Molecular cloning of a unique CMP-sialic acid synthetase that effectively utilizes both deaminoneuraminic acid (KDN) and N-acetylneuraminic acid (Neu5Ac) as substrates

Abstract

2-keto-3-deoxy-D-glycero-D-galacto-nononic acid (KDN) is a sialic acid (Sia) that is ubiquitously expressed in vertebrates during normal development and tumorigenesis. Its expression is thought to be regulated by multiple biosynthetic steps catalyzed by several enzymes, including CMP-Sia synthetase. Using crude enzyme preparations, it was shown that mammalian CMP-Sia synthetases had very low activity to synthesize CMP-KDN from KDN and CTP, and the corresponding enzyme from rainbow trout testis had high activity to synthesize both CMP-KDN and CMP-N-acetylneuraminic acid (Neu5Ac) (Terada et al. [1993] J. Biol. Chem., 268, 2640-2648). To demonstrate if the unique substrate specificity found in the crude trout enzyme is conveyed by a single enzyme, cDNA cloning of trout CMP-Sia synthetase was carried out by PCR-based strategy. The trout enzyme was shown to consist of 432 amino acids with two potential nuclear localization signals, and the cDNA sequence displayed 53.8% identity to that of the murine enzyme. Based on the Vmax/Km values, the recombinant trout enzyme had high activity toward both KDN and Neu5Ac (1.1 versus 0.68 min(-1)). In contrast, the recombinant murine enzyme had 15 times lower activity toward KDN than Neu5Ac (0.23 versus 3.5 min(-1)). Northern blot analysis suggested that several sizes of the mRNA are expressed in testis, ovary, and liver in a tissue-specific manner. These results indicate that at least one cloned enzyme has the ability to utilize both KDN and Neu5Ac as substrates efficiently and is useful for the production of CMP-KDN.

Nuclear Localization Signal of Murine CMP-Neu5Ac Synthetase Includes Residues Required for Both Nuclear Targeting and Enzymatic Activity

“…The photos are merged in Fig. 6A shows a comparison of bacterial CMP-sialic acid synthetases (19 -24), murine CMP-Neu5Ac-syn (25), and the recently cloned rainbow trout CMP-Kdn-syn, which exhibits a similar affinity for Neu5Ac and Kdn (26). Additionally, the sequences of putative CMP-sialic acid synthetases that have been identified based on homology were aligned in Evolutionary Conserved Residues in BC2 Are Important for Enzymatic Activity-Our finding that Arg 202 is part of the active site in murine CMP-Neu5Ac-syn is in excellent agreement with crystal structure data recently obtained for the NmB CMP-Neu5Ac-syn (45).…”

“…The nucleotide sequences of six bacterial (19 -24) and two vertebrate CMP-sialic acid-syn (25,26) are available. The alignment of the deduced protein sequences revealed five highly conserved motifs indicating a common ancestor (25,26). Thus the CMP-sialic acid synthetases provide the first example for an evolutionary conservation from bacteria to vertebrates among the sialic acid metabolizing enzymes.…”

Identification and partial characterization of soluble and membrane-bound KDN(deaminoneuraminic acid)-glycoproteins in human ovarian teratocarcinoma PA-1, and enhanced expression of free and bound KDN in cells cultured in mannose-rich media

“…CMP-KDN synthase that effectively activates KDN has been identified in rainbow trout testis [14,15]. KDN-transferase that is involved in the formation of α2 →8 KDN-linkage was also suggested in the ovary of rainbow trout [16].…”

“…In mammals no enzyme that specifically recognizes KDN is known. However, it has been shown, both in vivo and in vitro, that KDN and KDN-containing glycans can be synthesized via reactions catalyzed by the enzymes involved in the biosynthesis of Neu5Ac and Neu5Ac-containing glycans in mammals, although the formation of KDN and CMP-KDN is far less efficient than that of Neu5Ac and CMP-Neu5Ac [13][14][15]17]. In vitro experiment using a commercial α2, 6-sialyltansferase showed transfer of KDN from CMP-KDN to other sugar residues appeared to proceed efficiently [18].…”

Identification and Biochemical Characterization of Two Functional CMP-Sialic Acid Synthetases in Danio rerio

“…KDN, however, was the preferred substrate for dreCmas2, which showed poor in vitro activity with Neu5Ac. This observation was unexpected because dreCmas2 resembles omyCmas in substrate specificity (KDNϾ ϾNeu5AcϾNeu5Gc), whereas on the amino acid level, omyCmas and dreCmas1 are more closely related (36). With further identification and characterization of cmas genes from other fish and higher vertebrate species, the apparent discrepancies between sequence homologies and similarities in enzymatic properties may be resolved, and the importance of single amino acids in functional domains may be elucidated.…”