Characterization of a small chondroitin sulfate proteoglycan isolated from the mucus surrounding the embryos of Viviparus ater (Mollusca Gastropoda)

A small chondroitin sulfate proteoglycan was isolated and partially characterized for core protein and glycosaminoglycan structures from the mucus surrounding embryos in the developmental pouch of Viviparus ater (Mollusca Gastropoda). The protein bearing polysaccharide nature was confirmed by gel-permeation chromatography separation of fractions positive to the uronic acid dosage, 7.5% SDS-PAGE under reducing conditions, sequential staining with alcian blue and ammoniacal silver. Its molecular mass was calculated at about 228,800. After degradation of the galactosaminoglycan components by chondroitinase ABC in the presence of proteinase inhibitors, the molecular mass of the core protein was determined at about 72,200. Treatment of the proteoglycan with keratanase did not modify its electrophoretic migration. Isoelectric focusing of the core protein demonstrated a micro-heterogeneity with the presence of two isoforms with different isoelectric point, pI = 8.2 and 6.6, in a ratio of about 1:2.2. The glycosaminoglycan component of the proteoglycan was characterized as chondroitin sulfate with a molecular mass of about 30,750 composed of 5% non-sulfated unsaturated disaccharide, 94% monosulfated disaccharides (4-monosulfated to 6-monosulfated disaccharide ratio of 1.36) and 1.5% disulfated disaccharides (in particular 1.3% 2,6-disulfated disaccharide) with a sulfate to carboxyl ratio of 0.96. Degradation of the chondroitin sulfate with chondroitinase ABC and ACII permitted to determine a percentage of glucuronic acid of about 78.4. The proteoglycan isolated from the mucus surrounding the embryos of Viviparus ater is formed by a small core protein bearing about five chondroitin sulfate chains (80% chondroitin sulfate/20% dermatan sulfate) with potential function in the developmental processes of molluscs embryos.