Structural and Molecular Mechanisms of B Cell Co-Receptor Function
Access StatusFull text of the requested work is not available in DASH at this time ("dark deposit"). For more information on dark deposits, see our FAQ.
MetadataShow full item record
CitationSusa, Katherine. 2022. Structural and Molecular Mechanisms of B Cell Co-Receptor Function. Doctoral dissertation, Harvard University Graduate School of Arts and Sciences.
AbstractB cells are central to the adaptive immune response, serving as the basis for generation of humoral immunity to foreign agents. To initiate an immune response, an antigen binds to a cell surface B cell receptor (BCR), leading to a coordinated cellular response that culminates in the secretion of neutralizing antibodies. To enhance the sensitivity of B cell signaling, the BCR functions in association with a multicomponent co-receptor complex that contains the complement receptor CD21, the signaling protein CD19, and CD81. CD81 belongs to the tetraspanin family of proteins and is critical for proper B cell function by acting both as a chaperone to enable cell surface expression of properly folded CD19 and as a signaling scaffold to regulate the localization of CD19 in the membrane during B cell signaling.
The work presented in this dissertation makes key advances toward understanding the functional partnership between CD19 and CD81 through structural and mechanistic studies. In Chapter 2, I show that CD81 relies on its ectodomain to traffic CD19 to the cell surface, and then use cell-based assays and crystallography to illuminate the binding mechanism of the anti-CD81 antibody 5A6. I show that the 5A6 antibody binds selectively to activated B cells and recognizes a conformational epitope on CD81 that is masked when CD81 is bound to CD19 on resting B cells, revealing that the CD19-CD81 interaction is dynamically regulated upon B cell activation. In Chapter 3, I engineer a stable CD19-CD81 complex and report the cryo-EM structure of this complex bound to a therapeutic antigen binding fragment (Fab). This structure, the first high-resolution structure of any tetraspanin bound to a partner protein, shows a tetraspanin captured in the “open” conformation, highlighted by a major reorganization of the transmembrane helices to occlude the cholesterol binding pocket present in apo-CD81. This structure reveals that tetraspanins are a conformationally regulated family of membrane proteins and provides a structural explanation for how changes in tetraspanin structure facilitate shuttling of protein partners in and out of cholesterol-enriched lipid rafts to regulate signal transduction. In Chapter 4, I engineer a cell-based system that enables parallel quantification of B cell co-receptor interactions with local phosphosite information in the seconds to hours after BCR stimulation. Using this system, I determine the kinetics of the response of proteins previously implicated in signaling events downstream of BCR engagement, including the BCR itself, Akt, PKC, PI3K, and Raf isoforms. I also identity many proteins with no previous connection to B cell signaling that are responsive to BCR stimulation and verify SWAP70 and the glutamate transporter SLC1A1 as novel regulators of the early B cell response. Collectively, the work described here provides a molecular understanding of B cell co-receptor function and offers new insight into approaches to modulate the B cell signaling pathway.
Citable link to this pagehttps://nrs.harvard.edu/URN-3:HUL.INSTREPOS:37371933
- FAS Theses and Dissertations