Intracellular Sorting of Aspartylglucosaminidase: The Role ofN-Linked Oligosaccharides and Evidence of Man-6-P-Independent Lysosomal Targeting (2025)

Activation and Oligomerization of Aspartylglucosaminidase

Journal of Biological Chemistry, 1998

Secretory, membrane, and lysosomal proteins undergo covalent modifications and acquire their secondary and tertiary structure in the lumen of the endoplasmic reticulum (ER). In order to pass the ER quality control system and become transported to their final destinations, many of them are also assembled into oligomers. We have recently determined the three-dimensional structure of lysosomal aspartylglucosaminidase (AGA), which belongs to a newly discovered family of homologous amidohydrolases, the N-terminal nucleophile hydrolases. Members of this protein family are activated from an inactive precursor molecule by an autocatalytic proteolytic processing event whose exact mechanism has not been thoroughly determined. Here we have characterized in more detail the initial events in the ER required for the formation of active AGA enzyme using transient expression of polypeptides carrying targeted amino acid substitutions. We show that His 124 at an interface between two heterodimers of AGA is crucial for the thermodynamically stable oligomeric structure of AGA. Furthermore, the side chain of Thr 206 is essential both for the proteolytic activation and enzymatic activity of AGA. Finally, the proper geometry of the residues His 204 -Asp 205 seems to be crucial for the activation of AGA precursor polypeptides. We propose here a reaction mechanism for the activation of AGA which could be valid for homologous enzymes as well.

Functional analyses of active site residues of human lysosomal aspartylglucosaminidase: implications for catalytic mechanism and autocatalytic activation

The EMBO Journal, 1996

Aspartylglucosaminidase (AGA) is a lysosomal asparaginase that participates in the breakdown of glycoproteins by cleaving the amide bond between the asparagine and the oligosaccharide chain. Active AGA is an (ap)2 heterotetramer of two non-identical subunits that are cleaved proteolytically from an enzymatically inactive precursor polypeptide. On the basis of the three-dimensional structure recently determined by us, we have here mutagenized the putative active site amino acids of AGA and studied by transient expression the effect of targeted substitutions on the enzyme activity and catalytic properties of AGA. These analyses support the novel type of catalytic mechanism, suggested previously by us, in which AGA utilizes as the nucleophile the N-terminal residue of the ,B subunit and most importantly its a-amino group as a base that increases the nucleophilicity of the OH group. We also provide evidence for autocatalytic activation of the inactive AGA precursor and putative involvement of active site amino acids in the proteolytic processing. The data obtained on the structure and function of AGA would indicate that AGA is a member of a recently described novel class of hydrolytic enzymes (amidohydrolases) sharing a common structural determinant in their three-dimensional structure and whose catalytic mechanisms with an N-terminal nucleophile seem basically to be similar.

Primary Folding of Aspartylglucosaminidase. SIGNIFICANCE OF DISULFIDE BRIDGES AND EVIDENCE OF EARLY MULTIMERIZATION

Journal of Biological Chemistry, 1996

Processing by Endoplasmic Reticulum Mannosidases Partitions a Secretion-impaired Glycoprotein into Distinct Disposal Pathways

Journal of Biological Chemistry, 2000

In the early secretory pathway, a distinct set of processing enzymes and family of lectins facilitate the folding and quality control of newly synthesized glycoproteins. In this regard, we recently identified a mechanism in which processing by endoplasmic reticulum mannosidase I, which attenuates the removal of glucose from asparagine-linked oligosaccharides, sorts terminally misfolded ␣ 1 -antitrypsin for proteasome-mediated degradation in response to its abrogated physical dissociation from calnexin (Liu, Y., Choudhury, P., Cabral, C., and Sifers, R. N. (1999) J. Biol. Chem. 274, 5861-5867). In the present study, we examined the quality control of genetic variant PI Z, which undergoes inappropriate polymerization following biosynthesis. Here we show that in stably transfected hepatoma cells the additional processing of asparagine-linked oligosaccharides by endoplasmic reticulum mannosidase II partitions variant PI Z away from the conventional disposal mechanism in response to an arrested posttranslational interaction with calnexin. Intracellular disposal is accomplished by a nonproteasomal system that functions independently of cytosolic components but is sensitive to tyrosine phosphatase inhibition. The functional role of ER mannosidase II in glycoprotein quality control is discussed.

Ser72Pro active-site disease mutation in human lysosomal aspartylglucosaminidase: abnormal intracellular processing and evidence for extracellular activation

Human Molecular Genetics, 1996

Aspartylglucosaminuria (AGU) is a lysosomal storage disease caused by deficient activity of aspartylglucosaminidase (AGA). We report here a T214C mutation leading to a Ser72Pro substitution in four Arab families. This is the first naturally occurring AGU mutation involving an active-site amino acid of this recently crystallized hydrolase and it seems to represent the second most common AGU mutation worldwide. The intracellular consequences of the Ser72Pro mutation were analyzed by transient expression in COS-1 cells and we were able to demonstrate that this active-site mutation most probably does not destroy the enzyme activity per se, but specifically prevents the proteolytic activation cleavage of AGA in the endoplasmic reticulum (ER). The mutant enzyme is, however, folded correctly enough to allow mannose-6-phosphorylation and targeting to lysosomes. The overexpressed mutant enzyme remained inactive intracellularly, but the secreted mutant precursor was proteolytically activated extracellularly, resulting in a similar subunit composition to that in the wild-type AGA in the ER. The partially activated mutant enzyme was endocytosed further by the recipient cells. These data demonstrate that the proteolytic activation of AGA can also occur extracellularly and suggest that the driving mechanism of AGA precursor cleavage is autocatalytic.

A novel aspartylglucosaminuria mutation affects translocation of aspartylglucosaminidase

Human mutation, 2004

The AGA gene is mutated in patients with aspartylglucosaminuria (AGU), a lysosomal storage disease enriched in the Finnish population. The disease mechanism of AGU and the biochemistry and cell biology of the lysosomal aspartylglucosaminidase (AGA) enzyme are well characterized. Here, we have investigated a novel AGU mutation found in a Finnish patient. The mutation was detected as a compound heterozygote with the Finnish major mutation in the other allele. The novel point mutation, c.44T>G, causes the L15R amino acid substitution in the signal sequence of the AGA enzyme. The mutated AGA enzyme was here analyzed by over expression in BHK and COS-1 cells. The L15R AGA protein was only faintly detectable by immunofluorescence analysis and observed in the endoplasmic reticulum. Metabolic labeling and immunoprecipitation revealed only a small amount of AGA polypeptides but the specific activity of the mutant enzyme was surprisingly high, 37% of the wild type. The amino acid substitution probably affects translocation of AGA polypeptides by altering a critical hydrophobic core structure of the signal sequence. It appears that the small amounts of active enzyme are not able to reach the lysosomes thus explaining the development of AGU disease in the patient. © 2004 Wiley-Liss, Inc.

The effects of N-glycosylation sites and the N-terminal region on the biological function of [beta] 1, 3-N-acetylglucosaminyltransferase 2 and its secretion

Biochemical and biophysical …, 2005

Fate and Sorting of Acid β-Glucosidase in Transgenic Mammalian Cells

Molecular Genetics and Metabolism, 2000

Gaucher disease (GD) is associated with mutations at the acid ␤-glucosidase (GCase) locus and the resultant defective activity of the enzyme product. GCase is a membrane-associated glycoprotein that requires detergents for extraction and phospholipid interfaces for full catalytic activity. Normal human fibroblasts and overexpressing transgenic cell lines were used to evaluate the intracellular disappearance, degradation, and secretion of human GCase, including GD fibroblasts and C2C12 cells transduced with MFG-GCase retrovirus and CHO cells stably transfected with the tetracycline transactivation conditional expression system (tet-CHO-GCase). Compared to HF, the disappearance of GCase from the transgenic cells was 12-30 times greater, and had degradative and secretory components. In tet-CHO-GCase cells the majority of GCase was secreted. Intracellular degradation occurred in compartments sensitive to monensin and brefeldin A, and the ALLN or leupeptin protease inhibitors, i.e., ER, Golgi, and lysosomes. In tet-CHO-GCase cells, GCase degradation and secretion rates were inversely related to expression level. Saponin permeabilization analyses of tet-CHO-GCase cells showed that a majority of GCase was soluble, with a rapid disappearance via secretion and degradation. A progressively increasing proportion of GCase became saponin insoluble with a t 1/2 ‫؍‬ 2-3 h. Intracellular saponin-soluble and-insoluble GCases were degraded with t 1/2 ϳ2 and 14 h, respectively. Confocal microscopy showed colocalization of glycosylated or unglycosylated GCase with LAMP-2, an integral lysosomal membrane protein, to vesicular bodies. These studies show that GCase secretion was N-linked glycosylation dependent, whereas sorting to and membrane attachment in the lysosome were N-linked glycosylation independent.

β-Hexosaminidase over-expression affects lysosomal glycohydrolases expression and glycosphingolipid metabolism in mammalian cells

Molecular and Cellular Biochemistry, 2011

Aspartylglycosaminuria: protein chemistry and molecular biology of the most common lysosomal storage disorder of glycoprotein degradation

The FASEB Journal, 1993

Aspartylglycosaminuria (AGU) (McKusick 20840) is the most common disorder of glycoprotein degradation caused by the failure of lysosomes to digest the protein-to-carbohydrate linkage of Asn-linked glycoproteins. During the past few years there has been significant progress in our understanding of both the protein chemistry and molecular biology of glycosylasparaginase (EC 3.5.1.26) as well as the molecular changes underlying the storage disease AGUthat results from deficiency of this lysosomal hydrolase. Modern clinical assays have been developed for the diagnosis and carrier detection of this disease. Detailed structure, substrate specificity, mechanism of action, and a part of the active site of glycosylasparaginase have been defined. Molecular biology of glycosylasparaginase has progressed rapidly and already some mutations in the glycosylasparaginase gene resulting in AGUhave been identified. Evolutionary aspects based on sequence data indicate a mechanistic relationship between mammalian glycosylasparaginases and bacterial/plant asparaginases.-Mononen, I.,