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@ARTICLE{Achilles:1051951,
      author       = {Achilles, Sophie and Tomczak, Jan-Niklas and Baumann,
                      Fabiane-Samira and Haddad, Bassam G. and Oswald, Stefan and
                      Machtens, Jan-Philipp and Geertsma, Eric R. and Wittig, Ilka
                      and Lamprecht, Georg},
      title        = {{N} -glycans on {SLC}26{A}3 do not significantly alter
                      plasma membrane or lipid raft trafficking, but appear to
                      stabilize interdomain contacts to stimulate transport},
      journal      = {American journal of physiology / Gastrointestinal and liver
                      physiology},
      volume       = {329},
      number       = {5},
      issn         = {0193-1857},
      address      = {Bethesda, Md.},
      publisher    = {American Physiological Society},
      reportid     = {FZJ-2026-00632},
      pages        = {G628 - G638},
      year         = {2025},
      note         = {The mass spectrometric analysis of this project was
                      supportedby funding from the German Research Foundation
                      (ProjectNumber: 505943254) to S.O. This work was supported
                      by theGerman Research Foundation as parts of the Research
                      Unit FOR5046 (Project Number: 426950122) to J.-P.M. (Project
                      P2), toE.R.G. (Project P1), and to I.W. and G.L. (Project
                      P5). The authorsgratefully acknowledge computing time on the
                      supercomputerJURECA (1) at Forschungszentrum J€ulich under
                      Grant No.jara0177.},
      abstract     = {DRA (Downregulated in adenoma, SLC26A3) is a major apical
                      intestinal Cl/HCO3 exchanger, which is expressed in
                      complexand hybrid N-glycosylated forms. Although the
                      importance of N-glycosylation is evident from the
                      significantly reduced transportactivity of
                      non-N-glycosylated DRA constructs (DRA-N0), the underlying
                      molecular mechanisms are controversial. Therefore,plasma
                      membrane expression and lipid raft localization of
                      glycosylation-deficient DRA-N0 were analyzed in HEK cells.
                      The activityof DRA-N0 was reduced by $70\%$ compared with
                      the wild-type construct. Absolute expression of DRA-N0 was
                      significantlyreduced by $57\%$ in the cell lysate and by 34
                      and $45\%$ in the plasma membrane and in plasma
                      membrane-derived lipid rafts,respectively. These amounts are
                      insufficient to account for the reduction in activity.
                      Furthermore, the statistical analysis did notsupport a
                      difference in the relative expression of DRA and DRA-N0 in
                      the plasma membrane and in plasma membrane-derivedlipid
                      rafts, indicating that N-glycosylation does not affect
                      transport activity through trafficking and localization in
                      these cell compartments.To gain insight into potential
                      intramolecular effects of N-glycosylation on DRA, its
                      three-dimensional structure waspredicted using AlphaFold3
                      with complex N-glycans covalently attached to N153, N161,
                      and N164 in the transport domain. Thisrevealed multiple
                      inward- and outward-facing conformations of the protein. The
                      number of interdomain contacts of the transportdomain-bound
                      glycans with the scaffold domain was higher in the
                      inward-facing state. Because substrate release to the
                      cytoplasmrepresents the rate-limiting step in many transport
                      proteins, this suggests that in DRA, glycans stabilize the
                      inward-facingstate facilitating anion transport.NEW $\&$
                      NOTEWORTHY Deficient N-glycosylation decreases DRA transport
                      activity but does not significantly affect trafficking tothe
                      plasma membrane or to lipid rafts. Meanwhile, molecular
                      modeling predicts stabilizing interdomain contacts of the
                      glycans,covalently attached to the transport domain, with
                      the scaffold domain having more contacts in the
                      inward-facing state. Favoringthe inward-facing state may
                      facilitate more efficacious anion transport, as substrate
                      release from this state into the cytoplasm isa rate limiting
                      step for numerous transport proteins.},
      cin          = {IBI-1},
      ddc          = {610},
      cid          = {I:(DE-Juel1)IBI-1-20200312},
      pnm          = {5243 - Information Processing in Distributed Systems
                      (POF4-524) / DFG project G:(GEPRIS)426950122 - FOR 5046:
                      Integrative Analyse epithelialer SLC26 Anionentransporter
                      – von der molekularen Struktur zur Pathophysiologie
                      (426950122)},
      pid          = {G:(DE-HGF)POF4-5243 / G:(GEPRIS)426950122},
      typ          = {PUB:(DE-HGF)16},
      pubmed       = {41071686},
      UT           = {WOS:001620711300001},
      doi          = {10.1152/ajpgi.00362.2024},
      url          = {https://juser.fz-juelich.de/record/1051951},
}