tailieunhanh - Báo cáo khoa học: Disruption of transport activity in a D93H mutant thiamine transporter 1, from a Rogers Syndrome family

Rogers syndrome is an autosomal recessive disorder result-ing in megaloblastic anemia, diabetes mellitus, and sensori-neural deafness. The gene associated with this disease encodes for thiamine transporter 1 (THTR1), a member of the SLC19 solute carrier family including THTR2 and the reduced folate carrier (RFC). Using transient transfections into NIH3T3 cells of a D93H mutant THTR1derived from a Rogers syndrome family, we determined the expression, post-translational modification, plasma mem-brane targeting and thiamine transport activity. We also explored the impact on methotrexate (MTX) transport activity of a homologous missense D88H mutation in the human RFC, a close homologue of THTR1 | Eur. J. Biochem. 270 4469-4477 2003 FEBS 2003 oi 2-l 2 i0i0 4 i 9jc Disruption of transport activity in a D93H mutant thiamine transporter 1 from a Rogers Syndrome family Dana Baron1 Yehuda G. Assaraf2 Stavit Drori2 and Ami Aronheim1 -Department of Molecular Genetics The Rappaport Institute for Research in the Medical Sciences and the B. Rappaport Faculty of Medicine and department of Biology The Technion-Israel Institute of Technology Haifa Israel Rogers syndrome is an autosomal recessive disorder resulting in megaloblastic anemia diabetes mellitus and sensorineural deafness. The gene associated with this disease encodes for thiamine transporter - THTR- a member of the SLC-9 solute carrier family including THTR2 and the reduced folate carrier RFC . Using transient transfections into NIH3T3 cells of a D93H mutant THTR1 derived from a Rogers syndrome family we determined the expression post-translational modification plasma membrane targeting and thiamine transport activity. We also explored the impact on methotrexate MTX transport activity of a homologous missense D88H mutation in the human RFC a close homologue of THTR-. Western blot analysis revealed that the D93H mutant THTR- was normally expressed and underwent a complete N-glycosylation. However while this mutant THTR- was targeted to the plasma membrane it was completely devoid of thiamine transport activity. Consistently introduction into MTX transport null cells of a homologous D88H mutation in the hRFC did not result in restoration of MTX transport activity thereby suggesting that D88 is an essential residue for MTX transport activity. These results suggest that the D93H mutation does not interfere with transporter expression glycosylation and plasma membrane targeting. However the substitution of this negatively charged amino acid Asp93 by a positively charged residue His in an extremely conserved region the border of transmembrane domain 2 intracellular loop 2 in the SLC-9 family presumably .