Human α-galactosidase A: glycosylation site 3 is essential for enzyme solubility

Abstract

Human alpha-galactosidase A (EC 3.2.1.22; alpha-Gal A) is the homodimeric glycoprotein that hydrolyses the terminal alpha-galactosyl moieties from glycolipids and glycoproteins. The type, site occupancy and function of the N-linked oligosaccharide chains on this lysosomal hydrolase were determined. Endoglycosidase treatment of the purified recombinant enzyme and mutagenesis studies indicated that three (Asn-139, Asn-192 and Asn-215) of the four potential N-glycosylation consensus sequences were occupied by complex, high-mannose and hybrid-type oligosaccharides respectively. When expressed in COS-1 cells, glycoforms with glycosylation site 1 or 2 obliterated had more than 70% of wild-type activity, and both glycoforms were secreted. In contrast, the glycoform with only site 3 eliminated had decreased activity (less than 40%); little, if any, was secreted. Expressed mutant glycoforms in which site 3 and site 1 or 2 were obliterated had little, if any, intracellular or secreted enzymic activity, and immunofluorescence microscopy revealed that the expressed mutant glycoforms were retained in the endoplasmic reticulum, presumably where they were degraded. Thus glycosylation at site 3 was crucial to the formation of soluble, active enzyme, as well as transport to the lysosome. Absence of the site 3 hybrid-type oligosaccharide exposed an adjacent, normally protected, hydrophobic region, resulting in aggregation of the enzyme polypeptide in the endoplasmic reticulum. In support of this concept, endoglycosidase H-treated enzyme or mannose-terminated enzyme expressed in Autographa californica cells also aggregated when concentrated, emphasizing that site 3 occupancy by a hybrid-type oligosaccharide was required for enzyme solubility.

Role of Glycosylation in Conformational Stability, Activity, Macromolecular Interaction and Immunogenicity of Recombinant Human Factor VIII

“…Deletion or substitutions of Asn residues from the Nlinked consensus sequences Asn-Xxx-Ser/Thr of lysosomal enzymes (β-glucuronidase, α-galactosidase, heparan Nsulfatase) resulted in substantial loss of activity (37)(38)(39). Furthermore, it has been reported that ∼90% of the activity of hCG was lost as a result of the deletion of the N-glycan linked to the α52 Asn residues (40).…”

Biosynthesis, Glycosylation, and Enzymatic Processingin Vivo of Human Tripeptidyl-peptidase I

“…61). Oligosaccharides also ensure stability and resistance to protease digestion of some lysosomal hydrolases and lysosomal membrane proteins (62-64) as well as intrinsic enzyme activity (65) and solubility (66), although the removal of the carbohydrate moiety from a variety of glycoproteins had no apparent effect on their biological activity or chemical properties (67).…”

UniProt Genomic Mapping for Deciphering Functional Effects of Missense Variants

“…However due to UniProt's focus on protein structural and functional annotation the publications that review the molecular details on the function of glycosylation at this and other sites are annotated in UniProtKB (Garman and Garboczi 2004;Chen et al 2009). Evidence here shows that the oligomannose-containing carbohydrate at this Asn215 site plus the Asn192 site (not shown) are responsible for secretion of the active enzyme (Ioannou et al 1998) and targeting to the lysosome (Ghosh et al 2003). Mutation of Asn215 to Ser eliminates the carbohydrate attachment site, causing inefficient trafficking of the .…”