Degree of Polymerization (DP) of Polysialic Acid (PolySia) on Neural Cell Adhesion Molecules (N-CAMs)

Abstract

␣2,8-Linked polysialic acid (polySia) is a structurally unique antiadhesive glycotope that covalently modifies N-linked glycans on neural cell adhesion molecules (N-CAMs). These sugar chains play a key role in modulating cell-cell interactions, principally during embryonic development, neural plasticity, and tumor metastasis. The degree of polymerization (DP) of polySia chains on N-CAM is postulated to be of critical importance in regulating N-CAM function. There are limitations, however, in the conventional methods to accurately determine the DP of polySia on N-CAM, the most serious being partial acid hydrolysis of internal ␣2,8-ketosidic linkages that occur during fluorescent derivatization, a step necessary to enhance chromatographic detection. To circumvent this problem, we have developed a facile method that combines the use of Endo-␤-galactosidase to first release linear polySia chains from N-CAM, with high resolution high pressure liquid chromatography profiling. This strategy avoids acid hydrolysis prior to chromatographic profiling and thus provides an accurate determination of the DP and distribution of polySia on N-CAM. The potential of this new method was evaluated using a nonpolysialylated construct of N-CAM that was polysialylated in vitro using a soluble construct of ST8Sia II or ST8Sia IV. Whereas most of the oligosialic acid/polySia chains consisted of DPs ϳ50 -60 or less, a subpopulation of chains with DPs ϳ150 to ϳ180 and extending to DP ϳ400 were detected. The DP of this subpopulation is considerably greater than reported previously for N-CAM. Endo-␤-galactosidase can also release polySia chains from polysialylated membranes expressed in the neuroblastoma cell line, Neuro2A, and native N-CAM from embryonic chick brains.

Polysialylated Neuropilin-2 Is Expressed on the Surface of Human Dendritic Cells and Modulates Dendritic Cell-T Lymphocyte Interactions

“…Given the relatively homogenous band centered at 150 kDa, but ranging from 130 to 200 kDa, that is detected on an immunoblot of protein in a cell lysate probed with mAb 735, though, we propose that NRP-2 is the predominant carrier of PSA in dendritic cells. The broadness of a band of polysialylated protein on immunoblot does not necessarily indicate the presence of multiple proteins, as PSA chains vary greatly in size and generate heterogeneity in protein molecular mass (42,43). It is of note that although NRP-1 and NRP-2 share extensive amino acid sequence homology, we found that NRP-1 is not polysialylated in dendritic cells (data not shown).…”

Polysialic Acid Profiles of Mice Expressing Variant Allelic Combinations of the Polysialyltransferases ST8SiaII and ST8SiaIV

“…However, Nakata and Troy (37) reported the presence of small amounts of polySia with DPs of up to ϳ400 in Neuro2A cells stably transfected with ST8SiaIV using endo-␤-galactosidase for enzymatic release of polySia. Further studies are required to investigate whether such long polymers are restricted to cellular systems overexpressing polysialyltransferases or whether more sensitive methods are required for the detection of polySia chains directly from mouse brain.…”

Structural and Functional Impairments of Polysialic Acid by a Mutated Polysialyltransferase Found in Schizophrenia

“…2 is a polymer of sialic acid with a degree of polymerization (DP) ranging from 8 to 400 (1)(2)(3)(4) and is mainly attached to neural cell adhesion molecule (NCAM) in vertebrates (1). PolySia is expressed in brains during embryonic and postneonatal development and mostly disappears in adult brains, although NCAM expression levels remain unchanged (1,5,6).…”