Biosynthesis and Biological Significances of LacdiNAc Group on N- and O-Glycans in Human Cancer Cells.

An increasing number of studies have shown that the disaccharide GalNAcß1→4GlcNAc (LacdiNAc) group bound to N- and O-glycans in glycoproteins is expressed in a variety of mammalian cells. Biosynthesis of the LacdiNAc group was well studied, and two ß4-N-acetylgalactosaminyltransferases, ß4GalNAcT3 and ß4GalNAcT4, have been shown to transfer N-acetylgalactosamine (GalNAc) to N-acetylglucosamine (GlcNAc) of N- and O-glycans in a ß-1,4-linkage. The LacdiNAc group is often sialylated, sulfated, and/or fucosylated, and the LacdiNAc group, with or without these modifications, is recognized by receptors and lectins and is thus involved in the regulation of several biological phenomena, such as cell differentiation. The occurrences of the LacdiNAc group and the ß4GalNAcTs appear to be tissue specific and are closely associated with the tumor progression or regression, indicating that they will be potent diagnostic markers of particular cancers, such as prostate cancer. It has been demonstrated that the expression of the LacdiNAc group on N-glycans of cell surface glycoproteins including ß1-integrin is involved in the modulation of their protein functions, thus affecting cellular invasion and other malignant properties of cancer cells. The biological roles of the LacdiNAc group in cancer cells have not been fully understood. However, the re-expression of the LacdiNAc group on N-glycans, which is lost in breast cancer cells by transfection of the ß4GalNAcT4 gene, brings about the partial restoration of normal properties and subsequent suppression of malignant phenotypes of the cells. Therefore, elucidation of the biological roles of the LacdiNAc group in glycoproteins will lead to the suppression of breast cancers.

LacdiNAcylation of N-glycans in MDA-MB-231 human breast cancer cells results in changes in morphological appearance and adhesive properties of the cells.

We demonstrated previously that the expression of the disaccharide, GalNAcß1 → 4GlcNAc (LacdiNAc), on N-glycans of cell surface glycoproteins in MDA-MB-231 human breast cancer cells suppresses their malignant properties such as tumor formation in nude mice. Here, we report changes in the morphological appearance and adhesive properties of two kinds of clonal cells of MDA-MB-231 cells overexpressing ß4-N-acetyl-galactosaminyltransferase 4. The clonal cells exhibited a cobble stone-like shape as compared to a spindle-like shape of the mock-transfected cells and the original MDA-MB-231 cells. This was associated with an increased expression of cell surface E-cadherin, a marker of epithelial cells, and a decreased expression of N-cadherin, vimentin, α-smooth muscle actin and ZEB1, markers of mesenchymal cells. In addition, the clonal cells showed a lower migratory activity compared to the mock-transfected cells by wound-healing assay. These results suggest that mesenchymal-epithelial transition may be occurring in these clonal cells. Furthermore, increased adhesion to extracellular matrix proteins such as fibronectin, collagen type I, collagen type IV, and laminin was observed. The clonal cells spread and enlarged, whereas the mock-transfected cells demonstrated poor spreading on laminin-coated plates in the absence of fetal calf serum, indicating that expression of LacdiNAc on cell surface glycoproteins results in changes in cell adhesive and spreading properties particularly to laminin.

Enhanced expression of the ß4-N-acetylgalactosaminyltransferase 4 gene impairs tumor growth of human breast cancer cells.

Two ß4-N-acetylgalactosaminyltransferases (ß4GalNAcTs), ß4GalNAcT3 and ß4GalNAcT4, have been shown to be involved in the synthesis of the GalNAcß1 â†’ 4GlcNAc (LacdiNAc) group expressed on the outer branches of N- and/or O-glycans, and only ß4GalNAcT4 is expressed in human mammary gland. We found that the expression level of the LacdiNAc group decreases as human breast cancers progress. To investigate biological significances of this disaccharide in human breast cancers, we transfected the FLAG-tagged ß4GalNAcT4 cDNA into MDA-MB-231 cells, and obtained several clones showing enhanced expression of the gene. Clones 1 and 2 showed 15 and 9 times more transcript than mock-transfected cells. The FLAG-ß4GalNAcT4 protein and its product, the LacdiNAc group, were detected in clone 1 and 2 cells. No change was observed in their growth rates while significant decreases in colony forming and invasive abilities were observed for clone 1 and 2 cells. When clone 1 cells were transplanted subcutaneously into nude mice, no tumors were formed while tumors were formed with mock-transfected cells. These results indicate that the expression of the LacdiNAc group is quite important for the suppression of malignancies of the MDA-MB-231 cells.

Expression of LacdiNAc groups on N-glycans among human tumors is complex.

Aberrant glycosylation of proteins and lipids is one of the characteristic features of malignantly transformed cells. The GalNAc ß 1 → 4GlcNAc (LacdiNAc or LDN) group at the nonreducing termini of both N- and O-glycans is not generally found in mammalian cells. We previously showed that the expression level of the LacdiNAc group in N-glycans decreases dramatically during the progression of human breast cancer. In contrast, the enhanced expression of the LacdiNAc group has been shown to be associated with the progression of human prostate, ovarian, and pancreatic cancers. Therefore, the expression of the disaccharide group appears to be dependent on types of tumors. The mechanism of formation of the LacdiNAc group in human tumors and cancer cells has been studied, and two ß 4-N-acetylgalacto-saminyltransferases ( ß 4GalNAcTs), ß 4GalNAcT3 and ß 4GalNAcT4, have been shown to be involved in the biosynthesis of this disaccharide group in a tissue-dependent manner. Transfection of the ß 4GalNAcT3 gene brought about significant changes in the malignant phenotypes of human neuroblastoma, indicating that this disaccharide group is important for suppressing the tumor growth.

A cell culture system for the induction of Mallory bodies: Mallory bodies and aggresomes represent different types of inclusion bodies.

Mallory bodies (MBs) represent keratin-rich inclusion bodies observed in human alcoholic liver disease and in several chronic non-alcoholic liver diseases. The mechanism of their formation and their relationship to other inclusion bodies such as aggresomes is incompletely understood. We could induce keratin aggregates typical of MBs in cultured clone 9 rat hepatocytes by transgenic expression of wild-type and mutant aquaporin2 or alpha1-antitrypsin and under various forms of other cellular stress. By immunocytochemical analysis, p62 and poly-ubiquitin, components of classical MBs, could be demonstrated in the keratin aggregates of clone 9 hepatocytes. In addition, histone deacetylase 6, a microtubule-associated deacetylase, was identified as a novel component of the keratin aggregates. Thus, together with their ultrastructural appearance as randomly oriented, organelle-free aggregates of keratin filaments, the keratin aggregates in clone 9 hepatocytes correspond to MBs. An imbalance in keratin 8 to 18 with very low levels of keratin 18 appears to be the underlying cause for their formation. The formation of MBs was microtubule-dependent although not depending on the activity of histone deacetylase 6. Forskolin-induced MBs in clone 9 hepatocytes were reversible structures which disappeared upon drug withdrawal. The MBs were not related to aggresomes since overexpressed misfolded transgenic proteins were undetectable in the keratin aggregates and no vimentin fiber cage was detectable, both of which represent hallmarks of aggresomes. Thus, cultured clone 9 hepatocytes are a useful system to study further aspects of the pathobiology of MBs.

Protein quality control: the who's who, the where's and therapeutic escapes.

In cells the quality of newly synthesized proteins is monitored in regard to proper folding and correct assembly in the early secretory pathway, the cytosol and the nucleoplasm. Proteins recognized as non-native in the ER will be removed and degraded by a process termed ERAD. ERAD of aberrant proteins is accompanied by various changes of cellular organelles and results in protein folding diseases. This review focuses on how the immunocytochemical labeling and electron microscopic analyses have helped to disclose the in situ subcellular distribution pattern of some of the key machinery proteins of the cellular protein quality control, the organelle changes due to the presence of misfolded proteins, and the efficiency of synthetic chaperones to rescue disease-causing trafficking defects of aberrant proteins.

The proteasome is involved in the degradation of different aquaporin-2 mutants causing nephrogenic diabetes insipidus.

Mutations in the water channel aquaporin-2 (AQP2) can cause congenital nephrogenic diabetes insipidus. To reveal the possible involvement of the protein quality control system in processing AQP2 mutants, we created an in vitro system of clone 9 hepatocytes stably expressing endoplasmic reticulum-retained T126M AQP2 and misrouted E258K AQP2 as well as wild-type AQP2 and studied their biosynthesis, degradation, and intracellular distribution. Mutant and wild-type AQP2 were synthesized as 29-kd nonglycosylated and 32-kd core-glycosylated forms in the endoplasmic reticulum. The wild-type AQP2 had a t(1/2) of 4.6 hours. Remarkable differences in the degradation kinetics were observed for the glycosylated and nonglycosylated T126M AQP2 (t(1/2) = 2.0 hours versus 0.9 hours). Moreover, their degradation was depending on proteasomal activity as demonstrated in inhibition studies. Degradation of E258K AQP2 also occurred rapidly (t(1/2) = 1.8 hours) but in a proteasome- and lysosome-dependent manner. By triple confocal immunofluorescence microscopy misrouting of E258K to lysosomes via the Golgi apparatus could be demonstrated. Notwithstanding the differences in degradation kinetics and subcellular distribution such as endoplasmic reticulum-retention and misrouting to lysosomes, both T126M and E258K AQP2 were efficiently degraded. This implies the involvement of different protein quality control processes in the processing of these AQP2 mutants.

Expression of a mutant ER-retained polytope membrane protein in cultured rat hepatocytes results in Mallory body formation.

Mallory bodies represent cytokeratin-rich inclusion bodies which occur characteristically but not exclusively in human alcoholic liver disease and experimentally in mice during chronic intoxication with drugs. We report the first in vitro cell system of Mallory body induction. In clone 9 rat hepatocytes stably transfected to express an ER-retained T126M-aquaporin-2 (AQP2), on the mean 40% of the cells contained cytokeratin-rich inclusion bodies. By electron microscopy, their structure corresponded to that of genuine Mallory bodies. Such inclusion bodies were not detectable in clone 9 rat hepatocytes stably expressing a Golgi apparatus/lysosome-retained E258K-aquaporin-2. Proteasome inhibition increased the number of Mallory body-containing T126M-AQP2-expressing clone 9 hepatocytes to 60% on average. Proteasome inhibition in non-transfected, cytokeratin meshwork-forming clone 9 hepatocytes resulted in Mallory body formation on average in 6% of cells. Collectively, these data suggest that in the described in vitro cell system, Mallory body formation is induced by the presence of non-native protein conformers and point to the involvement of the proteasomal digestive system. The here reported in vitro system will be useful in studies about the biogenesis and progression of Mallory bodies, their relationship to aggresomes, and the role of inclusion bodies in the pathogenesis of cell damage.