title: Automated N-glycan profiling of a mutant Trypanosoma rangeli sialidase expressed in Pichia pastoris, using tandem mass spectrometry and bioinformatics.
abstract: A mutant Trypanosoma rangeli sialidase, Tr7, expressed in Pichia pastoris, exhibits significant trans-sialidase activity, and has been used for analytical-scale production of sialylated human milk oligosaccharides. Mass spectrometry-based site-specific N-glycoprofiling of Tr7 showed that heterogeneous high-mannose type N-glycans were present at all the five potential N-linked glycosites. N-linked glycans in Tr7 were predominantly neutral oligosaccharides with compositions Man(8-16)GlcNA(c2), but also mono- and di-phosphorylated oligosaccharides in the forms of Man(9-15)P(1)GlcNA(c2) and Man(9-14)P(2)GlcNA(c2), respectively. Some phosphorylated N-linked glycans further contained an additional HexNAc, which has not previously been reported in P. pastoris-expressed proteins. We compiled a method pipeline that combined hydrophilic interaction liquid chromatography enrichment of glycopeptides, high accuracy mass spectrometry and automated interpretation of the mass spectra with in-house developed "MassAI" software, which proved efficient in glycan site microheterogeneity analysis. Functional analysis showed that the deglycosylated Tr7 retained more than 90% of both the sialidase and trans-sialidase activities relative to the glycosylated Tr7.
authors: Department of Biochemistry and Molecular Biology, University of Southern Denmark, 5230 Odense M, Denmark.
authors: Haiying
authors: H
authors: Li
authors: Department of Biochemistry and Molecular Biology, University of Southern Denmark, 5230 Odense M, Denmark.
authors: Morten I
authors: MI
authors: Rasmussen
authors: Department of Biochemistry and Molecular Biology, University of Southern Denmark, 5230 Odense M, Denmark.
authors: Martin R
authors: MR
authors: Larsen
authors: Center for BioProcess Engineering, Department of Chemical and Biochemical Engineering, Technical University of Denmark, Building 229, 2800 Kgs, Lyngby, Denmark.
authors: Yao
authors: Y
authors: Guo
authors: Center for BioProcess Engineering, Department of Chemical and Biochemical Engineering, Technical University of Denmark, Building 229, 2800 Kgs, Lyngby, Denmark.
authors: Carsten
authors: C
authors: Jers
authors: Department of Biochemistry and Molecular Biology, University of Southern Denmark, 5230 Odense M, Denmark Department of Parasitology, Institute of Biomedical Sciences, University of Sao Paulo, Sao Paulo, Brazil.
authors: Giuseppe
authors: G
authors: Palmisano
authors: Center for BioProcess Engineering, Department of Chemical and Biochemical Engineering, Technical University of Denmark, Building 229, 2800 Kgs, Lyngby, Denmark.
authors: Jørn D
authors: JD
authors: Mikkelsen
authors: Department of Biochemistry and Molecular Biology, University of Southern Denmark, 5230 Odense M, Denmark f.kir@bmb.sdu.dk.
authors: Finn
authors: F
authors: Kirpekar
mesh_terms: D006031:Glycosylation; D008895:Milk, Human / Q000737:chemistry; D009154:Mutation; D009439:Neuraminidase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010843:Pichia / Q000235:genetics / Q000378:metabolism; D011134:Polysaccharides / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D015800:Protozoan Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins; D057509:Trypanosoma rangeli / Q000201:enzymology* / Q000235:genetics
chemical_list: D011134:Polysaccharides; D015800:Protozoan Proteins; D011994:Recombinant Proteins; D009439:Neuraminidase
keywords: N-glycan; P. pastoris; T. rangeli sialidase mutant Tr7; mass spectrometry; trans-sialidase activity