Site-specific glycosylation analysis of the bovine lysosomal α-mannosidase

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Abstract

Lysosomal α-mannosidase is a broad specificity exoglycosidase involved in the ordered degradation of glycoproteins. The bovine enzyme is used as an important model for understanding the inborn lysosomal storage disorder α-mannosidosis. This enzyme of about 1000 amino acids consists of five peptide chains, namely a- to e-peptides and contains eight N-glycosylation sites. The N497 glycosylation site of the c-peptide chain is evolutionary conserved among LAMANs and is very important for the maintenance of the lysosomal stability of the enzyme. In this work, relying on an approach based on mass spectrometric techniques in combination with exoglycosidase digestions and chemical derivatizations, we will report the detailed structures of the N-glycans and their distribution within six of the eight N-glycosylation sites of the bovine glycoprotein. The analysis of the PNGase F-released glycans from the bovine LAMAN revealed that the major structures fall into three classes, namely high-mannose-type (Fuc0–1Glc0–1Man4–9GlcNAc2), hybrid-type (Gal0–1Man4–5GlcNAc4), and complex-type (Fuc0–1Gal0–2Man3GlcNAc3–5) N-glycans, with core fucosylation and bisecting GlcNAc. To investigate the exact structure of the N-glycans at each glycosylation site, the peptide chains of the bovine LAMAN were separated using SDS–PAGE and in-gel deglycosylation. These experiments revealed that the N497 and N930 sites, from the c- and e-peptides, contain only high-mannose-type glycans Glc0–1Man5–9GlcNAc2, including the evolutionary conserved Glc1Man9GlcNAc2 glycan, and Fuc0–1Man3–5GlcNAc2, respectively. Therefore, to determine the microheterogeneity within the remaining glycosylation sites, the glycoprotein was reduced, carboxymethylated, and digested with trypsin. The tryptic fragments were then subjected to concanavalin A (Con A) affinity chromatography, and the material bound by Con A-Sepharose was purified using reverse-phase high-performance liquid chromatography (HPLC). The tandem mass spectrometry (ESI-MS/MS) and the MALDI analysis of the PNGase F-digested glycopeptides indicated that (1) N692 and N766 sites from the d-peptide chain both bear glycans consisting of high-mannose (Fuc0–1Man3–7GlcNAc2), hybrid (Fuc0–1 Gal0–1Man4–5GlcNAc4), and complex (Fuc0–1Gal0–2Man3GlcNAc4–5) structures; and (2) the N367 site, from the b-peptide chain, is glycosylated only with high-mannose structures (Fuc0–1Man3–5GlcNAc2). Taking into consideration the data obtained from the analysis of either the in-gel-released glycans from the abc- and c-peptides or the tryptic glycopeptide containing the N367 site, the N133 site, from the a-peptide, was shown to be glycosylated with truncated and high-mannose-type (Fuc0–1Man4–5GlcNAc2), complex-type (Fuc0–1Gal0–1Man3GlcNAc5), and hybrid-type (Fuc0–1Gal0–1Man5GlcNAc4) glycans.

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