Involvement of beta-1,4-galactosyltransferase V in malignant transformation-associated changes in glycosylation

In spite of marked changes in the glycosylation upon malignant transformation of cells, no biological significance of beta-1, 4-galactosyltransferase (beta-1,4-GalT) activities has been elucidated. When beta-1,4-GalT activities toward 1 mM GlcNAcbeta-S-pNP were determined using homogenates of NIH3T3 and its transformant, MTAg, MTAg contained 1.3 times higher activities. Northern blot analysis, however, revealed that the beta-1,4-GalT V gene expression increases by three times with a decrease in that of beta-1,4-GalT II by one-fifth and without significant changes in those of other beta-1,4-GalTs in MTAg. Analysis of beta-1,4-GalT V acceptor-specificity showed that the GlcNAcbeta1-->6Man group of the GlcNAcbeta1-->6(GlcNAbeta1-->2)Manalpha1- branch is galactosylated. These results indicate that changes in beta-1,4-GalT II and V activities are important for the altered glycosylation.     

Correlated gene expression between beta-1,4-galactosyltransferase V and N-acetylglucosaminyltransferase V in human cancer cell lines

Since our previous study showed that the gene expression level of beta-1,4-galactosyltransferase (beta-1,4-GalT) V is only increased in mouse NIH3T3 transformant and that beta-1,4-GalT V preferentially galactosylates the GlcNAcbeta1 --> 6Man branch of oligosaccharides [Shirane et al. (1999) Biochem. Biophys. Res. Commun. 265, 434-438], whether its gene expression is correlated with malignant transformation was investigated. Northern blot analysis of beta-1, 4-GalTs I, II, III, IV, V, and VI and N-acetylglucosaminyltransferase (GlcNAcT)V in human cancer cell lines showed that the gene expression levels of beta-1,4-GalT V but not other beta-1,4-GalTs are strongly correlated with those of GlcNAcT V whose activity was shown to increase by malignant transformation. These results indicate that beta-1,4-GalT V is involved in the galactosylation of highly branched oligosaccharides characteristic of malignantly transformed cells.     

Galactosylation of N-linked oligosaccharides by human beta-1,4-galactosyltransferases I, II, III, IV, V, and VI expressed in Sf-9 cells

Several studies showed that Sf-9 cells can synthesize the galactosylated N-linked oligosaccharides if beta-1,4-galactosyltransferase (beta-1,4-GalT) is supplied. The full-length human beta-1,4-GalT I, II, III, IV, V, and VI cDNAs were independently transfected into Sf-9 cells, and the galactosylation of endogenous membrane glycoproteins was examined by lectin blot analysis using Ricinus communis agglutinin-I (RCA-I), which preferentially interacts with oligosaccharides terminated with Galbeta1-->4GlcNAc group. Several RCA-I-reactive bands appeared in all of the gene-transfected cells, and disappeared on treatment of blots with beta-1,4-galactosidase or N-glycanase prior to incubation with lectin. Introduction of the antisense beta-1,4-GalT II and V cDNAs separately into human colorectal adenocarcinoma SW480 cells, in which beta-1,4-GalT I, II, and V genes were expressed, resulted in the reduction of RCA-I binding toward N-linked oligosaccharides of the membrane glycoproteins. Differences were found in their K(m) values toward UDP-Gal and GlcNAcbeta-S-pNP and in their acceptor specificities toward oligosaccharides with the GlcNAcbeta1-->4(GlcNAcbeta1-->2)Man branch and with the GlcNAcbeta1-->6(GlcNAcbeta1-->2)Man branch. These results indicate that beta-1,4-GalTs II, III, IV, V, and VI are involved in the N-linked oligosaccharide biosynthesis cooperatively but not in a redundant manner with beta-1,4-GalT I within cells.     

Control of bisecting GlcNAc addition to N-linked sugar chains

In the present study, experimental control of the formation of bisecting GlcNAc was investigated, and the competition between beta-1,4-GalT (UDP-galactose:N-acetylglucosamine beta-1, 4-galactosyltransferase) and GnT-III (UDP-N-acetylglucosamine:beta-d-mannoside beta-1, 4-N-acetylglucosaminyltransferase) was examined. We isolated a beta-1,4-GalT-I single knockout human B cell clone producing monoclonal IgM and several transfectant clones that overexpressed beta-1,4-GalT-I or GnT-III. In the beta-1,4-GalT-I-single knockout cells, the extent of bisecting GlcNAc addition to the sugar chains of IgM was increased, where beta-1,4-GalT activity was reduced to about half that in the parental cells, and GnT-III activity was unaltered. In the beta-1,4-GalT-I transfectants, the extent of bisecting GlcNAc addition was reduced although GnT-III activity was not altered significantly. In the GnT-III transfectants, the extent of bisecting GlcNAc addition increased along with the increase in levels of GnT-III activity. The extent of bisecting GlcNAc addition to the sugar chains of IgM was significantly correlated with the level of intracellular beta-1,4-GalT activity relative to that of GnT-III. These results were interpreted as indicating that beta-1, 4-GalT competes with GnT-III for substrate in the cells.     

Normal levels of serum glycoproteins maintained in beta-1, 4-galactosyltransferase I-knockout mice

The galactose-mediated clearance of serum glycoproteins from the circulation was evaluated using beta-1,4-galactosyltransferase (beta-1,4-GalT) I-knockout mice. Partial structural study of the oligosaccharides released from mouse serum glycoproteins revealed that 77.4% of the oligosaccharides from beta-1,4-GalT I(+/+) mouse contain galactose, while 7.7% of those from beta-1,4-GalT I(-/-) mouse were galactosylated. Under the conditions, no significant change in serum protein concentrations was observed between the normal and mutant mice. The results indicate that the hepatic asialoglycoprotein receptor-mediated system is not functioning in the clearance of endogenous serum glycoproteins.     

Occurrence of poly-N-acetyllactosamine synthesis in Sf-9 cells upon transfection of individual human beta-1,4-galactosyltransferase I, II, III, IV, V and VI cDNAs

Lectin blot analysis of membrane glycoprotein samples from Sf-9 cells upon transfection of individual human beta-1,4-galactosyltransferase (beta-1,4-GalT) I, II, III, IV, V et VI cDNAs showed that the endogenous N-linked oligosaccharides are galactosylated (Guo et al., Glycobiology (2001), in press). Further analysis revealed that membrane glycoprotein samples from all the gene-transfected cells are also reactive to Lycopersicon esculentum agglutinin (LEA) et Datura stramonium agglutinin (DSA), both of which bind to oligosaccharides with poly-N-acetyllactosamine chains while no lectin reactive protein bands are detected when blots are pretreated with a mixture of diplococcal beta-1,4-galactosidase et jack bean beta-N-acetylhexosaminidase or N-glycanase. Analysis of endo-beta-galactosidase-digestion products revealed the presence of the Gal1-->GlcNAc1-->Gal and/or GlcNAc1-->Gal structures in the gene-transfected cells. When the homogenates of the gene-transfected cells were used as enzyme sources towards oligosaccharides with the GlcNAc beta 1-->(3Gal beta 1-->4GlcNAc)(1-3) structures, human recombinant beta-1,4-GalTs I et II galactosylated these oligosaccharides more effectively than other beta-1,4-GalTs. These results indicate that beta-1,4-GalTs I-VI can synthesize poly-N-acetyllactosamine chains with beta-1,3-N-acetylglucosaminyltransferase.     

Selective inhibition of beta-1,4- and alpha-1,3-galactosyltransferases: donor sugar-nucleotide based approach

A combined rational and library approach was used to identify bisphosphonates (IC50 = 20 microM) and galactose type 1-N-iminosugar (IC50=45 microM) as novel motifs for selective inhibition of beta-1,4-galactosyltransferase (beta-1,4-GalT) and alpha-1,3-galactosyltransferase (alpha-1,3-GalT), respectively. Our results demonstrate that, though these two galactosyltransferases both utilize the same donor sugar-nucleotide (UDP-Gal), the difference in their mechanisms can be utilized to design donor sugar or nucleotide analogues with inhibitory activities selective for only one of the galactosyltransferases. Investigation of beta-1,4-GalT inhibition using UDP-2-deoxy-2-fluorogalactose (UDP-2-F-Gal), UDP, and bisphosphonates, also led to the observation of metal dependent inhibition of beta-1,4-GalT. These observations and the novel inhibitor motifs identified in this study pave the way for the design and identification of even more potent and selective galactosyltransferase inhibitors.     

Beta-1,4-galactosylation of N-glycans is a complex process

Most beta-1,4-galactosyltransferase (beta-1,4-GalT)-knockout mice die after birth. Although several defects were found transiently in these animals, the primary cause of death is obscure. Not only beta-1,4-linked galactose residues on N-glycans, but also beta-1, 4-GalT activities were found in some of the tissues. Recently, five human genes which encode beta-1,4-GalTs have been cloned, and the possible presence of such novel beta-1,4-GalTs in mice is considered to bring about survival of the mutant animal beyond birth. In order to understand the semi-lethal nature of this animal, it is inevitable to clarify how individual novel beta-1,4-GalTs are involved in the biosynthesis of glycoconjugates based on their acceptor-substrate specificities.     

Differential expression of thymosin beta-10 by early passage and senescent vascular endothelium is modulated by VPF/VEGF: evidence for senescent endothelial cells in vivo at sites of atherosclerosis.

VPF/VEGF acts selectively on the vascular endothelium to enhance permeability, induce cell migration and division, and delay replicative senescence. To understand the changes in gene expression during endothelial senescence, we investigated genes that were differentially expressed in early vs. late passage (senescent) human dermal endothelial cells (HDMEC) using cDNA array hybridization. Early passage HDMEC cultured with or without VPF/VEGF overexpressed 9 and underexpressed 6 genes in comparison with their senescent counterparts. Thymosin beta-10 expression was modulated by VPF/VEGF and was strikingly down-regulated in senescent EC. The beta-thymosins are actin G-sequestering peptides that regulate actin dynamics and are overexpressed in neoplastic transformation. We have also identified senescent EC in the human aorta at sites overlying atherosclerotic plaques. These EC expressed senescence-associated neutral beta-galactosidase and, in contrast to adventitial microvessel endothelium, exhibited weak staining for thymosin beta-10. ISH performed on human malignant tumors revealed strong thymosin beta-10 expression in tumor blood vessels. This is the first report that Tbeta-10 expression is significantly reduced in senescent EC, that VPF/VEGF modulates thymosin beta-10 expression, and that EC can become senescent in vivo. The reduced expression of thymosin beta-10 may contribute to the senescent phenotype by reducing EC plasticity and thus impairing their response to migratory stimuli.     

Expression change of β-1,4 galactosyltransferase I, V mRNAs and Galβ1,4GlcNAc group in rat sciatic nerve after crush

Glycosylation is one of the most important post-translational modifications. It is clear that the single step of β-1,4-galactosylation is performed by a family of β-1,4-galactosyltransferases (β-1,4-GalTs), and that each member of this family may play a distinct role in different tissues and cells. β-1,4-GalT I and V are involved in the biosynthesis of N-linked oligosaccharides. In the present study, Real-time PCR revealed that the β-1,4-GalT I and V mRNAs reached peaks at 2 w after sciatic nerve crush. In situ hybridization showed that at 1 d after sciatic nerve crush, the expression levels of β-1,4-GalT I and V mRNAs were strong at the crush site, and decreased gradually from crush site to the distal segments. In addition, combined in situ hybridization for β1,4-GalT I and V mRNAs and immunohistochemistry for S100 showed that β1,4-GalT I and V mRNAs were mainly located in Schwann cells. Lectin blot showed that the expression of Galβ1,4GlcNAc group increased at 6 h immediately, reached a peak at 12 h and remained elevated up to 4 w after sciatic nerve crush. In conclusion, β1,4-GalT I and V might play important roles in the regeneration of the injuried sciatic nerve, and upregulation of Galβ1,4GlcNAc group might be correlated with the process of the sciatic nerve injury.     

The widespread effect of beta 1,4-galactosyltransferase on N-glycan processing

We investigated beta 1,4-GalT (UDP-galactose: beta-d-N-acetylglucosaminide beta 1,4-galactosyltransferase) in terms of intracellular competition with GnT-IV (UDP-N-acetylglucosamine: alpha1,3-d-mannoside beta1,4-N-acetylglucosaminyltransferase) and GnT-V (UDP-N-acetylglucosamine: alpha1,6-d-mannoside beta 1,6-N-acetylglucosaminyltransferase). The beta 1,4-GalT-I gene was introduced into Chinese hamster ovary (CHO) cells producing human interferon (hIFN)-gamma (IM4/V/IV cells) and five clones expressing various levels of beta 1,4-GalT were isolated. As we previously reported, parental IM4/V/IV cells express high levels of GnT-IVa and -V and produce hIFN-gamma having primarily tetraantennary sugar chains. The branching of sugar chains on hIFN-gamma was suppressed in the beta 1,4-GalT-enhanced clones to a level corresponding to the intracellular activity of beta 1,4-GalT relative to GnTs. Moreover, the contents of hybrid-type and high-mannose-type sugar chains increased in these clones. The results showed that beta 1,4-GalT widely affects N-glycan processing by competing with GnT-IV, GnT-V, and alpha-mannosidase II in cells and also by some other mechanisms that suppress the conversion of high-mannose-type sugar chains to the hybrid type.     

Involvement of murine β-1,4-galactosyltransferase V in lactosylceramide biosynthesis

Human β-1,4-galactosyltransferase (β-1,4-GalT) V was shown to be involved in the biosynthesis of N-glycans, O-glycans and lactosylceramide (Lac-Cer) by in vitro studies. To determine its substrate specificity, enzymatic activity and its products were analyzed using mouse embryonic fibroblast (MEF) cells from β-1,4-GalT V (B4galt5)-mutant mice. Analysis of expression levels of the β-1,4-GalT I-VI genes revealed that the expression of the β-1,4-GalT V gene in B4galt5 ( +/- ) - and B4galt5 ( -/- ) -derived MEF cells are a half and null when compared to that of B4galt5 ( +/+ )-derived MEF cells without altering the expression levels of other β-1,4-GalT genes. These MEF cells showed no apparent difference in their growth. When β-1,4-GalT activities were determined towards GlcNAcβ-S-pNP, no significant difference in its specific activity was obtained among B4galt5 ( +/+ )-, B4galt5 ( +/- ) - and B4galt5 ( -/- ) -derived MEF cells. No significant differences were obtained in structures and amounts of N-glycans and lectin bindings to membrane glycoproteins among B4galt5 ( +/+ )-, B4galt5 ( +/- ) - and B4galt5 ( -/- ) -derived MEF cells. However, when cell homogenates were incubated with glucosylceramide in the presence of UDP-[(3)H]Gal, Lac-Cer synthase activity in B4galt5 ( +/- ) - and B4galt5 ( -/- ) -derived MEF cells decreased to 41% and 11% of that of B4galt5 ( +/+ )-derived MEF cells. Consistent with this, amounts of Lac-Cer and its derivative GM3 in B4galt5 ( -/- ) -derived MEF cells decreased remarkably when compared with those of B4galt5 ( +/+ )-derived MEF cells. These results indicate that murine β-1,4-GalT V is involved in Lac-Cer biosynthesis.     

CHIP-dependent p53 regulation occurs specifically during cellular senescence

p53 regulates several biological processes, including senescence. Its protein stability is regulated by ubiquitination and proteasomal degradation, mainly mediated by Mdm2. However, other E3 ligases have been identified, such as the chaperone-associated ligase CHIP, although their precise function regarding p53 degradation remains elusive. Interestingly, CHIP deficiency has been recently shown to result in accelerated aging in mice, although the molecular basis of this phenotype was not completely understood. In this study, we explore the role of CHIP in regulating p53 in senescence. We demonstrate that in senescent human fibroblasts, CHIP is up-regulated concomitant with a significant down-regulation of p53. Moreover, CHIP partially translocates to the nucleus and acquires higher ubiquitination levels in senescent cells. Notably, CHIP overexpression in young cells, to levels similar to those recorded during senescence, leads to p53 degradation to below its basal levels. In addition, whereas CHIP silencing has no effect on p53 stability in young cells, a considerable p53 accumulation occurs in their senescent counterparts. Finally, we have observed an attenuation of the CHIP-associated molecular folding-refolding machinery during senescence, and supportively, inhibition of Hsp90 activity leads to rapid p53 degradation only in senescent cells. Taking these results together, we conclude that CHIP-dependent p53 regulation occurs specifically during senescence.     

EZH2在衰老细胞中表达下调引起Netrin-1表达增加进而促进交感神经轴突生长

交感神经在一些疾病动物模型和老年小鼠模型组织中呈现发芽生长和密度增加的趋势。这提示,交感神经与衰老密切相关。本研究采用免疫组织化学检测老年小鼠肝、肺、脾组织和人结肠腺瘤组织衰老模型中交感神经标记蛋白酪氨酸羟化酶（TH）的表达情况。结果证实,衰老组织中的交感神经密度显著增加。然而,目前对衰老组织的这种生物学改变机制尚不明确。本研究以人成纤维细胞系2BS为实验材料,经转录组测序发现,分泌性轴突导向因子导蛋白-1在衰老细胞中上调表达。ELISA和实时定量PCR结果显示,在博来霉素、离子射线、癌蛋白RasV12过表达所诱导的早衰及复制性衰老的2BS细胞中,导蛋白-1的蛋白质和mRNA表达量均比年轻对照组高。DNA损伤反应（DNA damage response,DDR）是调控衰老细胞分泌蛋白质表达的重要途径。为明确导蛋白-1在衰老细胞的上调表达是否与DDR相关,使用小分子化合物抑制DDR的重要效应分子ATM（ataxia telangiectasia mutated）和CHK2（checkpoint kinase 2）在博来霉素诱导的衰老2BS细胞中的活性。ELISA和实时定量PCR结果显示,与博来霉素诱导衰老组相比,阻断衰老细胞中的DDR活性未引起导蛋白-1表达水平的改变。提示导蛋白-1在衰老细胞中的表达不依赖于DDR途径。Western印迹检测发现组蛋白甲基转移酶EZH2在衰老细胞中的蛋白质表达明显少于年轻细胞;实时定量PCR结果表明使用小分子化合物抑制EZH2在年轻细胞中的活性引起导蛋白-1表达增加;CHIP实验检测发现,EZH2对年轻细胞的导蛋白-1启动子区DNA片段富集显著而在衰老细胞未见富集。以上结果提示EZH2在衰老细胞中的下调表达是导蛋白-1表达增加的重要原因。大鼠DRG与衰老细胞共培养结果证实,衰老细胞对交感神经具有导向作用,并且该现象依赖于导蛋白-1的分泌。根据以上结果得出结论,导蛋白-1在衰老细胞中的上调表达不依赖于DDR途径而与EZH2的下调表达相关,衰老细胞通过分泌导蛋白-1促进交感神经轴突定向生长。本研究为深入探讨衰老相关疾病的发生机制以及防治提供了新线索,具有一定的科学意义和潜在的转化应用价值。     

The Role of TNF-<Emphasis Type="Italic">α</Emphasis> and its Receptors in the Production of <Emphasis Type="Italic">β</Emphasis>-1,4-galactosyltransferase I mRNA by Rat Primary Type-2 Astrocytes

β-1,4-galactosyltransferase I (β-1,4-GalT I) plays an important role in the synthesis of the backbone structure of adhesion molecules involved in leukocyte–endothelial cell interaction. The expression of β-1,4-GalT I mRNA increased in primary human endothelial cells after exposure to tumor necrosis factor-α (TNF-α). In the central nervous system (CNS), astrocytes play a pivotal role in immunity as immunocompetent cells by secreting cytokines and inflammatory mediators, there are two types of astrocytes. Type-1 astrocytes can secrete TNF-α when stimulated with Lipopolysaccharide (LPS), while the responses of type-2 astrocytes during inflammation are unknown. So we examined the expression change of β-1,4-GalT I mRNA in type-2 astrocytes after exposure to TNF-α and LPS. Real-time PCR showed that TNF-α or LPS affected β-1,4-GalT I mRNA expression in a time- and dose-dependent manner. RT-PCR analysis revealed that TNFR1 and TNFR2 were present in normal untreated type-2 astrocytes, and that TNF-α, TNFR1 and TNFR2 increased in type-2 astrocytes after exposure to TNF-α or LPS. Immunocytochemistry showed that TNFR1 was expressed in the cytoplasm, nucleus and processes of normal untreated type-2 astrocytes, and distributed mainly in the cytoplasm and processes after exposure to LPS. TNFR2 was mainly expressed in the nucleus of normal untreated type-2 astrocytes, and distributed mainly in the processes of type-2 astrocytes after exposure to LPS. Both anti-TNFR1 and anti-TNFR2 antibodies suppressed β-1,4-GalT I mRNA expression induced by TNF-α or LPS. From these results, we conclude that TNF-α signaling via both TNFR1 and TNFR2 translocated from nucleus to cytoplasm or processes is sufficient to induce β-1,4-GalT I mRNA. In addition, we observed that not only exogenous TNF-α but also TNF-α produced by type-2 astrocytes affected β-1,4-GalT I mRNA production in type-2 astrocytes. These results suggest that an autocrine loop involving TNF-α contributes to the production of β-1,4-GalT I mRNA in response to inflammation. Chunlin Xia is the co-first author.     

衰老相关新基因CSIG的cDNA克隆和功能

为了获得 2BS细胞衰老过程中表达下降的差异基因片段Y6 2的编码序列 ,以cDNA末端快速扩增法获得细胞衰老相关新基因CSIG(cellularsenescenceinhibitedgene ,细胞衰老抑制基因 )的cDNA全长 .CSIGcDNA长 196 1bp ,编码 4 90个氨基酸 ,在多种重要组织中都有不同程度的表达 ;蛋白产物位于细胞核内特定位点 ,可能在核仁中聚集 .细胞转染表明 :CSIG可抑制细胞衰老并延长细胞寿限 ,可能通过核糖体生物合成过程或基因转录调节来调控细胞衰老过程     

Generation of novel chimeric LacdiNAcS by gene fusion of α-lactalbumin and β1,4-galactosyltransferase 1

Novel chimeric lacdiNAc (GalNAc(β1-4)GlcNAc) synthase (c-LacdiNAcS) was generated by gene fusion of α-lactalbumin (α-LA) and β1,4-galactosyltransferase 1 (β1,4-GalT1). c-LacdiNAcS was expressed in Lec8 Chinese hamster ovary (Lec8 CHO) cells and exhibited N-acetylgalactosaminyltransferase (GalNAcT) activity in the absence of exogenous α-LA as well as other glycosyltransferase activities including lactose synthase (LacS), and β1,4-GalT. These glycosyltransferase activities of c-LacdiNAcS were compared to those activities induced in LacS system under the co-presence of bovine β1,4-GalT1 and α-LA, indicating that each domain of α-LA and β1,4-GalT1 on c-LacdiNAcS is not only folding correctly, but also interacting together. Furthermore, c-LacdiNAcS was found to be auto-lacdiNAcylated and can synthesize lacdiNAc structures on cellular glycoproteins, demonstrating that GalNAcT activity of c-LacdiNAcS is functional in Lec8 CHO cells.