U of Nebraska Medical Center Gundry Lab - Sickle Cell Manuscript

Characterization and statistical modeling of glycosylation changes in sickle cell disease and trait
Data License: CC BY 4.0
  • Organism: Homo sapiens
  • Instrument: LTQ Orbitrap Velos
  • SpikeIn: No
  • Keywords: RBC, red blood cell, glycomics, N-glycan, O-glycan, glycosylation
  • Lab head: Rebekah Gundry Submitter: Christopher Ashwood
Abstract
Sickle cell disease (SCD) is an inherited genetic disorder that causes anemia, pain crises, organ infarction, and infections in 13 million people worldwide. Previous studies have revealed changes in sialic acid levels associated with RBCs' sickling, and stressed RBCs bear clustered terminal surface mannose structures mediating hemolysis, but detailed glycan structures and anti-glycan antibody in SCD remain understudied. Here, we compiled results obtained through lectin arrays, glycan arrays, and mass spectrometry to interrogate red blood cell glycoproteins and glycan-binding proteins found in the plasma of healthy individuals and patients with SCD and sickle cell trait (SCT). Lectin arrays and mass spectrometry revealed an increase in α2,6 sialylation and a decrease in α2,3 sialylation and blood group antigens displayed on red blood cells. Increased plasma glycan protein binding in SCD to immunogenic asialo and sialyl core 1, Lewis A, and Lewis Y, suggesting a heightened anti-glycan immune response, was observed in plasma from patients with SCD. Predictive modeling affirmed glycan expression changes and glycan protein binding in SCD but revealed additional changes in ABO blood group expression and binding to α2,8 sialylated glycans. Our data provide detailed insight into glycan changes associated with SCD and imply glycans as potential therapeutic targets.
Experiment Description
Structural analysis of released N- and O- glycans from delipidated red blood cells from healthy donors and donors with sickle cell disease. PGC-LC-ESI-MS/MS (PGC-liquid chromatography-electrospray ionization-MS/MS) experiments were performed using a nanoLC-2D high performance liquid chromatography system (Eksigent) interfaced with an LTQ Orbitrap Velos mass spectrometer (Thermo Fisher Scientific). Glycans were separated on a PGC-LC column (3 μm, 100 mm × 0.18 mm, Hypercarb, Thermo Fisher Scientific) maintained at 80 °C for N-glycans and 40 °C for O-glycans. To enhance ionization and improve detection of low intensity glycans, post-column make-up flow consisting of 100% methanol was utilized.
Sample Description
Lipid extracted RBCs were prepared for mass spectrometry (MS) from erythrocyte ghosts, as prepared above. Proteins were reduced with 5 mM final concentration of tris(2-carboxyethyl) phosphine at 37 °C for 45 minutes and alkylated with a 10 mM final concentration of iodoacetamide for 45 minutes in the dark at RT, followed by Qubit analysis for protein quantitation (Thermo Fisher Scientific).
Created on 8/17/20, 12:12 PM
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180921_RBC_NG.sky.zip2020-08-17 12:12:5181251286666
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