Degree of polymerization (DP) of polysialic acid (polySia) on neural cell adhesion molecules (N-CAMS): development and application of a new strategy to accurately determine the DP of polySia chains on N-CAMS.
Alpha2,8-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 alpha2,8-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-beta-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 approximately 50-60 or less, a subpopulation of chains with DPs approximately 150 to approximately 180 and extending to DP approximately 400 were detected. The DP of this subpopulation is considerably greater than reported previously for N-CAM. Endo-beta-galactosidase can also release polySia chains from polysialylated membranes expressed in the neuroblastoma cell line, Neuro2A, and native N-CAM from embryonic chick brains.
Nakata
Daisuke
D
Department of Biochemistry and Molecular Medicine, University of California School of Medicine, Davis, California 95616.
Troy
Frederic A
FA
D000818:Animals; D019556:COS Cells; D045744:Cell Line, Tumor; D002642:Chick Embryo; D002522:Chlorocebus aethiops; D002845:Chromatography; D002851:Chromatography, High Pressure Liquid; D005353:Fibronectins / Q000737:chemistry; D005470:Fluorometry; D005690:Galactose / Q000737:chemistry; D006026:Glycoside Hydrolases / Q000737:chemistry; D006031:Glycosylation; D006801:Humans; D006868:Hydrolysis; D019158:N-Acetylneuraminic Acid / Q000737:chemistry; D009362:Neoplasm Metastasis; D019006:Neural Cell Adhesion Molecules / Q000737:chemistry*; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase / Q000737:chemistry; D010957:Plasmids / Q000378:metabolism; D011108:Polymers / Q000737:chemistry; D017434:Protein Structure, Tertiary; D012794:Sialic Acids / Q000737:chemistry*; D013997:Time Factors; D014162:Transfection
D005353:Fibronectins; D019006:Neural Cell Adhesion Molecules; D011108:Polymers; D012794:Sialic Acids; C021319:polysialic acid; D006026:Glycoside Hydrolases; C026093:keratan-sulfate endo-1,4-beta-galactosidase; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; D019158:N-Acetylneuraminic Acid; D005690:Galactose