The isolation and partial characterization of the major glycoprotein (LGP-I) from the articular lubricating fraction from bovine synovial fluid

Abstract

The articular lubricating fraction from bovine synovial fluid was prepared by repeated fractionation in three consecutive CsCl density gradients to remove completely traces of hyaluronic acid. The major glycoprotein consituent (LGP-I) was then isolated by repeated gel-permeation chromatography. The yield of the LGP-I component was about 20 mg/litre of synovial fluid. Sedimentation-equilibrium measurements showed that this glycoprotein was homogeneous and the mol.wt. was calculated to be 227500. Amino acids represented 43% (w/w) and carbohydrate constituents 44% (w/w) of the molecule. Threonine, glutamic acid, proline and lysine (224, 127, 242 and 128 residues/1000 residues respectively) were the major amino acids. Galactosamine, galactose and N-acetylneuraminic acid (202, 162 and 114 residues/molecule of LGP-I component respectively) accounted for 98% of the total carbohydrate residues present. Small amounts of mannose and glucosamine (1 and 9mol respectively/mol of LGP-I component) were also present. After treatment of LGP-I component with alkali and NaB3H4 radioactivity was incorporated into alpha-aminobutyric acid and alanine in a molar ratio of 4:1, and radioactive galactosaminitol was isolated by ion-exchange chromatography from a cleaved oligosaccharide fraction. These data demonstrate the presence of threonine and serine -O-GalNAc linkages, but only 25% of the theoretical likages involving threonine were cleaved by a beta-elimination reaction. Digestion of LGP-I component with Pronase followed by chromatography on DEAE-cellulose yielded glycopeptide fractions with a similar amino acid and carbohydrate composition to the intact molecule. Treatment of desialylated and intact LGP-I component with galactose oxidase followed by reduction with NaB3H4 revealed the presence of 52mol of terminal galactose in the intact molecule and 153mol of galactose/mol of LGP-I component after treatment with neuraminidase. The data indicate the LGP-I component is composed of a single polypeptide chain containg more than 150 oligaosaccharide side chains composed of O-GaINAc-Gal distributed over the length of the peptide chain and that terminal sialic acid residues are linked to galactose in two-thirds of these side chains.