Abstract
Mucins function as a protective layer rendering the apical surface of epithelial cells nonadhesive to a variety of microorganisms and macromolecules. Muc-1 is a transmembrane mucin expressed at the apical cell surface of mouse uterine epithelial cells (UEC) that disappears as UEC become receptive for embryo implantation (Surveyor, G. A., Gendler, S. J., Pemberton, L., Das, S. K., Chakraborty, I., Julian, J., Pimental, R. A., Wegner, C. W., Dey, S. K., and Carson, D. D. (1995) Endocrinology 136, 3639-3647). In the present study, the kinetics of Muc-1 assembly, cell surface expression, release, and degradation were examined in polarized mouse UEC in vitro. Mucins were identified as the predominant glycoconjugates synthesized, apically expressed, and vectorially released in both wild-type and Muc-1 null mice. When mucins were released, greater than 95% were directed to the apical compartment. Approximately half of the cell-associated mucins lost during a 24-h period were found in the apical compartment. Vectorial biotinylation detected apically disposed, cell-surface mucin and indicated that at least 34% of these mucins are released apically within 24 h. This suggests that release of mucin ectodomains is part of the mechanism of mucin removal from the apical cell surface of UEC. The half-lives of total cell-associated mucins and Muc-1 were 19.5 +/- 1 and 16.5 +/- 0.8 h, respectively. Muc-1 represented approximately 10% of the [3H]glucosamine-labeled, cell-associated mucins. Studies of the kinetics of intracellular transport of Muc-1 indicated transit times of 21 +/- 15 min from the rough endoplasmic reticulum to Golgi apparatus and 111 +/- 28 min from the Golgi apparatus to the cell surface. Collectively, these studies provide the first comprehensive description of Muc-1 and mucin maturation, metabolism, and release by polarized cells, as well as defining a major metabolic fate for mucins expressed by UEC. Normal metabolic processing appears to be sufficient to account for the removal of Muc-1 protein during the transition of UEC to a receptive state.
Highlights
Mucins are a heterogenous class of large molecular weight glycoproteins that function in vertebrates to protect epithelial cell surfaces from pathogens and digestive enzymes as well as to lubricate these surfaces [1,2,3,4]
We demonstrate that mouse uterine epithelial cells (UEC) express other mucins, that these other mucins are associated with the apical cell surface, and that their rate of protein clearance is similar to that of Muc-1
Other prominent 3H-labeled bands were detected in the total protein extract. These components were present in total protein extracts of UEC from Muc-1 null mice indicating that these components
Summary
Materials—Female mice (CF-1) were purchased from Sasco (Omaha, NE) and Muc-1 null female mice (129SVJxC57BL6) were generated as described previously [16]. 5,000 – 10,000 dpm of 3H-labeled Nonidet P-40-extractable, cell-associated apically released glycoconjugates or 3,000 –5,000 dpm of 3H-labeled Muc-1 was used for these assays except where otherwise indicated These suspensions were incubated for 2 h at room temperature with constant rotary agitation. The immunoprecipitated Muc-1 was eluted from the protein A by two sequential incubations for 3 h at 25 °C using 8 M guanidine hydrochloride, 0.5% (w/v) CHAPS, 20 mM Tris acetate, pH 7.0, 0.025% (w/v) sodium azide, and 2 mM EDTA This extract was dialyzed and dried under rotary vacuum with the other samples. The nonimmunoprecipitable Nonidet P-40 extract, the residual extract, and the media fractions were dialyzed (Mr cut-off 1,000) at 4 °C against 0.1 ϫ D-PBS containing 0.02% (w/v) sodium azide, 1 mM EDTA, 1:1,000 PIMS, and 1 mM glucosamine with four changes of dialysis buffer. Other Procedures—- Protein concentrations were determined by the procedure of Lowry et al [32] using bovine serum albumin as a standard
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