Structure and biological function of ENPP6, a choline-specific glycerophosphodiester-phosphodiesterase

Junko Morita,Osamu Nureki,Hiroyuki Arai,Emiko Mihara,Junichi Takagi,Hirofumi Ueda,Kuniyuki Kano,Kazuki Kato,Hiroshi Nishimasu,Junken Aoki,Hiroyuki Takita,Hiroaki Asou,Hideki Sakagami,Hidetoshi Tokuyama,Takanao Sato,Yasuo Yamamoto,Ryuichiro Ishitani

doi:10.1038/srep20995

Abstract

Choline is an essential nutrient for all living cells and is produced extracellularly by sequential degradation of phosphatidylcholine (PC). However, little is known about how choline is produced extracellularly. Here, we report that ENPP6, a choline-specific phosphodiesterase, hydrolyzes glycerophosphocholine (GPC), a degradation product of PC, as a physiological substrate and participates in choline metabolism. ENPP6 is highly expressed in liver sinusoidal endothelial cells and developing oligodendrocytes, which actively incorporate choline and synthesize PC. ENPP6-deficient mice exhibited fatty liver and hypomyelination, well known choline-deficient phenotypes. The choline moiety of GPC was incorporated into PC in an ENPP6-dependent manner both in vivo and in vitro. The crystal structure of ENPP6 in complex with phosphocholine revealed that the choline moiety of the phosphocholine is recognized by a choline-binding pocket formed by conserved aromatic and acidic residues. The present study provides the molecular basis for ENPP6-mediated choline metabolism at atomic, cellular and tissue levels.

Highlights

Unlike myelin basic protein (MBP), which was expressed both in Schwann cells and oligodendrocytes, ENPP6 was not detected in Schwann cells in femoral nerves (Fig. 1B)
At postnatal day 2 (P2) and P4, ENPP6 was immunohistochemically detected in immature oligodendrocytes, where it was predominantly found both in the processes and cell bodies (Fig. 1D)
The expression of ENPP6 was not observed in immature oligodendrocyte precursor cells (OPCs), but it was dramatically increased after the treatment with thyroid hormones (T3 and T4), which induce OPC differentiation into oligodendrocytes (Fig. 1E–G)

Summary

Results

When D-α -GPC was added to the culture medium, PC-containing D-choline was detected almost exclusively in ENPP6-expressing cells (Fig. 3I), indicating that D-choline was incorporated into ENPP6-expressing but not control cells These results suggested that at the cellular level, ENPP6 hydrolyzes α -GPC to produce phosphocholine as a choline source, thereby contributing to the choline needed by the liver and needed for myelin synthesis. The side-chain Cβ atom of Ser[71] forms van der Waals interactions with the side chain of Tyr[75], and contributes to the choline-binding pocket formation (Fig. S13D), suggesting that the side-chain methyl group of a threonine residue at this position would generate steric clashes with Tyr[75] These observations provided a structural explanation for why only ENPP6 has a serine residue as a catalytic nucleophile among the ENPP family members. Our structural and mutational analyses reveal that specific recognition of the choline moiety of GPC is required for ENPP6-mediated choline metabolism

Discussion

Author Contributions

Additional Information