The rainbow trout CMP-sialic acid synthetase utilises a nuclear localization signal different from that identified in the mouse enzyme

Abstract

The terminal sugar sialic acid (Sia) plays a pivotal role in cell-cell interaction and recognition. A prerequisite for the biosynthesis of sialoglycoconjugates is the activation of Sia to cytidine monophosphate-Sia (CMP-Sia), by CMP-Sia synthetases (CMP-Sia-syn). CMP-Sia-syn are conserved from bacteria to man, and have been found to reside in the nucleus of all vertebrate species analysed to date. We previously cloned the CMP-Sia-syn from rainbow trout (rt) and identified three clusters of basic amino acids (BC) that might act as nuclear localization signals (NLS). Here, we utilised chimeric proteins and rt CMP-Sia-syn mutants in which putative NLS sequences were deleted, to identify the nuclear transport signal. Divergent from the mouse enzyme, where the crucial NLS is part of the enzyme's active site, in the rt CMP-Sia-syn the NLS and active site are disparate. The crucial NLS in the fish enzyme is bipartite and the functionality depends on a free N-terminus. Comparative analysis of all putative rt NLS in mouse and fish cells identified a second inferior motif (rtBC5-6), which was functional only in fish cells suggesting some differences in transport mechanism or folding variabilities in fish. Moreover, based on computational analyses of putative CMP-Sia-syn from distant deuterostomian organisms it was concluded that CMP-Sia-syn nuclear localization is a relatively recent invention, originating in echinoderms. In summary, our data describing structural differences in the NLS of vertebrate CMP-Sia-syn, and the independence of Sia activation from the subcellular localization of the enzyme, provide supporting evidence that nuclear localization is linked to a second yet unknown function.

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

“…Deletion of the central basic cluster (Figs. 1 and 7) abolished enzymatic activity as reported for mouse (14), rainbow trout (15), and zebrafish CMAS enzymes (this study). X-ray analysis of the enzymatically active domain of murine CMAS confirmed that this amino acid stretch is part of the active site (31).…”

“…So far, vertebrate CMAS enzymes, independent of tissue or species, have been predominantly found in nuclear fractions with just minor amounts in other compartments (11,13). Nuclear sequestration has been confirmed with recombinant proteins in different cellular systems (15,41,42). In this study, we identified and characterized the first cytosolic vertebrate CMAS.…”

“…This unusual intracellular localization was first recognized in the lens epithelial layer by E. L. Kean in 1969 (12) and later confirmed in a variety of biochemical studies using different tissues and species (11,13). Although the nuclear localization signals have been identified in recombinant mouse and rainbow trout CMAS (14,15), the biological relevance of this unusual intracellular localization still remains an enigma. In this study, we identified and cloned two distinct cmas genes in zebrafish.…”

“…The five conserved CMAS motifs essential for enzymatic activity are marked with dashed lines. The nuclear localization signals of mmuCMAS and omyCmas are underlined (14,15). Basic clusters identified in dreCmas1 (BC1 to BC4) and dreCmas2 (dre2-BC) are boxed in gray and white, respectively.…”

Humanization of recombinant glycoproteins expressed in insect cells

“…The amino acid sequence of this UDP-Gal transporter is similar to that of human UDP-GlcNAc transporter (3); however, it does not transport UDP-GlcNAc (20). CMPNeu5Ac is synthesized in the nucleus in mammalian cells (22)(23)(24), and it is believed that CMP-Neu5Ac is transported through the cytoplasm to the Golgi by a speciˆc transporter. However, since Drosophila CMP-SAS is localized to the Golgi (19), insect cells may not require a speciˆc transporter for CMP-Neu5Ac.…”