WGA‐binding, mucin glycoproteins protect the apical cell surface of mouse uterine epithelial cells

Abstract

Expression of apical cell surface proteins and glycoproteins was examined in polarized primary cultures of mouse uterine epithelial cells (UEC). Lectin-gold cytochemistry revealed that wheat germ agglutinin (WGA) bound specifically to the components of the apical glycocalyx as well as intracellular vesicles. Double labeling with the pH sensitive dye 3-(2,4-dinitroanilino)-3′amino-N-methyldipropylamine (DAMP) demonstrated the acidic nature of the WGA-staining intracellular vesicles. The enzymatic and chemical sensitivities of the WGA binding sites on the apical cell surface were monitored both by WGA-gold staining as well as by ¹²⁵I-WGA binding assays. In thin sections, a large fraction of these sites were removed by pronase; however, application of a wide variety of proteases, glycosidases, or chemical treatments to the apical surface of intact UEC failed to reduce WGA binding. In no case did treatments designed to remove sialic acids reduce ¹²⁵I-WGA binding more than 12%. In contrast, endo-β-galactosidase as well as a combination of β-galactosidase with β-hexosaminidase succeeded in removing 28% and 77% of these sites, respectively. These studies suggested that the majority of the apically disposed WGA binding sites involved N-acetylglucosamine residues rather than sialic acids and included lactosaminoglycans. Many of the proteins detected at the apical cell surface by lactoperoxidase-catalyzed radioiodination were WGA-binding glycoproteins. A major class of these glycoproteins displayed Mr > 200 kDa by SDS-PAGE and was heavily labeled metabolically by ³H-glucosamine or by vectorial labeling at the apical cell surface with galactosyl transferase and UDP-³H-galactose. Analyses of the ³H-labeled oligosaccharides labeled by either procedure indicated that a large fraction of the apically disposed WGA-binding oligosaccharides consisted of neutral, O-linked mucin-type structures with median MW of approximately 1,500. Oligosaccharides in this fraction were partially (15%) sensitive to endo-β-galactosidase digestion and bound to Datura stramonium agglutinin (68%), demonstrating the presence of lactosaminoglycan sequences. UEC were an extremely effective barrier to attachment or invasion by either a highly invasive melanoma cell line, B16-BL6, or implantation-competent mouse blastocysts. In contrast, neither uterine stromal cells nor a non-polarizing UEC cell line, RL95, prevented B16-BL6 attachment. Treatment of UEC with 2-acetamido-2-deoxy-α-D-galactopyranoside (GalNAcide), an inhibitor of mucin biosynthesis, decreased apical binding of ¹²⁵I-WGA by 66%, increased accessibility of apically disposed tryptic sites by 1.75-fold, and increased apical expression of ³H-heparin binding sites by almost threefold; however, Gal-NAcide-treated UEC remained resistant to invasion by either B16-BL6 cells or mouse blastocysts. Collectively, these data indicate that apical mucin glycoproteins of UEC provide an enzymatically resistant barrier which can limit accessibility to other lumenally disposed agents. Drastic reduction of mucin expression is likely to be required to permit cellular access to the apical cell surface of UEC.