Evidence for two catabolic endoglycosidase activities in beta-mannosidase-deficient goat fibroblasts.

Cultured skin fibroblasts derived from Nubian goats deficient in lysosomal beta-mannosidase, which had previously been shown to accumulate storage oligosaccharides with the structures Man beta 4GlcNAc beta 4GlcNAc and Man beta 4GlcNAc (in the ratio of 2.7:1) were evaluated for their ability to catabolize exogenous [3H]GlcN-labelled glycoproteins isolated from the secretions of cultured goat or human fibroblasts. Regardless of the source of exogenous labelled glycoprotein, affected goat fibroblasts took up the labelled glycoprotein from the culture medium and subsequently accumulated the same major labelled oligosaccharide, identified as Man beta 4GlcNAc beta 4GlcNAc; no such oligosaccharide accumulated in normal goat fibroblasts under the same conditions. Tunicamycin-treated affected fibroblasts also took up labelled exogenous glycoprotein and accumulated labelled storage trisaccharide, further suggesting the direct accumulation of storage trisaccharide from impaired glycoprotein-associated oligosaccharide catabolism. Treatment of metabolically labelled affected fibroblasts with leupeptin, an inhibitor of lysosomal cathepsins, resulted in the 2- to 6-fold inhibition of trisaccharide accumulation, while having little effect on the uptake of [3H]GlcN or the accumulation of labelled disaccharide. The results are most consistent with the presence of two endoglycosidases, an endo-beta-N-acetylglucosaminidase and an endo-aspartylglucosaminidase, in goat fibroblasts. These two activities, rather than heterogeneous core oligosaccharide structures, are responsible for the ultimate accumulation of storage oligosaccharides with one and two GlcNAc residues at their reducing terminus.