N-linked glycosylation and its impact on the electrophoretic mobility and function of the human proton-coupled folate transporter (HsPCFT)

Ersin Selcuk Unal, Rongbao Zhao, Andong Qiu, I. David Goldman

Research output: Contribution to journalArticle

54 Citations (Scopus)

Abstract

The human proton-coupled folate transporter (HsPCFT, SLC46A1) mediates intestinal absorption of folates and transport of folates into the liver, brain and other tissues. On Western blot, HsPCFT migrates as a broad band (~ 55 kDa), higher than predicted (~ 50 kDa) in cell lines. Western blot analysis required that membrane preparations not be incubated in the loading buffer above 50 °C to avoid aggregation of the protein. Treatment of membrane fractions from HsPCFT-transfected HeLa cells with peptidyl N-glycanase F, or cells with tunicamycin, resulted in conversion to a ~ 35 kDa species. Substitution of asparagine residues of two canonical glycosylation sites to glutamine, individually, yielded a ~ 47 kDa protein; substitution of both sites gave a smaller (~ 35 kDa) protein. Single mutants retained full transport activity; the double mutant retained a majority of activity. Transport function and molecular size were unchanged when the double mutant was hemagglutinin (HA) tagged at either the NH2 or COOH terminus and probed with an anti-HA antibody excluding degradation of the deglycosylated protein. Wild-type or deglycosylated HsPCFT HA, tagged at amino or carboxyl termini, could only be visualized on the plasma membrane when HeLa cells were first permeabilized, consistent with the intracellular location of these domains.

Original languageEnglish (US)
Pages (from-to)1407-1414
Number of pages8
JournalBiochimica et Biophysica Acta - Biomembranes
Volume1778
Issue number6
DOIs
StatePublished - Jun 2008

Fingerprint

Glycosylation
Electrophoretic mobility
Hemagglutinins
HeLa Cells
Folic Acid
Western Blotting
Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase
Tunicamycin
Proteins
Substitution reactions
Membranes
Asparagine
Intestinal Absorption
Glutamine
Proteolysis
Anti-Idiotypic Antibodies
Buffers
Cell membranes
Cell Membrane
Liver

Keywords

  • Folate transport
  • HCP1
  • Hereditary folate malabsorption (HFM)
  • Intestinal folate absorption
  • PCFT glycosylation
  • PCFT secondary structure
  • PCFT, proton-coupled folate transporter
  • PCFT/HCP1
  • SLC46A1

ASJC Scopus subject areas

  • Biochemistry
  • Cell Biology
  • Biophysics

Cite this

@article{b86fca0ca3fb49aea2af0f7a46c83b93,
title = "N-linked glycosylation and its impact on the electrophoretic mobility and function of the human proton-coupled folate transporter (HsPCFT)",
abstract = "The human proton-coupled folate transporter (HsPCFT, SLC46A1) mediates intestinal absorption of folates and transport of folates into the liver, brain and other tissues. On Western blot, HsPCFT migrates as a broad band (~ 55 kDa), higher than predicted (~ 50 kDa) in cell lines. Western blot analysis required that membrane preparations not be incubated in the loading buffer above 50 °C to avoid aggregation of the protein. Treatment of membrane fractions from HsPCFT-transfected HeLa cells with peptidyl N-glycanase F, or cells with tunicamycin, resulted in conversion to a ~ 35 kDa species. Substitution of asparagine residues of two canonical glycosylation sites to glutamine, individually, yielded a ~ 47 kDa protein; substitution of both sites gave a smaller (~ 35 kDa) protein. Single mutants retained full transport activity; the double mutant retained a majority of activity. Transport function and molecular size were unchanged when the double mutant was hemagglutinin (HA) tagged at either the NH2 or COOH terminus and probed with an anti-HA antibody excluding degradation of the deglycosylated protein. Wild-type or deglycosylated HsPCFT HA, tagged at amino or carboxyl termini, could only be visualized on the plasma membrane when HeLa cells were first permeabilized, consistent with the intracellular location of these domains.",
keywords = "Folate transport, HCP1, Hereditary folate malabsorption (HFM), Intestinal folate absorption, PCFT glycosylation, PCFT secondary structure, PCFT, proton-coupled folate transporter, PCFT/HCP1, SLC46A1",
author = "{Selcuk Unal}, Ersin and Rongbao Zhao and Andong Qiu and Goldman, {I. David}",
year = "2008",
month = "6",
doi = "10.1016/j.bbamem.2008.03.009",
language = "English (US)",
volume = "1778",
pages = "1407--1414",
journal = "Biochimica et Biophysica Acta - Biomembranes",
issn = "0005-2736",
publisher = "Elsevier",
number = "6",

}

TY - JOUR

T1 - N-linked glycosylation and its impact on the electrophoretic mobility and function of the human proton-coupled folate transporter (HsPCFT)

AU - Selcuk Unal, Ersin

AU - Zhao, Rongbao

AU - Qiu, Andong

AU - Goldman, I. David

PY - 2008/6

Y1 - 2008/6

N2 - The human proton-coupled folate transporter (HsPCFT, SLC46A1) mediates intestinal absorption of folates and transport of folates into the liver, brain and other tissues. On Western blot, HsPCFT migrates as a broad band (~ 55 kDa), higher than predicted (~ 50 kDa) in cell lines. Western blot analysis required that membrane preparations not be incubated in the loading buffer above 50 °C to avoid aggregation of the protein. Treatment of membrane fractions from HsPCFT-transfected HeLa cells with peptidyl N-glycanase F, or cells with tunicamycin, resulted in conversion to a ~ 35 kDa species. Substitution of asparagine residues of two canonical glycosylation sites to glutamine, individually, yielded a ~ 47 kDa protein; substitution of both sites gave a smaller (~ 35 kDa) protein. Single mutants retained full transport activity; the double mutant retained a majority of activity. Transport function and molecular size were unchanged when the double mutant was hemagglutinin (HA) tagged at either the NH2 or COOH terminus and probed with an anti-HA antibody excluding degradation of the deglycosylated protein. Wild-type or deglycosylated HsPCFT HA, tagged at amino or carboxyl termini, could only be visualized on the plasma membrane when HeLa cells were first permeabilized, consistent with the intracellular location of these domains.

AB - The human proton-coupled folate transporter (HsPCFT, SLC46A1) mediates intestinal absorption of folates and transport of folates into the liver, brain and other tissues. On Western blot, HsPCFT migrates as a broad band (~ 55 kDa), higher than predicted (~ 50 kDa) in cell lines. Western blot analysis required that membrane preparations not be incubated in the loading buffer above 50 °C to avoid aggregation of the protein. Treatment of membrane fractions from HsPCFT-transfected HeLa cells with peptidyl N-glycanase F, or cells with tunicamycin, resulted in conversion to a ~ 35 kDa species. Substitution of asparagine residues of two canonical glycosylation sites to glutamine, individually, yielded a ~ 47 kDa protein; substitution of both sites gave a smaller (~ 35 kDa) protein. Single mutants retained full transport activity; the double mutant retained a majority of activity. Transport function and molecular size were unchanged when the double mutant was hemagglutinin (HA) tagged at either the NH2 or COOH terminus and probed with an anti-HA antibody excluding degradation of the deglycosylated protein. Wild-type or deglycosylated HsPCFT HA, tagged at amino or carboxyl termini, could only be visualized on the plasma membrane when HeLa cells were first permeabilized, consistent with the intracellular location of these domains.

KW - Folate transport

KW - HCP1

KW - Hereditary folate malabsorption (HFM)

KW - Intestinal folate absorption

KW - PCFT glycosylation

KW - PCFT secondary structure

KW - PCFT, proton-coupled folate transporter

KW - PCFT/HCP1

KW - SLC46A1

UR - http://www.scopus.com/inward/record.url?scp=43649088482&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=43649088482&partnerID=8YFLogxK

U2 - 10.1016/j.bbamem.2008.03.009

DO - 10.1016/j.bbamem.2008.03.009

M3 - Article

VL - 1778

SP - 1407

EP - 1414

JO - Biochimica et Biophysica Acta - Biomembranes

JF - Biochimica et Biophysica Acta - Biomembranes

SN - 0005-2736

IS - 6

ER -