Despite the profound retinal degeneration observed in the mice lacking
GNPTAB, the activity of five acid hydrolases in whole-eye extracts was not diminished compared with the wild-type levels
(Table 2) . We next examined cathepsin D, one of the critical lysosomal enzymes involved in photoreceptor outer segment processing in the RPE. Lack of this protease in mice results in retinal degeneration with marked apoptosis of photoreceptors.
25 Whole-eye lysates from wild-type, heterozygous, and homozygous mice were subjected to immunoblot analysis with anti-cathepsin D antibodies. The major cathepsin D isoform detected in both the wild-type and the heterozygous lysates was approximately 43 kDa, indicative of the mature species
(Fig. 6) . The lysate of the
GNPTAB −/− mice showed this band as well as an additional slower migrating band. To determine whether the cathepsin D molecules in the three lysates differed in the nature of their two N-linked glycans, aliquots were treated with Endo H or PNGase before electrophoresis. Endo H selectively cleaves high-mannose–type N-linked glycans whereas PNGase cleaves both high mannose and complex-type species. As shown in
Figure 6 , Endo H treatment caused essentially all the cathepsin D from wild-type and heterozygous eye samples to migrate faster on the gels and PNGase increased the migration only slightly more. This result indicates that most of the cathepsin D in these lysates contains two high-mannose units, whereas a minority of the molecules contains one high-mannose unit and one complex-type unit. In contrast, the upper band of the cathepsin D from the
GNPTAB −/− lysate was resistant to Endo H, whereas approximately half of the lower band migrated faster after incubation with this enzyme. After treatment with PNGase, almost all the cathepsin D migrated faster on the gel to the same position as that observed with the wild-type lysate. These findings show that most of the cathepsin D of the
GNPTAB −/− mice contains two complex-type glycans, whereas a minority of the molecules contains one high-mannose and one complex-type glycan. This difference in processing of the two N-linked glycans of cathepsin D reflects the fact that the presence of Man-6-P residues on high-mannose glycans prevents processing to complex-type species. In the wild-type and heterozygous samples, the glycans would be expected to be mostly phosphorylated high-mannose units.
26 In the absence of GlcNAc-1-phosphotransferase activity as occurs in the mice lacking
GNPTAB, these glycans are processed to complex-type species that migrate slower in SDS-PAGE.