Cysteine Oxidation in Human Galectin-1 Occurs Sequentially via a Folded Intermediate to a Fully Oxidized Unfolded Form

Hans Ippel, Michelle C. Miller, Ruud P. M. Dings, Anna-Kristin Ludwig, Hans-Joachim Gabius, Kevin H. Mayo*

*Corresponding author for this work

Research output: Contribution to journalArticleAcademicpeer-review

Abstract

Galectins are multifunctional effectors in cellular homeostasis and dysregulation. Oxidation of human galectin-1 (Gal-1) with its six sulfhydryls produces a disulfide-bridged oxidized form that lacks normal lectin activity yet gains new glycan-independent functionality. Nevertheless, the mechanistic details as to how Gal-1 oxidation occurs remain unclear. Here, we used 15N and 13C HSQC NMR spectroscopy to gain structural insight into the CuSO4-mediated path of Gal-1 oxidation and identified a minimum two-stage conversion process. During the first phase, disulfide bridges form slowly between C16-C88 and/or C42-C66 to produce a partially oxidized, conformationally flexible intermediate that retains the ability to bind lactose. Site-directed mutagenesis of C16 to S16 impedes the onset of this overall slow process. During the second phase, increased motional dynamics of the intermediate enable the relatively distant C2 and C130 residues to form the third and final disulfide bond, leading to an unfolded state and consequent dimer dissociation. This fully oxidized end state loses the ability to bind lactose, as shown by the hemagglutination assay. Consistent with this model, we observed that the Gal-1 C2S mutant maintains intermediate-state structural features with a free sulfhydryl group at C130. Incubation with dithiothreitol reduces all disulfide bonds and allows the lectin to revert to its native state. Thus, the sequential, non-random formation of three disulfide bridges in Gal-1 in an oxidative environment acts as a molecular switch for fundamental changes to its functionality. These data inspire detailed bioactivity analysis of the structurally defined oxidized intermediate in, e.g., acute and chronic inflammation.
Original languageEnglish
Article number6956
Number of pages24
JournalInternational Journal of Molecular Sciences
Volume25
Issue number13
DOIs
Publication statusPublished - 1 Jul 2024

Keywords

  • agglutination
  • alarmin
  • disulfide bridges
  • inflammation
  • lectin
  • GALACTOSIDE-BINDING-PROTEIN
  • PROMOTES AXONAL REGENERATION
  • CELL-GROWTH
  • CARBOHYDRATE-RECOGNITION
  • GLYCAN INTERACTIONS
  • LECTIN
  • ACID
  • IDENTIFICATION
  • SEQUENCE
  • HMGB1

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