Characterization of the glycosylation of recombinant Endopolygalacturonase I from Aspergillus niger

Abstract

The carbohydrate chains of recombinant endopolygalacturonase I (EPG I) from Aspergillus niger were characterized using a combination of mass spectrometric techniques. High performance liquid chromatography (HPLC) in conjunction with electrospray ionization mass spectrometry was used to separate the components of EPG I liberated by trypsin digestion. In-source collision-induced dissociation (CID) was utilized to fragment the digestion products entering the mass spectrometer, and the generation of carbohydrate fragment ions allowed for the identification of glycopeptides. The masses of the resulting glycans were calculated and entered into a carbohydrate database to search for possible structures. The primary sequences of the carbohydrate chains were confirmed by digesting aliquots of the intact glycopeptide with endo- and exoglycosidases and then analyzing the digestion products using matrix-assisted laser desorption/ionization mass spectrometry. These experiments demonstrated that one of the two N-linked sites of EPG I was occupied by a series of high-mannose structures, the second N-linked site was not occupied, and no O-linked sites were detected.

Oligosaccharide analysis using anion attachment in negative mode electrospray mass spectrometry

“…Another interesting feature of saccharides is that they appear to play important roles in cell-surface recognition phenomena [1,2]. An understanding of the saccharide structure is essential for understanding the varied functions of carbohydrates in biological systems.Mass spectrometry has emerged as an important technique for oligosaccharide analysis [3][4][5][6][7][8][9][10][11][12][13][14][15][16][17]. Important innovations came through the introduction of electrospray (ES) and matrix-assisted laser desorption/ionization (MALDI).…”

Mapping Glycans onto Specific N-Linked Glycosylation Sites of Pyrus communis PGIP Redefines the Interface for EPG−PGIP Interactions

“…We have previously discovered that several of the EPGs we have studied (PG I, PGA, PGC from Aspergillus niger, along with PG 2 and PG 3 from Botrytis cinerea) contain O-linked mannose. [31][32][33] These modifications are located near the N-terminus in close proximity to the sites where we hypothesize PGIP binding occurs. This has led us to speculate that O-mannosylation may play an important role in EPG/PGIP interaction, as the O-Man-initiated modification has been previously observed to have a dramatic impact on the R-dystroglycan/laminin interaction in animals.…”

“…These single site changes are unlikely to account for the wide spectrum of binding and inhibitory activities observed, and little emphasis has been placed on the potential for glycosylation to effect the ability of PGIPs to bind, and/or to inhibit EPGs. We have previously discovered that several of the EPGs we have studied (PG I, PGA, PGC from Aspergillus niger , along with PG 2 and PG 3 from Botrytis cinerea ) contain O-linked mannose. − These modifications are located near the N-terminus in close proximity to the sites where we hypothesize PGIP binding occurs. This has led us to speculate that O-mannosylation may play an important role in EPG/PGIP interaction, as the O-Man-initiated modification has been previously observed to have a dramatic impact on the α-dystroglycan/laminin interaction in animals …”

Genomics of protein folding in the endoplasmic reticulum, secretion stress and glycosylation in the aspergilli

“…In some cases, elongation of the Man 8-9 GlcNAc 2 ER-structure with a limited number of a-1,2-linked mannose residues can also occur, as seen for acid carboxy peptidase of Aspergillus saitoi (Chiba et al, 1993;Ichishima, 2000) and endopolygalacturonase I of A. niger (Colangelo et al, 1999a), carrying sugar chains-like Man 10-11 GlcNAc 2 and Man 13 GlcNAc 2 , respectively. Reports on hyperglycosylation in filamentous fungi are rare and the range of mannose residues is rather limited: Man 18 GlcNAc 2 has been observed on pectin methylesterase of A. niger (Warren et al, 2002) and Hex 7-26 GlcNAc 2 on A. niger a-galactosidase A (Wallis et al, 2001).…”