The occurrence of α-mannosidase activity at the surface of hamster embryo (NIL) fibroblasts is indicated by the following findings: (a) When NIL cells were incubated on the glass surfaces on which ovalbumin glycopeptides were covalently linked, a rapid release of free mannose from ovalbumin glycopeptides was observed as evidenced by analysis on gas chromatography/mass spectrometry. (b) Cell suspensions as well as intact cell monolayers hydrolyzed rapidly p-nitrophenyl-α-D-mannoside, and the time-course of the hydrolytic cleavage was linear from the moment of mixing of the substrate with the cells. The hydrolysis of the nitrophenyl glycosides of β-D-mannose, α-D-galactose, β-D-galactose, α-L-fucose, β-D-glucose, β-D-N-acetylgalactosamine and β-D-N-acetylglucosamine was negligible or more than ten times lower as compared with the hydolysis of α-D-mannoside. (c) No released or secreted activity of mannosidase could be detected under the conditions used. (d) Studies using known proportions of broken cells in the incubation mixture indicated that more than 90 percent of the mannosidase activity measured was attributable to intact cells and not to broken cells or cell fragments. (e) Hydrolysis of p-nitrophenyl-α-D-mannoside by cell monolayers was inhibited, in the order of decreasing inhibitory activity, by yeast mannan, ovalbumin, α-1,4-L-mannonolactone, α-methylmannoside, and mannose-6-phosphate. High inhibitory activity of the mannan polysaccharide and of ovalbumin favored the presence of the mannosidase activity at the cell surface, as these substrates may not penetrate rapidly into the cells.
The following findings indicated that the cell surface mannosidase is mediating the cell adhesion based on the recognition of high-mannose-type glycopeptide: (a) Ovalbumin- coated plastic surfaces strongly promoted attachment and spreading of NIL fibroblasts, whereas the same ovalbumin coat did not promote attachment and spreading of some other cell types (BALB/c 3T3 fibroblasts and freshly prepared rat liver cells). (b) Digestion of ovalbumin with α-mannosidase greatly reduced the adhesion-mediating activity. (c) Cell adhesion to ovalbumin-coated surfaces was strongly inhibited by mannose tetrasaccharides, moderately by α-1,4-L-mannonolactone, and weakly by α- methylmannoside and mannose-6-phosphate. This order of the inhibitory activity for cell attachment is the same as that for the inhibition of mannosidic hydrolysis. The interpretation that the cell surface mannosidase is able to mediate cell adhesion is in agreement with previous studies suggesting that polyvalent glycosidase surfaces can promote cell adhesion to a degree similar to that caused by fibronectin and several lectins by interacting with their cell surface substrate site (the accompanying papers of this series).