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GaHyeon Kim,Jung A Seo,Myung Jin Oh,Hyun Joo An 한국당과학회 2021 한국당과학회 학술대회 Vol.2021 No.01
Agalsidase beta, recombinant enzyme therapeutic, which is a glycoprotein that treats lysosome accumulation disorders caused by deficiency of the enzyme alpha galactosidase A has 3 glycosylation sites(N108, N161, N184). In this protein, there are various glycans from acidic glycans to high mannose, so accurate monitoring is required for proper afficacy and function of drug. Recently, intact analysis is heavily used due to the advantage of simple preparation, short MS analysis time, glycan occupancy and micro-heterogeneity confirmation. However, numerous glycosylation sites, high molecular weight and glycan heterogeneity complicate the mass spectrum, making data interpretation and glycan assignment difficult. Here, we obtained the mass value of intact agalsidase beta, which eased the complexity of the mass spectrum by lowering the high molecular weight of the glycoprotein through stepwise enzymatic treatment. To facilitate the assignment of sialic acid and phosphorylated glycans at each glycosylation sites, deconvoluted spectra of sialidase and phosphatase-treated and untreated agalsidase beta were compared. Possible glycoforms could be assigned for each peak in the deconvoluted spectra within the accepted mass tolerance of ±5 Da. In the monomer and dimer structures of agalsidase beta, a glycosylation pattern was confirmed in which HexNAcNeuAc residues(494.1748 Da) and 2HexNAc2NeuAc residues (988.3496 Da) were increased, respectively. Our approach can be a efficient tool to confirm glycosylation pattern of therapeutic glycoproteins in biosimilar development.
Gahyeon Jin,Il-Hwan Kim,Yonggyun Kim 한국응용곤충학회 2023 한국응용곤충학회 학술대회논문집 Vol.2023 No.10
Two bacterial genera, Xenorhabdus and Photorhabdus, are mutually symbiotic to the entomopathogenic nematodes, Steinernema and Heterorhabditis, respectively. Success parasitism of the nematode-bacterial complex depends on the host immunosuppression by the bacteria via their secondary metabolites. Lrp (Leucine-responsive regulatory protein) is a global transcriptional factor of the bacteria and play a crucial role in the parasitism. However, its regulatory targets to suppress the insect immunity were not clearly determined. This study investigated the regulatory target genes and subsequent secondary metabolites by Lrp in Xenorhabdus hominickii. Lrp expression occurred at the early infection stage in a target insect, Spodoptera exigua. Among eight non-ribosomal peptide synthetase (NRPS1-NRPS8) genes, six gene (NRPS3-NRPS8) expressions were positively correlated with Lrp expression in the infected larvae of S. exigua. Exchange of the Lrp promoter with an inducible promoter altered the production of the secondary metabolites along with alteration of the NRPS expression levels. The immunosuppressive activities of X. hominickii depended on the Lrp expression level. The metabolites produced by Lrp expression possessed the eicosanoid-biosynthesis inhibitors and hemolytic factors. A cyclic dipeptide (= cPF) was produced under Lrp control and identified to inhibit phospholipase A2 activity of S. exigua in a competitive inhibitory manner. These results suggest that Lrp is a global transcriptional factor of X. hominickii and plays crucial role in insect immunosuppression by modulating NRPS expressions.