The widely used immunosuppressant cyclosporine A (CSA) blocks nuclear translocation of the transcription factor, NF-AT (nuclear factor of activated T cells), preventing its activity. mRNA for several NF-AT isoforms has been shown to exist in cells outside of the immune system, suggesting a possible mechanism for side effects associated with CSA treatment. In this study, we demonstrate that CSA inhibits biochemical and morphological differentiation of skeletal muscle cells while having a minimal effect on proliferation. Furthermore, in vivo treatment with CSA inhibits muscle regeneration after induced trauma in mice. These results suggest a role for NF-AT-mediated transcription outside of the immune system. In subsequent experiments, we examined the activation and cellular localization of NF-AT in skeletal muscle cells in vitro. Known pharmacological inducers of NF-AT in lymphoid cells also stimulate transcription from an NF-AT-responsive reporter gene in muscle cells. Three isoforms of NF-AT (NF-ATp, c, and 4/x/c3) are present in the cytoplasm of muscle cells at all stages of myogenesis tested. However, each isoform undergoes calcium-induced nuclear translocation from the cytoplasm at specific stages of muscle differentiation, suggesting specificity among NF-AT isoforms in gene regulation. Strikingly, one isoform (NF-ATc) can preferentially translocate to a subset of nuclei within a single multinucleated myotube. These results demonstrate that skeletal muscle cells express functionally active NF-AT proteins and that the nuclear translocation of individual NF-AT isoforms, which is essential for the ability to coordinate gene expression, is influenced markedly by the differentiation state of the muscle cell.

FCP1 [transcription factor IIF (TFIIF)-associated carboxyl-terminal domain (CTD) phosphatase] is the only identified phosphatase specific for the phosphorylated CTD of RNA polymerase II (RNAP II).The phosphatase activity of FCP1 is enhanced in the presence of the large subunit of TFIIF (RAP74 in humans). It has been demonstrated that the CTD of RAP74 (cterRAP74; residues 436 -517) directly interacts with the highly acidic CTD of FCP1 (cterFCP; residues 879 -961 in human). In this manuscript, we have determined a high-resolution solution structure of a cterRAP74͞cterFCP complex by NMR spectroscopy. Interestingly, the cterFCP protein is completely disordered in the unbound state, but forms an ␣-helix (H1 ; E945-M961) in the complex. The cterRAP74͞cterFCP binding interface relies extensively on van der Waals contacts between hydrophobic residues from the H2 and H3 helices of cterRAP74 and hydrophobic residues from the H1 helix of cterFCP. The binding interface also contains two critical electrostatic interactions involving aspartic acid residues from H1 of cterFCP and lysine residues from both H2 and H3 of cterRAP74. There are also three additional polar interactions involving highly conserved acidic residues from the H1 helix. The cterRAP74͞cterFCP complex is the first highresolution structure between an acidic residue-rich domain from a holoenzyme-associated regulatory protein and a general transcription factor. The structure defines a clear role for both hydrophobic and acidic residues in protein͞protein complexes involving acidic residue-rich domains in transcription regulatory proteins. R NA polymerase II (RNAP II) is a multisubunit enzyme complex that enters the initiation complex with the carboxylterminal domain (CTD) of its largest subunit in an unphosphorylated form (RNAP IIA). The CTD contains a heptapeptide repeat (YSPTSPS) that becomes extensively phosphorylated (RNAP IIO) primarily at serine-2 and -5 during early stages of transcription (1-3). In the last several years, numerous protein kinases have been implicated in the phosphorylation of the CTD (4-8). This phosphorylation of the CTD enables RNAP II to progress from the initiation phase to a stable elongation complex, and the CTD remains extensively phosphorylated throughout the elongation phase of transcription (4-6). After completion of the transcription cycle, this same RNAP II must be in the unphosphorylated form (RNAP IIA) to be recruited back to the initiation complex (9). Therefore, dephosphorylation of the CTD by a phosphatase(s) is essential to generating a form of the polymerase (RNAP IIA) that is capable of reinitiating transcription.FCP1 [transcription factor IIF (TFIIF)-associating component of the CTD phosphatase], the only known RNAP II CTD-specific phosphatase, was originally partially purified from HeLa cell (10) and yeast (11) extracts. From experiments with this partially purified CTD phosphatase, it was determined that both general transcription factors IIB (TFIIB) and IIF (TFIIF) play important roles in regulating its activity (...

Glycosylation is a dynamic post-translational modification that changes during the development and progression of various malignancies. During the oncogenesis of breast carcinoma, the glycosyltransferase known as N-acetylglucosaminyltransferase Va (GnT-Va) transcript levels and activity are increased due to activated oncogenic signaling pathways. Elevated GnT-V levels leads to increased beta(1,6)-branched N-linked glycan structures on glycoproteins that can be measured using a specific carbohydrate binding protein or lectin known as L-PHA. L-PHA does not bind to nondiseased breast epithelial cells, but during the progression to invasive carcinoma, cells show a progressive increase in L-PHA binding. We have developed a procedure for intact protein L-PHA-affinity enrichment, followed by nanospray ionization mass spectrometry (NSI-MS/MS), to identify potential biomarkers for breast carcinoma. We identified L-PHA reactive glycoproteins from matched normal (nondiseased) and malignant tissue isolated from patients with invasive ductal breast carcinoma. Comparison analysis of the data identified 34 proteins that were enriched by L-PHA fractionation in tumor relative to normal tissue for at least 2 cases of ductal invasive breast carcinoma. Of these 34 L-PHA tumor enriched proteins, 12 are common to all 4 matched cases analyzed. These results indicate that lectin enrichment strategies targeting a particular glycan change associated with malignancy can be an effective method of identifying potential biomarkers for breast carcinoma.

Epithelial ovarian cancer is the deadliest female reproductive tract malignancy in Western countries. Less than 25% of cases are diagnosed when the cancer is confined, however, pointing to the critical need for early diagnostics for ovarian cancer. Identifying the changes that occur in the glycome of ovarian cancer cells may provide an avenue to develop a new generation of potential biomarkers for early detection of this disease. We performed a glycotranscriptomic analysis of endometrioid ovarian carcinoma using human tissue, as well as a newly developed mouse model that mimics this disease. Our results show that the N-linked glycans expressed in both non-diseased mouse and human ovarian tissues are similar; moreover, malignant changes in the expression of N-linked glycans in both mouse and human endometrioid ovarian carcinoma are qualitatively similar. Lectin reactivity was used as a means for rapid validation of glycan structural changes in the carcinomas that were predicted by the glycotranscriptome analysis. Among several changes in glycan expression noted, the increase of bisected N-linked glycans and the transcripts of the enzyme responsible for its biosynthesis, GnT-III, was the most significant. This study provides evidence that glycotranscriptome analysis can be an important tool in identifying potential cancer biomarkers.

O-Mannosyl-linked glycosylation is abundant within the central nervous system, yet very few glycoproteins with this glycan modification have been identified. Congenital diseases with significant neurological defects arise from inactivating mutations found within the glycosyltransferases that act early in the O-mannosyl glycosylation pathway. The N-acetylglucosaminyltransferase known as GnT-Vb or -IX is highly expressed in brain and branches O-mannosyl-linked glycans. Our results using SH-SY5Y neuroblastoma cells indicate that GnT-Vb activity promotes the addition of the O-mannosyl-linked HNK-1 modification found on the developmentally regulated and neuronspecific receptor protein-tyrosine phosphatase ␤ (RPTP␤). These changes in glycosylation accompany decreased cell-cell adhesion and increased rates of migration on laminin. In addition, we show that expression of GnT-Vb promotes its dimerization and inhibits RPTP␤ intrinsic phosphatase activity, resulting in higher levels of phosphorylated ␤-catenin, suggesting a mechanism by which GnT-Vb glycosylation couples to changes in cell adhesion. GnT-Vb-mediated glycosylation of RPTP␤ promotes galectin-1 binding and RPTP␤ levels of retention on the cell surface. N-Acetyllactosamine, but not sucrose, treatment of cells results in decreased RPTP retention, showing that galectin-1 binding contributes to the increased retention after GnT-Vb expression. These results place GnT-Vb as a regulator of RPTP␤ signaling that influences cell-cell and cell-matrix interactions in the developing nervous system. Glycosylation is regulated spatially and temporally during the development of the nervous system (1). In particular, sulfated glycoconjugates, such as the human natural killer-1 epitope, are important for proper migration and adhesion during neural development (2, 3). The human natural killer-1 (HNK-1) 3 epitope was originally discovered using a monoclonal antibody raised against a specific T-lymphoblastoid cell type (4). Since its discovery in lymphocytes, the HNK-1 (also known as CD57) antibody has been shown to react with many neural cell types, including glial, neuroectoderm, and neuroendocrine cells (5-8). The HNK-1 epitope consists of a glucuronic acid, transferred by glucuronyltransferases (GlcATs), that is 3-sulfated by HNK-1 sulfotransferase (HNK1st), linked to a precursor N-acetyllactosamine structure (Fig. 1). Numerous glycoproteins bearing the HNK-1 epitope have been identified in the nervous system, such as neural cell adhesion molecule (9), L1 (10), neural-glia cell adhesion molecule (11), myelinassociated glycoprotein (12), tenascin R (13), and RPTP (receptor protein-tyrosine phosphatase) (14, 15). In certain cell types, such as neural crest cells, the expression of the HNK-1 antigen is highly regulated during development (2) and is often found on migratory cells (3). The HNK-1 antigen is linked to critical functions during the formation of the nervous system, such as cell-matrix interactions (16), cell-cell adhesion (17), memory, and synaptic plasticity (15,18,19...

Epithelial ovarian cancer is diagnosed less than 25% of the time when the cancer is confined to the ovary, leading to 5-year survival rates of less than 30%. Therefore, there is an urgent need for early diagnostics for ovarian cancer. Our study using glycotranscriptome comparative analysis of endometrioid ovarian cancer tissue and normal ovarian tissue led to the identification of distinct differences in the transcripts of a restricted set of glycosyltransferases involved in N-linked glycosylation. Utilizing lectins that bind to glycan structures predicted to show changes, we observed differences in lectin-bound glycoproteins consistent with some of the transcript differences. In this study, we have extended our observations by the use of selected lectins to perform a targeted glycoproteomic analysis of ovarian cancer and normal ovarian tissues. Our results have identified several glycoproteins that display tumor-specific glycosylation changes. We have verified these glycosylation changes on glycoproteins from tissue using immunoprecipitation followed by lectin blot detection. The glycoproteins that were verified were then analyzed further using existing microarray data obtained from benign ovarian adenomas, borderline ovarian adenocarcinomas, and malignant ovarian adenocarcinomas. The verified glycoproteins found to be expressed above control levels in the microarray data sets were then screened for tumor-specific glycan modifications in serum from ovarian cancer patients. Results obtained from two of these glycoprotein markers, periostin and thrombospondin, have confirmed that tumor-specific glycan changes can be used to distinguish ovarian cancer patient serum from normal serum.

Biomarkers capable of detecting and targeting epithelial ovarian cancer cells for diagnostics and therapeutics would be extremely valuable. Ovarian cancer is the deadliest reproductive malignancy among women in the U.S., killing over 14 000 women each year. Both the lack of presenting symptoms and high mortality rates illustrate the need for earlier diagnosis and improved treatment of this disease. The glycosyltransferase enzyme GnT-III encoded by the Mgat3 gene is responsible for the addition of GlcNAc (N-acetylglucosamine) to form bisecting N-linked glycan structures. GnT-III mRNA expression is amplified in ovarian cancer tissues compared with normal ovarian tissue. We use a lectin capture strategy coupled to nano-ESI–RPLC–MS/MS to isolate and identify the membrane glycoproteins and unique glycan structures associated with GnT-III amplification in human ovarian cancer tissues. Our data illustrate that the majority of membrane glycoproteins with bisecting glycosylation are common to both serous and endometrioid histological subtypes of ovarian cancer, and several have been reported to participate in signaling pathways such as Notch, Wnt, and TGFβ.

Background: GPI-anchored proteins are elevated in breast carcinoma. Results: We utilized mass spectrometry and molecular biology techniques to capture and identify GPI-anchored proteins from breast carcinoma. Conclusion: Increased levels of GPI anchor addition contributes to the dedifferentiation of malignant breast epithelial cells. Significance: We have identified new potential diagnostic and therapeutic targets for breast carcinoma.