Identification and partial characterization of soluble and membrane-bound KDN(deaminoneuraminic acid)-glycoproteins in human ovarian teratocarcinoma PA-1, and enhanced expression of free and bound KDN in cells cultured in mannose-rich media.

KDN (Deaminoneuraminic acid, or deaminated neuraminic acid) is a minor but biosynthetically independent member of the sialic acid. Human occurrence of KDN has already been established, although its level is so little that it is often undetectable by conventional sialic acid analysis. Elevated expression of KDN in fetal cord blood cells and some malignant tumor cells have been reported. However, in mammalian cells and tissues KDN mostly occurs as the free sugar and little occurred conjugated to glycolipids and/or glycoproteins. A positive correlation between the ratio of free KDN/free Neu5Ac in ovarian adenocarcinomas and the stage of malignancy has been noted for diagnostic use. We hypothesized that elevated expression of KDN in mammalian systems may be closely related to elevated activities of enzymes involved in the formation of sialoglycoconjugates and/or aberrant supply of the precursor sugar, mannose, used in the biosynthesis of KDN. In this study we used human ovarian teratocarcinoma cells PA-1 to further analyze KDN expression in human cells. Major findings reported in this paper are, (i) a 30 kDa KDN-glycoprotein immunostainable with monoclonal antibody, mAb.kdn3G, (specific for the KDNalpha2 --> 3Galbeta1--> epitope) and sensitive to KDNase was identified in the membrane fraction of the cell: (ii) a 49 kDa KDN-glycoprotein that is not reactive with mAb.kdn3G but is sensitive to KDNase was identified in the soluble fraction: and (iii) PA-1 cells showed unique response to mannose added to the growth medium in that the levels of both free and bound forms of KDN are elevated. This is the first report on the identification of mammalian KDN-glycoproteins by chemical and biochemical methods.

Discovery of an alpha 2,9-PolyNeu5Ac glycoprotein in C-1300 murine neuroblastoma (clone NB41A3).

alpha2,8-PolyNeu5Ac is expressed on neural cell adhesion molecules during embryogenesis and also re-expressed on certain tumors. PolyNeu5Ac is therefore an oncodevelopmental antigen, has important regulatory effects on the adhesive and migratory behavior of neural cells, and is thus crucial to synaptic plasticity. Until now, alpha2,9-polyNeu5Ac, a linkage isomer of alpha2,8-polyNeu5Ac, has long been thought to occur only in capsules of neuroinvasive Neisseria meningitidis group C bacteria. Here we report the unexpected discovery of alpha2,9-polyNeu5Ac in a new cell adhesion-related glycoprotein on the membrane of C-1300 murine neuroblastoma cells (clone NB41A3). We also report the expression of alpha2,9-polyNeu5Ac was affected by cell growth and retinoic acid-induced differentiation. Occurrence of the linkage isomer of alpha2,8-polyNeu5Ac has been left unrecognized by conventional methods using biological diagnostic probes for alpha2,8-polyNeu5Ac. Thus, our discovery may change contemporary views of biology and pathology of polysialic acid and open new avenues for the development of anti-neural tumor drugs.

Dynamic change of neural cell adhesion molecule polysialylation on human neuroblastoma (IMR-32) and rat pheochromocytoma (PC-12) cells during growth and differentiation.

Polysialic acid (PSA) is a regulatory epitope of neural cell adhesion molecule (NCAM) in homophilic adhesion of neural cells mediated by NCAM, is also known to be re-expressed in several human tumors, thus serves as an oncodevelopmental antigen. In this study, using a recently developed ultrasensitive chemical method in addition to immunochemical methods, growth stage-dependent and retinoic acid (RA)-induced differentiation-dependent changes of PSA expression in human neuroblastoma (IMR-32) and rat pheochromocytoma (PC-12) cells were analyzed both qualitatively and quantitatively. Both IMR-32 and PC-12 cells expressed PSA on NCAM, and the level of PSA expressed per unit weight of cells increased with post-inoculation incubation time. The most prominent feature was seen at the full confluence stage. RA induced neuronal differentiation in both IMR-32 and CP-12 cells that paralleled the change in the PSA level. Chemical analysis revealed the presence of NCAM glycoforms differing in the degree of polymerization (DP) of oligo/polysialyl chains, whose DP was smaller than 40. DP distribution of PSA was different between the cell lines and was changed by the growth stage and the RA treatment. Thus DP analysis of PSA is important in understanding both mechanism and biological significance of its regulated expression.