MOLECULAR-CLONING OF CYTIDINE MONOPHOSPHO-N-ACETYLNEURAMINIC ACID HYDROXYLASE - REGULATION OF SPECIES-SPECIFIC AND TISSUE-SPECIFIC EXPRESSION OF N-GLYCOLYLNEURAMINIC ACID
T. Kawano et al., MOLECULAR-CLONING OF CYTIDINE MONOPHOSPHO-N-ACETYLNEURAMINIC ACID HYDROXYLASE - REGULATION OF SPECIES-SPECIFIC AND TISSUE-SPECIFIC EXPRESSION OF N-GLYCOLYLNEURAMINIC ACID, The Journal of biological chemistry, 270(27), 1995, pp. 16458-16463
Cytidine monophospho-N-acetylneuraminic acid (CMP-NeuAc) hydroxylase,
which is the key enzyme for the synthesis of N-glycolylneuraminic acid
(NeuGc), has been purified from the cytosolic fraction of mouse liver
, as described in our previous paper. The amino acid sequences of the
purified CMP-NeuAc hydroxylase, and peptides obtained by lysylendopept
idase digestion, were used to synthesize specific oligonucleotide prim
ers. A mouse cDNA clone of the enzyme was obtained by a combination of
the polymerase chain reaction and rapid amplification of cDNA ends. T
he sequence of the clone contained an open reading frame coding for a
protein of 577 amino acids with a predicted molecular mass of 66 kDa.
The deduced sequence included the amino acid sequences obtained for th
e purified enzyme and peptides, and a complete match was obtained for
159 residues. The enzyme has neither a signal peptide sequence nor a m
embrane spanning domain, which is consistent with localization of the
enzyme in the cytosol. Transfection of a cDNA construct to COS-1 cells
increased the enzyme activity and the amount of NeuGc. Comparison of
the sequence with GenBank data indicated that no similar sequence has
been reported so far. Northern blot analysis of various mouse tissues
with the enzyme cDNA as a probe indicated that expression of NeuGc is
related to the level of CMP-NeuAc hydroxylase mRNA. On Southern blot a
nalysis with the same probe, cross-hybridizing bands were detected in
the human and fish genomes.