Figure 9.

α-Man-II mediates Rh1 deglycosylation in vivo. (A) The MW of Rh1 was reduced after treatment with α1-6 mannosidase or α1-2,3 mannosidase. Purified Rh1 was incubated with different glycosidases at 37°C for 16 h. (B) The MW of Rh1 was reduced after digestion with recombinant α-Man-II. Recombinant dMPPE and GST-α-Man-II were purified as described in Materials and methods. Purified Rh1 was incubated with GST–α-Man-II alone or with the mixture of GST–α-Man-II and dMPPE in 37°C for 16 h. rMPPE represents purified recombinant dMPPE and rMan-II represents purified recombinant GST–α-Man-II. Note that the activity of recombinant α-Man-II does not depend on the presence of dMPPE. (C) The MW of Rh1 from the α-Man-II mutant was similar to that of the dmppe mutant. (D) The MW of Rh1 from α-Man-II mutant was reduced after digestion with PNGase F. (E) Expression of the mutant form α-Man-II^S73G, but not wild-type α-Man-II, restored the MW of Rh1 in the dmppe mutant background. α-Man-II (Myc) and dMPPE expression levels are shown. (F) The role of dMPPE in Rh1 deglycosylation. dMPPE directly dephosphorylates α-Man-II, which in turn removes two mannoses from the oligosaccharide chain of Rh1. N-acetylglucosamine residues are shown as circles and mannose residues are shown as squares.