Cellular Site of Synthesis and Dynamics of Cell Surface Re‐Expression of Polysialic Acid of the Neural Cell Adhesion Molecule

Abstract

Homopolymers of a-2,8-ketosidically linked sialic acid (polysialic acid) represent a posttranslational modification which, in mammals, appears to be unique for the neural cell adhesion molecule and the a subunit of sodium channels in brain. Under steady-state conditions, polysialic acid is detectable in the plasma membrane of different cell types but not in the cytoplasm. We have studied the site of synthesis and the cell surface re-expression of polysialic acid in a clonal subline of small cell lung carcinoma using the monoclonal antibody 735 and bacteriophage endosialidase, both specific reagents for polysialic acid. After enzymic removal, cell surface polysialic acid re-expression reached control levels only after 5 days. When Golgi to plasma membrane transport of endosialidase-treated cells was blocked by culture at 20°C or in the presence of monensin at 37"C, de-novosynthesized polysialic acid became detectable in the Golgi apparatus. Our data show that synthesis of polysialic acid of the neural cell adhesion molecule with a degree of polymerization of at least nine occurs intracellular in the Golgi apparatus of a human small cell lung carcinoma cell line.Sialic acids are ubiquitous in oligosaccharide side chains of glycoconjugates of a wide variety of phyla [ 11. Homopolymers of a-2,8-ketosidically linked sialic acid (polySia) occur more specifically [2, 31 and, in mammals, have been reported to be associated with only two proteins, the neural cell adhesion molecule (NCAM) [4] and the a subunit of the voltagegated sodium channels in rat brain [5]. PolySia of NCAM modulates the homophilic binding properties of this and other cell-adhesion molecules [6-91 and, thereby, is involved in various cellular phenomena during organogenesis [lo] and regenerative processes [ll], as well as malignant growth [12].The genetics [13, 141 and molecular mechanisms [15, 161 of the synthesis of polySia in bacteria are well known. A multigenic cluster encodes for various proteins required for the synthesis, translocation and export of polySia. Synthesis and activation of sialic acid occurs in the cytosol, followed by the formation of polySia linked to undecaprenol at the cytoplasmic surface of the inner membrane by a membraneassociated polysialyltransferase. Phospholipid substitution at the inner membrane of the polySia is preceding by the translocation of fully assembled polySia across the membranes to the surface. In contrast, little is known about the mechanism and site of polySia biosynthesis in eukaryotes. In brain, the presence of a developmentally regulated sialyltransferase activity, which is required for the polysialylation of exogenous NCAM, has been demonstrated in a smooth membrane fraction containing Golgi membranes . From the results of chemical analysis [20] and overexpression of P-galactoside a-2,6-sialyltransferase during embryogenesis of Xenopus [21], it was suggested the pol- The present study was undertaken to obtain more direct information about the cellular site of synthesis of polySia. For this purpose, w...

Expression of tetraspanins in human lung cancer cells: frequent downregulation of CD9 and its contribution to cell motility in small cell lung cancer

“…Although a3b1 does not mediate strong adhesion to FN, a3b1-dependent cell migration on FN has been previously reported (Yauch et al, 1998). SCLC also expresses another adhesion molecule, N-CAM, which mediates homophilic cell-cell adhesion, and may be involved in SCLC metastasis (Scheidegger et al, 1994). In the present study, however, the exogenous CD9 and In contrast to NSCLC, SCLC easily spreads into submucosal tissues and metastasizes to regional lymph nodes.…”

“…CD9 associates with b1 integrins but not with N-CAM SCLC reportedly expresses adhesion proteins, b1 integrins and N-CAM, and both of these molecules are likely to regulate motility and metastasis of SCLC (Scheidegger et al, 1994;Bredin et al, 1998). To explore the mechanisms involved in the reduction of OS3-R5-pZCD9 motility, coimmunoprecipitation experiments using MoAbs against these molecules were performed (Figure 4b,left panel).…”

A nude mice model of human rhabdomyosarcoma lung metastases for evaluating the role of polysialic acids in the metastatic process

“…inoculated mice but in none of the s.c. injected ones probably because the s.c. environment is not favorable. In keeping with this hypothesis, other cell lines expressing PSA-NCAM such as the SCLC line NCL-H69 does not lead to lung metastases if injected s.c. (Scheiddeger et al, 1994). Moreover our recent work (Daniel et al, 2000) indicates that the PSA expressing SMtTW4 cells did not metastasize if grafted under the rat neck skin but did if implanted under the kidney capsule.…”

“…PSA-NCAM is expressed by several human neurectodermal tumors, including medulloblastomas, subclasses of neuroblastomas, small cell lung carcinomas, and rhabdomyosarcomas (Figarella-Branger et al, 1990;Molenaar et al, 1991;Scheiddeger et al, 1994). These tumors are known for their aggressive behavior and their ability to form metastases.…”

Biochemical Engineering of Surface α2–8 Polysialic Acid for Immunotargeting Tumor Cells

“…Therefore we propose to introduce modified carbohydrate antigens on the surface of cancer cells to which a strong immunogenic response can be induced. We chose ␣2-8 polysialic acid (polysialic acid) as our target antigen, because although not a universal cancer antigen, it is found on a number of important cancers (11)(12)(13), and there is strong evidence that it is associated with metastasis (12,14). In addition we have previously demonstrated that in its N-propionylated form (NPr polysialic acid) 1 it is an excellent immunogen (15,16).…”