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

Abstract

α-Mannosidosis is caused by the genetic defect of the lysosomal α-d-mannosidase (LAMAN), which is involved in the breakdown of free α-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 α-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 α-mannosidosis as indicated by the predominance of extended Man3GlcNAc2 oligosaccharides. Treatment with recombinant human α-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 asparagine-linked oligosaccharides on native lysosomal proteins.

We thank Nicole Eiselt and Klaus Neifer for excellent technical assistance and Kurt von Figura for critical reading of the manuscript.

This work was supported by the HUE-MAN consortium (European Commission FP VI contract LHSM-CT-2006-018692) as well as by the Centre National de la Recherche (Unité Mixte de Recherche CNRS/USTL 8576) and the Ministère de la Recherche et de l'Enseignement Supérieur. The Mass Spectrometry facility in Lille was funded by the European Community (FEDER), the Région Nord-Pas de Calais (France), and the Université des Sciences et Technologies de Lille.