The protective role of conjunctival goblet cell mucin sialylation

**Background:** Conjunctival goblet cells secrete gel-forming mucins (e.g., MUC5AC) that are heavily O-glycosylated, with sialic acid residues contributing to their negative charge and anti-adhesive properties. While alterations in mucin glycosylation have been observed in ocular surface diseases like dry eye and allergic conjunctivitis, the functional role of sialylation in ocular surface defense was poorly understood. This study aimed to investigate the role of sialylated mucins, particularly those modified by the sialyltransferase ST6GALNAC1, in protecting the ocular surface from allergens and particulate matter.

**Methods:** The authors compared conjunctival goblet cell sialylation in humans, C57BL/6J (B6J) mice, and Balb/c mice using Alcian Blue (AB) and Periodic acid-Schiff (PAS) staining, with and without neuraminidase treatment. Gene expression microarrays and cloning of St6galnac1 transcripts were performed to identify the genetic basis for differential sialylation. An intact St6galnac1 allele was transferred from B6J to Balb/c mice via backcrossing (generating Ao mice), and St6galnac1-knockout mice were generated on the B6J background using CRISPR/Cas9. Mucus secretion was stimulated by instilling ragweed pollen shells, and the resulting aggregates were analyzed for size, penetrability (using 0.5 μm fluorescent beads), and sialyl-Tn content. A murine model of ragweed pollen-induced allergic conjunctivitis was used to assess clinical scores, eosinophil infiltration (by flow cytometry and histology), allergen-specific IgE, and dendritic cell migration to draining lymph nodes. Human impression cytology specimens from healthy controls and atopic keratoconjunctivitis (AKC) patients were stained for sialyl-Tn, and ST6GALNAC1 expression was analyzed in a published pterygium RNA-seq dataset (GSE155776).

**Key Results:** Human and B6J mouse conjunctival goblet cells were AB-positive due to sialic acid, while Balb/c goblet cells were AB-negative. This difference was attributed to a point mutation (G>A) at the 3' splice site of St6galnac1 in Balb/c mice, leading to aberrant splicing and loss of the catalytic sialyl motif L. Intact St6galnac1 expression was confirmed in B6J and Ao mice, and sialyl-Tn was detected in their goblet cells and secreted mucus. Ao mouse mucus formed significantly larger pollen aggregates (p=0.0009 for fraction ≥1 mm) and encapsulated pollen shells with an impenetrable gel-like layer that excluded 0.5 μm fluorescent beads (p=0.0101, 0.0101, 0.0233 for different focus levels). Neuraminidase treatment reduced aggregate size (p=0.0344) and abolished the gel-like layer. In the allergic conjunctivitis model, Ao mice showed significantly lower clinical scores (p=0.0093), fewer scratch bouts (p=0.0449), reduced eosinophil infiltration (p=0.0379 for 100 mg/mL RW), and a higher antigen dose threshold for eosinophil accumulation (p=0.0091 for 50 mg/mL, p=0.0006 for 100 mg/mL). RW-specific IgE was elevated in WT but not Ao mice after challenges. The amount of remaining pollen in the conjunctival sac was significantly lower in Ao mice (p=0.0325), and the fraction of allergen-bearing dendritic cells in draining lymph nodes was reduced (p=0.0037). In humans, sialyl-Tn-positive goblet cells were increased in AKC patients (p=0.0443), and ST6GALNAC1 expression was upregulated in pterygium biopsies (p=0.0103). St6galnac1-KO mice on the B6J background showed reduced aggregate size and exacerbated eosinophilic inflammation compared to WT.

**Clinical Implications:** This study identifies sialylation of conjunctival goblet cell mucins as a critical mechanism for trapping and removing allergens and particulate matter from the ocular surface, thereby protecting against allergic inflammation. The findings suggest that enhancing mucin sialylation, for example by targeting ST6GALNAC1, could represent a novel therapeutic strategy for allergic conjunctivitis and other ocular surface diseases characterized by chronic inflammation or mechanical irritation. The upregulation of ST6GALNAC1 and sialyl-Tn in human AKC and pterygium may represent a protective response to chronic stimuli. The study also highlights the importance of considering genetic background (e.g., Balb/c vs. B6J mice) in ocular surface research.