Human proton coupled folic acid transporter is a monodisperse oligomer in the lauryl maltose neopentyl glycol solubilized state

TY - JOUR

T1 - Human proton coupled folic acid transporter is a monodisperse oligomer in the lauryl maltose neopentyl glycol solubilized state

AU - Aduri, Nanda G

AU - Ernst, Heidi A.

AU - Prabhala, Bala K.

AU - Bhatt, Shweta

AU - Boesen, Thomas

AU - Gajhede, Michael

AU - Mirza, Osman

N1 - Copyright © 2017. Published by Elsevier Inc.

PY - 2018/1/8

Y1 - 2018/1/8

N2 - The human proton coupled folic acid transporter PCFT is the major import route for dietary folates. Mutations in the gene encoding PCFT cause hereditary folic acid malabsorption, which manifests itself by compromised folate absorption from the intestine and also in impaired folate transport in to the central nervous system. Since its recent discovery, PCFT has been the subject of numerous biochemical studies aiming at understanding its structure and mechanism. One major focus has been its oligomeric state, with some reports supporting oligomers and others a monomer. Here, we report the overexpression and purification of recombinant PCFT. Following detergent screening n-Dodecyl β-D-maltoside (DDM) and lauryl maltose neopentyl glycol (LMNG) were chosen for further work as they exhibited the most optimal solubilization. We found that purified detergent solubilized PCFT was able to bind folic acid, thus indicating a functionally active protein. Size exclusion chromatography showed that PCFT in DDM was polydisperse; the LMNG preparation was clearly monodisperse but with shorter retention time than the major DDM peak. To assess the oligomeric state negative stain electron microscopy was performed which showed a particle with the size of a PCFT dimer.

AB - The human proton coupled folic acid transporter PCFT is the major import route for dietary folates. Mutations in the gene encoding PCFT cause hereditary folic acid malabsorption, which manifests itself by compromised folate absorption from the intestine and also in impaired folate transport in to the central nervous system. Since its recent discovery, PCFT has been the subject of numerous biochemical studies aiming at understanding its structure and mechanism. One major focus has been its oligomeric state, with some reports supporting oligomers and others a monomer. Here, we report the overexpression and purification of recombinant PCFT. Following detergent screening n-Dodecyl β-D-maltoside (DDM) and lauryl maltose neopentyl glycol (LMNG) were chosen for further work as they exhibited the most optimal solubilization. We found that purified detergent solubilized PCFT was able to bind folic acid, thus indicating a functionally active protein. Size exclusion chromatography showed that PCFT in DDM was polydisperse; the LMNG preparation was clearly monodisperse but with shorter retention time than the major DDM peak. To assess the oligomeric state negative stain electron microscopy was performed which showed a particle with the size of a PCFT dimer.

KW - Journal Article

U2 - 10.1016/j.bbrc.2017.12.008

DO - 10.1016/j.bbrc.2017.12.008

M3 - Journal article

C2 - 29208467

SN - 0006-291X

VL - 495

SP - 1738

EP - 1743

JO - Biochemical and Biophysical Research Communications

JF - Biochemical and Biophysical Research Communications

IS - 2

ER -