MDA-MB-231 breast cancer cell viability, motility and matrix adhesion are regulated by a complex interplay of heparan sulfate, chondroitin-/dermatan sulfate and hyaluronan biosynthesis.

Proteoglycans and glycosaminoglycans modulate numerous cellular processes relevant to tumour progression, including cell proliferation, cell-matrix interactions, cell motility and invasive growth. Among the glycosaminoglycans with a well-documented role in tumour progression are heparan sulphate, chondroitin/dermatan sulphate and hyaluronic acid/hyaluronan. While the mode of biosynthesis differs for sulphated glycosaminoglycans, which are synthesised in the ER and Golgi compartments, and hyaluronan, which is synthesized at the plasma membrane, these polysaccharides partially compete for common substrates. In this study, we employed a siRNA knockdown approach for heparan sulphate (EXT1) and heparan/chondroitin/dermatan sulphate-biosynthetic enzymes (ß4GalT7) in the aggressive human breast cancer cell line MDA-MB-231 to study the impact on cell behaviour and hyaluronan biosynthesis. Knockdown of ß4GalT7 expression resulted in a decrease in cell viability, motility and adhesion to fibronectin, while these parameters were unchanged in EXT1-silenced cells. Importantly, these changes were associated with a decreased expression of syndecan-1, decreased signalling response to HGF and an increase in the synthesis of hyaluronan, due to an upregulation of the hyaluronan synthases HAS2 and HAS3. Interestingly, EXT1-depleted cells showed a downregulation of the UDP-sugar transporter SLC35D1, whereas SLC35D2 was downregulated in ß4GalT7-depleted cells, indicating an intricate regulatory network that connects all glycosaminoglycans synthesis. The results of our in vitro study suggest that a modulation of breast cancer cell behaviour via interference with heparan sulphate biosynthesis may result in a compensatory upregulation of hyaluronan biosynthesis. These findings have important implications for the development of glycosaminoglycan-targeted therapeutic approaches for malignant diseases.

N-Glycosylation of an IgG antibody secreted by Nicotiana tabacum BY-2 cells can be modulated through co-expression of human ß-1,4-galactosyltransferase.

Nicotiana tabacum BY-2 suspension cells have several advantages that make them suitable for the production of full-size monoclonal antibodies which can be purified directly from the culture medium. Carbohydrate characterization of an antibody (Lo-BM2) expressed in N. tabacum BY-2 cells showed that the purified Lo-BM2 displays N-glycan homogeneity with a high proportion (>70%) of the complex GnGnXF glycoform. The stable co-expression of a human ß-1,4-galactosyltransferase targeted to different Golgi sub-compartments altered Lo-BM2N-glycosylation and resulted in the production of an antibody that exhibited either hybrid structures containing a low abundance of the plant epitopes (α-1,3-fucose and ß-1,2-xylose), or a large amount of galactose-extended N-glycan structures. These results demonstrate the suitability of stable N-glycoengineered N. tabacum BY-2 cell lines for the production of human-like antibodies.

Beta-1,4-galactosyltransferase-3 deficiency suppresses the growth of immunogenic tumors in mice.

Background: Beta-1,4-galactosyltransferase-3 (B4GALT3) belongs to the family of beta-1,4-galactosyltransferases (B4GALTs) and is responsible for the transfer of UDP-galactose to terminal N-acetylglucosamine. B4GALT3 is differentially expressed in tumors and adjacent normal tissues, and is correlated with clinical prognosis in several cancers, including neuroblastoma, cervical cancer, and bladder cancer. However, the exact role of B4GALT3 in the tumor immune microenvironment (TIME) remains unclear. Here, we aimed to elucidate the function of B4GALT3 in the TIME. Methods: To study the functions of B4GALT3 in cancer immunity, either weakly or strongly immunogenic tumor cells were subcutaneously transplanted into wild-type (WT) and B4galt3 knockout (KO) mice. Bone marrow transplantation and CD8+ T cell depletion experiments were conducted to elucidate the role of immune cells in suppressing tumor growth in B4galt3 KO mice. The cell types and gene expression in the tumor region and infiltrating CD8+ T cells were analyzed using flow cytometry and RNA sequencing. N-glycosylated proteins from WT and B4galt3 KO mice were compared using the liquid chromatography tandem mass spectrometry (LC-MS/MS)-based glycoproteomic approach. Results: B4galt3 KO mice exhibited suppressed growth of strongly immunogenic tumors with a notable increase in CD8+ T cell infiltration within tumors. Notably, B4galt3 deficiency led to changes in N-glycan modification of several proteins, including integrin alpha L (ITGAL), involved in T cell activity and proliferation. In vitro experiments suggested that B4galt3 KO CD8+ T cells were more susceptible to activation and displayed increased downstream phosphorylation of FAK linked to ITGAL. Conclusion: Our study demonstrates that B4galt3 deficiency can potentially boost anti-tumor immune responses, largely through enhancing the influx of CD8+ T cells. B4GALT3 might be suppressing cancer immunity by synthesizing the glycan structure of molecules on the CD8+ T cell surface, as evidenced by the changes in the glycan structure of ITGAL in immune cells. Importantly, B4galt3 KO mice showed no adverse effects on growth, development, or reproduction, underscoring the potential of B4GALT3 as a promising and safe therapeutic target for cancer treatment.

Association of polymorphism of the ß(1, 4)-galactosyltransferase-I gene with milk production traits in Holsteins.

The ß(1,4)-galactosyltransferase-I gene (ß4galt1) encodes the catalytic part of the enzyme lactose synthase, responsible of lactose synthesis in the mammary gland. The complete coding region of the gene was screened for the presence of allelic variation among a sample of 1,200 Iranian Holstein cows, using PCR-SSCP technique followed by sequencing. Nine polymorphic nucleotide sites were identified-one in exons I and VI, two in exons II and III, and three in exon V. Altogether 18 different genotypes were assigned. Statistical analysis showed that the genotypes of Β4GALT1 significantly affect milk, lactose, protein and total solid productions in both the first and second lactation (P < 0.001). Variance component analysis considering restricted maximum likelihood showed that the major factor making differences in milk, lactose, protein and total solid productions among the studied cow is the ß4galt1 genotype. We concluded that the ß4galt1 gene is potentially associated with milk production traits in dairy cows and should be considered for further studies on genetics of the milk production traits.

Assays for Evaluation of Substrates for and Inhibitors of ß-1,4-Galactosyltransferase 7.

ß-1,4-Galactosyltransferase 7 (ß4GalT7) is a key enzyme in the synthesis of two classes of glycosaminoglycans (GAG), i.e., heparan sulfate (HS) and chondroitin/dermatan sulfate (CS/DS). GAG chains are linear polysaccharides of alternating hexuronic acid and N-acetylhexosamine residues, commonly linked to core proteins to form proteoglycans with important roles in the regulation of a range of biological processes. The biosynthesis of GAGs is initiated by xylosylation of a serine residue of the core protein followed by galactosylation, catalyzed by ß4GalT7. The biosynthesis can also be initiated by xylosides carrying hydrophobic aglycons, such as 2-naphthyl ß-D-xylopyranoside. We have cloned and expressed ß4GalT7, and designed a cell-free assay to measure the activity of this enzyme. The assay employs a 96-well plate format for high throughput. In this chapter, we describe the cloning, expression, and purification of ß4GalT7, as well as assays proposed for development of substrates for GAG priming and for investigating inhibitors of ß4GalT7.

B-cell-specific ablation of ß-1,4-galactosyltransferase 1 prevents aging-related IgG glycans changes and improves aging phenotype in mice.

IgG N-glycans levels change with advancing age, making it a potential biomarker of aging. ß-1,4-galactosyltransferase (B4GALT) gene expression levels also increase with aging. Ultra performance liquid chromatography (UPLC) was used to examine changes inserum IgG N-glycans at six time points during the aging process. Most serum IgG N-glycans changed with aging in WT but not in CD19-cre B4GALT1 floxed mice. The relative abundance of fucosylated biantennary glycans with or without Neu5Gc structures changed with aging in heterozygous B4GALT1 floxed mice but not in homozygous B4GALT1 floxed mice. Additionally, the aging phenotype was more apparent in WT mice than in B4GALT1 floxed mice. These results demonstrate that fucosylated biantennary glycans and fucosylated biantennary glycans containing N-glycolylneuraminic acid (Neu5Gc)-linked N-acetyllactosamine (LacNAc) were highly associated with aging and were affected by the B4GALT1 floxed mouse genotype. The changing levels of fucosylated monoantennary glycans observed with aging in WT mice was reversed in B4GALT1 floxed mice and was not sex specific. In summary, B-cell-specific ablation of B4GALT1 from a glycoproteomic perspective prevented age-related changes in IgG N-glycans in mice. SIGNIFICANCE: In this study, serum IgG glycoproteomic data in wild-type (WT) and B-cell-specific ablation of ß-1,4-galactosyltransferase 1 mice (B4GALT) were analyzed. Results showed that fucosylated biantennary glycans with or without N-glycolylneuraminic acid (Neu5Gc)-linked N-acetyllactosamine (LacNAc) were highly associated with aging and were also affected by the B4GALT1 floxed mouse genotype. In terms of gender-specific information, the trend towards elevated fucosylated monoantennary glycans in WT mice was not seen in CD19-cre B4GALT1 floxed mice in either sex. B-cell-specific ablation of B4GALT1 plays an important role in age-related glycan changes; its specific functions and mechanisms are worthy of in-depth study. Our data suggest that investigating the relationship between galactosylation and aging may help advance the field of glycoproteomics and aging research.

Crystal structure of the catalytic domain of Drosophila beta1,4-Galactosyltransferase-7.

The beta1,4-galactosyltransferase-7 (beta4Gal-T7) enzyme, one of seven members of the beta4Gal-T family, transfers in the presence of manganese Gal from UDP-Gal to an acceptor sugar (xylose) that is attached to a side chain hydroxyl group of Ser/Thr residues of proteoglycan proteins. It exhibits the least protein sequence similarity with the other family members, including the well studied family member beta4Gal-T1, which, in the presence of manganese, transfers Gal from UDP-Gal to GlcNAc. We report here the crystal structure of the catalytic domain of beta4Gal-T7 from Drosophila in the presence of manganese and UDP at 1.81 A resolution. In the crystal structure, a new manganese ion-binding motif (HXH) has been observed. Superposition of the crystal structures of beta4Gal-T7 and beta4Gal-T1 shows that the catalytic pocket and the substrate-binding sites in these proteins are similar. Compared with GlcNAc, xylose has a hydroxyl group (instead of an N-acetyl group) at C2 and lacks the CH(2)OH group at C5; thus, these protein structures show significant differences in their acceptor-binding site. Modeling of xylose in the acceptor-binding site of the beta4Gal-T7 crystal structure shows that the aromatic side chain of Tyr(177) interacts strongly with the C5 atom of xylose, causing steric hindrance to any additional group at C5. Because Drosophila Cd7 has a 73% protein sequence similarity to human Cd7, the present crystal structure offers a structure-based explanation for the mutations in human Cd7 that have been linked to Ehlers-Danlos syndrome.

Golgi localisation of GMAP210 requires two distinct cis-membrane binding mechanisms.

BACKGROUND: The Golgi apparatus in mammals appears as a ribbon made up of interconnected stacks of flattened cisternae that is positioned close to the centrosome in a microtubule-dependent manner. How this organisation is achieved and retained is not well understood. GMAP210 is a long coiled-coil cis-Golgi associated protein that plays a role in maintaining Golgi ribbon integrity and position and contributes to the formation of the primary cilium. An amphipathic alpha-helix able to bind liposomes in vitro has been recently identified at the first 38 amino acids of the protein (amphipathic lipid-packing sensor motif), and an ARF1-binding domain (Grip-related Arf-binding domain) was found at the C-terminus. To which type of membranes these two GMAP210 regions bind in vivo and how this contributes to GMAP210 localisation and function remains to be investigated. RESULTS: By using truncated as well as chimeric mutants and videomicroscopy we found that both the N-terminus and the C-terminus of GMAP210 are targeted to the cis-Golgi in vivo. The ALPS motif was identified as the N-terminal binding motif and appeared concentrated in the periphery of Golgi elements and between Golgi stacks. On the contrary, the C-terminal domain appeared uniformly distributed in the cis-cisternae of the Golgi apparatus. Strikingly, the two ends of the protein also behave differently in response to the drug Brefeldin A. The N-terminal domain redistributed to the endoplasmic reticulum (ER) exit sites, as does the full-length protein, whereas the C-terminal domain rapidly dissociated from the Golgi apparatus to the cytosol. Mutants comprising the full-length protein but lacking one of the terminal motifs also associated with the cis-Golgi with distribution patterns similar to those of the corresponding terminal end whereas a mutant consisting in fused N- and C-terminal ends exhibits identical localisation as the endogenous protein. CONCLUSION: We conclude that the Golgi localisation of GMAP210 is the result of the combined action of the two N- and C-terminal domains that recognise different sub-regions of the cis-GA. Based on present and previous data, we propose a model in which GMAP210 would participate in homotypic fusion of cis-cisternae by anchoring the surface of cisternae via its C-terminus and projecting its distal N-terminus to bind the rims or to stabilise tubular structures connecting neighbouring cis-cisternae.

Transient co-expression for fast and high-yield production of antibodies with human-like N-glycans in plants.

Plant-based transient expression is potentially the most rapid and cost-efficient system for the production of recombinant pharmaceutical proteins, but safety concerns associated with plant-specific N-glycosylation have hampered its adoption as a commercial production system. In this article, we describe an approach based on the simultaneous transient co-expression of an antibody, a suppressor of silencing and a chimaeric human beta1,4-galactosyltransferase targeted for optimal activity to the early secretory pathway in agroinfiltrated Nicotiana benthamiana leaves. This strategy allows fast and high-yield production of antibodies with human-like N-glycans and, more generally, provides solutions to many critical problems posed by the large-scale production of therapeutic and vaccinal proteins, specifically yield, volume and quality.

Characterization of a novel ubiquitin-conjugating enzyme that regulates beta1,4-galactosyltransferase-1 in embryonic stem cells.

In this study we identified a novel galactosyltransferase 1-associating protein (GTAP) by cDNA cloning from a murine embryonic cDNA library using the two-hybrid yeast system. GTAP is expressed in early embryonic tissues, as well as in adult tissues with active cell turnover, and belongs to the class III ubiquitin-conjugating (E2) enzyme family. Its COOH-terminal domain contains a consensus sequence for ubiquitin binding shared by all the ubiquitin-conjugating enzymes, whereas its NH(2)-terminal domain appears critical for the binding and internalization of cell surface galactosyltransferase 1 (GalT1) in embryonic stem cells through a monensin- and MG132-dependent pathway. We have found that GTAP regulates GalT1-associated, laminin-dependent embryonic cell adhesion and the formation of embryoid bodies. Thus, GTAP functions as an evolutionarily conserved E2 enzyme, which may participate in intercellular adhesion and embryonic development. Disclosure of potential conflicts of interest is found at the end of this article.

Golgi clusters and vesicles mediate mitotic inheritance independently of the endoplasmic reticulum.

We have examined the fate of Golgi membranes during mitotic inheritance in animal cells using four-dimensional fluorescence microscopy, serial section reconstruction of electron micrographs, and peroxidase cytochemistry to track the fate of a Golgi enzyme fused to horseradish peroxidase. All three approaches show that partitioning of Golgi membranes is mediated by Golgi clusters that persist throughout mitosis, together with shed vesicles that are often found associated with spindle microtubules. We have been unable to find evidence that Golgi membranes fuse during the later phases of mitosis with the endoplasmic reticulum (ER) as a strategy for Golgi partitioning (Zaal, K.J., C.L. Smith, R.S. Polishchuk, N. Altan, N.B. Cole, J. Ellenberg, K. Hirschberg, J.F. Presley, T.H. Roberts, E. Siggia, et al. 1999. Cell. 99:589-601) and suggest that these results, in part, are the consequence of slow or abortive folding of GFP-Golgi chimeras in the ER. Furthermore, we show that accurate partitioning is accomplished early in mitosis, by a process of cytoplasmic redistribution of Golgi fragments and vesicles yielding a balance of Golgi membranes on either side of the metaphase plate before cell division.

Enhanced sialylation of recombinant erythropoietin in CHO cells by human glycosyltransferase expression.

Sialylation, the attachment of sialic acid residues to a protein, can affect the biological activity and in vivo circulatory half-life of glycoproteins. Human alpha2,3- sialyltransferase (alpha2,3-ST) and beta1,4-galactosyltransferase (beta1,4-GT) are responsible for terminal sialylation and galactosylation, respectively. Enhanced sialylation of human erythropoietin (EPO) by the expression of alpha2,3-ST and beta1,4-GT was achieved using recombinant Chinese hamster ovary (CHO) cells (EC1). The sialic acid content and sialylation of N-glycans were evaluated by HPLC. When alpha2,3-ST was expressed in CHO cells (EC1-ST2), the sialic acid content (moles of sialic acid/mole of EPO) increased from 6.7 to 7.5. In addition, the amount of trisialylated glycans increased from 17.3% to 26.1%. When alpha2,3-ST and beta1,4-GT were coexpressed in CHO cells (EC1-GTST15), the degree of sialylation was greater than that in EC1-ST2 cells. In the case of EC1-GTST15 cells, the sialic acid content increased to 8.2 and the proportion of trisialylated glycans was markedly increased from 17.3% to 35.5%. Interestingly, the amount of asialoglycans decreased only in the case of GTST15 cells (21.4% to 14.2%). These results show that coexpression of alpha2,3- ST and beta1,4-GT is more effective than the expression of alpha2,3-ST alone. Coexpression of alpha2,3-ST and beta1,4-GT did not affect CHO cell growth and metabolism or EPO production. Thus, coexpression of alpha2,3-ST and beta1,4-GT may be beneficial for producing therapeutic glycoproteins with enhanced sialylation in CHO cells.

Tumor-derived exosomal miR-1247-3p induces cancer-associated fibroblast activation to foster lung metastasis of liver cancer.

The communication between tumor-derived elements and stroma in the metastatic niche has a critical role in facilitating cancer metastasis. Yet, the mechanisms tumor cells use to control metastatic niche formation are not fully understood. Here we report that in the lung metastatic niche, high-metastatic hepatocellular carcinoma (HCC) cells exhibit a greater capacity to convert normal fibroblasts to cancer-associated fibroblasts (CAFs) than low-metastatic HCC cells. We show high-metastatic HCC cells secrete exosomal miR-1247-3p that directly targets B4GALT3, leading to activation of ß1-integrin-NF-κB signaling in fibroblasts. Activated CAFs further promote cancer progression by secreting pro-inflammatory cytokines, including IL-6 and IL-8. Clinical data show high serum exosomal miR-1247-3p levels correlate with lung metastasis in HCC patients. These results demonstrate intercellular crosstalk between tumor cells and fibroblasts is mediated by tumor-derived exosomes that control lung metastasis of HCC, providing potential targets for prevention and treatment of cancer metastasis.

Structural snapshots of beta-1,4-galactosyltransferase-I along the kinetic pathway.

During the catalytic cycle of beta1,4-galactosyltransferase-1 (Gal-T1), upon the binding of Mn(2+) followed by UDP-Gal, two flexible loops, a long and a short loop, change their conformation from open to closed. We have determined the crystal structures of a human M340H-Gal-T1 mutant in the open conformation (apo-enzyme), its Mn(2+) and Mn(2+)-UDP-Gal-bound complexes, and of a pentenary complex of bovine Gal-T1-Mn(2+)-UDP-GalNAc-Glc-alpha-lactalbumin. These studies show that during the conformational changes in Gal-T1, the coordination of Mn(2+) undergoes significant changes. It loses a coordination bond with a water molecule bound in the open conformation of Gal-T1 while forming a new coordination bond with another water molecule in the closed conformation, creating an active ground-state structure that facilitates enzyme catalysis. In the crystal structure of the pentenary complex, the N-acetylglucosamine (GlcNAc) moiety is found cleaved from UDP-GalNAc and is placed 2.7A away from the O4 oxygen atom of the acceptor Glc molecule, yet to form the product. The anomeric C1 atom of the cleaved GalNAc moiety has only two covalent bonds with its non-hydrogen atoms (O5 and C2 atoms), similar to either an oxocarbenium ion or N-acetylgalactal form, which are crystallographically indistinguishable at the present resolution. The structure also shows that the newly formed, metal-coordinating water molecule forms a hydrogen bond with the beta-phosphate group of the cleaved UDP moiety. This hydrogen bond formation results in the rotation of the beta-phosphate group of UDP away from the cleaved GalNAc moiety, thereby preventing the re-formation of the UDP-sugar during catalysis. Therefore, this water molecule plays an important role during catalysis in ensuring that the catalytic reaction proceeds in a forward direction.

Cell surface and Golgi pools of beta-1,4-galactosyltransferase are differentially regulated during embryonal carcinoma cell differentiation.

beta-1,4-Galactosyltransferase (GalTase) has two functionally distinct subcellular distributions. In the Golgi apparatus, GalTase participates in the glycosylation of secretory and membrane-bound glycoproteins, whereas on the cell surface it mediates specific aspects of intercellular adhesion. For this study, a murine GalTase clone was obtained by screening a lambda gt10 cDNA library made from F9 embryonal carcinoma cells with a heterologous bovine GalTase cDNA probe. The murine GalTase cDNA probe was used in conjunction with assays of GalTase activity to investigate the expression and distribution of GalTase during differentiation of F9 stem cells into secretory endodermal epithelium. During the initial phase of F9 cell differentiation, GalTase mRNA levels remained relatively constant; however, as differentiation progressed, as assayed by expression of the differentiation-specific marker laminin B1, GalTase mRNA levels and enzyme activity rose dramatically. Furthermore, subcellular fractionation of these cells showed that the increased GalTase levels were specifically associated with the Golgi apparatus, whereas GalTase specific activity on the plasma membrane remained constant. These results show that levels of cell surface and Golgi GalTase change relative to one another during F9 cell differentiation and suggest that these functionally distinct pools of GalTase are independently and differentially regulated.

Modifying the insect cell N-glycosylation pathway with immediate early baculovirus expression vectors.

The baculovirus-insect cell expression system is well-suited for recombinant glycoprotein production because baculovirus vectors can provide high levels of expression and insect cells can modify newly synthesized proteins in eucaryotic fashion. However, the N-glycosylation pathway of baculovirus-infected insect cells differs from the pathway found in higher eucaryotes, as indicated by the fact that glycoproteins produced in the baculovirus system typically lack complex biantennary N-linked oligosaccharide side chains containing penultimate galactose and terminal sialic acid residues. We recently developed a new type of baculovirus vector that can express foreign genes immediately after infection under the control of the viral ie1 promoter. These immediate early baculovirus expression vectors can be used to modify the insect cell N-glycosylation pathway and produce a foreign glycoprotein with more extensively processed N-linked oligosaccharides. These vectors can also be used to study the influence of the late steps in N-linked oligosaccharide processing on glycoprotein function. Further development could lead to baculovirus-insect cell expression systems that can produce recombinant glycoproteins with complex biantennary N-linked oligosaccharides structurally identical to those produced by higher eucaryotes.

Scattered Golgi elements during microtubule disruption are initially enriched in trans-Golgi proteins.

We have addressed the question of whether or not Golgi fragmentation, as exemplified by that occurring during drug-induced microtubule depolymerization, is accompanied by the separation of Golgi subcompartments one from another. Scattering kinetics of Golgi subcompartments during microtubule disassembly and reassembly following reversible nocodazole exposure was inferred from multimarker analysis of protein distribution. Stably expressed alpha-2,6-sialyltransferase and N-acetylglucosaminyltransferase-I (NAGT-I), both C-terminally tagged with the myc epitope, provided markers for the trans-Golgi/trans-Golgi network (TGN) and medial-Golgi, respectively, in Vero cells. Using immunogold labeling, the chimeric proteins were polarized within the Golgi stack. Total cellular distributions of recombinant proteins were assessed by immunofluorescence (anti-myc monoclonal antibody) with respect to the endogenous protein, beta-1,4-galactosyltransferase (GalT, trans-Golgi/TGN, polyclonal antibody). ERGIC-53 served as a marker for the intermediate compartment). In HeLa cells, distribution of endogenous GalT was compared with transfected rat alpha-mannosidase II (medial-Golgi, polyclonal antibody). After a 1-h nocodazole treatment, Vero alpha-2,6-sialyltransferase and GalT were found in scattered cytoplasmic patches that increased in number over time. Initially these structures were often negative for NAGT-I, but over a two- to threefold slower time course, NAGT-I colocalized with alpha-2,6-sialyltransferase and GalT. Scattered Golgi elements were located in proximity to ERGIC-53-positive structures. Similar trans-first scattering kinetics was seen with the HeLa GalT/alpha-mannosidase II pairing. Following nocodazole removal, all cisternal markers accumulated at the same rate in a juxtanuclear Golgi. Accumulation of cisternal proteins in scattered Golgi elements was not blocked by microinjected GTPgammaS at a concentration sufficient to inhibit secretory processes. Redistribution of Golgi proteins from endoplasmic reticulum to scattered structures following brefeldin A removal in the presence of nocodazole was not blocked by GTPgammaS. We conclude that Golgi subcompartments can separate one from the other. We discuss how direct trafficking of Golgi proteins from the TGN/trans-Golgi to endoplasmic reticulum may explain the observed trans-first scattering of Golgi transferases in response to microtubule depolymerization.

Differentially expressed long non­coding RNAs and mRNAs in patients with IgA nephropathy.

Long non­coding RNAs (lncRNAs) have been reported to serve a crucial role in renal diseases; however, their role in immunoglobulin A nephropathy (IgAN) remains unclear. In the present study, peripheral blood mononuclear cells (PBMCs) were collected from both patients with IgAN and healthy controls. A microarray analysis was then performed to identify differentially expressed lncRNAs and mRNAs in PBMCs, which were confirmed by quantitative polymerase chain reaction. In addition, Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway and lncRNA­mRNA co­expression network analyses were conducted. The present study identified 167 differentially expressed lncRNAs and 94 differentially expressed mRNAs. Numerous GO terms, including innate immune response, inflammatory response, IPAF inflammasome complex and UDP­galactose:ß­N­acetylglucosamine ß­1, and 3­galactosyltransferase activity, were significantly enriched in the differentially expressed mRNAs. The top five KEGG signaling pathways included nucleotide­binding oligomerization domain­like receptor signaling pathway, hematopoietic cell lineage, inflammatory bowel disease, tumor necrosis factor signaling pathway and other types of O­glycan biosynthesis. In addition, a total of 149 lncRNAs were shown to interact with 7 mRNAs that were associated with the 'innate immune response' GO term. The results of the present study demonstrated that differentially expressed lncRNAs and mRNAs may have a role in the development of IgAN. These results may aid in the elucidation of a basic pathogenic mechanism, the identification of possible biomarkers and the generation of potential novel treatment strategies for IgAN.

Computational design and experimental evaluation of glycosyltransferase mutants: engineering of a blood type B galactosyltransferase with enhanced glucosyltransferase activity.

Glycosyltransferases are an enormous and diverse class of enzyme encompassing 1% of all sequenced genomes. They catalyze the transfer of a monosaccharide from an activated donor such as a sugar-nucleotide to an acceptor molecule. Though the primary sequences of glycosyltransferases have little homology, X-ray structural studies on glycosyltransferases have revealed that there are two main folds and that the orientation of the sugar donors with respect to the folds is highly conserved. It seems that glycosyltransferases have evolved diversified specificities toward donor sugars by changing the amino acids around the monosaccharide moiety without altering the orientation of the nucleotide moiety. In this study, we designed new glycosyltransferases with altered donor specificities by use of a novel empirical model called the Epimer Propensity Index (EPI). The EPI was constructed using 221 carbohydrate-protein complex structures in the Protein Data Bank with either galactose or glucose in the complex. The blood type B synthesizing glycosyltransferase GTB, a galactosyltransferase was our target enzyme. Two GTB mutants designed to exhibit enhanced glucosyltransferase activity were cloned, expressed and characterized experimentally. The predicted GTB mutants, Ser185Asn and Ser185Cys, exhibited 4.3- and 4.8-fold elevations in k(cat)/K(m) for UDP-Glc relative to that of wild-type enzyme.