Combinational Analyses with Multiple Methods Reveal the Existence of Several Forms of Polysialylated Neural Cell Adhesion Molecule in Mouse Developing Brains

Abstract

Polysialic acid (polySia/PSA) is an anionic glycan polymer of sialic acid, and it mostly modifies the neural cell adhesion molecule (NCAM) in mammalian brains. Quality and quantity of the polySia of the polySia–NCAM is spatio-temporally regulated in normal brain development and functions, and their impairments are reported to be related to diseases, such as psychiatric disorders and cancers. Therefore, precise understanding of the state of polySia–NCAM structure would lead to the diagnosis of diseases for which their suitable evaluation methods are necessary. In this study, to develop these evaluation methods, structures of polySia–NCAM from mouse brains at six different developmental stages were analyzed by several conventional and newly developed methods. Integrated results of these experiments clearly demonstrated the existence of different types of polySia–NCAMs in developing brains. In addition, combinational analyses were shown to be useful for precise understanding of the quantity and quality of polySia, which can provide criteria for the diagnosis of diseases.

Comparative Studies of Polysialic Acids Derived from Five Different Vertebrate Brains

“…To gain a more insight into the polySia profiles observed on the blots, we performed SDS-PAGE/Native-PAGE (S/N) Map analysis as described previously [ 41 ]. Briefly, the start and end points of the smear on the SDS-PAGE and those on the native-PAGE were plotted to obtain the linear line, and the slope value for the line was obtained as the qualitative index of polySia.…”

“…In addition, it is interesting to point out that the KS-modified polySia-NCAM is more abundant in chicken and goldfish than in mouse, because the goldfish and chicken polySia-NCAM remains in 0.4–0.6 M NaCl fractions even after Endo-N treatment. It should be noted that adult pig brains, 8 w and retired mouse brains tend to show more KS-modified polySia-NCAM than embryonic brains [ 40 , 41 ]. The function of KS-modified polySia-NCAM needs to be further studied.…”

“…Samples were adjusted to 5 µg/mL (as BSA) with PBS (the concentration of Triton X-100 should be below 0.03%) and 50 µL of the solutions was absorbed onto a 96-well immunoplate, and other procedures were performed as described [ 41 ].…”

“…In contrast, our laboratory has pioneered a novel method for determination of the qualitative differences in polySia in regard to length and molecular size [ 21 , 38 , 39 , 40 ]. In our latest research, we demonstrated an integrated analysis method that combined several conventional analytical methods and a newly developed method [ 41 ]. We have already demonstrated this method to be useful for distinguishing different types of polySia in mouse brains at different developmental stages.…”

Polysialic Acid in the Immune System

“…The semiquantitative or qualitative analysis represented by Western blot (WB) and quantitative analysis such as ELISA and flow cytometry are mostly based in immunodetection of polySia (138,139). A disadvantage of ELISA is that it cannot distinguish polySia of different chain lengths and WB only offers relative quantification of polySia based on differential densitometry associated with chemiluminescent and fluorescent signal obtained from blots.…”