Detection of sialomucin complex (MUC4) in human ocular surface epithelium and tear fluid.
OBJECTIVE
To evaluate human ocular surface epithelium and tear fluid for the presence of sialomucin complex (MUC4), a high-molecular-weight heterodimeric glycoprotein composed of mucin (ASGP-1) and transmembrane (ASGP-2) subunits.


METHODS
Reverse transcription-polymerase chain reaction (RT-PCR) and Northern blot analysis assays were used to identify sialomucin complex RNA in ocular surface epithelia. Immunoprecipitation and immunoblot analysis were used to identify immunoreactive species in human tears and in the corneal and conjunctival epithelia using antibodies specific for carbohydrate and peptide epitopes on the sialomucin complex subunits. Immunofluorescence staining was used to detect sialomucin complex in frozen sections and impression cytology specimens of human cornea and conjunctival epithelia.


RESULTS
ASGP-1- and ASGP-2-specific sequences were amplified from RNA extracted from both conjunctival and corneal epithelial biopsies by RT-PCR. Sialomucin complex transcripts were also detected in these tissues by Northern blot analysis, with a greater level of RNA detected in the peripheral than the central corneal epithelium. Sialomucin complex was immunoprecipitated from tear fluid samples and both corneal and conjunctival epithelia and detected by immunoblot analysis with specific anti-ASGP-1 and anti-ASGP-2 antibodies. The ASGP-1 peptide antibody HA-1 stained the full thickness of the corneal and conjunctival epithelia. In contrast, antibody 15H10, which reacts against a carbohydrate epitope on ASGP-1, stained only the superficial epithelial layers of these tissues. No staining was observed in the conjunctival goblet cells.


CONCLUSIONS
Sialomucin complex was originally identified in rat mammary adenocarcinoma cells and has recently been shown to be produced by the ocular surface epithelia of rats. Furthermore, it has been identified as the rat homologue of human MUC4 mucin. The present studies show that it is expressed in the stratified epithelium covering the surface of the human eye and is present in human tear fluid. Expression of a carbohydrate-dependent epitope on the mucin subunit (ASGP-1) of sialomucin complex occurs in a differentiation-dependent fashion. Sialomucin complex joins MUC1 as another membrane mucin produced by the human ocular surface epithelia but is also found in the tear fluid, presumably in a soluble form, as found on the rat ocular surface.
Pflugfelder
S C
SC
Ocular Surface and Tear Center, Bascom Palmer Eye Institute, University of Miami School of Medicine, Florida 33136, USA. spflugfelder@bpei.med.miami.edu
Liu
Z
Z
Monroy
D
D
Li
D Q
DQ
Carvajal
M E
ME
Price-Schiavi
S A
SA
Idris
N
N
Solomon
A
A
Perez
A
A
Carraway
K L
KL
D000918:Antibody Specificity; D015152:Blotting, Northern; D003228:Conjunctiva / Q000737:chemistry*; D004848:Epithelium / Q000737:chemistry; D019573:Epithelium, Corneal / Q000737:chemistry*; D019084:Fluorescent Antibody Technique, Indirect; D006801:Humans; D055264:Mucin-4; D009077:Mucins / Q000032:analysis* / Q000235:genetics; D012313:RNA / Q000032:analysis; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012795:Sialoglycoproteins / Q000032:analysis; D013666:Tears / Q000737:chemistry*
C524658:MUC4 protein, human; C524660:Muc4 protein, rat; D055264:Mucin-4; D009077:Mucins; D012795:Sialoglycoproteins; D012313:RNA