The role of stomatin in erythropoiesis and a study of erythrocyte vesiculation

Abstract

Stomatin, a member of the stomatin/prohibitin/flotillin/HflK/C (SPFH) domain protein family, is widely expressed and conserved. It regulates erythrocyte GLUT-1 in humans, although its broader functions remain unclear. A rare disease-causing mutation in the STOM gene, including aberrant splicing around exon 3, led to a patient suffering severe erythrocyte defects and lifelong transfusion dependency. Structural studies show that stomatin forms large homo-oligomers and can form hetero-oligomers with related SPFH homology domain proteins. We found that STOM coding variants are classified as rare (<0.001%), with 166 of 288 amino acids, including Phe66, showing no missense mutations: underscoring their functional importance. Comparative analyses indicated conservation beyond the canonical SPFH domain. This encompassed cholesterol-associating ORA and CRAC/CARC motifs, a hydrophobic residue, an Ala-Glu-rich coiled coil, the IDML motif (a type II PDZ-binding domain), and verified palmitoylation sites, Cys30 and Cys87. Network analysis of the erythroid SPFH homology domain proteins STOM, STOML2, FLOT1, FLOT2, ERLIN 1, and ERLIN 2—confirmed to be present in multiple erythroblast, erythrocyte, and erythrocyte-derived vesicle proteomics datasets, unlike STOML1 or STOML3—revealed over 800 interacting proteins. Most were found in erythroblasts, with a preserved subnetwork of over 200 proteins in mature erythrocytes, all of which are documented in the Vesiclopedia database, suggesting a role in erythrocyte vesicle formation. CRISPR-Cas9-mediated editing of STOM; the homozygous deletion of 3 nucleotides encoding Phe66 and a heterozygous deletion on STOM exon 2, showed that these modifications are tolerated during proliferation but impact erythropoiesis, particularly during the late polychromatic to orthochromatic transition. Homozygous Phe66-edited cells exhibited reduced nucleus polarisation and impaired enucleation. This was somewhat compensated for in the heterozygous cell type, demonstrating a recessive loss-of-function pattern. The proteomic analysis of erythrocyte ghosts from individuals with Familial Pseudohyperkalemia highlights the involvement of the proteasomal network and AAA+ ATPases, such as VCP, in the stress response. In Pearson correlation analysis of fold change proteomics data representing vesicles released each week over a 5-week storage period of erythrocyte concentrates, stomatin showed close correlation with VCP, TAOK3, a serine-threonine kinase, and ZDHHC5, a palmitoyl transferase. Our data suggests that stomatin acts as a multifunctional scaffold and chaperone, has a role in protein remodeling, quality control, and vesicular transport with significance in erythropoiesis and the maintenance of mature erythrocytes.

Date of Award	1 Oct 2024
Original language	English
Awarding Institution	University of Bristol
Supervisor	Lesley Bruce (Supervisor) & Jon D Lane (Supervisor)