Sialidase L, a novel NeuAc(alpha)2--3Gal-specific sialidase from the leech, Macrobdella decora: Purification, characterization, cloning and expression
Description
Sialidase L releases 2,7-anhydro-NeuAc from sialoglyco-conjugates (Li, Y. -T. Nakagawa, H., Ross, S. A., Hansson, G., and Li, S. -C. (1990) J. Biol. Chem. 265, 21629-21633). This enzyme has been purified from Macrobdella leeches. The final preparation gives a single protein band on SDS-PAGE with a molecular mass of 84 kDa. The pI is determined to be 6.0 using isoelectric focusing. With 4-methylumbelliferyl-$\alpha$-NeuAc as substrate, the pH optimum is between pH 5.5 and 7.0. Unlike regular sialidases, sialidase L is not inhibited by 2-deoxy-2,3-dehydro-NeuAc. Among various sialoglycoconjugates tested, sialidase L cleaves only the NeuAc$\alpha2\to3$Gal linkage. NeuAc$\alpha2\to6$Gal, NeuAc$\alpha2\to6$GalNAc, NeuAc$\alpha2\to6$GlcNAc, NeuAc$\alpha2\to8$NeuAc and NeuAc$\alpha2\to9$NeuAc linkages are not hydrolyzed. It should become useful for the selective cleavage of NeuAc$\alpha2\to3$Gal linkages in sialoglycoconjugates without destroying other sialosyl linkages A 2.5-kilobase cDNA of sialidase L was cloned by a combination of methods based on polymerase chain reactions. The composite cDNA sequence reveals an open reading frame coding for 762 amino acids, including a putative 28-residue signal peptide at the N-terminus. The result suggests that sialidase L is a secretory enzyme. The coding sequence excluding the putative signal peptide of sialidase L was over-expressed in Escherichia coli. The purified recombinant enzyme was characterized to be as active as the enzyme isolated from the leech. It also possessed the strict NeuAc$\alpha2\to3$Gal linkage specificity and released the unique cleavage product, 2,7-anhydro-NeuAc from sialoglycoconjugates. The deduced amino acid sequence of sialidase L exhibits little similarity with other reported sialidases. However, sialidase L contains a conserved 'FRIP region' and four repeating 'Asp box' motifs that align well with the corresponding positions of bacterial sialidases. The predicted $\beta$-strand structures near the conserved motifs of sialidase L are similar to those of Salmonella typhimurium sialidase. Several conserved single amino acid residues of bacterial sialidases, including those known to be involved in the active site of Salmonella enzyme, are conserved in the deduced amino acid sequence of sialidase L. This observation suggests that part of the catalytic mechanism of sialidase L may be similar to the ordinary sialidase