Characterization of theN-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.

Structural Investigations of Glycoconjugates at High Sensitivity

“…Since then, the glycosylation sites of several glycoproteins have been characterized by LC/ESI/MS and LC/ESI/MS/ MS, including mouse monocolonal immunoglobulin (IgG2b), 166 murine scrapie prion protein (PrP sc ), 167 cyclooxygenase-2, 156 lutropin receptor glycoprotein, 168 endopolygalacturonase I, 169 and gelatinase B. 170 Also, several determinations of the glycosylation sites of recombinant glycoproteins have been communicated, including recombinant endo-polygalacturonase I from Aspergillus niger, 171 pectate lyase, 172 human coagulation factor VIIa, 173 human erythropoietin (rhEPO), 174,175 and corticotropin-releasing factor binding protein. 176 In 1993, Carr and co-workers devised an MS method for the selective detection of glycopeptides at the low picomole level during the chromatography of glycoprotein digests.…”

Characterization of the Glycosylation Sites in Cyclooxygenase-2 Using Mass Spectrometry

“…Mass spectrometry (MS) has been used as an effective method for the analysis of protein glycosylation ( − ); and tandem mass spectrometry (MS/MS), in combination with electrospray ionization, has been valuable in identifying glycopeptides. Through the use of precursor (parent) ion scans and marker ions such as m / z 163 [protonated hexose residue (Hex)], m / z 204 [protonated N -acetylhexosamine residue (HexNAc)], or m / z 366 (HexHexNAc) ( − ), it is possible to easily identify glycopeptides. Dissociation of protonated glycopeptides in a collision-induced decomposition (CID) experiment ( − ) also provides structural information regarding the amino acid sequence of the peptide, the types of sugars attached, and the residue that carries the glycosyl group.…”

On‐target endoglycosidase digestion matrix‐assisted laser desorption/ionization mass spectrometry of glycopeptides

“…Once the procedure had proven successful on known glycan structures, the digestion procedure was then applied to N ‐linked glycans with unknown structures, such as the N ‐linked glycan of pectate lyase. The digestion products of trypsin‐digested pectate lyase were separated by HPLC, and one LC fraction was identified as containing a glycopeptide 16. When analyzed by MALDI‐MS, this fraction was found to contain four peaks (Fig.…”