5-N-Acetylneuraminic acid (Neu5Ac) is the major sialic acid derivative found in animal cells. As a component of cell surface glycoconjugates, Neu5Ac is pivotal to numerous cellular recognition and communication processes including host-parasite interactions. A prerequisite for the synthesis of sialylated glycoconjugates is the activation of Neu5Ac to cytidine-monophosphate N-acetylneuraminic acid (CMP-Neu5Ac). The reaction is catalyzed by CMP-Neu5Ac-synthetase (syn), which, for unknown reasons, resides in the nucleus. Sequence analysis of the cloned murine CMP-Neu5Ac synthetase identified three clusters of basic amino acids (BC1-BC3) that might function as nuclear localization signals (NLS). In the present study chimeric protein and mutagenesis strategies were used to show that BC1 and BC2 are active NLS sequences when attached to the green fluorescent protein (enhanced GFP), but only BC2 is necessary and sufficient to mediate the nuclear import of CMP-Neu5Ac synthetase. Site-directed mutations identified the residues K 198 RXR to be essential for nuclear transport and Arg 202 to be necessary to complete the transport process. Cytoplasmic forms of CMPNeu5Ac synthetase generated by single site mutations in BC2 demonstrated that (i) enzyme activity is independent of nuclear localization, and (ii) Arg 199 and Arg 202 are involved in both nuclear transport and synthetase activity. Comparison of all known and predicted CMPsialic acid synthetases reveals Arg 202 and Gln 203 as highly conserved in evolution and critically important for optimal synthetase activity but not for nuclear localization. Combined, the data demonstrate that nuclear transport and enzyme activity are independent functions that share some common amino acid requirements in CMP-Neu5Ac synthetase.Sialic acids acids are a family of negatively charged, 9-carbon sugars that form terminal residues on cell surface glycoproteins and glycolipids and provide the bulk of the negative charge, which is characteristic for animal cell surfaces (for (8), as well as development (6, 9) and progression of malignancies (10 -12) are accompanied by alterations in the cellular sialylation pattern. In bacteria sialic acids are found as components of capsules and lipooligosaccharides and often are important virulence factors, mediating resistance to host defense mechanisms (reviewed in Refs. 13-15). For example, Neisseria meningitidis serogroup B (NmB), the major cause of meningitis outbreaks in the western hemisphere, expresses a capsular polysaccaride consisting of ␣2,8-linked sialic acid residues, exclusively. The polysialic acid (polySia) of the NmB capsule is identical to host expressed polySia, which represents a specific posttranslational modification of the neural cell adhesion molecule (for review, see Ref. 16). This classical example of antigenic mimicry explains the low serum response caused by NmB in infected individuals (17).A prerequisite for the incorporation of sialic acids into glycoconjugates is their activation as cytidine-monophosphate diester (CMP-Neu...

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.