Expression cloning of cDNA encoding a human β-1,3-N-acetylglucosaminyltransferase that is essential for poly-N-acetyllactosamine synthesis

The structure and biosynthesis of poly-N-acetyllactosamine display a dramatic change during development and oncogenesis. Poly-N-acetyllactosamines are also modified by various carbohydrate residues, forming functional oligosaccharides such as sialyl Lex. Herein we describe the isolation and functional expression of a cDNA encoding β-1,3-N-acetylglucosaminyltransferase (iGnT), an enzyme that is essential for the formation of poly-N-acetyllactosamine. For this expression cloning, Burkitt lymphoma Namalwa KJM-1 cells were transfected with cDNA libraries derived from human melanoma and colon carcinoma cells. Transfected Namalwa cells overexpressing the i antigen were continuously selected by fluorescence-activated cell sorting because introduced plasmids containing Epstein–Barr virus replication origin can be continuously amplified as episomes. Sibling selection of plasmids recovered after the third consecutive sorting resulted in a cDNA clone that directs the increased expression of i antigen on the cell surface. The deduced amino acid sequence indicates that this protein has a type II membrane protein topology found in almost all mammalian glycosyltransferases cloned to date. iGnT, however, differs in having the longest transmembrane domain among glycosyltransferases cloned so far. The iGnT transcript is highly expressed in fetal brain and kidney and adult brain but expressed ubiquitously in various adult tissues. The expression of the presumed catalytic domain as a fusion protein with the IgG binding domain of protein A enabled us to demonstrate that the cDNA encodes iGnT, the enzyme responsible for the formation of GlcNAcβ1 → 3Galβ1 → 4GlcNAc → R structure and poly-N-acetyllactosamine extension.

A Specific Light Chain of Kinesin Associates with Mitochondria in Cultured Cells

The motor protein kinesin is implicated in the intracellular transport of organelles along microtubules. Kinesin light chains (KLCs) have been suggested to mediate the selective binding of kinesin to its cargo. To test this hypothesis, we isolated KLC cDNA clones from a CHO-K1 expression library. Using sequence analysis, they were found to encode five distinct isoforms of KLCs. The primary region of variability lies at the carboxyl termini, which were identical or highly homologous to carboxyl-terminal regions of rat KLC B and C, human KLCs, sea urchin KLC isoforms 1–3, and squid KLCs. To examine whether the KLC isoforms associate with different cytoplasmic organelles, we made an antibody specific for a 10-amino acid sequence unique to B and C isoforms. In an indirect immunofluorescence assay, this antibody specifically labeled mitochondria in cultured CV-1 cells and human skin fibroblasts. On Western blots of total cell homogenates, it recognized a single KLC isoform, which copurified with mitochondria. Taken together, these data indicate a specific association of a particular KLC (B type) with mitochondria, revealing that different KLC isoforms can target kinesin to different cargoes.

Synthesis, Storage, and Release of Vascular Endothelial Growth Factor/Vascular Permeability Factor (VEGF/VPF) by Human Mast Cells: Implications for the Biological Significance of VEGF206

Mast cells have been implicated in various diseases that are accompanied by neovascularization. The exact mechanisms by which mast cells might mediate an angiogenic response, however, are unclear and therefore, we have investigated the possible expression of vascular endothelial growth factor/vascular permeability factor (VEGF/VPF) in the human mast cell line HMC-1 and in human skin mast cells. Reverse transcription-polymerase chain reaction (RT-PCR) analysis revealed that mast cells constitutively express VEGF121, VEGF165, and VEGF189. After a prolonged stimulation of cells for 24 h with phorbol 12-myristate 13-acetate (PMA) and the ionophore A23187, an additional transcript representing VEGF206 was detectable, as could be verified by sequence analysis. These results were confirmed at the protein level by Western blot analysis. When the amounts of VEGF released under unstimulated and stimulated conditions were compared, a significant increase was detectable after stimulation of cells. Human microvascular endothelial cells (HMVEC) responded to the supernatant of unstimulated HMC-1 cells with a dose-dependent mitogenic effect, neutralizable up to 90% in the presence of a VEGF-specific monoclonal antibody. Flow cytometry and postembedding immunoelectron microscopy were used to detect VEGF in its cell-associated form. VEGF was exclusively detectable in the secretory granules of isolated human skin mast cells. These results show that both normal and leukemic human mast cells constitutively express bioactive VEGF. Furthermore, this study contributes to the understanding of the physiological role of the strongly heparin-binding VEGF isoforms, since these were found for the first time to be expressed in an activation-dependent manner in HMC-1 cells.

Saccharomyces cerevisiae Cells with Defective Spindle Pole Body Outer Plaques Accomplish Nuclear Migration via Half-Bridge–organized Microtubules

Cnm67p, a novel yeast protein, localizes to the microtubule organizing center, the spindle pole body (SPB). Deletion of CNM67 (YNL225c) frequently results in spindle misorientation and impaired nuclear migration, leading to the generation of bi- and multinucleated cells (40%). Electron microscopy indicated that CNM67 is required for proper formation of the SPB outer plaque, a structure that nucleates cytoplasmic (astral) microtubules. Interestingly, cytoplasmic microtubules that are essential for spindle orientation and nuclear migration are still present in cnm67Δ1 cells that lack a detectable outer plaque. These microtubules are attached to the SPB half- bridge throughout the cell cycle. This interaction presumably allows for low-efficiency nuclear migration and thus provides a rescue mechanism in the absence of a functional outer plaque. Although CNM67 is not strictly required for mitosis, it is essential for sporulation. Time-lapse microscopy of cnm67Δ1 cells with green fluorescent protein (GFP)-labeled nuclei indicated that CNM67 is dispensable for nuclear migration (congression) and nuclear fusion during conjugation. This is in agreement with previous data, indicating that cytoplasmic microtubules are organized by the half-bridge during mating.

Signals transducers and activators of transcription (STAT)-induced STAT inhibitor-1 (SSI-1)/suppressor of cytokine signaling-1 (SOCS-1) suppresses tumor necrosis factor α-induced cell death in fibroblasts

Signal transducers and activators of transcription (STAT)-induced STAT inhibitor-1 [SSI-1; also known as suppressor of cytokine signaling-1 (SOCS-1)] was identified as a negative feedback regulator of Janus kinase-STAT signaling. We previously generated mice lacking the SSI-1 gene (SSI-1 −/−) and showed that thymocytes and splenocytes in SSI-1 −/− mice underwent accelerated apoptosis. In this paper, we show that murine embryonic fibroblasts lacking the SSI-1 gene are more sensitive than their littermate controls to tumor necrosis factor-α (TNF-α)-induced cell death. In addition, L929 cells forced to express SSI-1 (L929/SSI-1), but not SSI-3 or SOCS-5, are resistant to TNF-α-induced cell death. Furthermore L929/SSI-1 cells treated with TNF-α sustain the activation of p38 mitogen-activated protein (MAP) kinase. In contrast, SSI-1 −/− murine embryonic fibroblasts treated with TNF-α show hardly any activation of p38 MAP kinase. These findings suggest that SSI-1 suppresses TNF-α-induced cell death, which is mediated by p38 MAP kinase signaling.

Converting enzyme-independent release of tumor necrosis factor α and IL-1β from a stimulated human monocytic cell line in the presence of activated neutrophils or purified proteinase 3

Two important cytokines mediating inflammation are tumor necrosis factor α (TNFα) and IL-1β, both of which require conversion to soluble forms by converting enzymes. The importance of TNFα-converting enzyme and IL-1β-converting enzyme in the production of circulating TNFα and IL-1β in response to systemic challenges has been demonstrated by the use of specific converting enzyme inhibitors. Many inflammatory responses, however, are not systemic but instead are localized. In these situations release and/or activation of cytokines may be different from that seen in response to a systemic stimulus, particularly because associations of various cell populations in these foci allows for the exposure of procytokines to the proteolytic enzymes produced by activated neutrophils, neutrophil elastase (NE), proteinase 3 (PR3), and cathepsin G (Cat G). To investigate the possibility of alternative processing of TNFα and/or IL-1β by neutrophil-derived proteinases, immunoreactive TNFα and IL-1β release from lipopolysaccharide-stimulated THP-1 cells was measured in the presence of activated human neutrophils. Under these conditions, TNFα and IL-1β release was augmented 2- to 5-fold. In the presence of a specific inhibitor of NE and PR3, enhanced release of both cytokines was largely abolished; however, in the presence of a NE and Cat G selective inhibitor, secretory leucocyte proteinase inhibitor, reduction of the enhanced release was minimal. This finding suggested that the augmented release was attributable to PR3 but not NE nor Cat G. Use of purified enzymes confirmed this conclusion. These results indicate that there may be alternative pathways for the production of these two proinflammatory cytokines, particularly in the context of local inflammatory processes.

Cell Cycle-Dependent Changes in Microtubule Dynamics in Living Cells Expressing Green Fluorescent Protein-α TubulinV⃞

LLCPK-1 cells were transfected with a green fluorescent protein (GFP)-α tubulin construct and a cell line permanently expressing GFP-α tubulin was established (LLCPK-1α). The mitotic index and doubling time for LLCPK-1α were not significantly different from parental cells. Quantitative immunoblotting showed that 17% of the tubulin in LLCPK-1α cells was GFP-tubulin; the level of unlabeled tubulin was reduced to 82% of that in parental cells. The parameters of microtubule dynamic instability were compared for interphase LLCPK-1α and parental cells injected with rhodamine-labeled tubulin. Dynamic instability was very similar in the two cases, demonstrating that LLCPK-1α cells are a useful tool for analysis of microtubule dynamics throughout the cell cycle. Comparison of astral microtubule behavior in mitosis with microtubule behavior in interphase demonstrated that the frequency of catastrophe increased twofold and that the frequency of rescue decreased nearly fourfold in mitotic compared with interphase cells. The percentage of time that microtubules spent in an attenuated state, or pause, was also dramatically reduced, from 73.5% in interphase to 11.4% in mitosis. The rates of microtubule elongation and rapid shortening were not changed; overall dynamicity increased 3.6-fold in mitosis. Microtubule release from the centrosome and a subset of differentially stable astral microtubules were also observed. The results provide the first quantitative measurements of mitotic microtubule dynamics in mammalian cells.

Human tumor suppressor EXT gene family members EXTL1 and EXTL3 encode α1,4- N-acetylglucosaminyltransferases that likely are involved in heparan sulfate/ heparin biosynthesis

The tumor suppressors EXT1 and EXT2 are associated with hereditary multiple exostoses and encode bifunctional glycosyltransferases essential for chain polymerization of heparan sulfate (HS) and its analog, heparin (Hep). Three highly homologous EXT-like genes, EXTL1–EXTL3, have been cloned, and EXTL2 is an α1,4-GlcNAc transferase I, the key enzyme that initiates the HS/Hep synthesis. In the present study, truncated forms of EXTL1 and EXTL3, lacking the putative NH2-terminal transmembrane and cytoplasmic domains, were transiently expressed in COS-1 cells and found to harbor α-GlcNAc transferase activity. EXTL3 used not only N-acetylheparosan oligosaccharides that represent growing HS chains but also GlcAβ1–3Galβ1-O-C2H4NH-benzyloxycarbonyl (Cbz), a synthetic substrate for α-GlcNAc transferase I that determines and initiates HS/Hep synthesis. In contrast, EXTL1 used only the former acceptor. Neither EXTL1 nor EXTL3 showed any glucuronyltransferase activity as examined with N-acetylheparosan oligosaccharides. Heparitinase I digestion of each transferase-reaction product showed that GlcNAc had been transferred exclusively through an α1,4-configuration. Hence, EXTL3 most likely is involved in both chain initiation and elongation, whereas EXTL1 possibly is involved only in the chain elongation of HS and, maybe, Hep as well. Thus, their acceptor specificities of the five family members are overlapping but distinct from each other, except for EXT1 and EXT2 with the same specificity. It now has been clarified that all of the five cloned human EXT gene family proteins harbor glycosyltransferase activities, which probably contribute to the synthesis of HS and Hep.

Human blood-mobilized hematopoietic precursors differentiate into osteoclasts in the absence of stromal cells.

Osteoclastogenesis is a complex process that is facilitated by bone marrow stromal cells (SCs). To determine if SCs are an absolute requirement for the differentiation of human hematopoietic precursors into fully mature, osteoclasts (OCs), CD34+ cells were mobilized into the peripheral circulation with granulocyte colony-stimulating factor, harvested by leukapheresis, and purified by magnetic-activated cell sorting. This procedure yields a population of CD34+ cells that does not contain SC precursors, as assessed by the lack of expression of the SC antigen Stro-1, and that differentiates only into hematopoietic cells. We found that CD34+, Stro-1- cells cultured with a combination of granulocyte/macrophage colony-stimulating factor, interleukin 1, and interleukin 3 generated cells that fulfill current criteria for the characterization of OCs, including multinucleation, presence of tartrate-resistant acid phosphatase, and expression of the calcitonin and vitronectin receptors and of pp60c-src tyrosine kinase. These OCs also expressed mRNA for the noninserted isoform of the calcitonin receptor and excavated characteristic resorption pits in devitalized bone slices. These data demonstrate that accessory SCs are not essential for human osteoclastogenesis and that granulocyte colony-stimulating factor treatment mobilizes OC precursors into the peripheral circulation.

Expression of calbindin-D28K in motoneuron hybrid cells after retroviral infection with calbindin-D28K cDNA prevents amyotrophic lateral sclerosis IgG-mediated cytotoxicity.

Calbindin-D28K and/or parvalbumin appear to influence the selective vulnerability of motoneurons in amyotrophic lateral sclerosis (ALS). Their immunoreactivity is undetectable in motoneurons readily damaged in human ALS, and in differentiated motoneuron hybrid cells [ventral spinal cord (VSC 4.1 cells)] that undergo calcium-dependent apoptotic cell death in the presence of ALS immunoglobulins. To provide additional evidence for the role of calcium-binding proteins in motoneuron vulnerability, VSC 4.1 cells were infected with a retrovirus carrying calbindin-D28K cDNA under the control of the promoter of the phosphoglycerate kinase gene. Differentiated calbindin-D28K cDNA-infected cells expressed high calbindin-D28K and demonstrated increased resistance to ALS IgG-mediated toxicity. Treatment with calbindin-D28K antisense oligodeoxynucleotides, which significantly decreased calbindin-D28K expression, rendered these cells vulnerable again to ALS IgG toxicity.

Distinct pharmacological properties and distribution in neurons and endocrine cells of two isoforms of the human vesicular monoamine transporter.

A second isoform of the human vesicular monoamine transporter (hVMAT) has been cloned from a pheochromocytoma cDNA library. The contribution of the two transporter isoforms to monoamine storage in human neuroendocrine tissues was examined with isoform-specific polyclonal antibodies against hVMAT1 and hVMAT2. Central, peripheral, and enteric neurons express only VMAT2. VMAT1 is expressed exclusively in neuroendocrine, including chromaffin and enterochromaffin, cells. VMAT1 and VMAT2 are coexpressed in all chromaffin cells of the adrenal medulla. VMAT2 alone is expressed in histamine-storing enterochromaffin-like cells of the oxyntic mucosa of the stomach. The transport characteristics and pharmacology of each VMAT isoform have been directly compared after expression in digitonin-permeabilized fibroblastic (CV-1) cells, providing information about substrate feature recognition by each transporter and the role of vesicular monoamine storage in the mechanism of action of psychopharmacologic and neurotoxic agents in human. Serotonin has a similar affinity for both transporters. Catecholamines exhibit a 3-fold higher affinity, and histamine exhibits a 30-fold higher affinity, for VMAT2. Reserpine and ketanserin are slightly more potent inhibitors of VMAT2-mediated transport than of VMAT1-mediated transport, whereas tetrabenazine binds to and inhibits only VMAT2. N-methyl-4-phenylpyridinium, phenylethylamine, amphetamine, and methylenedioxymethamphetamine are all more potent inhibitors of VMAT2 than of VMAT1, whereas fenfluramine is a more potent inhibitor of VMAT1-mediated monamine transport than of VMAT2-mediated monoamine transport. The unique distributions of hVMAT1 and hVMAT2 provide new markers for multiple neuroendocrine lineages, and examination of their transport properties provides mechanistic insights into the pharmacology and physiology of amine storage in cardiovascular, endocrine, and central nervous system function.

A defect in glycosylphosphatidylinositol (GPI) transamidase activity in mutant K cells is responsible for their inability to display GPI surface proteins.

The final step in the pathway that provides for glycosylphosphatidylinositol (GPI) anchoring of cell-surface proteins occurs in the lumen of the endoplasmic reticulum and consists of a transamidation reaction in which fully assembled GPI anchor donors are substituted for specific COOH-terminal signal peptide sequences contained in nascent polypeptides. In previous studies we described a human K562 cell mutant line, designated class K, which assembles all the known intermediates of the GPI pathway but fails to display GPI-anchored proteins on its surface membrane. In the present study, we used mRNA encoding miniPLAP, a truncated form of placental alkaline phosphatase (PLAP), in in vitro assays with rough microsomal membranes (RM) of mutant K cells to further characterize the biosynthetic defect in this line. We found that RM from mutant K cells supported NH2-terminal processing of the nascent translational product, preprominiPLAP, but failed to show any detectable COOH-terminal processing of the resulting prominiPLAP to GPI-anchored miniPLAP. Proteinase K protection assays verified that NH2-terminal processed prominiPLAP was appropriately translocated into the endoplasmic reticulum lumen. The addition of hydrazine or hydroxylamine, which can substitute for GPI donors, to RM from wild-type or mutant cells defective in various intermediate biosynthetic steps in the GPI pathway produced large amounts of the hydrazide or hydroxamate of miniPLAP. In contrast, the addition of these nucleophiles to RM of class K cells yielded neither of these products. These data, taken together, lead us to conclude that mutant K cells are defective in part of the GPI transamidase machinery.

Localization, trafficking, and temperature-dependence of the Aequorea green fluorescent protein in cultured vertebrate cells.

The localization, trafficking, and fluorescence of Aequorea green fluorescent protein (GFP) in cultured vertebrate cells transiently transfected with GFP cDNA were studied. Fluorescence of GFP in UV light was found to be strongest when cells were incubated at 30 degrees C but was barely visible at an incubation temperature of 37 degrees C. COS-1 cells, primary chicken embryonic retina cells, and carp epithelial cells were fluorescently labeled under these conditions. GFP was distributed uniformly throughout the cytoplasm and nucleus independent of cell type examined. When GFP was fused to PML protooncogene product, fluorescence was detected in a unique nuclear organelle pattern indistinguishable from that of PML protein, showing the potential use of GFP as a fluorescent tag. To analyze both function and intracellular trafficking of proteins fused to GFP, a GFP-human glucocorticoid receptor fusion construct was prepared. The GFP-human glucocorticoid receptor efficiently transactivated the mouse mammary tumor virus promoter in response to dexamethasone at 30 degrees C but not at 37 degrees C, indicating that temperature is important, even for function of the GFP fusion protein. The dexamethasone-induced translocation of GFP-human glucocorticoid receptor from cytoplasm to nucleus was complete within 15 min; the translocation could be monitored in a single living cell in real time.

ETS1 suppresses tumorigenicity of human colon cancer cells.

We have ectopically expressed transcription factor ETS1 in two different highly tumorigenic human colon cancer cell lines, DLD-1 and HCT116, that do not express endogenous ETS1 protein and have obtained several independent clones. The expression of wild-type ETS1 protein in these colon cancer cells reverses the transformed phenotype and tumorigenicity in a dose-dependent manner. By contrast, expression in DLD-1 cells of a variant form of ETS1, lacking transcriptional activity, did not alter the tumorigenic properties of the cells, suggesting that the reduction in tumorigenicity in these clones was specific for the wild-type ETS1 gene products. Since these colon cancer cells have multiple genetic alterations, the system described in this paper could be a good model to study the suppression of tumorigenicity at a transcriptional level, which could lead to the design and development of novel drugs for cancer treatment.

MLN64 contains a domain with homology to the steroidogenic acute regulatory protein (StAR) that stimulates steroidogenesis

MLN64 is a protein that is highly expressed in certain breast carcinomas. The C terminus of MLN64 shares significant homology with the steroidogenic acute regulatory protein (StAR), which plays a key role in steroid hormone biosynthesis by enhancing the intramitochondrial translocation of cholesterol to the cholesterol side-chain cleavage enzyme. We tested the ability of MLN64 to stimulate steroidogenesis by using COS-1 cells cotransfected with plasmids expressing the human cholesterol side-chain cleavage enzyme system and wild-type and mutant MLN64 proteins. Wild-type MLN64 increased pregnenolone secretion in this system 2-fold. The steroidogenic activity of MLN64 was found to reside in the C terminus of the protein, because constructs from which the C-terminal StAR homology domain was deleted had no steroidogenic activity. In contrast, removal of N-terminal sequences increased MLN64’s steroidogenesis-enhancing activity. MLN64 mRNA was found in many human tissues, including the placenta and brain, which synthesize steroid hormones but do not express StAR. Western blot analysis revealed the presence of lower molecular weight immunoreactive MLN64 species that contain the C-terminal sequences in human tissues. Homologs of both MLN64 and StAR were identified in Caenorhabditis elegans, indicating that the two proteins are ancient. Mutations that inactivate StAR were correlated with amino acid residues that are identical or similar among StAR and MLN64, indicating that conserved motifs are important for steroidogenic activity. We conclude that MLN64 stimulates steroidogenesis by virtue of its homology to StAR.