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.

Phospholipid composition of articular cartilage boundary lubricant

“…Though earlier models considered hyaluronic acid (HA) as the predominant articular boundary lubricant [28], later work [16,23] showed that HA affected the viscosity of the synovial fluid with insignificant cartilage boundary lubrication. Subsequently, a highly purified glycoprotein fraction isolated from synovial fluid named lubricin, was observed to impart the lubricating properties of synovial fluid [33,[35][36][37][38][39][40]. Swann et al determined that lubricin consisted of 41.544.3% protein and 45.9-55.3% carbohydrate [35].…”

Role of lubricin and boundary lubrication in the prevention of chondrocyte apoptosis

“…Boundary lubrication dominates during periods of high load and low velocity, when the lubricant film layer is thinner than the surface roughness (16). In healthy articular joints, a layer of lubricin molecules covers the surface of cartilage and acts as an antiadhesive and boundary lubricant to prevent cartilage damage as surface asperities move against each other (17)(18)(19)(20). Patients with the autosomal recessive disease camptodactyly-arthropathy-coxa vara-pericarditis syndrome (CACP) lack functional lubricin and develop precocious cartilage failure.…”

The presence and distribution of lubricin in the caprine intervertebral disc

“…1,[4][5][6][7] Investigators working with synovial fluid were first to propose that it is a specific molecule-a large glycoprotein, which they termed ''lubricin''-that primarily imparts to synovial fluid its ability to act as a boundary lubricant for articular cartilage. 9,10 Another group working directly with cartilage explants described a similar molecule in the superficial zone of articular cartilage, which they named ''superficial zone protein (SZP).'' Both superficial zone chondrocytes and cells lining the synovial cavity were found capable of synthesizing and secreting this protein.…”