Abstract
Purpose :
The surface of every cell is decorated by a unique glycocalyx, which undergoes changes in its composition during cell growth, differentiation, and disease progression. Lectins are carbohydrate-binding proteins used to detect specific glycan signatures. In this study, we aim to characterize the glycocalyx composition of the different cell types present in the human ocular surface by combining the use of lectins with single-cell RNA sequencing (scRNAseq).
Methods :
Nine lectins, targeting major glycan structures, were conjugated to DNA oligonucleotides using click chemistry. Optimal conditions for lectin staining were determined by flow cytometry on cultured human corneal epithelial cells. Human donor corneas, obtained from Saving Sight, were processed into single cells through enzymatic digestions. These cells were incubated with the DNA-conjugated lectins and analyzed by scRNAseq using 10X genomics platform. Data analysis was conducted using the Seurat R package.
Results :
We identified a total of 14 different clusters based on canonical gene expression markers, representing conjunctival and corneal epithelial cells, goblet cells and a smaller proportion of melanocytes, fibroblasts, and immune cells. The analysis of lectin-derived barcodes revealed a significant heterogeneity in the glycocalyx of the different clusters. We found that the glycocalyx composition of epithelial cells differed depending on both their origin (conjunctival or corneal) and their differentiation stage (basal or apical). Goblet cells presented the most distinct glycan pattern, showing the highest levels of mucin-type O-glycans. Finally, we performed unbiased clustering of the cells using the cell surface glycan composition. As a result, we obtained a distribution of clusters that effectively discriminated between goblet cells, basal epithelium, and the suprabasal epithelial cells from both the conjunctiva and the cornea.
Conclusions :
We have defined the glycocalyx composition of the ocular surface at a single-cell level, revealing a heterogeneous and distinctive pattern of cell surface glycans across different cell types. This technique opens new possibilities for analyzing variations in the glycan pattern in ocular diseases and identifying new biomarkers.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.