Young-Choon Lee

Molecular Cloning and Functional Expression of Two Members of Mouse NeuAcα2,3Galβ1,3GalNAc GalNAcα2,6-Sialyltransferase Family, ST6GalNAc III and IV

Two cDNA clones encoding NeuAcα2,3Galβ1,3GalNAc GalNAcα2,6-sialyltransferase have been isolated from mouse brain cDNA libraries. One of the cDNA clones is a homologue of previously reported rat ST6GalNAc III according to the amino acid sequence identity (94.4%) and the substrate specificity of the expressed recombinant enzyme, while the other cDNA clone includes an open reading frame coding for 302 amino acids. The deduced amino acid sequence is not identical to those of other cloned mouse sialyltransferases, although it shows the highest sequence similarity with mouse ST6GalNAc III (43.0%). The expressed soluble recombinant enzyme exhibited activity toward NeuAcα2, 3Galβ1,3GalNAc, fetuin, and GM1b, while no significant activity was detected toward Galβ1,3GalNAc or asialofetuin, or the other glycoprotein substrates tested. The sialidase sensitivity of the14C-sialylated residue of fetuin, which was sialylated by this enzyme with CMP-[14C]NeuAc, was the same as that of ST6GalNAc III. These results indicate that the expressed enzyme is a new type of GalNAcα2,6-sialyltransferase, which requires sialic acid residues linked to Galβ1,3GalNAc residues for its activity; therefore, we designated it mouse ST6GalNAc IV. Although the substrate specificity of this enzyme is similar to that of ST6GalNAc III, ST6GalNAc IV prefers O-glycans to glycolipids. Glycolipids, however, are better substrates for ST6GalNAc III.

Molecular cloning and expression of chick embryo Galβ1,4GlcNAcα2,6‐sialyltransferase

DNA clones encoding beta-galactoside alpha 2,6-sialyltransferase have been isolated from chick embryonic cDNA libraries using sequence information obtained from the conserved amino acid sequence of the previously cloned enzymes. The cDNA sequence revealed an open-reading frame coding for 413 amino acids, and the deduced amino acid sequence showed 57.6% identity with the sequence of rat liver Gal beta 1,4GlcNAc alpha 2,6-sialyltransferase. The primary structure of this enzyme suggested a putative domain structure, similar to structures found in other glycosyltransferases, consisting of a short N-terminal cytoplasmic domain, a signal-membrane anchor domain, a proteolytically sensitive stem region and a large C-terminal active domain. The identity of this enzyme was confirmed by construction of a recombinant sialyltransferase in which the N-terminus part including the cytoplasmic tail, signal anchor domain and stem region was replaced with an immunoglobulin signal peptide sequence. The expression of this recombinant protein in COS-7 cells resulted in secretion of a catalytically active and soluble form of the enzyme into the medium. The expressed enzyme exhibited activity only towards the disaccharide moiety of Gal beta 1,4GlcNAc in glycoproteins.

Two step single primer mediated polymerase chain reaction. Application to cloning of putative mouse, β-galactoside α2,6-sialyltransferase cDNA

Abstract Using the 2 step single primer mediated polymerase chain reaction(PCR), mouse β-galactoside (α2,6-sialyltransferase cDNA was cloned. Single primer mediated PCR is a method to amplify a particular DNA fragment beyond its known sequence region. It employs only one primer for the reaction. Compared to other PCR methods to amplify an adjacent sequence of known DNA fragment, this method requires no enzymatic manipulation on template DNA and is applicable to a template on long DNA fragment. First, a short DNA fragment of the enzyme was obtained from mouse cDNA by the usual PCR method using degenerate primers synthesized according to a relatively conserved region in rat and human β-galactoside (α2,6-sialyltransferase. Four primers were synthesized based on this sequence, then 2 step single primer mediated PCR were performed to obtain 5′ and 3′ flanking sequences of this short fragment resulting in 1.0 kb and 1.3 kb fragments being amplified respectively. The integrity of the two fragments was confirmed by an additional PCR using primers synthesized according to the joined sequence, which contained 1.2kb complete putative mouse β-galactoside α2,6-sialyltransferase coding region. The result showed that the specificity and consequently applicability of the single primer mediated PCR for amplifying a particular DNA fragment beyond known sequence region was remarkably improved by the successive 2nd reaction.

Expression of mouse Gal,ß14GlcNAc α2,6-sialyl-transferase in an insoluble form in Escherichia coli and partial renaturation☆

Abstract Mouse Galsl,4GlcNAc α2,6-sialyltransferase was produced in an insoluble form in Escherichia coli cells harboring expression plasmids. The insoluble protein was solubilized with 8 M urea and diluted for renaturation of the enzyme. The substrate specificity and kinetic parameters, except for the specific activity, of the renatured enzyme were similar to those of the enzyme obtained from rat liver. These results suggest that a bacterial expression system is a potentially powerful tool for the large scale production of sialyltransferases and for elucidating the molecular mechanisms of sialyltransferases.

Cloning and Sequence Analysis of Another Shiga-Like Toxin IIe Variant Gene (slt-IIera) from an Escherichia coli R107 Strain Isolated from Rabbit

An Escherichia coli R107 strain (O26 serotype) producing a Shiga‐like toxin IIe variant (SLT‐IIera) was isolated from the mesenteric lymph node of a freshly dead rabbit carcass. The entire structural gene for this SLT‐IIera was cloned from chromosomal DNA by PCR using primers based on previously published slt‐IIe sequences. Nucleotide sequence analysis indicated that the slt‐llera gene was very similar to slt‐IIe (formerly called slt‐IIy) from E. coli strains S1191 and 412; five and one nucleotide changes were detected in A and B subunits, respectively, which resulted in changes in amino acid sequences of the corresponding subunits by three and one residues. Recombinant SLT‐IIera and SLT‐IIe produced using an E. coli host‐vector system showed similar cytotoxicity, suggesting that the variations in the structural gene of SLT‐IIera have no significant effect on cytotoxic level.