pKa Modulation of the Acid/Base Catalyst within GH32 and GH68: A Role in Substrate/Inhibitor Specificity?

Glycoside hydrolases of families 32 (GH32) and 68 (GH68) belong to clan GH-J, containing hydrolytic enzymes (sucrose/fructans as donor substrates) and fructosyltransferases (sucrose/fructans as donor and acceptor substrates). In GH32 members, some of the sugar substrates can also function as inhibitors, this regulatory aspect further adding to the complexity in enzyme functionalities within this family. Although 3D structural information becomes increasingly available within this clan and huge progress has been made on structure-function relationships, it is not clear why some sugars bind as inhibitors without being catalyzed. Conserved aspartate and glutamate residues are well known to act as nucleophile and acid/bases within this clan. Based on the available 3D structures of enzymes and enzyme-ligand complexes as well as docking simulations, we calculated the pKa of the acid-base before and after substrate binding. The obtained results strongly suggest that most GH-J members show an acid-base catalyst that is not sufficiently protonated before ligand entrance, while the acid-base can be fully protonated when a substrate, but not an inhibitor, enters the catalytic pocket. This provides a new mechanistic insight aiming at understanding the complex substrate and inhibitor specificities observed within the GH-J clan. Moreover, besides the effect of substrate entrance on its own, we strongly suggest that a highly conserved arginine residue (in the RDP motif) rather than the previously proposed Tyr motif (not conserved) provides the proton to increase the pKa of the acid-base catalyst

CD8 and CD4 Positive NKT Subpopulations and Immune-Checkpoint Pathways in Early-Onset Preeclampsia and Healthy Pregnancy

Although many studies have investigated the clinical aspect of early-onset preeclampsia, our knowledge about the immunological consequences of improper placenta development is scarce. The maternal immunotolerance against the fetus is greatly influenced by the Th1 predominance developed by the mother’s immune system. Thirty-two early-onset preeclamptic and fifty-one healthy pregnant women with appropriately matched gestational age were involved in our study. Mononuclear cells were separated from peripheral venous blood and the frequency of CD8⁺, CD4⁺, double positive (DP), and double negative (DN) NKT cell subpopulations was determined using multicolor flow cytometry. Following the characterization, the expression levels of different immune checkpoint receptors and ligands were also defined. Soluble CD226 levels were quantified by ELISA. Novel and significant differences were revealed among the ratios of the investigated NKT subsets and in the expression patterns of PD-1, LAG-3, TIGIT and CD226 receptors. Further differences were determined in the expression of CD112, PD-1, LAG-3 and CD226 MFI values between the early-onset preeclamptic and the healthy pregnant groups. Our results suggest that the investigated NKT subpopulations act differently in the altered immune condition characteristic of early-onset preeclampsia and indicate that the different subsets may contribute to the compensation or maintenance of Th1 predominance

The immunological effect of Galectin-9/TIM-3 pathway after low dose Mifepristone treatment in mice at 14.5 day of pregnancy

<div><p>The abortifacient Mifepristone (RU486) has proven to be a safe, effective, acceptable option for millions of women seeking abortion during the first and second trimester of pregnancy although its precise mechanism of action is not well understood. The main objective of this study was to investigate the impact of low dose Mifepristone administration on placental Galectin-9 (Gal-9) expression, as well as its effect on the cell surface expression of Gal-9, TIM-3 and CD107a molecules by different T and NK cell subsets. A model of Mifepristone-induced immunological changes was established in syngeneic pregnant BALB/c mice. RU486-induced alteration in placental Gal-9 expression was determined by immunohistochemistry. For immunophenotypic analysis, mid-pregnancy decidual lymphocytes and peripheral mononuclear cells were obtained from Mifepristone treated and control mice at the 14.5 day of gestation. TIM-3 and Gal-9 expression by peripheral and decidual immune cells were examined by flow cytometry. Our results revealed a dramatically decreased intracellular Gal-9 expression in the spongiotrophoblast layer of the haemochorial placenta in Mifepristone treated pregnant mice. Although low dose RU486 treatment did not cause considerable change in the phenotypic distribution of decidual and peripheral immune cells, it altered the Gal-9 and TIM-3 expression by different NK and T cell subsets. In addition, the treatment significantly decreased the CD107a expression by decidual TIM-3+ NK cells, but increased its expression by decidual NKT cell compared to the peripheral counterparts. These findings suggest that low dose Mifepristone administration might induce immune alterations in both progesterone dependent and independent way.</p></div

Examination of the TIGIT-CD226-CD112-CD155 Immune Checkpoint Network during a Healthy Pregnancy

Background: The importance of immune checkpoint molecules is well known in tumor and transplantation immunology; however, much less information is available regarding human pregnancy. Despite the significant amount of information about the TIGIT and CD226 immune checkpoint receptors in immune therapies, very little research has been conducted to study the possible role of these surface molecules and their ligands (CD112 and CD155) during the three trimesters of pregnancy. Methods: From peripheral blood, immune cell subpopulations were studied, and the surface expression of immune checkpoint molecules was analyzed by flow cytometry. Soluble immune checkpoint molecule levels were measured by ELISA. Results: Notable changes were observed regarding the percentage of monocyte subpopulation and the expression of CD226 receptor by CD4+ T and NKT cells. Elevated granzyme B content by the intermediate and non-classical monocytes was assessed as pregnancy proceeded. Furthermore, we revealed an important relationship between the CD226 surface expression by NKT cells and the serum CD226 level in the third trimester of pregnancy. Conclusions: Our results confirm the importance of immune checkpoint molecules in immunoregulation during pregnancy. CD226 seems to be a significant regulator, especially in the case of CD4+ T and NKT cells, contributing to the maternal immune tolerance in the late phase of pregnancy

Representative Gal-9 immunohistochemical staining of pregnant mouse placentae from untreated control and RU486 treated pregnant mice.

<p>IHC was performed on the placenta-sections of healthy control (Fig 1A) and RU486 treated (Fig 1B) mice. Yellow arrows indicate Gal-9 positive cells (magnification 350x). The lower circle graphs show the percentage of cytoplasmic Gal-9 positive cells (data are shown as mean). Differences were considered statistically significant for p-values ≤0.05. The images were captures utilizing the Pannoramic DESK scanner (3DHISTECH Ltd.) and analysis was performed by the Pannoramic viewer software (3DHISTECH Ltd.).</p

Gal-9 expression by Tregs and the proportion of Gal-9+ Th cell population in decidua and periphery from untreated control and RU486 treated pregnant mice.

<p>Box plot of the median, the 25th and 75th percentiles, range, and individual data values for the cell surface expression of Gal-9 by Treg cells and the frequency of Gal-9 positive Th cells in decidua and periphery from untreated control and RU486 treated pregnant mice. The middle line within the box represents the median, the middle dot within the box represents the mean, the boxes indicate the interquartile ranges and the whiskers show the most extreme observations. Differences were considered statistically significant for p-values ≤0.05.</p

TIM-3 expression by decidual and peripheral mononuclear cells from untreated control and RU486 treated pregnant mice.

<p>Box plot of the median, the 25th and 75th percentiles, range, and individual data values for the expression of TIM-3 by NK cells, NKT cells, γ/δT and CD4 T cells in periphery and decidua in untreated and RU486 treated pregnant mice. The middle line within the box represents the median, the middle dot within the box represents the mean, the boxes indicate the interquartile ranges and the whiskers show the most extreme observations. Differences were considered statistically significant for p-values ≤0.05.</p

Gal-9 expression by decidual and peripheral mononuclear cells from untreated control and RU486 treated pregnant mice.

<p>Box plot of the median, the 25th and 75th percentiles, range, and individual data values for the cell surface expression of Gal-9 by NK cells, NKT cells, γ/δT, CD4+ T cells in periphery and decidua of untreated and RU486 treated pregnant mice. The middle line within the box represents the median, the middle dot within the box represents the mean, the boxes indicate the interquartile ranges and the whiskers show the most extreme observations. Differences were considered statistically significant for p-values ≤0.05.</p

CD107a expression by different immune cell subsets and TIM-3 positive immune cell subsets in the periphery and in the decidua of untreated control and RU486 treated pregnant mice.

<p>Box plot of the median, the 25th and 75th percentiles, range, and individual data values for the he expression of CD107a by NK cells, NKT cells and γ/δT cells in the periphery and in the decidua of untreated control and RU486 treated pregnant mice (Fig 5A–5C). The expression of CD107a by TIM-3 positive NK cells, NKT cells and γ/δT cells in the periphery and in the decidua of untreated control and RU486 treated pregnant mice (Fig 5D–5F). The middle line within the box represents the median, the middle dot within the box represents the mean, the boxes indicate the interquartile ranges and the whiskers show the most extreme observations. Differences were considered statistically significant for p-values ≤0.05.</p