Human STX Polysialyltransferase Forms the Embryonic Form of the Neural Cell Adhesion Molecule

Abstract

PST and STX are polysialyltransferases that form polysialic acid in the neural cell adhesion molecule (N-CAM), although it is not known why these two polysialyltransferases exist. In the present study, we have first isolated cDNA encoding human STX, which includes 5-untranslated sequence. Northern blot analysis, using this cDNA and PST cDNA previously isolated by us, demonstrated that PST and STX are expressed in different fetal and adult tissues. STX is primarily expressed in embryonic tissues, but only modestly in adult heart, brain, and thymus. PST, on the other hand, is continuously expressed in adult heart, brain, thymus, spleen, small and large intestines, and peripheral blood leukocytes. In various parts of adult brain, the relative amount of PST and STX appears to be substantially different depending on the regions. The analysis by in situ hybridization of mouse adult brain, however, suggests that polysialic acid in the hippocampal formation is synthesized by both STX and PST. HeLa cells doubly transfected with the isolated STX cDNA and N-CAM cDNA supported neurite outgrowth much better than HeLa cells expressing N-CAM alone. However, polysialic acid synthesized by PST appears to be a better substratum than that synthesized by STX. Moreover, the genes for PST and STX were found to reside at chromosome 5, band p21 and chromosome 15, band q26, respectively. These results, taken together, strongly suggest that PST and STX are expressed distinctly in tissue-specific and cell-specific manners and that they apparently have distinct roles in development and organogenesis.Polysialic acid is a developmentally regulated glycan composed of a linear homopolymer of ␣-2,8-linked sialic acid residues. Polysialic acid is mainly attached to the neural cell adhesion molecule (N-CAM) 1 and is more abundant in embryonic brain than adult brain (1-3). Presence of this large negatively charged carbohydrate modulates the adhesive property of N-CAM, and removal of polysialic acid increases binding between N-CAMs (4, 5). During embryonic development, the polysialylated embryonic from of N-CAM is restricted to migrating cells (6, 7), and the removal of polysialic acid from the N-CAM during embryonic development affects motor-axon projection patterns (8). Polysialylated N-CAM was also shown to attenuate cell-cell interactions mediated by other cell adhesion molecules (9 -11).Recently, we and others have cloned cDNAs encoding human, hamster, and mouse polysialyltransferases (PST for human, PST-1 for hamster, and ST8Sia IV for mouse, respectively) (12-14). The amino acid sequences of PST and PST-1 are more than 97% identical. Both PST and PST-1 directed the expression of polysialic acid on the cell surface. The same studies also revealed that PST is highly homologous to STX (sialyltransferase X, or ST8Sia II) (12, 13), and STX and PST have 59% identity at the amino acid level. STX was originally cloned as a developmentally regulated sialyltransferase from the rat fetal brain (15). Although the substrate specificity of STX was not kno...

Differential Effects of Unnatural Sialic Acids on the Polysialylation of the Neural Cell Adhesion Molecule and Neuronal Behavior

“…We examined the effects of ManBut, a PSA synthesis inhibitor, on the neurite outgrowth of chick dorsal root ganglia neurons when cultured on a substratum of HeLa-NCAM/STX cells. Consistent with earlier studies, we found that neurite outgrowth was promoted by the polysialylation of NCAM on HeLa cell substrata (7,8,40). In one such experiment, summarized in Fig.…”

“…Root Ganglion Neurons-The expression of polysialic acid on neural cells has a significant effect on cell migration and axon outgrowth (8,14,39). We examined the effects of ManBut, a PSA synthesis inhibitor, on the neurite outgrowth of chick dorsal root ganglia neurons when cultured on a substratum of HeLa-NCAM/STX cells.…”

“…Cell Lines and Culture-Clonal HeLa cell lines that stably expressed NCAM-140 with or without concomitant expression of either polysialyltransferases STX or PST were a generous gift from Dr. Minoru Fukuda (The Burnham Institute) (7,8). The cells were maintained in Dulbecco's modified Eagle's medium/10% heat-inactivated fetal bovine serum supplemented with 400 g/ml G418 and 400 g/ml hygromycin (HeLa-NCAM/STX and HeLa-NCAM/PST only).…”

“…Therefore, the effect of PSA expression on neurite growth was only examined using HeLa substratum cells that stably expressed NCAM and STX. Sensory neurons from the dorsal root ganglia were isolated from 10-day-old chick embryos, dissociated with 0.5% trypsin, and seeded onto confluent cultures of HeLa monolayer in minimal essential medium containing 10% fetal bovine serum and 10 ng/ml nerve growth factor (7,8). The neuron/HeLa co-cultures were grown for 15 h, fixed using 4% formaldehyde in PBS, stained with antineurofilament mAb RM0270 (37), and visualized by immunofluorescence.…”

“…There are two major polysialyltransferases, STX and PST, which are responsible for polysialylation (4). STX (ST8SiaII) is predominately expressed during development, whereas PST (ST8SiaIV) is the major polysialyltransferase expressed in the adult central nervous system (5)(6)(7)(8)(9)(10)(11). The neural cell adhesion molecule (NCAM) appears to be, by far, the primary substrate for polysialylation because the absence of NCAM expression results in the almost complete loss of PSA immunoreactivity (12).…”