The carbohydrate Galα1-3Galβ1-(3)4GlcNAc-R (α-Gal) is produced in all mammals except for humans, apes and old world monkeys that lost the ability to synthetize this carbohydrate. Therefore, humans can produce high antibody titers against α-Gal. Anti-α-Gal IgE antibodies have been associated with tick-induced allergy (i.e. α-Gal syndrome) and anti-α-Gal IgG/IgM antibodies may be involved in protection against malaria, leishmaniasis and Chagas disease. The α-Gal on tick salivary proteins plays an important role in the etiology of the α-Gal syndrome. However, whether ticks are able to produce endogenous α-Gal remains currently unknown. In this study, the Ixodes scapularis genome was searched for galactosyltransferases and three genes were identified as potentially involved in the synthesis of α-Gal. Heterologous gene expression in α-Gal-negative cells and gene knockdown in ticks confirmed that these genes were involved in α-Gal synthesis and are essential for tick feeding. Furthermore, these genes were shown to play an important role in tick-pathogen interactions. Results suggested that tick cells increased α-Gal levels in response to Anaplasma phagocytophilum infection to control bacterial infection. These results provided the molecular basis of endogenous α-Gal production in ticks and suggested that tick galactosyltransferases are involved in vector development, tick-pathogen interactions and possibly the etiology of α-Gal syndrome in humans.

• MeSH
• alfa-galaktosidasa genetika   metabolismus   MeSH
• Anaplasma phagocytophilum patogenita   MeSH
• ehrlichióza genetika   metabolismus   MeSH
• galaktosyltransferasy metabolismus   MeSH
• genom genetika   MeSH
• HL-60 buňky MeSH
• infekce přenášené vektorem MeSH
• interakce hostitele a patogenu genetika   MeSH
• klíště mikrobiologie   MeSH
• lidé MeSH
• nádorové buněčné linie MeSH
• proteiny členovců metabolismus   MeSH
• sekvence aminokyselin MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

N-linked glycosylation of proteins has both functional and structural significance. Importantly, the glycan structure of a therapeutic protein influences its efficacy, pharmacokinetics, pharmacodynamics and immunogenicity. In this work, we developed glycosylation flux analysis (GFA) for predicting intracellular production and consumption rates (fluxes) of glycoforms, and applied this analysis to CHO fed-batch immunoglobulin G (IgG) production using two different media compositions, with and without additional manganese feeding. The GFA is based on a constraint-based modeling of the glycosylation network, employing a pseudo steady state assumption. While the glycosylation fluxes in the network are balanced at each time point, the GFA allows the fluxes to vary with time by way of two scaling factors: (1) an enzyme-specific factor that captures the temporal changes among glycosylation reactions catalysed by the same enzyme, and (2) the cell specific productivity factor that accounts for the dynamic changes in the IgG production rate. The GFA of the CHO fed-batch cultivations showed that regardless of the media composition, galactosylation fluxes decreased with the cultivation time more significantly than the other glycosylation reactions. Furthermore, the GFA showed that the addition of Mn, a cofactor of galactosyltransferase, has the effect of increasing the galactosylation fluxes but only during the beginning of the cultivation period. The results thus demonstrated the power of the GFA in delineating the dynamic alterations of the glycosylation fluxes by local (enzyme-specific) and global (cell specific productivity) factors.

• MeSH
• CHO buňky MeSH
• Cricetulus MeSH
• galaktosyltransferasy genetika   metabolismus   MeSH
• glykosylace MeSH
• imunoglobulin G biosyntéza   genetika   MeSH
• křečci praví MeSH
• rekombinantní proteiny biosyntéza   genetika   MeSH
• zvířata MeSH
• Check Tag
• křečci praví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

IgA nephropathy (IgAN), the most common primary glomerulonephritis, is characterized by renal immunodeposits containing IgA1 with galactose-deficient O-glycans (Gd-IgA1). These immunodeposits originate from circulating immune complexes consisting of anti-glycan antibodies bound to Gd-IgA1. As clinical disease onset and activity of IgAN often coincide with mucosal infections and dysregulation of cytokines, we hypothesized that cytokines may affect IgA1 O-glycosylation. We used IgA1-secreting cells derived from the circulation of IgAN patients and healthy controls and assessed whether IgA1 O-glycosylation is altered by cytokines. Of the eight cytokines tested, only IL-6 and, to a lesser degree, IL-4 significantly increased galactose deficiency of IgA1; changes in IgA1 O-glycosylation were robust for the cells from IgAN patients. These cytokines reduced galactosylation of the O-glycan substrate directly via decreased expression of the galactosyltransferase C1GalT1 and, indirectly, via increased expression of the sialyltransferase ST6GalNAc-II, which prevents galactosylation by C1GalT1. These findings were confirmed by siRNA knockdown of the corresponding genes and by in vitro enzyme reactions. In summary, IL-6 and IL-4 accentuated galactose deficiency of IgA1 via coordinated modulation of key glycosyltransferases. These data provide a mechanism explaining increased immune-complex formation and disease exacerbation during mucosal infections in IgAN patients.

• MeSH
• buněčné linie MeSH
• cytokiny * farmakologie   MeSH
• dospělí MeSH
• galaktosa metabolismus   nedostatek   MeSH
• galaktosyltransferasy * metabolismus   MeSH
• genový knockdown MeSH
• glykosylace účinky léků   MeSH
• IgA nefropatie enzymologie   patologie   MeSH
• imunoglobulin A * metabolismus   MeSH
• interleukin-4 farmakologie   MeSH
• interleukin-6 farmakologie   MeSH
• kyselina N-acetylneuraminová metabolismus   MeSH
• lidé MeSH
• malá interferující RNA metabolismus   MeSH
• polysacharidy metabolismus   MeSH
• Check Tag
• dospělí MeSH
• lidé MeSH
• mužské pohlaví MeSH
• ženské pohlaví MeSH

Binding of promastigotes to the sand fly midgut epithelium is regarded as an essential part of the Leishmania life cycle in the vector. Among Leishmania surface molecules putatively involved in attachment to the sand fly midgut, two GPI-anchored molecules are the most prominent: lipophosphoglycan (LPG) and promastigote surface protease gp63. In this work, we examined midgut attachment of Leishmania lines mutated in GPI-anchored molecules and compared results from 2 different techniques: in vivo development in sand flies and in vitro competitive binding assays using fluorescently labelled parasites. In combination with previous studies, our data provide additional support for (1) an LPG-independent parasite-binding mechanism of Leishmania major within the midgut of the permissive vector Phlebotomus perniciosus, and provide strong support for (2) the crucial role of L. major LPG in specific vector Phlebotomus papatasi, and (3) a role for Leishmania amazonensis gp63 in Lutzomyia longipalpis midgut binding. Moreover, our results suggest a critical role for GPI-anchored proteins and gp63 in Leishmania mexicana attachment to L. longipalpis midguts, as the wild type (WT) line accounted for over 99% of bound parasites.

• MeSH
• galaktosyltransferasy genetika   metabolismus   MeSH
• glykokonjugáty genetika   metabolismus   MeSH
• glykosfingolipidy genetika   metabolismus   MeSH
• hmyz - vektory parazitologie   MeSH
• kompetitivní vazba MeSH
• Leishmania fyziologie   MeSH
• lidé MeSH
• metaloendopeptidasy genetika   metabolismus   MeSH
• mutace MeSH
• Phlebotomus parazitologie   MeSH
• protozoální proteiny genetika   metabolismus   MeSH
• Psychodidae parazitologie   MeSH
• stadia vývoje MeSH
• trávicí systém parazitologie   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

This work reveals new structural relationships in the complex process of the interaction between activation receptors of natural killer cells (rat NKR-P1, human CD69) and novel bivalent carbohydrate glycomimetics. The length, glycosylation pattern and linker structure of receptor ligands were examined with respect to their ability to precipitate the receptor protein from solution, which simulates the in vivo process of receptor aggregation during NK cell activation. It was found that di-LacdiNAc triazole compounds show optimal performance, reaching up to 100% precipitation of the present protein receptors, and achieving high immunostimulatory activities without any tendency to trigger activation-induced apoptosis. In the synthesis of the compounds tested, two enzymatic approaches were applied. Whereas a β-N-acetylhexosaminidase could only glycosylate one of the two acceptor sites available with yields below 10%, the Y284L mutant of human placental β1,4-galactosyltransferase-1 worked as a perfect synthetic tool, accomplishing even quantitative glycosylation at both acceptor sites and with absolute regioselectivity for the C-4 position. This work insinuates new directions for further ligand structure optimisation and demonstrates the strong synthetic potential of the mutant human placental β1,4-galactosyltransferase-1 in the synthesis of multivalent glycomimetics and glycomaterials.

• MeSH
• aktivace lymfocytů účinky léků   imunologie   MeSH
• beta-N-acetylhexosaminidasy metabolismus   MeSH
• biomimetika metody   MeSH
• buňky NK chemie   účinky léků   imunologie   metabolismus   MeSH
• CD antigeny imunologie   metabolismus   MeSH
• diferenciační antigeny T-lymfocytů imunologie   metabolismus   MeSH
• galaktosyltransferasy genetika   metabolismus   MeSH
• imunoprecipitace MeSH
• krysa rodu rattus MeSH
• lektiny typu C agonisté   imunologie   metabolismus   MeSH
• lidé MeSH
• ligandy MeSH
• molekulární mimikry MeSH
• mutace MeSH
• placenta enzymologie   MeSH
• polysacharidy chemická syntéza   farmakologie   MeSH
• receptory buněk NK agonisté   imunologie   metabolismus   MeSH
• rekombinantní proteiny genetika   metabolismus   MeSH
• těhotenství MeSH
• vazba proteinů účinky léků   imunologie   MeSH
• vazebná místa účinky léků   imunologie   MeSH
• zvířata MeSH
• Check Tag
• krysa rodu rattus MeSH
• lidé MeSH
• těhotenství MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Human placental beta1,4-galactosyltransferase-I (EC 2.4.1.38) transfers the galactosyl moiety from UDP-Gal to various GlcNAc or Glc acceptors in vivo. Here, we describe the construction of its Y284L mutant as a His(6)propeptide-catbeta4GalT1 construct, in which the Gal-transferase activity was totally abolished in favor of its GalNAc-transferase activity. We used this mutant in the synthesis of three mono- and bivalent LacdiNAc glycomimetics with good yields. These compounds proved to be powerful ligands of two activation receptors of natural killer cells, NKR-P1 and CD69. A synthetic bivalent tethered di-LacdiNAc is the best currently known precipitation agent for both of these receptors and has promising potential for the development of immunoactive glycodrugs.

• MeSH
• bakteriální proteiny metabolismus   MeSH
• Campylobacter jejuni enzymologie   MeSH
• CD antigeny metabolismus   MeSH
• diferenciační antigeny T-lymfocytů metabolismus   MeSH
• epimerázy sacharidů metabolismus   MeSH
• galaktosyltransferasy genetika   metabolismus   MeSH
• glykokonjugáty biosyntéza   chemická syntéza   metabolismus   MeSH
• laktosa analogy a deriváty   biosyntéza   chemická syntéza   metabolismus   MeSH
• lektinové receptory NK-buněk - podrodina B metabolismus   MeSH
• lektiny typu C MeSH
• lidé MeSH
• mutace MeSH
• placenta enzymologie   MeSH
• substrátová specifita MeSH
• těhotenství MeSH
• Check Tag
• lidé MeSH
• těhotenství MeSH
• ženské pohlaví MeSH
• Publikační typ
• práce podpořená grantem MeSH
• publikace stažené z tisku MeSH