A metabolic inhibitor blocks cellular fucosylation and enables production of afucosylated antibodies.

The fucosylation of glycoproteins regulates diverse physiological processes. Inhibitors that can control cellular levels of protein fucosylation have consequently emerged as being of high interest. One area where inhibitors of fucosylation have gained significant attention is in the production of afucosylated antibodies, which exhibit superior antibody-dependent cell cytotoxicity as compared to their fucosylated counterparts. Here, we describe ß-carbafucose, a fucose derivative in which the endocyclic ring oxygen is replaced by a methylene group, and show that it acts as a potent metabolic inhibitor within cells to antagonize protein fucosylation. ß-carbafucose is assimilated by the fucose salvage pathway to form GDP-carbafucose which, due to its being unable to form the oxocarbenium ion-like transition states used by fucosyltransferases, is an incompetent substrate for these enzymes. ß-carbafucose treatment of a CHO cell line used for high-level production of the therapeutic antibody Herceptin leads to dose-dependent reductions in core fucosylation without affecting cell growth or antibody production. Mass spectrometry analyses of the intact antibody and N-glycans show that ß-carbafucose is not incorporated into the antibody N-glycans at detectable levels. We expect that ß-carbafucose will serve as a useful research tool for the community and may find immediate application for the rapid production of afucosylated antibodies for therapeutic purposes.

Sulfation of sialic acid is ubiquitous and essential for vertebrate development.

Glycosylation of proteins and lipids occurs in vertebrates, usually terminating with sialylation, which regulates the physicochemical and biological properties of these glycoconjugates. Although less commonly known, sialic acid residues also undergo various modifications, such as acetylation, methylation, and sulfation. However, except for acetylation, the enzymes or functions of the other modification processes are unknown. To the best of our knowledge, this study is the first to demonstrate the ubiquitous occurrence of sulfated sialic acids and two genes encoding the sialate: O-sulfotransferases 1 and 2 in vertebrates. These two enzymes showed about 50% amino acid sequence identity, and appeared to be complementary to each other in acceptor substrate preferences. Gene targeting experiments showed that the deficiency of these genes was lethal for medaka fish during young fry development and accompanied by different phenotypes. Thus, the sulfation of sialic acids is essential for the vertebrate development.

Sialic acid sulfation is induced by the antibiotic treatment in mammalian cells.

Sialic acids (Sias) are an outermost-situated sugar of glycoproteins and glycolipids to play important roles in various biological phenomena. They are often modified by additional substituents, such as O-acetyl group, to display more than 50 different structures in nature. Of those modified Sia, nothing is known about the occurrence and biological functions of sulfated Sias (SiaSs) in mammals. To elucidate the significance of sialic acid sulfation, we investigated various mammalian-cultured cell lines for the expression of SiaS using the specific antibody 3G9. First, SiaS is expressed in a cell line-dependent and a cell density-dependent manner. Second, in CHO cells, the expression of SiaS is reversibly induced by treatment with the antibiotic G418. Taken together, the expression of SiaS is changed by intrinsic and extrinsic factors in mammalian cells. This is the first demonstration of regulated expression of SiaS.

Inflammation contributes to NKX3.1 loss and augments DNA damage but does not alter the DNA damage response via increased SIRT1 expression.

The oxidative stress response is a cellular defense mechanism that protects cells from oxidative damage and cancer development. The exact molecular mechanism by which reactive oxygen species (ROS) contribute to DNA damage and increase genome instability in prostate cancer merits further investigation. Here, we aimed to determine the effects of NKX3.1 loss on antioxidant defense in response to acute and chronic inflammation in an in vitro model. Oxidative stress-induced DNA damage resulted in increased H2AX((S139)) phosphorylation (a hallmark of DNA damage), along with the degradation of the androgen receptor (AR), p53 and NKX3.1, upon treatment with conditioned medium (CM) obtained from activated macrophages or H2O2. Furthermore, the expression and stability of SIRT1 were increased by CM treatment but not by H2O2 treatment, although the level of ATM((S1981)) phosphorylation was not changed compared with controls. Moreover, the deregulated antioxidant response resulted in upregulation of the pro-oxidant QSCN6 and the antioxidant GPX2 and downregulation of the antioxidant GPX3 after CM treatment. Consistently, the intracellular ROS level increased after chronic treatment, leading to a dose-dependent increase in the ability of LNCaP cells to tolerate oxidative damage. These data suggest that the inflammatory microenvironment is a major factor contributing to DNA damage and the deregulation of the oxidative stress response, which may be the underlying cause of the increased genetic heterogeneity during prostate tumor progression.

TNFα-mediated loss of ß-catenin/E-cadherin association and subsequent increase in cell migration is partially restored by NKX3.1 expression in prostate cells.

Inflammation-induced carcinogenesis is associated with increased proliferation and migration/invasion of various types of tumor cells. In this study, altered ß-catenin signaling upon TNFα exposure, and relation to loss of function of the tumor suppressor NKX3.1 was examined in prostate cancer cells. We used an in vitro prostate inflammation model to demonstrate altered sub-cellular localization of ß-catenin following increased phosphorylation of Akt(S473) and GSK3ß(S9). Consistently, we observed that subsequent increase in ß-catenin transactivation enhanced c-myc, cyclin D1 and MMP2 expressions. Consequently, it was also observed that the ß-catenin-E-cadherin association at the plasma membrane was disrupted during acute cytokine exposure. Additionally, it was demonstrated that disrupting cell-cell interactions led to increased migration of LNCaP cells in real-time migration assay. Nevertheless, ectopic expression of NKX3.1, which is degraded upon proinflammatory cytokine exposure in inflammation, was found to induce the degradation of ß-catenin by inhibiting Akt(S473) phosphorylation, therefore, partially rescued the disrupted ß-catenin-E-cadherin interaction as well as the cell migration in LNCaP cells upon cytokine exposure. As, the disrupted localization of ß-catenin at the cell membrane as well as increased Akt(S308) priming phosphorylation was observed in human prostate tissues with prostatic inflammatory atrophy (PIA), high-grade prostatic intraepithelial neoplasia (H-PIN) and carcinoma lesions correlated with loss of NKX3.1 expression. Thus, the data indicate that the ß-catenin signaling; consequently sub-cellular localization is deregulated in inflammation, associates with prostatic atrophy and PIN pathology.