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Effects of N-linked glycosylation on the creatine transporter

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Portland Press Ltd.
Publication Date
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PMC
Keywords
  • Research Article
Disciplines
  • Biology
  • Chemistry
  • Medicine

Abstract

bic274.dvi Biochem. J. (2006) 393, 459–469 (Printed in Great Britain) doi:10.1042/BJ20050857 459 Effects of N-linked glycosylation on the creatine transporter Nadine STRAUMANN, Alexandra WIND, Tina LEUENBERGER and Theo WALLIMANN1 From the Swiss Federal Institute of Technology, ETH Zu¨rich, Institute of Cell Biology, ETH Ho¨nggerberg, 8093 Zu¨rich, Switzerland The CRT (creatine transporter) is a member of the Na+- and Cl−- dependent neurotransmitter transporter family and is responsible for the import of creatine into cells, and thus is important for cellular energy metabolism. We established for CRT an expression system in HEK-293 cells that allowed biochemical, immuno- logical and functional analysis of CRT wild-type and glycosy- lation-deficient mutants. Analysis of HA (haemagglutinin)- tagged CRT-NN (wild-type rat CRT with an HA-tag at the C- terminus) revealed several monomeric immunoreactive species with apparent molecular masses of 58, 48 and 43 kDa. The 58 kDa species was shown to be plasma-membrane-resident by EndoHf (endoglycosidase Hf) and PNGase F (peptide N-glycosidase F) treatments and represents fully glycosylated CRT, whereas the 48 kDa and 43 kDa species were glycosylation intermediates and non-glycosylated CRT respectively. Glycosylation-deficient mutants (Asn192Asp, Asn197Asp and Asn192Asp/Asn197Asp) showed altered electrophoretic mobility, indicating that CRT is indeed N-glycosylated. In addition, a prominent CRT band in the range of 75–91 kDa was also detected. Pharmacological inhibition of N-linked glycosylation by tunicamycin in CRT- NN-expressing cells gave a similar reduction in molecular mass, corroborating the finding that Asn192 and Asn197 are major N-gly- cosylation sites in CRT. Although the apparent Km was not signi- ficantly affected in glycosylation-deficient mutants compared with CRT-NN, we measured reduced Vmax values for all mutants (21–28% residual activity), and 51% residual activity after enzymatic deglycosylation of surface proteins in intact CRT-

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