Characterization of the N‐linked glycosylation site of recombinant pectate lyase

Abstract

Recombinant pectate lyase from Aspergillus niger was overexpressed in Aspergillus nidulans. The two recombinant proteins produced differed in molecular mass by 1200 Da, which suggested that the larger molecular weight protein was glycosylated. The deduced amino acid sequence was searched for potential N-linked glycosylation sites, and one potential site was identified at residue 64. The proteins were analyzed for their ability to bind various lectins as an assay for the presence of carbohydrates. The proteins were then digested with trypsin to facilitate the isolation of the potential glycosylation site. The resulting digestion products were subsequently analyzed by liquid chromatography/mass spectrometry using in-source collision induced dissociation to detect glycopeptides. Once the glycopeptide had been identified, treatment with an endoglycosidase both verified the location of glycosylation and identified the mass of the glycan. The Complex Carbohydrate Structural Database was searched for possible N-linked structures with the same mass, and the suggested primary sequence was confirmed by an exoglycosidase digestion. The data demonstrated that the larger recombinant protein contained a high mannose N-linked structure (Man(5)GlcNAc(2)) attached to N-64, while this site was not occupied in the smaller protein.

Mode of Action Analysis and Structure-Function Relationships of Aspergillus Niger Pectinolytic Enzymes

“…The overexpressed A. niger plyA gene resulted in the production of an N-glycosylated form and an N-glycon free form [13] of PL Y A. These enzymes showed identical specific activities of 40.5 and 41.1 U/mg, respectively under standard conditions [5].…”

A glycoprotein with anti-inflammatory properties secreted by an Aspergillus nidulans modified strain

“…Also, its chromatographic behavior remained the same, which suggests that this protein is a typical N-glycosylated product. Other works using glycoproteins from the genus Aspergillus supported this hypothesis, as N-glycosylation is common in such fungi (7,16,21). Figure 6 shows the results of SDS-PAGE of the purified anti-inflammatory protein from A. nidulans under reduced conditions followed by silver staining.…”

“…This is the key amino acid for the action of N-glucosyltransferases, as described before (6,7,21). SDS-PAGE…”

Structures and Functions of Oligosaccharins: The Role of Endoglycanases

“…PGA and PGB are likely required during the early stages of pathogenicity, and it has been suggested that PGA and PGB are scouting enzymes which help the fungus sense the presence of pectin by generating low molecular weight inducers while other EPGs are subsequently expressed. Many of the PDEs produced by fungi have been identified as being glycosylated [30][31][32]. The glycosylation state of the various PDEs may impact pathogenesis; however, the effects of the carbohydrate side chains on the properties of glycosylated PDEs are not known.…”