INTRACELLULAR SORTING OF ASPARTYLGLUCOSAMINIDASE - THE ROLE OF N-LINKED OLIGOSACCHARIDES AND EVIDENCE OF MAN-6-P-INDEPENDENT LYSOSOMAL TARGETING

Aspartylglucosaminidase (AGA, E.C. 3.5.1.26) is a soluble lysosomal hy drolase 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 the ir phosphorylation in transport and activity of heterodimeric aspartyl glucosaminidase, using in vitro mutagenesis and transient expression o f mutant polypeptides in COS cells, The single N-glycosylation sites o f both the alpha and beta 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 tran sport, but the totally nonglycosylated enzyme, although active and pro cessed into subunits, was not transported into lysosomes and became tr apped in the endoplasmic reticulum (ER) or secreted, The intracellular targeting of AGA was partially disturbed by the lack of glycosylation in the beta subunit, resulting in accumulation of dimeric, active pol ypeptides in the ER, whereas lack of oligosaccharides in the alpha sub unit did not affect the intracellular targeting of AGA, N-glycans in t he beta subunit were found to be essential for the long-term stability of the polypeptide in the cell, but not for initial folding or subuni t processing into the active dimeric molecule, Both subunits have two glycosylation isoforms, Both forms of the alpha subunit were found to be phosphorylated, whereas only one of the two glycosylation isoforms of the beta subunit is phosphorylated, The mutant enzyme with nonglyco sylated alpha subunit and nonphosphorylated beta subunit is transporte d into lysosomes, suggesting that AGA is capable of using an alternati ve, mannose-6-phosphate receptor-independent routing into lysosomes.