Developmental regulation of polysialic acid synthesis in mouse directed by two polysialyltransferases, PST and STX

Edgar Ong 2 3 Jun Nakayama 1 2 3 Kiyohiko Angata 2 3 Luane Reyes 2 3 Tsutomu Katsuyama 1 2 Yasumasa Arai 0 2 Minoru Fukuda 2 3 0 Department of Anatomy, Juntendo University School of Medicine , Tokyo 113, Japan 1 Central Clinical Laboratories, Shinshu University Hospital , Matsumoto 390, Japan 2 10901 North Torrey Pines Road, La Jolla, CA 92037 3 Glycobiology Program, La Jolla Cancer Research Center, The Burnham Institute , La Jolla, CA 92037, USA - Received on September 10, 1997; revised on November 11, 1997; accepted on November 11, 1997 4These two authors contributed equally to this work Polysialic acid is a developmentally regulated carbohydrate attached to the neural cell adhesion molecule, N-CAM, and abundant in embryonic tissues. There is increasing evidence that polysialic acid reduces N-CAM adhesion, thereby promoting neurite outgrowth and cellular mobility. It has been shown that two enzymes, polysialyltransferase, PST, and sialyltransferase X, STX, form polysialic acid on N-CAM. However, it is not known how these two enzymes contribute to polysialylation. In order to determine how the expression of PST and STX leads to polysialic acid synthesis during mouse development, the expression of PST and STX transcripts were evaluated by Northern blot analysis, competitive reverse transcriptionpolymerase chain reaction and in situ hybridization, and those results were correlated to the expression of polysialic acid. The results obtained by these analyses demonstrated that both PST and STX transcripts were barely detected at embryonic day 8 (E8) but increased after E9. PST and STX transcripts were present in substantial quantity between E11 and E15, coinciding with the period when maximum synthesis of polysialic acid is required. Ten days after birth, the level of STX transcript declined substantially, whereas the level of PST transcript only gradually declined and persisted in the adult brain. These results, taken together, strongly suggest that PST and STX coordinately synthesize polysialic acid during development. At the same time, they are expressed differentially in tissue-specific and cell-type-specific manners, suggesting that PST and STX may have distinct roles in development and organogenesis. Introduction Polysialic acid is a developmentally regulated carbohydrate composed of a linear homopolymer of a -2,8-linked sialic acid (Finne, 1982). Polysialylated N-CAM is abundant in embryonic tissues, whereas the majority of N-CAM in adult tissues lacks this unique glycan. There is increasing evidence that polysialylated N-CAM may promote cell migration and enhance neurite outgrowth and branching during development and neural regeneration (Edelman, 1985; Rutishauser and Landmesser, 1996). Polysialic acid is thought to modulate the functional properties of N-CAM by rendering it less adhesive to itself (homophilic binding) (Hoffman and Edelman, 1983; Sadoul et al., 1983) or to other cell surface receptors (heterophilic binding). In the latter case, it has been shown that N-CAM engages in cooperative interactions with L1 on the same membrane (cis-interaction) (Kadman et al., 1990). The studies on N-CAM knock-out mice demonstrated a defect in spatial learning and memory, due to an anomaly in the olfactory bulb and hippocampus where polysialic acid is continuously synthesized in the adult brain (Tomasiewicz et al., 1993; Cremer et al., 1994). By using N-CAM knock-out mice and endoneuraminidase (endo-N) treatment, recent studies have demonstrated that polysialic acid is required for the migration of the cells in the subventricular zone of the olfactory bulb (Hu et al., 1996). Similarly, endo-N treatment of hippocampal cells in organotypic slice cultures was shown to prevent the induction of long-term potentiation, presumably by impairing the induction of synaptic plasticity (Muller et al., 1996). These results, taken together, strongly suggest that polysialylated N-CAM plays a critical role during development and neural regeneration. The cDNAs encoding human, hamster and mouse polysialyltransferases were cloned (PST for human, PST-1 for hamster, ST8Sia IV for mouse; Eckhardt et al., 1995; Nakayama et al., 1995; Yoshita et al., 1995). The amino acid sequences of PST and PST-1 are more than 97% identical, and both PST and PST-1 directed the expression of polysialic acid on the cell surface. The same studies also revealed that PST and PST-1 are highly homologous to sialyltransferase X, STX (ST8Sia II) which had been cloned from rat embryonic brain (Livingston and Paulson, 1993), suggesting that STX is a polysialyltransferase. In support of this speculation, STX was also shown to direct the synthesis of polysialic acid (Kojima et al., 1995; Scheidegger et al., 1995) and to form polysialic acid in both wild-type N-CAM as well as soluble chimeric N-CAM proteins (Kojima et al., 1996; Angata et al., 1997). Consistent with the presumed roles of polysialic acid, it has been shown that PST and STX facilitate neurite outgrowth (Nakayama et al., 1995; Angata et al., 1997). After transfecting HeLa cells with human PST or STX and N-CAM cDNAs or N-CAM cDNA alone, they were used as the substratum for the neurite outgrowth assay. When neurons derived from embryonic chick brains were grown on these substrata, neurites were much longer and more branched on the substratum cells expressing polysialic acid and N-CAM than those on the substratum expressing N-CAM alone. By using an in vitro assay system, both PST and STX were shown to add polysialic acid to fetuin and soluble chimeric Fig. 1. Northern blot analysis of the transcripts of mPST, mSTX, and N-CAM in various adult mouse tissues. The same blot was sequentially probed by 32P-labeled mPST cDNA (PST), mSTX cDNA (STX), mouse N-CAM cDNA (N-CAM), and b -actin cDNA (b -actin). The migration positions of RNA size markers are shown at the left. N-CAM (Kojima et al., 1996; Mhlenhoff et al., 1996; Nakayama and Fukuda, 1996). This demonstrates that either PST or STX alone can form polysialic acid by adding the first a -2,8-linked sialic acid to a -2,3-linked sialic acid in an acceptor, followed by multiple addition of a -2,8-linked sialic acid residues. In this context, PST and STX thus appear to share common enzymatic properties. Expression of PST and STX may directly regulate the function of N-CAM by controlling PSA synthesis. On the other hand, it is not known how the expression of these two enzymes leads to polysialic acid formation. In order to address this question, in the present study the expression of PST and STX transcripts were evaluated during mouse development by Northern blot analysis, RT-PCR and in situ hybridization of PST and STX transcripts, and these findings were correlated to the expression of polysialic acid. The results obtained strongly suggest that PST and STX are expressed differentially, but in some cases overlappingly in specific tissues, and coordinately contribute to polysialic acid synthesis. Results Iso (...truncated)