Title : Functional characterization of the N-glycosylation
sites of human
acid sphingomyelinase by site-directed mutagenesis
Abstract :
- Most soluble lysosomal enzymes require a mannose-6-phosphate recognition marker present on asparagine-linked oligosaccharides asparagine-linked oligosaccharides for proper targeting to lysosomes
- We have determined the influence of the six potential N-linked oligosaccharide chains of human acid sphingomyelinase ( ASM ) on catalytic activity, targeting, and processing of the enzyme
- Each N-glycosylation site was modified by site-directed mutagenesis and subsequently expressed in COS-1 cells
- Evidence is presented that five of these sites are used
- Elimination of the four N-terminal glycosylation sites does not disturb lysosomal targeting, processing, or enzymatic activity
- However, removal of the two C-terminal N-glycosylation sites inhibits the formation of mature enzyme
- Absence of glycosylation site five resulted in rapid cleavage of the primary translation product to an enzymatically inactive protein which accumulated inside the endoplasmic reticulum/Golgi, whereas deletion of glycosylation site six led to the formation of an inactive ASM precursor , also retained inside the endoplasmic reticulum/Golgi
- Our results also provide evidence that the site of early proteolytic cleavage of newly synthesized ASM must be located between the second and third glycosylation sites