Immunohistochemical localization of beta-1,4-galactosyltransferase in human pancreatic tissues.

beta-1,4-Galactosyltransferase (GalTase) is the glycosyltransferase in the Golgi apparatus that transfers galactose from UDP-galactose to terminal N-acetylglucosamine residues in glycoconjugates with formation of a beta-1,4 linkage. Neoplasms undergo various changes in the carbohydrate moieties of their glycoconjugates. This process also indicates the possibility of changes in glycosyltransferases themselves. Therefore, we compared the binding pattern of a monoclonal antibody (MAb8628) against GalTase in both normal and neoplastic exocrine pancreatic tissues. Ten normal and 11 neoplastic human exocrine pancreatic tissues obtained from surgery were used. Frozen sections were incubated with this antibody. Supranuclear regions and terminal bars of normal duct cells and acinar cells revealed positive staining for GalTase at the light microscopic level. Centroacinar cells revealed positive staining in their perinuclear region. Neoplastic cells were also stained in their supranuclear regions and terminal bars. Supranuclear regions were well developed in neoplastic cells and intensely stained compared with those in normal cells. The supranuclear regions and the terminal bars corresponded to the trans cisternae of the Golgi apparatus and the junctional complex (i.e., tight junction and adherens junction), respectively, seen at the electron microscopic level. Pancreatic neoplastic changes thus led to an increase in the expression of GalTase in the Golgi apparatus, the increase of which may have an important effect on the intercellular adhesion and communication among pancreatic epithelial cells. Measurement of this enzyme is useful for diagnosis of exocrine pancreatic neoplastic changes from normal tissues.     

Molecular analysis of cell surface beta-1,4-galactosyltransferase function during cell migration.

Despite the identification and characterization of cell surface receptors for the extracellular matrix, it is unknown how their relative expression and cytoskeletal association regulate cell migration. Previous studies have identified beta-1,4-galactosyltransferase (GalTase; EC 2.4.1.38) on the surface of migrating cells, where it mediates cell migration on basal lamina matrices by associating with the cytoskeleton and binding to N-linked oligosaccharides in the E8 domain of laminin. In this study, the function of GalTase during cell migration was examined directly by analyzing the migration rate of stably transfected cell lines in which the relative level of surface GalTase and its ability to associate with the cytoskeleton were altered. We show here that the cytoskeleton contains a limiting, saturable, number of binding sites for surface GalTase. Furthermore, the rate of cell migration was inversely related to the ability of surface GalTase to associate with the cytoskeleton. Elevating surface GalTase in excess of the number of cytoskeleton-binding sites reduced the rate of cell migration, whereas decreasing the amount of surface GalTase available to bind the cytoskeleton increased migration rates. These results show that the rate of cell migration on basal lamina is directly dependent upon the expression of surface GalTase and the ability of this protein to associate with a limiting number of cytoskeleton-binding sites.     

人尿激酶原cDNA在牛内皮细胞中的表达

本实验将人的尿激酶原（Ｐｒｏ－ＵＫ）ｃＤＮＡ导人体外培养的胎牛主动脉内皮细胞（ＥＣ），得到了表达该基因的转化细胞，以期覆盖血管内支架或小口径人工血管（＜４ｍｍ）等移植物达到防止血栓形成，提高移植物通畅率的目的。利用磷酸钙盐沉淀法将重组逆转录病毒载体ｐＮ２－ＣＭＶ·ＵＫ导人包装细胞ＰＡ３１７，获得了含有重组逆转录病毒（Ｐｒｏ－ＵＫＲＮＡ序列）的培养上滑。用机械刮取胎牛主动脉内腔面分离ＥＣ，进行常规培养。用重组逆转录病毒感染７代以内的牛ＥＣ，并经Ｇ４ｌ８筛选得到抗性细胞。Ｓｏｕｔｈｅｒｎｂｌｏｔ分析表明ＰｒｏＵＫｃＤＮＡ已整合进ＥＣ基因组。以鼠抗人的Ｐｒｏ－ＵＫ单克隆抗体为一抗，作细胞免疫组化分析（Ｌ５Ａ８法），抗性细胞胞浆中出现阳性棕色颗粒，对照细胞为阴性结果。溶圈实验测得尿激酶原分泌量约为２３Ｕ／１０６细胞／２４小时。上述结果证明人Ｐｒｏ－ＵＫｃＤＮＡ已整合入牛ＥＣ基因组，表达产物具有免疫活性和纤溶酶原激活作用。     

An antibody produced in tobacco expressing a hybrid beta-1,4-galactosyltransferase is essentially devoid of plant carbohydrate epitopes

N-glycosylation of a mAb may have a major impact on its therapeutic merits. Here, we demonstrate that expression of a hybrid enzyme (called xylGalT), consisting of the N-terminal domain of Arabidopsis thaliana xylosyltransferase and the catalytic domain of human beta-1,4-galactosyltransferase I (GalT), in tobacco causes a sharp reduction of N-glycans with potentially immunogenic core-bound xylose (Xyl) and fucose (Fuc) residues as shown by Western blot and MALDI-TOF MS analysis. A radioallergosorbent test inhibition assay with proteins purified from leaves of WT and these transgenic tobacco plants using sera from allergic patients suggests a significant reduction of potential immunogenicity of xylGalT proteins. A mAb purified from leaves of plants expressing xylGalT displayed an N-glycan profile that featured high levels of galactose, undetectable xylose, and a trace of fucose. Hence, a transgenic plant expressing the hybrid GalT might yield more effective and safer monoclonals for therapeutic purposes than WT plants and even transgenic plants expressing the unchanged GalT.     

Stable expression of human beta1,4-galactosyltransferase in plant cells modifies N-linked glycosylation patterns

beta1,4-Galactosyltransferase (UDP galactose: beta-N-acetylglucosaminide: beta1,4-galactosyltransferase; EC 2.4.1. 22) catalyzes the transfer of galactose from UDP-Gal to N-acetylglucosamine in the penultimate stages of the terminal glycosylation of N-linked complex oligosaccharides in mammalian cells. Tobacco BY2 cells lack this Golgi enzyme. To determine to what extent the production of a mammalian glycosyltransferase can alter the glycosylation pathway of plant cells, tobacco BY2 suspension-cultured cells were stably transformed with the full-length human galactosyltransferase gene placed under the control of the cauliflower mosaic virus 35S promoter. The expression was confirmed by assaying enzymatic activity as well as by Southern and Western blotting. The transformant with the highest level of enzymatic activity has glycans with galactose residues at the terminal nonreducing ends, indicating the successful modification of the plant cell N-glycosylation pathway. Analysis of the oligosaccharide structures shows that the galactosylated N-glycans account for 47.3% of the total sugar chains. In addition, the absence of the dominant xylosidated- and fucosylated-type sugar chains confirms that the transformed cells can be used to produce glycoproteins without the highly immunogenic glycans typically found in plants. These results demonstrate the synthesis in plants of N-linked glycans with modified and defined sugar chain structures similar to mammalian glycoproteins.     

Localization of the long form of beta-1,4-galactosyltransferase to the plasma membrane and Golgi complex of 3T3 and F9 cells by immunofluorescence confocal microscopy.

beta-1,4-Galactosyltransferase (GalTase) is localized to two subcellular compartments, the Golgi complex, where it participates in cellular glycosylation, and the plasma membrane, where it functions as a receptor for oligosaccharide ligands on opposing cells or in the extracellular matrix. The gene for GalTase encodes two nearly identical proteins that differ only in their N-terminal cytoplasmic domains: both short and long GalTases share an 11-aa cytoplasmic tail, but long GalTase has an additional 13-aa sequence on its cytoplasmic domain. In this study, we investigated the subcellular distribution of endogenous long GalTase in untransfected F9 and 3T3 cells by using confocal microscopy and antibodies specific for the 13-aa sequence unique to long GalTase. Long GalTase was found in the Golgi complex as expected; long GalTase was also found on the plasma membrane in cell-type-specific distributions. In 3T3 cells, long GalTase was evident on the basal surface of cells possessing a migratory phenotype, being concentrated at the leading and trailing edges; nonmigratory cells had little detectable surface immunoreactivity. In F9 cells, long GalTase was localized on the plasma membrane, being concentrated at the apical aspect of intercellular junctions. These results demonstrate that in 3T3 and F9 cells, long GalTase is present on the cell surface in addition to the Golgi complex. The pattern of surface expression shows cell-type specificity that is consistent with GalTase function in cellular interactions.     

Pendrin does not increase sulfate uptake in mammalian COS-7 cells

Pendred's syndrome is characterized by goiter, sensorineural deafness and impaired iodide organification. It is one of the most frequent causes of congenital deafness accounting for about 10% of hereditary hearing loss. It is caused by mutations in the pendrin (PDS) gene, which was postulated to be a sulfate transporter, because of its homology with other genes. We tested sulfate transport in mammalian COS-7 cells that were transiently transfected with PDS cDNA. 35SO4 uptake increased in a time-dependent manner, but this phenomenon was similar in cells transfected with PDS and in mock-transfected cells (450 and 360 cpm/beta-gal units at 10 min, respectively; 38,250 and 31,000 cpm/beta-gal units, at 12 h, respectively). There was no significant increase in 35SO4 uptake using increasing amounts of PDS-containing plasmid (up to 12 microg per dish). These data indicate that pendrin is not a sulfate transporter. Additional functional studies on this protein are warranted to clarify its role in thyroid pathophysiology and inner ear development.     

Replication of a plasmid bearing a human Alu-family repeat in monkey COS-7 cells.

Monkey COS-7 cells were transformed with BLUR8 DNA, a pBR322 plasmid containing a human Alu-family sequence at the BamHI site. Within 24 hr of transformation 2-5% of the BLUR8 molecules recovered resisted cleavage by Dpn I, indicating they had replicated. Electron microscopy revealed appropriately sized circular molecules with replication bubbles whose centers were mapped to the Alu insert. A 16-base-pair deletion within the Alu sequence prevented replication. The results indicate that certain Alu sequences can serve as origins of replication in COS-7 cells.     

Isolation of a cDNA encoding a murine UDPgalactose:beta-D-galactosyl- 1,4-N-acetyl-D-glucosaminide alpha-1,3-galactosyltransferase: expression cloning by gene transfer.

We have developed a genetic approach to isolate cloned cDNA sequences that determine expression of cell surface oligosaccharide structures and their cognate glycosyltransferases. A cDNA library was constructed in a mammalian expression vector by using mRNA from a murine cell line known to express a UDPgalactose:beta-D-galactosyl-1,4-N-acetyl-D-glucosaminide alpha-1,3-galactosyltransferase [(alpha 1-3)GT; EC 2.4.1.151]. This library was transfected into COS-1 cells, which lack expression of (alpha 1-3)GT. Transfected cells containing functional (alpha 1-3)GT cDNAs were detected and isolated with a lectin that recognizes the surface-expressed glycoconjugate product of the (alpha 1-3)GT enzyme. One cloned (alpha 1-3)GT cDNA was rescued from lectin-positive transfected cells. This cDNA contains a single long open reading frame that predicts a 394-amino-acid protein. No significant primary structure similarities were identified between this protein and other known sequences. However, the protein predicts a type II transmembrane topology similar to two other mammalian glycosyltransferases. This topology places the large COOH-terminal domain within the Golgi lumen; this domain was shown to be catalytically active when expressed in COS-1 cells as a portion of a secreted protein A fusion peptide. Biochemical analysis confirmed that this enzyme catalyzes a transglycosylation reaction between UDP-Gal and Gal(beta 1-4)GlcNAc to form Gal(alpha 1-3)Gal(beta 1-4)GlcNAc. This cloning approach may be generally applicable to the isolation of cDNAs encoding other mammalian glycosyltransferases.     

人瘦素的基因克隆及其在COS-7细胞中的表达

目的 构建重组人瘦素哺乳细胞表达载体并在COS-7细胞表达重组人瘦素。方法 提取脂肪细胞总RNA，用RT-PCR扩增人瘦素cDNA并克隆至载体pUCm-T，并对克隆基因进行DNA序列分析。以克隆的人瘦素cDNA为模板，用特异引物扩增瘦素基因，经KpnI和BamH I酶切，插入相应酶切的哺乳细胞表达载体pcDNA3，构建重组哺乳细胞表达载体并转染COS-7细胞，RT-PCR和Western印迹检测其在COS-7细胞中的表达。结果 RT-PCR扩增的DNA片断和预期的人瘦素cDNA大小一致；序列分析显示，克隆的基因序列和文献报道的人瘦素基因序列一致；经RT-PCR和Western印迹鉴定，转染的COS-7细胞可表达、分泌人瘦素。结论 构建了人瘦素的哺乳动物细胞表达载体，并成功地在COS-7细胞中获得重组人瘦素的分泌表达。     

人破骨细胞分化因子基因的克隆及在COS-7细胞中的表达

目的 克隆人破骨细胞分化因子(ODF)基因并在真核细胞中表达。方法 以人骨肉瘤细胞系MG63的总RNA为模板，采用反转录-聚合酶链反应(RT-PCR)法得到ODF的编码区cDNA，构建真核表达载体pcDNA3．1( )-ODF，在脂质体介导下转染非洲绿猴肾细胞系(COS)-7，经G418压力筛选建立稳定转染人ODF的细胞系，Western印迹检测其在COS7细胞中的表达。结果获得的人ODFcDNA序列与文献报道的核苷酸序列一致，Western印迹证实稳定转染人ODF的COS7细胞系中有ODF的表达。结论 构建了人破骨细胞分化因子(ODF)的真核表达载体，并在COS7细胞中获得稳定表达。     

Expression cloning of a cDNA encoding the bovine histamine H1 receptor.

A functional cDNA clone for the histamine H1 receptor was isolated from a cDNA library of bovine adrenal medulla by a combination of molecular cloning in an expression vector and electrophysiological assay in Xenopus oocytes. The H1 receptor cDNA encodes a protein of 491 amino acids (Mr 55,954) with seven putative transmembrane domains, illustrating the similarity to other receptors that couple with guanine nucleotide-binding regulatory proteins (G protein-coupled receptors). The sequence homology between the H1 and H2 receptors is not higher than that between the histamine H1 and m1-muscarinic receptors. The cloned receptor protein expressed in COS-7 cells bound specifically to [3H]mepyramine, an H1 receptor antagonist, and this binding was displaced by H1 receptor antagonists and histamine with affinities comparable with those in membranes of bovine adrenal medulla. H1 receptor mRNA was shown to be expressed in brain and in peripheral tissues, including lung, small intestine, and adrenal medulla. This investigation discloses the molecular nature of the H1 receptor--a receptor that mediates diverse neuronal and peripheral actions of histamine and that may be of therapeutic importance in allergy.     

Expression of 17beta- and 3beta-hydroxysteroid dehydrogenases and steroidogenic acute regulatory protein in non-luteinizing bovine granulosa cells in vitro

Granulosa cells of small follicles differentiate in vitro in serum-free medium, resulting in increased estradiol secretion and abundance of mRNA encoding cytochrome P450aromatase (P450arom). We tested the hypothesis that differentiation in vitro also involves increased expression of 3beta- and 17beta-hydroxysteroid dehydrogenases (HSD) in the absence of steroidogenic acute regulatory protein (StAR) expression, as has been observed in vivo. Granulosa cells from small (<6 mm diameter) follicles were cultured for up to 6 days, and mRNA levels quantified by Northern hybridization or RT-PCR. Estradiol and progesterone concentrations in medium increased with time in culture, as did mRNA encoding P450arom, 3beta- and 17beta-HSD but not P450scc. Both P450arom and 17beta-HSD were significantly correlated with estradiol accumulation in culture medium. Progesterone secretion was correlated with 3beta-HSD but not P450scc mRNA levels. StAR mRNA was detectable by RT-PCR, did not change with duration of culture and was not correlated with progesterone secretion. FSH significantly stimulated P450arom and 17beta-HSD mRNA levels. Cell origin (from the antral or the basal layer of the membrana granulosa) did not affect steroidogenesis. We conclude that under the present cell culture system granulosa cells do not luteinize, and show expression of key steroidogenic enzymes in patterns similar to those occurring in differentiating follicles in vivo. Further, the data suggest that 17beta-HSD may be as important as P450arom in regulating estradiol secretion, and that 3beta-HSD is more important than P450scc as a regulator of progesterone secretion in non-luteinizing granulosa cells.     

Assembly of pericellular matrices by COS-7 cells transfected with CD44 lymphocyte-homing receptor genes.

The capacity to assemble and retain a pericellular matrix is correlated with the expression of the cell surface binding sites specific for the extracellular matrix macromolecule hyaluronan. These binding proteins have been termed hyaluronan receptors. The lymphocyte-homing receptor CD44 may have identity with these hyaluronan receptors. To determine whether hyaluronan receptors function independently in this capacity for matrix assembly, mammalian cells were transfected with cDNA encoding the putative hyaluronan receptor CD44. After transfection with CD44 cDNA, COS cells gained the capacity to assemble hyaluronan-dependent pericellular matrices in the presence of exogenously added hyaluronan and proteoglycan. Thus, CD44 receptors do function as matrix-organizing, matrix-anchoring hyaluronan-binding proteins. In addition, the expression of CD44/hyaluronan receptors alone is sufficient to direct this matrix assembly. If matrix assembly is a function of cells in vivo that express hyaluronan receptors, this raises interesting possibilities for the role of the receptors in cell migration, when new extracellular matrix environments are encountered.     

Expression of bovine vitamin K-dependent carboxylase activity in baculovirus-infected insect cells.

A vitamin K-dependent carboxylase has recently been purified from bovine liver microsomes and candidate cDNA clones have been isolated. Definitive identification of the carboxylase remains circumstantial since expression of candidate carboxylase cDNAs in mammalian cells is confounded by the presence of endogenous carboxylase activity. To overcome this problem, a recombinant strain of baculovirus (Autographa california nuclear polyhedrosis virus, AcMNPV) encoding a putative carboxylase (vbCbx/AcMNPV) was used to infect Sf9 insect cells, which we demonstrate have no endogenous carboxylase activity. Infection with vbCbx/AcMNPV conferred vitamin K-dependent carboxylase activity to Sf9 insect cells. Carboxylase activity was demonstrated to peak 2-3 days after infection with vbCbx/AcMNPV. Metabolic radiolabeling with L-[35S]methionine revealed that the 90-kDa recombinant protein is the major protein synthesized at the time of peak activity after infection. An anti-peptide antibody directed against residues 86-99 reacted with bovine liver carboxylase on Western blot analysis and immunoprecipitated recombinant carboxylase from infected Sf9 microsomal protein preparations. Since Sf9 insect cells lack endogenous vitamin K-dependent carboxylase activity, expression of carboxylase activity in Sf9 insect cells with recombinant baculovirus demonstrates that the protein encoded by this cDNA is a vitamin K-dependent gamma-glutamyl carboxylase.     

Expression of a synthetic bovine rhodopsin gene in monkey kidney cells.

We report here the high-level expression of a synthetic gene for bovine rhodopsin in transfected monkey kidney COS-1 cells. Rhodopsin is produced in these cells to a level of 0.3% of the cell protein, and it binds exogenously added 11-cis-retinal to generate the characteristic rhodopsin absorption spectrum. We describe a one-step immunoaffinity procedure for purification of the rhodopsin essentially to homogeneity. The COS-1 cell rhodopsin activates the GTPase activity of bovine transducin in a light-dependent manner with the same specific activity as that of purified bovine rhodopsin. Electron microscopy of immunogold-stained cells indicates that rhodopsin is located in the plasma membrane of the transfected cells and is oriented with the amino terminus on the extracellular side of the membrane. This orientation is analogous to that of rhodopsin in the disk membranes of photoreceptor cells in the bovine retina.