Intracellular Sorting of Aspartylglucosaminidase: The Role ofN-Linked Oligosaccharides and Evidence of Man-6-P-Independent Lysosomal Targeting

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Abstract
Aspartylglucosaminidase (AGA, E.C. 3.5.1.26) is a soluble lysosomal hydrolase that participates in the degradation of glycoproteins. Here we analyzed the special features in the intracellular targeting of this dimeric amidohydrolase, especially the role of N-linked sugars and their phosphorylation in transport and activity of heterodimeric aspartylglucosaminidase, using in vitro mutagenesis and transient expression of mutant polypeptides in COS cells. The single N-glycosylation sites of both the α and β subunits were destroyed individually and in combination. Just one remaining N-glycosylation site on either subunit was sufficient for normal processing into subunits and lysosomal transport, but the totally nonglycosylated enzyme, although active and processed into subunits, was not transported into lysosomes and became trapped in the endoplasmic reticulum (ER) or secreted. The intracellular targeting of AGA was partially disturbed by the lack of glycosylation in the β subunit, resulting in accumulation of dimeric, active polypeptides in the ER, whereas lack of oligosaccharides in the α subunit did not affect the intracellular targeting of AGA. N-glycans in the β subunit were found to be essential for the long-term stability of the polypeptide in the cell, but not for initial folding or subunit processing into the active dimeric molecule. Both subunits have two glycosylation isoforms. Both forms of the α subunit were found to be phosphorylated, whereas only one of the two glycosylation isoforms of the β subunit is phosphorylated. The mutant enzyme with nonglycosylated a subunit and nonphosphorylated β subunit is transported into lysosomes, suggesting that AGA is capable of using an alternative, mannose-6-phosphate receptor-independent routing into lysosomes.