Dmitri Kapitonov

Constitutive EGFR signaling confers a motile phenotype to neural stem cells

The epidermal growth factor receptor (EGFR) has been shown to play an important role in brain development, including stem and precursor cell survival, proliferation, differentiation, and migration. To further examine the temporal and spatial requirements of erbB signals in uncommitted neural stem cells (NSCs), we expressed the ligand-independent EGF receptor, EGFRvIII, in C17.2 NSCs. These NSCs are known to migrate and to evince a tropic response to neurodegenerative environments in vivo but for which an underlying mechanism remains unclear. We show that enhanced erbB signaling via constitutive kinase activity of EGFRvIII in NSCs sustains an immature phenotype and enhances NSC migration.

Expression of gangliosides in neuronal development of P19 embryonal carcinoma stem cells

Gangliosides are constituents of the cell membrane and are known to have important functions in neuronal differentiation. We employed an embryonal carcinoma stem cell line P19 as an in vitro model to investigate the expression of gangliosides during neuronal development. After treatment with retinoic acid, these cells differentiate synchronously into neuron‐like cells by a series of well‐defined events of development. We examined several aspects of ganglioside metabolism, including the changes of ganglioside pattern, the activities and gene expression of several enzymes at different stages of differentiation, and the distribution of gangliosides in differentiating neurons. Undifferentiated P19 cells express mainly GM3 and GD3. After P19 cells were committed to differentiation, the synthesis of complex gangliosides was elevated more than 20‐fold, coinciding with the stage of neurite outgrowth. During the maturation of differentiated cells, the expression of c‐series gangliosides was downregulated concomitantly with upregulation of the expression of a‐ and b‐series gangliosides. We also examined the distribution of gangliosides in differentiating neurons by confocal and transmission electron microscopy after cholera toxin B subunit and sialidase treatment. Confocal microscopic studies showed that gangliosides were distributed on the growth cones and exhibited a punctate localization on neurites and soma. Electron microscopic studies indicated that they also are enriched on the plasma membranes of neurites and the filopodia as well as on the lamellipodia of growth cones during the early stage of neurite outgrowth. Our data demonstrate that the expression of gangliosides in P19 cells during RA‐induced neuronal differentiation resembles that of the in vivo development of the vertebrate brain, and hence validates it as an in vitro model for investigating the function of gangliosides in neuronal development. J. Neurosci. Res. 62:363–373, 2000.

Combinatorial PCR approach to homology-based cloning: cloning and expression of mouse and human GM3-synthase.

GM3-synthase, also known as sialyltransferase I (ST-I), catalyzes the transfer of a sialic acid residue from CMP-sialic acid onto lactosylceramide to form ganglioside GM3. In order to clone this enzyme, as well as other sialyltransferases, we developed an approach that we termed combinatorial PCR. In this approach, degenerate primers were designed on the basis of conserved sequence motifs of the ST3 family of sialyltransferases (STs). The nucleotide sequence of the primers was varied to cover all amino acid variations occurring in each motif. In addition, in some primers the sequence was varied to cover possible homologous substitutions that are absent in the available motifs. A panel of cDNA from 12 mouse and 8 human tissues was used to enable cloning of tissue- and stage-specific sialyltransferases. Using this approach, the fragments of 11 new putative sialyltransferases were isolated and sequenced so far. Analysis of the expression pattern of a particular sialyltransferase across the panel of cDNA from the different tissues provided information about the tissue specificity of ST expression. We chose two new ubiquitously expressed human and mouse STs to clone full-length copies and to assay for GM3-synthase activity. One of the STs, which exhibited the highest homology to ST3 Gal III, showed activity toward lactosylceramide (LacCer) and was termed ST3 Gal V according to the suggested nomenclature [1]. The other ubiquitously expressed sialyltransferase was termed ST3Gal VI. All isolated sialyltransferases were screened for alternatively spliced forms (ASF). Such forms were found for both human ST3Gal V and ST3Gal VI in human fetal brain cDNA library. The detailed cloning strategy, functional assay, and full length cDNA and protein sequences of GM3 synthase (ST3Gal V, or ST-I) are presented.

Effect of N-glycosylation on turnover and subcellular distribution of N- acetylgalactosaminyltransferase I and sialyltransferase II in neuroblastoma cells

Abstract: Gangliosides are sialylated glycosphingolipids whose biosynthesis is catalyzed by a series of endoplasmic reticulum (ER)‐ and Golgi‐resident glycosyltransferases. Protein expression, processing, and subcellular localization of the key regulatory enzymes for ganglioside biosynthesis, sialyltransferase II (ST‐II) and N‐acetylgalactosaminyltransferase I (GalNAcT), were analyzed upon transient expression of the two enzymes in the neuroblastoma cell lines NG108‐15 and F‐11. The enzymes were endowed with a C‐terminal epitope tag peptide (FLAG) for immunostaining and immunoaffinity purification using a FLAG‐specific antibody. Mature ST‐II‐FLAG and GalNAcT‐FLAG were expressed as N‐glycoproteins with noncomplex oligosaccharides. ST‐II‐FLAG was distributed to the Golgi apparatus, whereas GalNAcT‐FLAG was found in the ER and Golgi. Inhibition of early N‐glycoprotein processing with castanospermine resulted in a distribution of ST‐II‐FLAG to the ER, whereas that of GalNAcT‐FLAG remained unaltered. In contrast to GalNAcT, the activity of ST‐II and the amount of immunostained enzyme were reduced concomitantly by 75% upon incubation with castanospermine. This was due to a fourfold increased turnover of ST‐II‐FLAG, which was not found with GalNAcT‐FLAG. The ER retention and increased turnover of ST‐II‐FLAG were most likely due to its inability to bind to calnexin upon inhibition of early N‐glycoprotein processing. Calnexin binding was not observed for GalNAcT‐FLAG, indicating a differential effect of N‐glycosylation on the turnover and subcellular localization of the two glycosyltransferases.

Transcriptional regulation of the human UDP-galactose:ceramide galactosyltransferase (hCGT) gene expression: Functional role of GC-box and CRE

UDP-galactose:ceramide galactosyltransferase (CGT, EC 2.4.1.45) is a key enzyme in the biosynthetic pathway of galactocerebroside (GalC), the most abundant glycolipid in myelin. Using a GalC expressing cell line, human oligodendroglioma (HOG), one which does not express GalC, human neuroblastoma (LAN-5), we previously demonstrated that the human CGT (hCGT) gene promoter functions in a cell-specific manner. Because the proximal (−292/−256) and distal (−747/−688) positive domains were shown to be critically involved in regulating the expression of several myelin-specific genes, we further investigated the functional roles of these two motifs in hCGT expression. Mutation analysis confirmed that a GC-box (−267/−259) and a CRE (−697/−690) were critical for hCGT expression. Electrophoretic mobility shift assay (EMSA) demonstrated that these motifs specifically bound to nuclear extracts from both cell lines. Using antibodies to Sp1, Sp3, pCREB-1, and ATF-1, these proteins were shown to be components of the EMSA complexes. However, the only difference between the HOG and LAN-5 cells was found in the EMSA profile of the CRE complexes. This difference may account for the differential transcription of the hCGT gene in the two cell types. Furthermore, the expression levels of ATF-1 detected were much higher in HOG cells than in LAN-5 cells. Thus, our data suggest that the GC-box and CRE function cooperatively, and that the CRE regulates the cell-specific expression of the hCGT gene. Published in 2004.

Transcriptional Regulation of Signal Regulatory Protein α1 Inhibitory Receptors by Epidermal Growth Factor Receptor Signaling

Signal regulatory protein (SIRP) α1 is a membrane glycoprotein and a member of the SIRP receptor family. These transmembrane receptors have been shown to exert negative effects on signal transduction by receptor tyrosine kinases via immunoreceptor tyrosine-based inhibitory motifs in the carboxyl domain. Previous work has demonstrated that SIRPs negatively regulate many signaling pathways leading to reduction in tumor migration, survival, and cell transformation. Thus, modulation of SIRP expression levels or activity could be of great significance in the field of cancer therapy. The aim of the present study was to determine the factors that regulate levels of SIRPα1 in human glioblastoma cells that frequently overexpress the epidermal growth factor receptor (EGFR) because SIRPs have been shown to negatively regulate EGFR signaling. Northern blot analysis and immunoprecipitation assays showed variable expression levels of endogenous SIRPα transcripts in nine well-characterized glioblastoma cell lines. We examined SIRPα1 regulation in U87MG and U373MG cells in comparison with clonal derivatives that express a truncated form of erbB2, which negatively regulates EGFR signaling by inducing the formation of nonfunctional heterodimeric complexes. Mutant erbB2-expressing cells contained more SIRPα1 mRNA when compared with the parental cells in presence or absence of serum. Similarly, immunoprecipitation assays showed increased SIRPα1 protein levels in erbB-inhibited cells when compared with parental cells. Messenger RNA stability assays revealed that the increased mRNA levels in EGFR-inhibited cells were due to an induction of transcription. Consistent with this finding, expression of the erbB2 mutant receptor up-regulated SIRPα1 promoter activity in all cell lines tested. Interestingly, pharmacological inhibition of the kinase activities of EGFR, erbB2, and src and activation of mitogen-activated protein kinase, but not phosphatidylinositol 3′-kinase, significantly up-regulated SIRPα1 promoter activity. Based on these observations, we hypothesize that down-modulation of EGFR signaling leads to transcriptional up-regulation of the inhibitory SIRPα1 gene. These data may be important in the application of erbB-inhibitory strategies and for design of therapies for the treatment of glial tumors and other epithelial malignancies.