S4B)
S4B). antibody receptor tripartite motif-containing protein 21 (TRIM21) and are positive for lysine-48 ubiquitin chains. IgA neutralizes adenovirus infection in a TRIM21- and proteasome-dependent manner in both human and mouse cells. Translocated IgA also potently activates NF-B signaling pathways in cells expressing TRIM21, whereas viral infection in the absence of antibody or TRIM21 is undetected. TRIM21 recognizes AN3365 an epitope in IgG Fc that is not conserved in IgA; however, fluorescence anisotropy experiments demonstrate that direct binding to IgA is maintained. We use molecular modeling to show that TRIM21 forms a nonspecific hydrophobic seal around a -loop structure that is present in IgG, IgM, and IgA, explaining how TRIM21 achieves such remarkable broad antibody specificity. The findings demonstrate that the antiviral protection afforded by IgA extends to the intracellular cytosolic environment. More IgA is produced per day in the human body than all of the other antibody isotypes combined (1). Humans express two IgA isotypes, IgA1 and IgA2, and each isotype is expressed in several oligomeric states including, monomeric (mIgA), dimeric (dIgA), and secretory (S-IgA) (1). S-IgA plays a key immune role at mucosal surfaces as the critical first line of defense against inhaled or ingested pathogens, as well as confining commensal bacteria to the intestinal lumen; however, serum IgA also plays a AN3365 significant but less Rabbit Polyclonal to CARD11 well understood immune role (2). Circulating serum IgA, which is predominantly mIgA1, is found at concentrations of 23 mg/mL, making it the second most prevalent antibody class in plasma after IgG (3). During infection, pathogens are bound by mIgA, which is able to interact with and aggregate Fc receptor I (FcRI) molecules. Receptor activation promotes phagocytosis, antigen presentation, antibody-dependent cellular cytotoxicity, cytokine, and superoxide release (4). However, FcRI, like most Fc receptors, is exclusively expressed on professional myeloid cells (5,6). Viral neutralization is mediated by antibodies whose in vitro binding to a virus can cause a reduction in infectious titer independently of effector mechanisms such as Fc-mediated phagocytosis or complement fixation (7). Recently, we discovered a neutralization pathway mediated by the cytosolic antibody receptor, tripartite motif-containing protein 21 (TRIM21), which is expressed in most tissue types and not just professional cells (8,9). TRIM21 binds to IgG molecules that have been carried inside cells by infecting virus particles (8). TRIM21 binds IgG Fc via its C-terminal PRYSPRY domain at subnanomolar affinity, making it one of the highest-affinity IgG Fc receptors in the human body (8,10). After binding a virion-associated antibody, TRIM21 targets the cytosolic antibodyvirus complex for proteasomal degradation in a process called antibody-dependent intracellular neutralization (ADIN) (8,11). In addition to mediating ADIN, TRIM21 stimulates the NF-B, activator protein 1 (AP-1), and interferon regulatory factors IRF3/IRF5/IRF7 immune signaling pathways and induces an antiviral state (12). Interestingly, TRIM21 can also perform these immune functions by binding to cytosolic virion-associated IgM molecules (8,12). AN3365 Use of both IgG and IgM is unusual as most antibody receptors are strongly isotype specific. In this study, we investigated whether TRIM21 can use IgA molecules to stimulate viral neutralization or innate immune signaling. We find that TRIM21 can bind directly to IgA and that it recognizes virion-associated IgA inside infected cells. By recruiting TRIM21, IgA mediates virus neutralization in the cytosol and potently activates NF-B. These data demonstrate that circulatory IgA has a broader role to play in combatting viral infection than previously identified and identifies TRIM21 like a distinctively broad isotype receptor. == Results == == IgA Enters Cells During Viral Illness and Is Detected by TRIM21. == To investigate whether IgA antibodies enter cells during viral illness, replication-deficient human being adenovirus type 5-GFP (AdV) was incubated with pooled human being serum IgA (hIgA) and added to HeLa cells. Thirty minutes after infection, cells were fixed and incubated with labeled anti-human IgA and anti-adenovirus IgG. hIgA was recognized inside HeLa cells and colocalized to intracellular AdV (Fig. 1A). To determine whether TRIM21 can bind directly to IgA, an in vitro fluorescence anisotropy experiment was performed. IgA was titrated into Alexa 488-labeled TRIM21 PRYSPRY, and the switch in anisotropy was measured. Fitting the producing data exposed that IgA binds TRIM21 with an affinity (KD) of 50 24 M (Fig. 1B). We also measured binding of purified IgA1 and IgA2 and acquired affinities of 55 M 23 and 53 39 M,.