Abstract
Chondroitin sulfate (CS)-D and CS-E, which are characterized by oversulfated disaccharide units, have been shown to regulate neuronal adhesion, cell migration, and neurite outgrowth. CS proteoglycans (CSPGs) consist of a core protein to which one or more CS chains are attached via a serine residue. Although several brain CSPGs, including mouse DSD-1-PG/phosphacan, have been found to contain the oversulfated D disaccharide motif, no brain CSPG has been reported to contain the oversulfated E motif. Here we analyzed the CS chain of appican, the CSPG form of the Alzheimer's amyloid precursor protein. Appican is expressed almost exclusively by astrocytes and has been reported to have brain- and astrocyte-specific functions including stimulation of both neural cell adhesion and neurite outgrowth. The present findings show that the CS chain of appican has a molecular mass of 25-50 kDa. This chain contains a significant fraction (14.3%) of the oversulfated E motif GlcUA beta 1-3GalNAc(4,6-O-disulfate). The rest of the chain consists of GlcUA beta 1-3GalNAc(4-O-sulfate) (81.2%) and minor fractions of GlcUA beta 1-3GalNAc and GlcUA beta 1-3GalNAc(6-O-sulfate). We also show that the CS chain of appican contains in its linkage region the 4-O-sulfated Gal structure. Thus, appican is the first example of a specific brain CSPG that contains the E disaccharide unit in its sugar backbone and the 4-O-sulfated Gal residue in its linkage region. The presence of the E unit is consistent with and may explain the neurotrophic activities of appican.
Highlights
Brain GAGs and PGs have attracted attention in connection with the pathology of Alzheimer’s disease (AD)
Because the core protein of amyloid precursor-like protein 2 (APLP2) PG has an SDS-polyacrylamide gel electrophoresis mobility distinct from the appican core protein [47, 48], our preparation had no significant contamination of the APLP2 PG
amyloid precursor protein (APP), but not the APLP2, sequence was found in the core protein of our appican preparations from C6 cells following chondroitinase digestion [1]
Summary
Materials—Appican was purified essentially as described in our previous report [1]. Briefly, rat C6 glioma cells transfected with the L-APP cDNA [6] were grown in Dulbecco’s modified Eagle’s medium with supplements. ABC was carried out using 5 mIU of the enzyme and 5 pmol of the 2AB-derivatized polysaccharide or 1 g of appican in a total volume of 20 l of 0.05 M Tris-HCl buffer, pH 8.0, containing 0.05 M sodium acetate at 37 °C for 20 min [45]. Chondroitinase AC-II digestion of the linkage hexasaccharides or 1 g of appican was conducted using 5 mIU of the enzyme in a total volume of 30 l of 0.03 M sodium acetate buffer, pH 6.0, at 37 °C for 10 min [45]. Chondro-4-O-sulfatase or chondro-6O-sulfatase digestion of the linkage hexasaccharides (5 pmol) was carried out using 20 mIU of the enzyme in a total volume of 30 l of 0.04 M Tris-HCl buffer, pH 7.5, containing 0.04 M sodium acetate and 100 g/ml bovine serum albumin, at 37 °C for 2 h [46]. Eluates were monitored by fluorescence intensity with excitation and emission wavelengths of 330 and 420 nm or by absorbance at 232 nm [44]
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