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Bovine Brain Myelin Glycerophosphocholine Choline Phosphodiesterase is an Alkaline Lysosphingomyelinase of the eNPP-Family, Regulated by Lysosomal Sorting

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Abstract

Glycerophosphocholine choline phosphodiesterase (GPC-Cpde) is a glycosylphosphatidylinositol (GPI)-anchored alkaline hydrolase that is expressed in the brain and kidney. In brain the hydrolase is synthesized by the oligodendrocytes and expressed on the myelin membrane. There are two forms of brain GPC-Cpde, a membrane-linked (mGPC-Cpde) and a soluble (sGPC-Cpde). Here we report the characterisation sGPC-Cpde from bovine brain. The amino acid sequence was identical to ectonucleotide pyrophosphatase/phosphodiesterase 6 (eNPP6) precursor, lacking the N-terminal signal peptide region and a C-terminal stretch, suggesting that the hydrolase was solubilised by C-terminal proteolysis, releasing the GPI-anchor. sGPC-Cpde existed as two isoforms, a homodimer joined by a disulfide bridge linking C414 from each monomer, and a monomer resulting from proteolysis N-terminally to this disulfide bond. The only internal disulfide bridge, linking C142 and C154, stabilises the choline-binding pocket. sGPC-Cpde was specific for lysosphingomyelin, displaying 1 to 2 orders of magnitude higher catalytic activity than towards GPC and lysophosphatidylcholine, suggesting that GPC-Cpde may function in the sphingomyelin signaling, rather than in the homeostasis of acylglycerophosphocholine metabolites. The truncated high mannose and bisected hybrid type glycans linked to N118 and N341 of sGPC-Cpde is a hallmark of glycans in lysosomal glycoproteins, subjected to GlcNAc-1-phosphorylation en route through Golgi. Thus, sGPC-Cpde may originate from the lysosomes, suggesting that lysosomal sorting contributes to the level of mGPC-Cpde on the myelin membrane.

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Abbreviations

LPC:

Lysophosphatidylcholine

SPC:

Lysosphingomyelin

GPC:

Glycerophosphocholine

GPC-Cpde:

Glycerophosphocholine choline phosphodiesterase (EC 3.1.4.38)

PLC:

Phospholipase C

eNPP:

Ectonucleotide pyrophosphatase/phosphodiesterase

endo H:

Mannosyl-glycoprotein endo-β-N-acetylglucosaminidase (EC 3.2.1.96)

PNGase F:

Peptide-N4-(N-acetyl-β-glucosaminyl)asparagine amidase (EC 3.5.1.52)

GPI:

Glycosylphosphatidylinositol

p-nPPC:

p-Nitrophenylphosphocholine

Con A:

Concanavalin A

DEAE:

Diethylaminoethyl

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Acknowledgments

We thank Bente Mortensen for technical support, Toril Anne Gronseth, Department of Pharmacy, University of Tromso for help on Mass Spectrometry analyses and prof. Knut Sletten, University of Oslo on Edman degradation. The work was supported by Institute of Medical Biology, the Health Faculty, University of Tromsø.

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Authors and Affiliations

  1. Institute of Medical Biology, University of Tromso, MH Building, 9037, Tromso, Norway

    Linn Greiner-Tollersrud, Thomas Berg, Gry Evjen & Ole K. Greiner-Tollersrud

  2. Division of Child and Adolescent Health, Department of Medical Genetics, University Hospital of Northern Norway, 9038, Tromso, Norway

    Hilde M. F. R. Stensland

Authors
  1. Linn Greiner-Tollersrud
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  2. Thomas Berg
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  5. Ole K. Greiner-Tollersrud
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Correspondence to Ole K. Greiner-Tollersrud.

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Greiner-Tollersrud, L., Berg, T., Stensland, H.M.F.R. et al. Bovine Brain Myelin Glycerophosphocholine Choline Phosphodiesterase is an Alkaline Lysosphingomyelinase of the eNPP-Family, Regulated by Lysosomal Sorting. Neurochem Res 38, 300–310 (2013). https://doi.org/10.1007/s11064-012-0921-z

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  • DOI: https://doi.org/10.1007/s11064-012-0921-z

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