Human Erythrocyte Surface Fucose Expression Increases with Age and Hyperglycemia

Abstract

BackgroundReactive oxygen species and other free radicals, together with glucose and its metabolites are believed to play important roles in the aging process. The carbohydrate components of glycosylated proteins are important in mediating cell-cell interactions and a role has been suggested for them in the aging process. Erythrocytes are critical cells in the human body, heavily glycosylated and relatively easily available and so are good candidates to yield insights into how patterns of glycosylation change with age and disease. It has been claimed, based on a periodic acid Schiff assay, that human aging is associated with a decline of erythrocyte surface sialic acids. Plant lectins allow for more specific assays for glycans, including determining the linkage of sialic acids and analysis of single cells by flow cytometry. ResultsSialic acid specific plant lectins, Maackia amurensis lectin II (MAL), binding to α-2,3 linked sialic acids and Sambucus nigra (SNA), α-2,6 sialic acids, were used to show that erythrocyte surface sialic acids have no significant associations with donor age. A combination of storage and cellular aging produces a specific loss of α-2,6 sialic acids. By contrast, erythrocyte surface terminal fucoses increase significantly with age of donor. In order to determine which aspects of aging are important in determining this change, we investigated whether this novel human aging biomarker is associated with higher plasma glucose values, assessed by glycated hemoglobin (HbA1c) and reactive oxygen species (ROS) generation. Fucose levels were associated with HbA1c levels, but not ROS generation. ConclusionOur study identifies novel glycan-based biomarkers for human aging and disease. The simplicity of lectins are presented here as an attractive tool to study cellular medication in aging and disease.

Human erythrocyte surface fucose expression increases with age and hyperglycemia

“…We also tried to reproduce these findings in murine erythrocytes, but found they lacked SNA binding (Supplemental Figure 3a, Extended data) 32 , suggesting poor lectin visibility of surface α-2,6 sialic acids. There were no changes in MAL or SNA binding when murine RBCs were fractionated by density (Supplemental Figure 3b and 3c, Extended data) 32 .…”

“…It is well known that during cellaging erythrocytes increase their density, become more rigid and fragile, and more deposition of C3 is observed. Our gravity based Percoll fractionation technique separates erythrocytes based on density into 5-7 fractions (Supplemental Figure 1a and 1b, Extended data) 32 , with approximately 1% cells represented in the densest fractions (5, 6, and 7). We confirmed higher fragilities (Supplemental Figure 1d, Extended data) 32 and more C3 deposition (Supplemental Figure 1c, Extended data) 32 in the densest fractions.…”

“…Our gravity based Percoll fractionation technique separates erythrocytes based on density into 5-7 fractions (Supplemental Figure 1a and 1b, Extended data) 32 , with approximately 1% cells represented in the densest fractions (5, 6, and 7). We confirmed higher fragilities (Supplemental Figure 1d, Extended data) 32 and more C3 deposition (Supplemental Figure 1c, Extended data) 32 in the densest fractions. Although we failed to observe any differences in MAL binding to RBCs from any fraction (Figure 2a-d), we observed highly specific SNA binding loss in the densest fractions (Figure 2a-d).…”

“…The data from Figure 2 are generated from erythrocytes stored for seven or more days prior to fractionation. Western blotting identifies the SNA binding loss as arising from bands around 70kDa and 100kDa, corresponding to dimeric glycophorin A (GPA) and Band 3, respectively (Supplemental Figure 2, Extended data) 32 . We also tried to reproduce these findings in murine erythrocytes, but found they lacked SNA binding (Supplemental Figure 3a, Extended data) 32 , suggesting poor lectin visibility of surface α-2,6 sialic acids.…”

“…Western blotting identifies the SNA binding loss as arising from bands around 70kDa and 100kDa, corresponding to dimeric glycophorin A (GPA) and Band 3, respectively (Supplemental Figure 2, Extended data) 32 . We also tried to reproduce these findings in murine erythrocytes, but found they lacked SNA binding (Supplemental Figure 3a, Extended data) 32 , suggesting poor lectin visibility of surface α-2,6 sialic acids. There were no changes in MAL or SNA binding when murine RBCs were fractionated by density (Supplemental Figure 3b and 3c, Extended data) 32 .…”