Cytokine-induced beta-galactoside alpha-2,6-sialyltransferase in human endothelial cells mediates alpha 2,6-sialylation of adhesion molecules and CD22 ligands

Sialic acids decorating blood and cell surface proteins can play important roles in various biological processes. The inflammatory cytokines tumor necrosis factor-alpha (TNF-alpha) and interleukin-1, as well as bacterial lipopolysaccharide, can activate vascular endothelium, increasing expression of several surface glycoproteins. Here we show that treatment of cultured human endothelial cells (HEC) with TNF-alpha, interleukin-1, or lipopolysaccharide causes increased expression of the enzyme beta-galactoside alpha-2,6-sialytransferase (alpha 2-6STN). TNF-alpha was most effective, inducing a 3.5-fold enhancement of cell-associated sialytransferase activity by 72 h. In addition, activated HEC secreted a large portion of the induced sialyltransferase activity into the medium. Analysis of labeled HEC showed both a relative and an absolute increase of alpha 2,6-linked sialic acid on N-linked oligosaccharides after TNF-alpha stimulation. This coincided with increased expression of endothelial glycoproteins bearing N-linked glycans with alpha 2,6-linked sialic acid detected by the lectin Sambucus nigra agglutinin. The cytokine-inducible endothelial cell adhesion molecules E-selectin, ICAM-1, and VCAM-1 are among these glycoprotein substrates for alpha 2-6STN. These changes also correlated with a substantial increase in binding sites for CD22 beta, a mammalian lectin known to recognize oligosaccharides carrying multiple copies of alpha 2,6-linked sialic acid. Northern analysis revealed increased levels of mRNA encoding alpha 2-6STN. Thus, activation of endothelial cells during inflammatory and immunological processes may induce alpha 2-6STN, which can participate in sialylation of other activation-dependent molecules.     

Cloning and expression of human Gal beta 1,3(4)GlcNAc alpha 2,3-sialyltransferase

Here we report the cDNA sequence of human Gal beta 1,3(4)GlcNAc alpha 2,3-sialyltransferase. The cDNA was isolated from a human placental cDNA library by screening with a 150bp probe generated by PCR using degenerate primers based on the sequences found in the sialyl motif. Comparative analysis of this cDNA with the rat liver alpha 2,3-sialyltransferase sequence indicates 91% nucleotide similarity between the two sequence in the predicted coding region. On the amino acid level, the degree of conservation is 97%. Surprisingly, Northern analysis indicated that the gene is expressed at low levels in human placenta but is abundantly expressed in skeletal muscle and fetal tissues.     

Molecular cloning and functional expression of feline thrombopoietin

Feline thrombopoietin (TPO) was molecularly cloned to establish a basis for cytokine therapy of thrombocytopenia in cats. cDNA clones covering the whole coding sequence of feline TPO were isolated from feline liver. The feline TPO cDNA obtained in this study contained an open reading frame encoding 349 amino acid residues. The predicted amino acid sequence of feline TPO shared 78.7, 69.9, 72.9 and 83.0% similarity with sequences of human, murine, rat and canine TPO, respectively. Four cysteine residues and two of four N-glycosylation sites that are conserved among species were also found at the corresponding positions in feline TPO. The feline TPO cDNA fragment encoding the whole amino acid coding region was recloned into an expression vector, and the resulting vector was transfected into 293T cells using the calcium phosphate method. The supernatant of the transfected 293T cells stimulated the proliferation of a human megakaryoblastic leukemia cell line (UT-7/TPO) cells in a dose dependent manner, indicating that the feline TPO cDNA obtained in this study encodes biologically active feline TPO.     

Molecular cloning and gene expression of chum salmon cystatin

A salmon cystatin cDNA clone was isolated from a chum salmon cDNA library. The clone encoded a full-length extracellular-type cystatin and its signal peptide and included 5'- and 3'-untranslated regions. The deduced amino acid sequence showed a high degree of sequence similarity to mammalian cystatin C, chicken egg cystatin, and chum salmon pituitary cystatin. By Northern blot analysis, the salmon cystatin was found to show apparently non-tissue specific expression. Because platyfish EHS cells transfected with a cystatin expression vector produced a 13 kDa mature cystatin in the culture medium, the salmon cystatin was considered to act as an extracellular type of cystatin in the fish cells. These findings indicate that the salmon cystatin is a homolog of mammalian cystatin C.     

Molecular cloning and expression of rabbit pancreatic cholesterol esterase

Rabbit pancreatic cholesterol esterase (CEase, carboxyl ester lipase, EC 3.1.1.3) has been cloned from a lambda gt11 library of adult rabbit pancreatic cDNA. The open reading frame consists of 1788 nucleotides which encodes 576 amino acids of the functional protein and a 20 amino acid leader peptide. When compared to other species, the greatest homology is observed between residues 82-248 with little or no homology at the C-terminal end where proline-glutamate-serine-threonine (PEST) segments are a characteristic feature of the human CEase. Rabbit CEase (RCEase) retains the active-site serine (gxsxg), the active-site histidine and the tentative heparin binding site (KKRCLQ) at similar positions in comparison to pancreatic CEases of other species. When rabbit CEase cDNA is expressed in monkey kidney (COS-7) cells, enzymatic hydrolytic activity is detected in the growth medium as is a 67 kDa protein by Western blotting with polyclonal anti-CEase antibody. Northern blot analysis shows two mRNA (2.2 and 3.2 kb) species.     

alpha2,3-sialyltransferase mRNA and alpha2,3-linked glycoprotein sialylation are increased in malignant gliomas

Microinjection of a substance P analogue (1 mM; 7 or 10 nl) into laminae I and II of the L7 dorsal horn of decerebrate cats significantly potentiated (P < 0.05) the increase in arterial pressure evoked by microinjection of L-glutamate (109 mM; 7 or 10 nl) into these spinal sites. Microinjection of the substance P analogues (i.e., GR73638 and [Sar9,Met(O2)11]-substance P) which were selective NK-1 receptor agonists, had no impact on the cardioacceleration evoked by microinjection of L-glutamate (P > 0.05). In addition, microinjection of these analogues had no effect on the modest and non-significant increase in phrenic nerve discharge evoked by L-glutamate. We conclude that stimulation of NK-1 receptors in the superficial laminae of the dorsal horn potentiates the pressor responses to microinjection of L-glutamate.     

Molecular cloning and functional expression of rat liver glutathione-dependent dehydroascorbate reductase

We have isolated a cDNA clone for a novel glutathione-dependent dehydroascorbate reductase from a rat liver cDNA library in lambdagt11 by immunoscreening. The authenticity of the clone was confirmed as follows: first, the antibody that had been purified through affinity for the protein expressed by the cloned lambdagt11 phage recognized only the enzyme in a crude extract from rat liver; and second, two internal amino acid sequences of purified enzyme were identified in the protein sequence predicted from the cDNA. The predicted protein consists of 213 amino acids with a molecular weight of 24,929, which is smaller by approximately 3,000 than the value obtained by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. This discrepancy of the molecular weight was explained by post-translational modification because the recombinant protein expressed by a mammalian system (Chinese hamster ovary cells) was of the same size as rat liver enzyme but larger than the protein expressed by a bacterial system (Escherichia coli). Chinese hamster ovary cells, originally devoid of glutathione-dependent dehydroascorbate reductase activity, was made to elicit the enzyme activity (1.5 nmol/min/mg of cytosolic protein) by expression of the recombinant protein. Additionally, the cells expressing the enzyme were found to accumulate 1.7 times as much ascorbate as the parental cells after incubation with dehydroascorbate. This result points to the importance of the dehydroascorbic acid reductase in maintaining a high concentration of ascorbate in the cell.     

Molecular cloning and functional expression of equine interleukin-1 receptor antagonist

Equine interleukin-1 receptor antagonist (IL-1ra) was molecularly cloned to establish a basis for cytokine therapy of acute and chronic inflammatory diseases in the horse. cDNA clones encoding the whole coding sequence of equine IL-1ra were isolated from equine peripheral blood mononuclear cells (PBMC) that had been stimulated with lipopolysaccharide (LPS). The equine IL-1ra cDNA obtained in this study contained an open reading frame encoding 177 amino acid residues. The predicted amino acid sequence of equine IL-1ra shared 75.7, 75.3 and 76.3% similarity with sequences of human, murine and rabbit IL-1ras, respectively. An N-glycosylation site and five cysteine residues conserved in human, murine and rabbit IL-1ras were also found at the corresponding positions in equine IL-1ra. Recombinant glutathione S-transferase (GST)-equine IL-1ra fusion protein produced by Escherichia coli was purified. This protein was shown to inhibit the cytostatic or cytotoxic activity of IL-1 on A375S2 cells, indicating that the equine IL-1ra cDNA obtained in this study encodes biologically active equine IL-1ra.     

Myxoma virus encodes an alpha2,3-sialyltransferase that enhances virulence

A 4.7-kb region of DNA sequence contained at the right end of the myxoma virus EcoRI-G2 fragment located 24 kb from the right end of the 163-kb genome has been determined. This region of the myxoma virus genome encodes homologs of the vaccinia virus genes A51R, A52R, A55R, A56R, and B1R; the myxoma virus gene equivalents have been given the prefix M. The MA55 gene encodes a protein belonging to the kelch family of actin-binding proteins, while the MA56 gene encodes a member of the immunoglobulin superfamily related to a variety of cellular receptors and adhesion molecules. A novel myxoma virus early gene, MST3N, is a member of the eukaryotic sialyltransferase gene family located between genes MA51 and MA52. Detergent lysates prepared from myxoma virus-infected cell cultures contained a virally encoded sialyltransferase activity that catalyzed the transfer of sialic acid (Sia) from CMP-Sia to an asialofetuin glycoprotein acceptor. Analysis of the in vitro-sialylated glycoprotein acceptor by digestion with N-glycosidase F and by lectin binding suggested that the MST3N gene encodes an enzyme with Galbeta1,3(4)GlcNAc alpha2,3-sialyltransferase specificity for the N-linked oligosaccharide of glycoprotein. Lectin binding assays demonstrated that alpha2,3-sialyltransferase activity is expressed by several known leporipoxviruses that naturally infect Sylvilagus rabbits. The sialyltransferase is nonessential for myxoma virus replication in cell culture; however, disruption of the MST3N gene caused attenuation in vivo. The possible implications of the myxoma virus-expressed sialyltransferase in terms of the host's defenses against infection are discussed.     

Two naturally occurring alpha2,6-sialyltransferase forms with a single amino acid change in the catalytic domain differ in their catalytic activity and proteolytic processing

The alpha2,6-sialyltransferase (ST) is a Golgi glycosyltransferase that adds sialic acid residues to glycoprotein N-linked oligosaccharides. Here we show that two forms of alpha2,6-sialyltransferase are expressed by the liver and are encoded by two different RNAs that differ by a single nucleotide. The ST tyr possesses a Tyr at amino acid 123, whereas the ST cys possesses a Cys at this position. The ST tyr is more catalytically active than the ST cys; however, both are functional when introduced into tissue culture cells. The proteolytic processing and turnover of the ST tyr and ST cys proteins differ dramatically. The ST cys is retained intact in COS-1 cells, whereas the ST tyr is rapidly cleaved and secreted. Analysis of the N-linked oligosaccharides of these proteins demonstrates that both proteins enter the late Golgi. However, differences in ST tyr and ST cys proteolytic processing may be related to differences in their localization, because ST tyr but not ST cys is expressed at low levels on the cell surface. The possibility that the ST tyr is cleaved in a post-Golgi compartment is supported by the observation that a 20 degrees C temperature block, which stops protein transport in the trans Golgi network, blocks both cleavage and secretion of the ST tyr.     

Molecular cloning and expression of a novel human cDNA containing CAG repeats

A novel human cDNA containing CAG repeats, designated B120, was cloned by PCR amplification. An approximately 300-bp 3' untranslated region in this cDNA was followed by a 3426-bp coding region containing the CAG repeats. A computer search failed to find any significant homology between this cDNA and previously reported genes. The number of CAG trinucleotide repeats appeared to vary from seven to 12 in analyses of genomic DNA from healthy volunteers. An approximately 8-kb band was detected in brain, skeletal muscle and thymus by Northern blot analysis. The deduced amino-acid sequence had a polyglutamine chain encoded by CAG repeats as well as glutamine- and tyrosine-rich repeats, which has also been reported for several RNA binding proteins. We immunized mice with recombinant gene product and established a monoclonal antibody to it. On Western immunoblotting, this antibody detected an approximately 120-kDa protein in human brain tissue. In addition, immunohistochemical staining showed that the cytoplasm of neural cells was stained with this antibody. These findings indicated that B120 is a novel cDNA with a CAG repeat length polymorphism and that its gene product is a cytoplasmic protein with a molecular mass of 120 kDa.     

Molecular cloning and expression of NlaIII restriction-modification system in E. coli

The NlaIII restriction enzyme isolated from Neisseria lactamica recognizes the sequence 5'-CATG-3', cleaving after the G to generate a four base 3' overhang. The NlaIII methylase and a portion of the NlaIII endonuclease gene were cloned into E. coli by the methylase selection method, and the remaining portion of the NlaIII endonuclease gene was cloned by inverse PCR. The nucleotide sequence of the endonuclease gene and the methylase gene were determined. The NlaIII endonuclease gene is 693 bp, encoding a protein with predicted molecular weight of 26487. The NlaIII methylase gene was identical with that previously reported [Labbe, D., Joltke, H.J. and Lau, P.C. (1990) Cloning and characterization of two tandemly arranged DNA methyltransferse genes of Neisseria lactamica: an adenine-specific M.NlaIII and a cytosine-type methylase. Mol. Gen. Genet. 224, 101-110]. The endonuclease and methylase genes overlap by four bases and are transcribed in the same orientation. The endonuclease gene was cloned into an improved T7 vector, and a high level of NlaIII endonuclease expression was achieved in E. coli.