotide pyrophosphatase/phosphodiesterase family with lysophospholipase C activity, catalysing the conversion of lysophosphatidylcholine to PCho with a monoacylglycerol by-product (19)(20)(21)(22) . ENPP6 may therefore act upstream of PHOSPHO1 to generate intravesicular PCho, however the role of ENPP6 in bone mineralization has yet to Accepted Article be investigated.…”

“…Ectonucleotide pyrophosphatase/phosphodiesterase 6 (ENPP6; or glycerophosphocholine cholinephosphodiesterase) is a member of the nucleotide pyrophosphatase/phosphodiesterase family with lysophospholipase C activity, catalyzing the conversion of lysophosphatidylcholine to PCho with a monoacylglycerol byproduct. ( 19‐22 ) Therefore, ENPP6 may act upstream of PHOSPHO1 to generate intravesicular PCho; however, the role of ENPP6 in bone mineralization has yet to be investigated. This study aims to characterize the skeletal phenotype of the global ENPP6 KO mouse to give insight into its role in bone formation.…”

How To Build a Bone: PHOSPHO1, Biomineralization, and Beyond

“…A second enzymatic processing phase is hypothesized to convert generated LPL to PCho for direct hydrolysis by PHOSPHO1, mediated by NPP6 . NPP6 is a member of the nucleotide pyrophosphatase/phosphodiesterase family and has been shown to possess lysophospholipase C activity, catalyzing the conversion of lysophosphocholine with a monoacylglycerol by‐product . Expression of NPP6 has been demonstrated in bone tissue lysate and was immunolocalized to hypertrophic chondrocytes and forming bone surfaces .…”

“…(82) NPP6 is a member of the nucleotide pyrophosphatase/phosphodiesterase family and has been shown to possess lysophospholipase C activity, catalyzing the conversion of lysophosphocholine with a monoacylglycerol by-product. (91)(92)(93) Expression of NPP6 has been demonstrated in bone tissue lysate and was immunolocalized to hypertrophic chondrocytes and forming bone surfaces. (82) Specific localization of NPP6 to MVs has yet to be established, however.…”