Dataset Information

Deciphering Minor Glycan Signatures: Cross-Serotype Recombinant Adeno-Associated Virus (rAAV) Glycan Profiling Using Multimodal Glycoproteomics Technologies

ABSTRACT: Glycan analysis of viral vectors is essential for pharmaceutical quality evaluation. Recent advances in mass spectrometry (MS)-based analytical technologies have identified glycosylation in adeno-associated viruses (AAVs). However, reported glycan structures vary between studies, due to the extremely low abundance (<1%) of glycosylated particles and the presence of hitchhiker molecules, making AAV glycosylation a topic of debate. This underscores the need for standardized and highly sensitive detection methods. Here, we present a multimodal glycoproteomic approach to characterize glycosylation across six serotypes of 28 recombinant AAVs (rAAVs). An ultrasensitive lectin microarray, using a broad-reactive anti-AAV antibody, detected O-glycan-binding lectin signals in rAAV6, consistent with previous findings. Notably, a unique binding signal was observed with Urtica dioica lectin (UDA). Liquid chromatography-tandem mass spectrometry (LC-MS/MS), combined with UDA-based glycoprotein/glycopeptide dual enrichment and glycopeptide identification, confirmed the predominant presence of a divalent high-mannose N-glycosylated peptide from a hitchhiking host-derived glycoprotein, the AAV receptor. In contrast, monovalent high-mannose N-glycosylated peptides were identified using a single-step protein-level enrichment approach, suggesting an avidity effect in UDA binding. These findings highlight the significance of advanced multimodal glycoproteomic in rAAV characterization and offer new insights into host-derived hitchhiker glycoproteins present in AAV preparations.

ORGANISM(S): Cloning Vector Aav

SUBMITTER: Atsushi Kuno

PROVIDER: PXD062588 | JPOST Repository | Sat Apr 04 00:00:00 BST 2026

REPOSITORIES: jPOST

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			Action	DRS
	6-8_UDA_AspN_3p100_Slot1-19_1_1207(F006292).xlsx	Xlsx
	6-8_UDA_AspN_3p100_Slot1-19_1_1207.d.zip	Other
	6-8_UDA_AspN_3p100_Slot1-19_1_1207_uncalib_6.1.452-(1)_Byonic(N&O).xlsx	Xlsx
	6-8_UDA_AspN_UDA_1p2_Slot1-21_1_1213.d.zip	Other
	6-8_UDA_AspN_UDA_1p2_Slot1-21_1_1213_uncalib_6.1.452_Byonic(N&O).xlsx	Xlsx

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Project description:In comprehensive glycome analysis with a high-density lectin microarray, we have previously shown that the recombinant N-terminal domain of the lectin BC2L-C from Burkholderia cenocepacia (rBC2LCN) binds exclusively to undifferentiated human induced pluripotent stem (iPS) cells and embryonic stem (ES) cells but not to differentiated somatic cells. Here we demonstrate that podocalyxin, a heavily glycosylated type1 transmembrane protein, is the predominant glycoprotein ligand of rBC2LCN on human iPS cells and ES cells. When analyzed by DNA microarray, podocalyxin was found to be highly expressed in both iPS cells and ES cells. Western and lectin blotting revealed that rBC2LCN binds predominantly to podocalyxin with a high molecular weight of more than 240 kDa in undifferentiated iPS cells of six different origins and four ES cell lines, but no binding was observed in either differentiated mouse feeder cells or somatic cells. The specific binding of rBC2LCN to podocalyxin prepared from a large set of iPS cells (138 types) and ES cells (15 types) was also confirmed using a high-throughput antibody-overlay lectin microarray. Alkaline digestion greatly reduced the binding of rBC2LCN to podocalyxin, indicating that the major glycan ligands of rBC2LCN are presented on O-glycans. Furthermore, rBC2LCN was found to exhibit significant affinity to a branched Oglycan comprising an H type3 structure as the most probable rBC2LCN glycan ligand (Kd, 4.0 x 10-5 M) prepared from human 201B7 iPS cells, suggesting that H type3 is a novel potential pluripotency marker. We conclude that podocalyxin is the predominant glycoprotein ligand of rBC2LCN on human iPS cells and ES cells. Gene expressions in human embryonic stem cells (ESCs) and human induced pluripotent stem cells (iPSCs). Human iPS cells (MRC5-iPS, AM-iPS, UtE-iPS, PAE-iPS) were induced from four different somatic cells by infection of the retroviral vectors pMXs encoding OCT3/4, SOX2, KLF4 and c-MYC, simultaneously. Human iPS cells (TIG/MKOS#19, #56, #106) were generated through reprogramming by Sendai virus infection-mediated expression of OCT4, SOX2, KLF4, and c-MYC.

Dataset Information

Deciphering Minor Glycan Signatures: Cross-Serotype Recombinant Adeno-Associated Virus (rAAV) Glycan Profiling Using Multimodal Glycoproteomics Technologies

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