Impaired Lysosomal Trimming of N-Linked Oligosaccharides Leads to Hyperglycosylation of Native Lysosomal Proteins in Mice with α-Mannosidosis

Abstract

␣-Mannosidosis is caused by the genetic defect of the lysosomal ␣-D-mannosidase (LAMAN), which is involved in the breakdown of free ␣-linked mannose-containing oligosaccharides originating from glycoproteins with N-linked glycans, and thus manifests itself in an extensive storage of mannose-containing oligosaccharides. Here we demonstrate in a model of mice with ␣-mannosidosis that native lysosomal proteins exhibit elongated N-linked oligosaccharides as shown by two-dimensional difference gel electrophoresis, deglycosylation assays, and mass spectrometry. The analysis of cathepsin B-derived oligosaccharides revealed a hypermannosylation of glycoproteins in mice with ␣-mannosidosis as indicated by the predominance of extended Man3GlcNAc2 oligosaccharides. Treatment with recombinant human ␣-mannosidase partially corrected the hyperglycosylation of lysosomal proteins in vivo and in vitro. These data clearly demonstrate that LAMAN is involved not only in the lysosomal catabolism of free oligosaccharides but also in the trimming of asparaginelinked oligosaccharides on native lysosomal proteins.The lysosomal ␣-D-mannosidase (LAMAN; EC 3.2.1.24) belongs to the group of at least seven lysosomal exoglycosidases which sequentially degrade oligosaccharides derived from glycoproteins (2, 31). These glycoproteins enter the lysosomal compartment by either endocytic pathways (extracellular and plasma membrane proteins) or autophagic processes (intracellular proteins). In addition, free oligosaccharides originating from lipid-linked oligosaccharides in the endoplasmic reticulum and from glycoproteins by the endoplasmic reticulumassociated protein degradation (ERAD) pathway are transported into the lysosome, where these oligosaccharides are subsequently degraded (9, 45). Inside the lysosome, the degradation of the glycoproteins is described as a bidirectional process in which on the one hand the polypeptide is hydrolyzed by a cohort of lysosomal endo-and exoproteases with partially overlapping specificities like cathepsins and other peptidases 40,52). On the other hand, the sugar moiety is stepwise hydrolyzed into its monosaccharides by exoglycosidases. The precise order of the bidirectional breakdown of glycoproteins is unclear, although assumptions can be made based on the analysis of the storage products of the different glycoproteinoses (31). Therefore, it is assumed that an efficient degradation of the oligosaccharide chain is highly dependent on the cleavage of the protein-oligosaccharide linkage by the glycosylasparaginase (2, 31). In contrast, the proteolysis of the polypeptide backbone is mainly unaffected by intact oligosaccharide structures on the glycoproteins (1).LAMAN has a broad substrate specificity, cleaving nonreducing terminal ␣1,2-, ␣1,3-, and ␣1,6-mannosyl linkages found in complex-type, hybrid-type, and high-mannose-type asparagine-linked glycans (30, 60). Additionally, a second lysosomal mannosidase (MAN2B2) specific for the core ␣1,6 branch was characterized and found to be dependent on the prior enzymati...

Mannose 6 Dephosphorylation of Lysosomal Proteins Mediated by Acid Phosphatases Acp2 and Acp5

“…In addition, extracellular lysosomal proteins can be internalized and transported along the endocytic pathway in a Man6P-dependent manner, which represents the therapeutic principle of enzyme replacement therapy of selected lysosomal storage disorders (38). Upon arrival in lysosomes, many lysosomal proteins undergo further modifications such as proteolytic activation and oligosaccharide processing (13,54). In addition to the Man6P-dependent transport, cell type-specific and distinct Man6P-independent pathways for the transport of lysosomal enzymes have been reported (7).…”

Structural and mechanistic insight into N-glycan processing by endo-α-mannosidase

“…These polysaccharide decorations play important roles in processes such as protein folding, targeting, antigenicity, and lectin interactions, with defects leading to cellular dysfunction (1). Aberrant N-glycan composition, through either incorrect or incomplete processing, is associated with various conditions, including Alzheimer's disease, congenital disorders of glycosylation, viral infection, and metastatic cancer progression (2)(3)(4). Alteration of N-glycan biosynthesis in cancerous cells and by human pathogenic viruses renders the various proteins involved in their biosynthesis and modification therapeutic targets.…”

Chronic enzyme replacement therapy ameliorates neuropathology in alpha‐mannosidosis mice

“…Therefore, transgenic and nontransgenic knockout mice as well as wild‐type mice were analyzed for storage and secondary pathological changes 8, 16. TLC analyses revealed comparable accumulation of oligosaccharides linked to 2‐9 mannose residues (Man 2 ‐Man 9 ) between both genotypes in brain (Fig.…”

“…To date, 125 different disease‐causing mutations were identified affecting enzymatic activity, stability and intracellular transport of LAMAN 3, 4, 5, 6. Lysosomal deficiency of LAMAN leads to the accumulation of free mannose‐containing oligosaccharides of asparagine ( N )‐linked glycans derived from proteolysis of glycoproteins within the lysosomal compartment7 as well as impaired trimming of N ‐linked oligosaccharides on native lysosomal proteins 8. Cells with storage pathology are found throughout the body, including the peripheral and central nervous system (CNS).…”