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 differentiationdependent 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.Sialic acid (Sia) 1 residues are found in monomeric form almost exclusively at the non-reducing ends of various glycan chains and also occur in polymeric forms (PSA) in nature less frequently, less abundantly, and less diversely than the monomeric residues (e.g. Ref. 1). ␣238-Linked poly(Neu5Ac) on neural cell adhesion molecule (NCAM) is a universal feature of vertebrates and is known to be developmentally regulated. NCAM glycoforms having PSA with higher DP are more abundant in the fetal brain of vertebrates, whereas the majority of NCAM in adult does not contain PSA. High-DP PSA is considered to be involved in developmental regulation of homophilic neural cell adhesion and migration during maturation of embryonic brain (2). High-DP PSA-NCAM is also known to be re-expressed in certain types of tumors, and thus PSA chains with high DP are referred to as an oncofetal or oncodevelopmental antigen (3).In our recent studies (4 -9), we have developed an ultrasensitive and selective method (DMB/HPLC-FD) for determination of the length or DP of PSA chains expressed on NCAM at various developmental stages of embryonic chicken brain (7). We found that DP of PSA chains on NCAM is indeed mostly Յ40 (7-9) and not Ͼ55 (or even Ͼ100) as estimated previously (10) for PSA-glycopeptides from human neuroblastoma cells. Furthermore, our previous results (7) supported the view that there exist numerous NCAM glycoforms differing in their DP of PSA chains. The presence of these PSA chains may be necessary for coarse and fine-tuning of the homophilic binding property of NCAM, and both the post-translational biosynthesis of PSA and the secretion of these various NCAM glycoforms are probably physiologically regulated. We therefore p...

␣2,8-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, ␣2,9-polyNeu5Ac, a linkage isomer of ␣2,8-polyNeu5Ac, has long been thought to occur only in capsules of neuroinvasive Neisseria meningitidis group C bacteria. Here we report the unexpected discovery of ␣2,9-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 ␣2,9-polyNeu5Ac was affected by cell growth and retinoic acid-induced differentiation. Occurrence of the linkage isomer of ␣2,8-polyNeu5Ac has been left unrecognized by conventional methods using biological diagnostic probes for ␣2,8-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.Polysialic acid (PSA) 1 is a unique cell surface homopolymer that is expressed by clinically important serogroups B and C Neisseria meningitidis in ␣2,8-and ␣2,9-linked isomers respectively (1) (Fig. 1). These molecules encapsulate the organisms, which are responsible for a dominant portion of all cases of meningococcal meningitis, and are critical to their pathogenesis. One of these linkage isomers, ␣2,8-polyNeu5Ac, which also forms the capsule of neuroinvasive Escherichia coli K1, is poorly immunogenic (2), probably due to the fact that it is a self-antigen occurring as an oncodevelopmentally regulated mammalian antigen in the neural cell adhesion molecule (NCAM) and certain tumors. In contrast ␣2,9-polyNeu5Ac is immunogenic enough to be used as a vaccine against meningitis caused by group C meningococci and is expressed by these organisms in both O-acetylated and O-deacetylated forms (2). Since the 1980s, the molecular biology, structural biology, and physiological and pathophysiological functions of ␣2,8-polyNeu5Ac on NCAM have already been the subject of major reviews (3-7). Usually detection and identification of ␣2,8-PSA in NCAM studies are made using monoclonal antibodies (mAbs) specific to ␣2,8-polyNeu5Ac. Although such immunological reagents are useful for studies of developmentally regulated dynamic expression of ␣2,8-polyNeu5Ac, they cannot be used to define DP or to detect either changes in the distribution of glycoforms differing in DP or the presence of PSA differing in sequences or inter-residue linkages. Prior to the identification of ␣2,8-polyNeu5Ac-NCAM, we discovered ␣2,8-polyNeu5Gc by chemical and biochemical methods, which was the first example of animal PSA (8, 9). Later we also unveiled divergent forms of PSA, such as an ␣2,8-poly(Neu5Ac, Neu5Gc) co-polymer, ␣2,8-polyKDN, and ␣2,5-Oglycolyl-linked oligo/polyNeu5Gc (10, 11). These PSA-glycoproteins (gp) were shown to play biological functions in fertilization and e...

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.

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