Abstract
Purpose::
Human corneal and conjunctival epithelia express three membrane-associated mucins (MAMs) at their apical surface. These mucins, MUC1, 4 and 16, are present in cytoplasmic vacuoles, in addition to being present in the surface glycocalyx. Their ectodomains are constitutively shed into the tear film. In order to measure trafficking of these mucins following shedding, a comparison of the amount of surface membrane versus cytoplasmic stores is needed. The purpose of this study was, therefore, to determine the proportion of cell surface to cytoplasmic stores of the MAMs in cultured ocular surface epithelia and then determine the amount of cell surface mucin removed by a test sheddase.
Methods::
Immortalized human corneal limbal (HCLE) and conjunctival (HCjE) epithelial cell lines were grown to confluence in serum free medium and switched to DMEM/F12 with serum for 7 days to optimize mucin production (Gipson et al., 2003). Some cell cultures were treated with a sheddase (neutrophil elastase) for 30 minutes. Proteins at the apical cell surface of cultures were labeled with a non-cell permeable biotin analog (Sulfo-NHS-SS-biotin). The reaction was quenched and cells lysed in buffer containing mild detergent. Immobilized NeutrAvidin Gel was used to capture biotin-labeled proteins. Cell surface and cytoplasmic proteins (unbiotinylated wash-through samples) were separated on 1% SDS-agarose gels for immunoblot with antibodies to MUC1, 4 and 16. Densitometry was done to determine amounts of MUC protein in cell surface and cytoplasmic fractions and were expressed as % of total +/- SEM.
Results::
The three MAMs expressed by the human ocular surface epithelial cells were found in both cell surface and cytoplasmic fractions with a differential compartmentalization of the MAMs between the two cell lines. In HCLE cells, 38 +/- 9% of MUC1 was on the cell surface and in HCjE cells, 49 +/- 0.6%. In contrast, 14 +/- 5% of MUC4 was on the cell surface in HCLE cells, but 40 +/- 6 % in HCjE cells. In HCLE cells, 55 +/- 10% of total MUC16 was present on the cell surface and in HCjE cells, 70 +/- 7%. In a preliminary experiment, there was a 55 +/- 6% decrease in the amount of MUC16 on the cell surface in response to treatment with neutrophil elastase, a sheddase of MUC16 but not MUC1 or 4.
Conclusions::
The membrane-associated mucins expressed by ocular surface epithelia are present in differing proportions on the cell surface and in cytoplasmic stores. These methods allow measurement of the MAMs following shedding experiments.
Keywords: cornea: epithelium • cornea: surface mucins • cornea: tears/tear film/dry eye