Abstract
Recent studies have demonstrated the involvement of two polysialyltransferases in neural cell adhesion molecule (N-CAM) polysialylation. The availability of cDNAs encoding these enzymes facilitated studies on polysialylation of N-CAM. However, there is a dearth of detailed structural information on the degree of polymerization (DP), DP ranges, and the influence of embryogenesis on the DP. It is also unclear how many polysialic acid (polySia) chains are attached to a single core N-glycan. In this paper we applied new, efficient, and sensitive high pressure liquid chromatography methods to qualitatively and quantitatively analyze the polySia structures expressed on embryonic and adult chicken brain N-CAM. Our studies resulted in the following new findings. 1) The DP of the polySia chains was invariably 40-50 throughout developmental stages from embryonic day 5 to 21 after fertilization. In contrast, glycopeptides containing polySia with shorter DPs, ranging from 15 to 35, were isolated from adult brain. 2) Chemical evidence showed glycan chains abundant in Neu5Acalpha2,8Neu5Ac were expressed during all developmental stages including adult. 3) Levels of both di- and polySia were found to show distinctive changes during embryonic development.
Highlights
Polysialic acid1 is a structurally and functionally unique glycotope expressed on the surface of living cells [1]
Despite extensive studies on the expression and function of Polysialic acid (polySia) on neural cell adhesion molecule (N-CAM), there is a dearth of structural information on the degree of polymerization (DP) and, importantly, how the chain length may change during embryonic development
As it is imperative to keep polySia chains intact and to accurately quantify the polySia, we exhaustively digested membrane-associated polySia-containing glycopeptides from delipidated homogenates of intact whole chicken brains with nonspecific bacterial protease to bring about solubilization
Summary
Small glycopeptides remaining in the supernatant were precipitated by adding one more volume of acetone (75% acetone precipitate) Both the 50% and 75% acetone precipitates were subjected to size fractionation on Sephacryl S-200 columns (1.6 ϫ 134 cm) equilibrated and eluted with 10 mM Tris-HCl (pH 8.0) containing 0.1 M NaCl. The elution was monitored by A230 nm and by determination of Neu5Ac using the fluorometric HPLC method, after hydrolysis in 0.1 N HCl for 2 h at 80 °C. To improve the yield of higher polymers, lyophilized samples (12 g of Neu5Ac) were first treated with 10 l of 1 M HCl at ambient temperature for 2 h to facilitate lactonization of polySia [32], dried on a centrifugal vacuum evaporator, and subjected to controlled hydrolysis in 100 l of 0.1 M acetic acid for 15 min at 60 °C. A major broad peak that eluted after the DP 32 peak was used as a high molecular weight colominic acid sample throughout this study
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