DIURNAL-VARIATIONS IN TEAR GLYCOPROTEINS - EVIDENCE FOR AN EPITHELIALORIGIN FOR THE MAJOR NON-REDUCIBLE GREATER-THAN-OR-EQUAL-TO-450 KDA SIALOGLYCOPROTEIN(S)

Purpose. To characterize the nature and origin of changes in tear glyc oproteins accompanying eye closure. Methods. Reflex (R) and overnight closed (C) eye tears collected by capillary tubes were centrifuged wit h the resulting R pellets (primarily desquamated epithelial cells) and C pellets (primarily PMN and some epithelial cells) extracted in acid ic PBS. Extracts and supernatants were separated by size-exclusion HPL C and/or SDS-PAGE. Gels were stained or blotted and immune- or lectin- probed. An HPLC glycoprotein fraction of greater than or equal to 450 kDa isolated from all four sources was characterized before and after partial deglycosylation, using antibodies specific to known mucin and carbohydrate epitopes. Immunofluorescence microscopy was carried out o n human conjunctiva, using as probe a MAb to salivary mucin specific f or a sialyl Le(a) epitope, which was found to cross-react specifically with the major non-reducible high molecular weight sialoglycoproteins (SGs) in tears. These SGs were immunoprecipitated and blot-probed alo ng with tissue extracts. Results. R fluid contained minor amounts of n umerous glycoproteins, including probably several of inducible lacrima l secretory origin. Results confirmed sIgA as the principal source of the intense reducible glycoprotein bands common to C fluid. Smaller am ounts of free secretory component and serum glycoproteins were also vi sualized. The HPLC fraction (2450 kDa) consisted of foul major non-red ucible glycoproteins. In R fluid, this fraction (<1% total protein) co nsisted primarily of two entities: a 450-500 kDa SG and a larger asial oglycoprotein. The SG accounts for as much as 85% of the total protein in the R pellet extract. C fluid was associated with a selective incr ease in SGs and a shift in distribution to two SGs >500 kDa. All SGs e xhibited a common antigenicity reacting specifically with the MAb for the sialyl Lee epitope. SGs indistinguishable in size and antigenicity were recovered iri epithelial extracts. Immunofluorescence microscopy revealed that reactivity was localized to the epithelial plasma membr ane, increasing in intensity from basal to apical cells. Although thes e SGs exhibited some properties in common with MUC1, immunological and other data suggest a unique SG. Conclusions. Tear glycoproteins are d erived from four principal sources. In R fluid, an inducible lacrimal secretion predominates. In C fluid, a constitutive sIgA secretion pred ominates, augmented by a serum exudate and SGs derived at least in par t from the epithelium. In R fluid and pellet extracts, the SGs consist primarily of a 450-500 kDa species that is most probably derived from the plasma membrane. Larger antigenically related SGs are prevalent i n C fluid.