Abstract:
:Glycosylasparaginase (EC 3.5.1.26) is a lysosomal amidase which hydrolyzes the bond between asparagine and the sugar moiety in N-linked glycoproteins. Deficiency of the enzyme results in aspartylglycosaminuria (AGU), the most common disorder of glycoprotein degradation. Mature enzyme is formed by two proteolytic cleavage steps subsequent to removal of its signal peptide: (1) an activation cleavage in the ER of the initial single-chain 49-kDa polypeptide into a 27-kDa alpha- and 19-kDa beta-subunit; (2) a cleavage in lysosomes which removes 10 amino acids from the C-terminus of the alpha-subunit without affecting enzyme activity. Each subunit of glycosylasparaginase contains one N-linked oligosaccharide (N38, alpha-subunit; N308, beta-subunit). Both oligosaccharides were phosphorylated and releasable by Endo-H digestion, indicating they were of the high-mannose type. These glycosylation sequenons were mutagenized to determine the role of the oligosaccharide at each site in proper folding and transport of glycosylasparaginase. An N38D mutant underwent the lysosomal processing step, indicating that targeting to lysosomes can be via the phosphorylated beta-subunit oligosaccharide alone. Deletion of the beta-subunit oligosaccharide oat N308 by an aspartic acid substitution resulted in very little protein or enzyme activity in the transfected cells, reemphasizing that glycosylation of the beta-subunit site is important for efficient folding and/or targeting. A different mutation to eliminate the same N-glycosylation sequenon (T310A) yielded more protein and enzyme activity, and a double mutant N38D/T310A yielded the same results as the single beta-subunit substitution. Yield of enzyme for all mutants was increased in cells treated with brefeldin A. The N308 glycosylation site of the beta-subunit appears to be more important in maintaining normal transport and stability of human glycosylasparaginase.
journal_name
Arch Biochem Biophysjournal_title
Archives of biochemistry and biophysicsauthors
Park H,Vettese-Dadey M,Aronson NN Jrdoi
10.1006/abbi.1996.0144subject
Has Abstractpub_date
1996-04-01 00:00:00pages
73-7issue
1eissn
0003-9861issn
1096-0384pii
S0003986196901447journal_volume
328pub_type
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