Assay of mucins in human tear fluid

Abstract

Mucin genes, both secreted (MUC2, MUC5AC, MUC5B, MUC7) and membrane associated (MUC1, MUC4, MUC16), have been reported to be expressed by ocular surface epithelia. The purpose of this study was to comprehensively assay the mucin content of human tear fluid using multiple antibodies for each mucin and to develop a sensitive, semi-quantitative method for the assay of mucins in tears. Tear washes were obtained by instillation of saline onto the ocular surface, followed by collection from the inferior fornix. Tear proteins were separated in 1% agarose gels, transferred to nitrocellulose membrane by vacuum blotting and probed with multiple antibodies recognizing MUC1, MUC2, MUC4, MUC5AC, MUC5B, MUC7 and MUC16. Binding was detected using chemiluminescence, and quantity was determined by densitometry. Serial dilutions of pooled tears from normal individuals were assayed to determine the linear range of detectability. MUC1, MUC4, MUC16, MUC5AC and low levels of MUC2 were consistently detected in human tear fluid, while MUC5B and MUC7 were not. Use of several antibodies recognizing different epitopes on the same mucin confirmed these findings. The antibodies to mucins bound to serial dilutions of tears in a linear fashion (r2 > 0.9), indicating the feasibility of semi-quantitation. MUC5AC in tear fluid had an increased electrophoretic mobility compared to MUC5AC isolated from conjunctival tissue. This study provides clear evidence that the mucin component of tears is a mixture of secreted and shed membrane-associated mucins, and for the first time demonstrates MUC16 in tear fluid. Immunoblots of tears using agarose gel electrophoresis and chemiluminescence detection provide a semi-quantitative assay for mucin protein that will be useful for comparisons with tears from diseased eyes or after pharmacological intervention.

Fig. 1

Detection of MUCs 1, 2, 4, 16 and 5AC in tear fluid was determined through the use of multiple mucin-specific antibodies on samples of pooled human tears (n=4, unless noted). In order to compare their binding patterns and determine which would be best for use in quantitative analyses, 2-to-3 antibodies with differing epitopes per membrane-associated mucin (A) and per secreted mucin (B, C) were screened. (A) Major differences in electrophoretic mobility were seen with the antibodies used to detect MUC1 and MUC4. Note, however, that faint bands with the same mobility as that detected with HMFG-1 can be seen in the HMFG-2 and 214D4 blots (arrowhead). No major differences were noted between any of the MUC16 antibodies. Note that multiple bands were detected for MUC16 and its carbohydrate epitope H185 when 100 μg of total tear proteins, pooled from 17 subjects, was examined. (B) Three MUC2-specific antibodies were tested on 100 μg of total protein per lane from tears pooled from multiple collections of a single individual (T1) or single collections of 21 normal individuals (T2). Weak binding (as compared to that of the positive control colon, (C)) was seen with 2 of the 3 antibodies (PMH1, PH1417), although the number and electrophoretic mobility of the positive bands was different (*-marks 75 kDa). In contrast, strong positive binding was seen with 10 μg of total protein loaded per lane for MUC5AC, with no major differences in electrophoretic mobility. (C) MUC5B and MUC7 were not detected in human tears, even with maximal loading of tear proteins (100 μg). One μg of purified human cervical mucins (M) was used as positive control for MUC5B and 10 μg of human saliva (S) as positive control for MUC7.

Fig. 2

The feasibility of quantitating mucins in tears with the immunoblot assay developed in this study is confirmed by the regression analysis of the antibody binding over a discrete range of protein concentrations. Tears were run non-reduced in these assays. Densitometric analyses of immunoblots (insets) of two-fold serial dilutions of tear proteins pooled from 2-to-4 normal subjects are illustrated for each MUC antibody considered for further studies. The lower limit of detectability was reached for each mucin (see insets). Density in background subtracted pixels was determined and plotted against protein concentration. In these analyses the regression coefficients (r²) confirmed the linearity of the responses.

Fig. 3

Pilot experiment to show feasibility that single collections of tears from individuals can be used to perform semi-quantitative analyses of the mucin content per μg total protein. Immunoblots of human tears from each of 4 to 5 individuals were probed with antibodies specific for MUCs 1, 4, 16 and 5AC. Note that differences in band density can be discerned between individuals when equivalent amounts of total protein are loaded per lane and that MUC1 showed the greatest variability between individuals. Blot (A) was probed for MUC1 with antibody 214D4, 32 μg of total protein was loaded per sample with 4 of 5 positive. (B) MUC4 was detected with antibody 1G8, 16 μg loaded per sample with 5 of 5 positive. (C) Antibody OC125 was used to detect MUC16 on 10 μg of total protein per sample, with 4 of 4 positive. (D) MUC5AC was detected with antibody CLH2, 12 μg of total protein, with 4 of 4 positive.

Fig. 4

MUC5AC mucin collected from tears and conjunctival epithelial cells have different electrophoretic mobility. (A) MUC5AC secreted into tear fluid (T) is of higher mobility than that isolated from fundus (F) or gall bladder (G) and in secreted human cervical mucus (M). To ensure visualization of any minor components of MUC5AC in tears, 200 μg of total protein were examined in this blot. Arrowhead indicates the loading wells at the top of the gel. (B) MUC5AC in tears (T) is also of higher mobility than that isolated from conjunctival epithelial cells (E) of the same subject. (C) The addition of protease inhibitors (T+) to tears in two individuals did not affect MUC5AC electrophoretic mobility as compared to tears from the contralateral eye with no inhibitors (T−). These results indicate lack of proteolytic cleavage post-collection. In these experiments, MUC5AC mucin was detected on immunoblots of non-reduced proteins with the MUC5AC-specific antibodies 791 (A) and CLH2 (B, C).