Title : Analysis of the glycosylation and phosphorylation of the alpha-
subunit of the lysosomal
enzyme ,
beta-hexosaminidase A, by site-directed mutagenesis
Abstract :
- The glycosylation and subsequent phosphorylation of mannose residues is a pivotal modification during the biosynthesis of lysosomal enzymes
- We have identified the sites of N-linked glycosylation and oligosaccharide phosphorylation on the alpha- subunit of beta-hexosaminidase and have determined the influence of the oligosaccharides on the folding and transport of the enzyme
- The potential glycosylation sequences , either singly or in combination, were eliminated through site-directed mutagenesis of the cDNA
- By expression of the mutant cDNAs in COS-1 cells, each of the three glycosylation sites on the alpha- subunit was found to be modified by an oligosaccharide
- One of the three oligosaccharides was the preferred site of phosphorylation
- The absence of any individual oligosaccharide did not diminish the expression of the catalytic activity associated with the alpha-chain chain , implying proper folding and assembly of subunits
- A profound effect was observed, however, when all three oligosaccharides were absent
- The unglycosylated alpha- subunit , resulting from genetic alteration of all three glycosylation sites or synthesis of the wild-type protein in the presence of tunicamycin, was catalytically inactive
- It was found to be improperly folded into an insoluble aggregate, linked through inappropriate disulfide bonds
- The unglycosylated protein was trapped in the lumen of the endoplasmic reticulum and was found in a complex with the Ig heavy chain-binding protein , BiP
- The properties of the nonglycosylated, misfolded alpha- subunit were similar to some mutant alpha- subunits in Tay-Sachs disease patients
- The results indicate that the oligosaccharides are essential, although not in a site-specific manner, for proper folding and cellular transport of the alpha- subunit