Human lysosomal α-glucosidase: functional characterization of the glycosylation sites

Abstract

N-linked glycosylation is one of the important events in the post-translational modification of human lysosomal alpha-glucosidase. Phosphorylation of mannose residues ensures efficient transport of the enzyme to the lysosomes via the mannose 6-phosphate receptor. The primary structure of lysosomal alpha-glucosidase, as deduced from the cDNA sequence, indicates that there are seven potential glycosylation sites. We have eliminated these sites individually by site-directed mutagenesis and thereby demonstrated that all seven sites are glycosylated. The sites at Asn-882 and Asn-925 were found to be located in a C-terminal propeptide which is cleaved off during maturation. Evidence is presented that at least two of the oligosaccharide side chains of human lysosomal alpha-glucosidase are phosphorylated. Elimination of six of the seven sites does not disturb enzyme synthesis or function. However, removal of the second glycosylation site at Asn-233 interferes dramatically with the formation of mature enzyme. The mutant precursor is synthesized normally and assembles in the endoplasmic reticulum, but immunoelectron microscopy reveals a deficiency of alpha-glucosidase in the Golgi complex and in the more distal compartments of the lysosomal transport pathway.

Structural modeling of mutant α-glucosidases resulting in a processing/transport defect in Pompe disease

“…Of further note, the seven Nlinked glycosylation sites predicted by the primary structure of human acid a-glucosidase and, indeed, occupied by carbohydrate side chains are all located at the outer surface of the molecule (Figure 2). 28,29 Then, we mapped the locations of amino acid residues involved in substitutions that were earlier shown to hamper the post-translational processing and transport of human acid a-glucosidase (Table 1). In these cases, the 110-kD precursor enzyme was synthesized, but defective processing and transport is indicated by the far less than normal amounts of the acid a-glucosidase species of 95 kDa and 76/ 70 kDa detectable by polyacrylamide gel electrophoresis in the presence of SDS-polyacrylamide gel electrophoresis (PAGE).…”

Human Small Intestinal Maltase-glucoamylase cDNA Cloning

“…This type of catalytic site (WIDMNE) has an aspartic acid (D) catalytic site (45,46). This catalytic site is conserved in other carbohydrate hydrolases including human, rabbit, and rat SIM (29, 30, 47); mouse and human (36, 48) lysosomal maltase; fungal maltases from Aspergillus niger, Mucor javanicus, Schwanniomyces occidentalis (49, 50, 51); and plant maltase from sugar beets and barley (52,53).…”

“…This type of catalytic site (WIDMNE) has an aspartic acid (D) catalytic site (45,46 and plant maltase from sugar beets and barley (52,53).…”

Silent exonic mutation in the acid-α-glycosidase gene that causes glycogen storage disease type II by affecting mRNA splicing

“…3,4 The enzyme is synthesized as a catalytically inactive precursor and undergoes posttranslational modification. 5,6 The precursor protein then undergoes a stepwise proteolysis at both termini, yielding the mature GAA consisting of four associated polypeptides. 4 We conducted a molecular genetic study of a Japanese patient with childhood-onset GSDII.…”