Biology subjects

Yin, V. C.

Publications and source records attributed to Yin, V. C..

3 recordsLinked to original sources

bioRxiv2023

CD5L is a canonical component of circulatory IgM

Immunoglobulin M (IgM) is an evolutionary conserved key component of humoral immunity, and the first antibody isotype to emerge during an immune response. IgM is a large (1 MDa), multimeric protein, for which both hexameric and pentameric structures have been described, the latter additionally containing a joining (J) chain. Using a combination of single-particle mass spectrometry and mass photometry, proteomics and immunochemical assays, we here demonstrate that circulatory (serum) IgM exclusively exists as a complex of J-chain-containing pentamers covalently bound to the small CD5 antigen-like (CD5L, 36 kDa) protein. In sharp contrast, secretory IgM in saliva and milk is principally devoid of CD5L. Unlike IgM itself, CD5L is not produced by B cells, implying that it associates with IgM in the extracellular space. We demonstrate that CD5L integration has functional implications, i.e., it diminishes IgM binding to two of its receptors, the Fc{micro}R and the polymeric Immunoglobulin receptor (pIgR). On the other hand, binding to Fc{micro}R as well as complement activation via C1q seem unaffected by CD5L integration. Taken together, we redefine the composition of circulatory IgM as a J-chain containing pentamer, always in complex with CD5L.

molecular biology↗

bioRxiv2022

Spectral Interferences Impede the High-Resolution Mass Analysis of Recombinant Adeno-Associated Viruses

Recombinant adeno-associated viruses (rAAVs) are the leading platform for in vivo delivery of gene therapies, with several already approved for clinical use. However, the heterogeneity and structural complexity of these viral particles render them challenging targets to characterize. Orbitrap-based native mass spectrometry (MS) is a method capable of directly characterizing intact megadalton protein assemblies. Here we used such an approach to characterize four different preparations of rAAV8 (two empty and two filled) differing in both their transgene and relative capsid protein isoform (i.e. VP1, VP2 and VP3) content. Interestingly, in native MS measurements of these samples, we observe complicated, unusual, and dramatically different spectral appearances between the four rAAV preparations that cannot be rationalized or interpreted using conventional approaches (i.e. charge state deconvolution). By combining high-resolution native MS, single particle charge detection MS, and spectral simulations, we reveal that these unexpected features result from a combination of stochastic assembly-induced heterogeneity and divergent gas phase charging behaviour between the four rAAV preparations. Our results stress the often-neglected heterogeneity of rAAVs, but also highlight the pitfalls of standard high-resolution mass analysis for such particles. Finally, we show that charge detection MS and spectral simulations can be used to tackle these challenges.

biochemistry↗

bioRxiv2021

Probing Affinity, Avidity, Anti-Cooperativity, and Competition in Antibody and Receptor Binding to the SARS-CoV-2 Spike by Single Particle Mass Analyses

Determining how antibodies interact with the spike (S) protein of the SARS-CoV-2 virus is critical for combating COVID-19. Structural studies typically employ simplified, truncated constructs that may not fully recapitulate the behaviour of the original complexes. Here, we combine two single particle mass analysis techniques (mass photometry and charge-detection mass spectrometry) to enable measurement of full IgG binding to the trimeric SARS-CoV-2 S ectodomain. Our experiments reveal that antibodies targeting the S-trimer typically prefer stoichiometries lower than the symmetry-predicted 3:1 binding. We determine that this behaviour arises from the interplay of steric clashes and avidity effects that are not reflected in common antibody constructs (i.e. Fabs). Surprisingly, these sub-stoichiometric complexes are fully effective at blocking ACE2 binding despite containing free receptor binding sites. Our results highlight the importance of studying antibody/antigen interactions using complete, multimeric constructs and showcase the utility of single particle mass analyses in unraveling these complex interactions.

immunology↗