Publication Date:
2015-11-06
Description:
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 GlcNAc 2 , but also mono- and di-phosphorylated oligosaccharides in the forms of Man 9-15 P 1 GlcNAc 2 and Man 9-14 P 2 GlcNAc 2 , 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.
Print ISSN:
0959-6658
Electronic ISSN:
1460-2423
Topics:
Biology
,
Medicine
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