Ig is a secreted glycoprotein that recognizes antigens through its antigen-binding fragment region. In addition, Ig connects with other components of the immune system through the crystallizable (Fc) region of its fragment to play its effector function. The Fc domain of Ig can also interact with complement protein. Antibody isotype and N- glycosylation pattern determined by constant heavy chain (CH) region are important determinants of antibody function.
Jagn 1 encodes a universally expressed transmembrane protein, which has been proved to be necessary for ER recombination in Drosophila and melanocyte development and the differentiation and survival of human neutrophils. So far, the defects described in mammals are limited to neutrophils, in which JAGN 1 regulates the process of particle formation. Because the differentiation of B cells into plasma cells secreting antibodies involves significant ER amplification to support high-speed antibody production and secretion, we set out to study the role of JAGN 1 in B cell biology.
The deletion of Jagn 1 in B cells will lead to the decrease of Ig level and humoral immunity.
In order to study the role of Jagn 1 in B cells, we bred Jagn 1 FL-MB1-cre mice (hereinafter referred to as Jagn 1δ B) to delete Jagn1in pro-B cells at an early stage. In order to eliminate the influence of Cre in Mb 1-Cre strain, we analyzed the development of B cells, the composition of B cell subsets and the serum Ig level in the same batch of Mb 1-Cre and control mice. In all the test parameters, we did not observe any significant difference between these queues, so we used Jagn 1fl/fl mice as control in the whole experiment.
By measuring the mRNA levels of Jagn 1 in B cells of B220+ spleen and plasma cells differentiated in vitro, the effective deletion of Jagn 1 in B cell lineage can be determined. The loss of protein was confirmed by flow cytometry and intracellular staining of plasma cells JAGN 1. In order to evaluate the subcellular localization of JAGN 1 in B cells, we established a plasmacytoma cell line (MPC- 1 1) by knocking out the expression of endogenous Jagn 1 and JAGN 1 fused with amino terminal V5 tag. Immunohistochemical analysis showed that V5-JAGN 1 was located in the endoplasmic reticulum of B cells.
The deletion of Jagn 1 mediated by Cb23 -Cre involves B cell development, B cell homeostasis and plasma cells, which excludes Mb 1- Cre effect and confirms the phenotype of Jagn 1 KO. (source: JEM)
Jagn 1 deficiency will affect the development of plasma cells.
In order to deeply understand the role of Jagn 1 in the development and homeostasis of B cells, we analyzed the bone marrow (BM) and spleen of JAGN1Δ B and the homeostasis level of the development and mature B cell subsets of the same batch of mice as control by flow cytometry. Jagn1Δ B mice showed normal early B cell development, but compared with the same batch of control mice, the level of BM plasma cells (Lin-CD28+CD 138+) decreased significantly.
Although we observed that the deficiency of Jagn 1 obviously affected the plasma cells in BM, which were longevity and the main source of serum antibody level, the short-lived plasma cells in the spleen of Jagn 1δ B mice only decreased slightly. In addition, compared with the control mice in the same batch, the spleen of JAGN1Δ B mice was enlarged, the proportion of B cells in the spleen margin was increased, and the proportion of follicular B cells was decreased by flow cytometry analysis and immunohistochemical detection. Therefore, the inactivation of Jagn 1 leads to obvious damage of BM plasma cells and changes of spleen B cells.
Normal development of B cells, but changed the steady state of spleen B cells and decreased the plasma cells Jagn 1δ B mice. (source: JEM)
Jagn 1 mutant plasma mother cells increased endoplasmic reticulum stress and changed endoplasmic reticulum structure.
In order to further analyze the consequences of deletion of antibody secreting cell Jagn 1, we isolated spleen B cells from control and JAGN 1δ B mice, and used LPS * * * days to induce IgM secretion and plasma cell differentiation. LPS induces newborn B cells to differentiate into activated B cells, plasma cells and final plasma cells. LPS * * * JAGN1Δ B-derived B cells led to a significant decrease, although the changes of cells and cells were small before antibody production.
In order to test the ability of Jagn 1 Kob cells to produce Ig in response to LPS *** in vitro, the intracellular and secreted IgM levels of Jagn 1 deficient and sufficient B cells were quantified. Compared with the control cells, the deletion of JAGN 1 leads to a large decrease of intracellular and secretory Ig. It is worth noting that the membrane (? M) and secretion (? S)S)mRNA has the same transcription level. Similar results were obtained by producing transformed B cells similar to the germinal center in a more physiologically meaningful in vitro differentiation system (iGB culture).
The control and Jagn 1 defective B cells were cultured on 40LB stromal cells, with CD40L expression membrane, which produced B cell activating factors to promote survival and supported the optimal proliferation of B cells. IL-4 *** cells were used for 4 days, and then IL-21* * for 4 days to produce plasma-forming cells with switched IgG 1 and IgE categories. Using this analysis, compared with the control cells, the proportion of early activated B cells and pre-plasma cell-forming cells produced by Jagn 1 defective B cells changed, but the number of plasma cell-forming cells was the same. This is consistent with our observation. It was found that the spleen cells of Jagn1Δ B mice were at a normal level. However, the production of IgE and IgG 1 in jagn1Δ b plasma cells decreased significantly, which reduced the number and size of spots in ELISPOT analysis.
Increasing the survival of antibody-producing cells can not restore immunoglobulin production in Jagn 1δ B mice. (source: JEM)
The rare disease mutation of JAGN 1 affects the human Ig glycospectrum. The mutation of human JAGN 1 gene leads to severe congenital neutropenia. Recent reports show that some patients with JAGN 1 also suffer from hypoglobulinemia. Therefore, we analyzed the serum of patients with homozygous missense mutation or frameshift deletion mutation in JAGN 1, which resulted in mutation and possible non-functional protein expression. Importantly, even if the serum Ig level is within the normal range, we have detected that the Ig glycospectrum of the most abundant IgG 1 subclass in the serum of patients with JAGN 1 mutation has changed.
In B lymphocytes transformed by Epstein-Barr virus from patients, we detected signs of increased endoplasmic reticulum stress, including increased splicing of XBP 1 and up-regulation of heat shock protein family A member 5. These data show that the deletion of functional JAGN 1 protein in patients with congenital neutropenia is related to the changes of Ig N- glycosylation, ER homeostasis and B cell function, which is similar to the phenotype we observed in JAGN 1δ B mouse model.
conclusion
JAGN 1 seems to affect the function of antibody production factory in cells, but what makes them incredible is that the change of sugar molecular structure may promote the antibody to better combine with other immune cells and enhance the immune defense response of the body; The researchers then clarified this mechanism in human samples. Rare genetic defects usually occur in a few people.
However, it may also be helpful to analyze the basic biological principles of the human body. In this case, the researchers found that specific genes may affect the function of endoplasmic reticulum in cells, thus affecting the production of antibodies. In addition, the researchers also revealed the changes of the sugar coating on the antibody surface, which has a very important impact on the actual function of the antibody in the body.