Circulating glycornas: A novel class of glycoconjugates in blood and implications for erythrocyte biology

Abstract

Abstract Background: The discovery of glycoRNAs introduced a groundbreaking paradigm, revealing RNA could be covalently modified with glycans and are present on the cell surface. These studies suggested that glycoRNAs could act as signaling molecules and play a role in extracellular communication and immune modulation through interaction with siglec receptors. Recently, glycoRNAs were also found to exist on the cell surface of neutrophils, mediate transendothelial migration, and serve as ligands for P-selectin. These findings raised questions about their presence and potential roles in other cells within the human circulatory system, which are known to have cell free RNA but also contains high concentrations of ribonucleases. Methods: GlycoRNAs in human blood fractions (leukocytes, erythrocytes, and plasma) were detected using RNA-optimized periodate oxidation and aldehyde ligation (rPAL). To confirm their surface localization, intact erythrocytes were treated with either RNase or live-cell sialidase prior to rPAL analysis. Blood samples from individuals with different ABO blood groups were also analyzed in triplicate. The molecular identity of glycan structures was determined using a mass spectrometry-based technique termed glycanDIA. For this analysis, total RNA and proteins were isolated from erythrocytes for comparative characterization. Flow cytometry was used for functional validation of surface glycoRNAs. Erythrocytes were treated with a mock solution or with RNase A and then stained with a lamprey monoclonal antibody specific for the blood group H antigen (OmcFL3-02). Antibodies against Glycophorin A (CD235a) and Rhesus D (RhD) were used as controls. Results: Using the rPAL method, robust glycoRNA signals were detected in all tested human blood fractions (leukocytes, erythrocytes, and plasma). The presence of glycoRNAs on erythrocytes was particularly surprising given their anucleate nature. To determine their location, intact erythrocytes were treated with sialidases, which resulted in a >90% loss of the glycoRNA signal, supporting their localization on the outer cell surface. GlycoRNAs were present in individuals from all ABO blood groups, in varying quantities. Mass spectrometry analysis of erythrocyte RNA revealed the presence of both N- and O-glycan motifs, decorated with fucose and sialic acid. Intriguingly, our analysis also identified glycan structures corresponding to the A, B, and H (O) blood group antigens directly on RNA molecules. This novel finding challenges the long-standing paradigm that these critical antigens exist exclusively on glycoproteins and glycolipids. Flow cytometry experiments provided functional validation for this discovery. Treatment of erythrocytes with RNase A led to a statistically significant decrease in the binding of a specific H-antigen-specific antibody (p<0.01). This effect was specific, as the same treatment did not affect the binding of antibodies against other major surface proteins, CD235a (Glycophorin A) or Rhesus D (RhD). These results indicate that surface RNAs are physically associated with blood group antigen structures, potentially modulating their presentation on the cell surface. Conclusions: The discovery of glycoRNAs in the circulatory system, particularly the association with erythrocyte blood group antigens, represent an advancement in our understanding of both RNA and erythrocyte biology. These findings may open new avenues of research toward exploring the functional implications of circulating glycoRNAs in hematology, immune surveillance, and pathogenesis of diseases, and yield potential new targets for diagnostics and therapeutics.