Site-Specific N-Glycosylation on the AAV8 Capsid Protein

Abstract

Adeno associated virus (AAV) is a versatile gene delivery tool, which has been approved as a human gene therapy vector for combating genetic diseases. AAV capsid proteins are the major components that determine the tissue specificity, immunogenicity and in vivo transduction performance of the vector. In this study, the AAV8 capsid glycosylation profile was systemically analyzed by peptide mass fingerprinting utilizing high-resolution mass spectrometry to determine the presence of capsid glycosylation. We identified N-glycosylation on the amino acid N499 of the capsid protein. We characterized the overall sugar profile for vector produced in 293 cells. Multiple N-glycosylated host-cell proteins (HCPs) copurified with AAV8 vectors and were identified by analyzing LC-MS data utilizing a human database and proteome discoverer search engine. The N-glycosylation analysis by MALDI-TOF MS, highlighted the probability of AAV8 interaction with terminal galactosylated N-glycans within the HCPs.

Keywords: Adeno associated virus; host cell protein analysis; mass spectrometry; site specific N-glycan analysis; virus-host interaction.

Figure 1

Intracellular or secreted Adeno associated virus (AAV)8 were processed in parallel and analyzed using multiple techniques. (A) Samples visualized on the SDS-PAGE gel by glycoprotein labelling. (B) N-glycan analysis using MALDI-MS identifies the type of glycosylation. The peptides generated by Glu C or trypsin digestion were enriched using HILIC cartridges. Deglycosylation of enriched glycosylated peptide in the presence of H₂¹⁸O labelled the glycosite with ¹⁸O. (C) The samples were injected to LC-MS and using the database of AAV8 VPs, the N-glycosite was identified (D) The same data was searched against the human database to discover which proteins were copurified with AAV8.

Figure 2

AAV8 Glycosylation detection. AAV8 derived from the cell (intracellular), and media (secreted) are separated on SDS-PAGE (a) protein bands visualized after glycoprotein staining. Glycosylated bands were observed near the AAV8 capsid protein (VP) region (55–100 kDa). Gel (b) is gel (a) stained with Coomassie brilliant blue to visualize the whole protein profile. (c) Independent Coomassie stained gel run after de-N- glycosylation of secreted sample. (+ and − the addition of PNGase F).

Figure 3

MALDI- MS spectra of 2-AA derived N-glycan in negative mode [M−H]⁻. (a) The whole glycome profile of intracellular AAV8. (b) The whole glycome analysis of secreted AAV8. The most intense peaks from both the spectra (m/z 2110.234, 1948.088, 2401.633, 1832.984 were further confirmed by MS/MS fragmentation spectra in MALDI-TOF MS in lift mode (supplemental Figure S2).

Figure 4

Two distinct tandem mass spectra of peptide sequence “VSTTTGQNNNSNFAWTAGTK” of the AAV8 capsid protein (in the common region of VPs). Characterized CID spectra of glycosylated peptide after ¹⁸O mediated digestion. (a) The position of de-N-glycosylated asparagine 499 (¹⁸O-incorporated aspartic acid) was attested by mass increment of 2.8547 Da (theoretical mass difference 2.98 Da) to the series of b and y ion (the precursor ion mass: m/z 2101.93193). (b) The position of Non-glycosylated asparagine 499 in the sequence was confirmed by y12/b8 ions (the precursor ion mass: m/z 2099.0766 Da). The highlighted ions (marked with a star) shows the mass differences from the non-glycosylated peptide mass spectrum. y11+ (highlighted) ions are same in both the spectra. (c) Capsid protein amino acid sequence and possible N-glycosylation sites predicted by NetNglyc software based on the consensus sequence (NXT/S, “X” can be any amino acid except proline). Different N-terminal sequence of VP1, VP2 and VP3 are marked in the sequence. The glycosite identified peptide is highlighted on the sequence.

Figure 5

Host-cell protein (HCP) identification in intracellular and secreted AAV8 using Proteome Discoverer^1.4.