Light fraction proteinpolysaccharide was prepared by water extraction of nucleus pulposus, annulus fibrosus, and costal cartilage obtained at autopsy from fifteen skeletally normal subjects ranging in age from four and one half to seventy-six years. Age dependent changes in glucosamine and galactosamine content of the crude tissue and of the light fraction proteinpolysaccharide were demonstrated. A decrease with aging in per cent of total hexosamine extractable in the light fraction proteinpolysaccharide and an increase in protein content was observed in all three tissues. Electrophoresis of the light fraction produced two main components differing in both polysaccharide and protein content.

Age-Related Variations in Proteinpolysaccharides from Human Nucleus Pulposus, Annulus Fibrosus, and Costal Cartilage

l. Proteoglycans (PGs) in rabbit corneal stroma and mouse sclera have been stained for electron microscopy with Cupromeronic blue in a critical electrolyte concentration (CEC) mode, with and without prior digestion of the tissue by keratanase or chondroitinase ABC to remove the keratan sulphate (KS) or chondroitin-dermatan sulphates (CS or DS) respectively.2. Two classes of PGs, located orthogonally to the corneal collagen fibrils at either the ‘step’ (band ‘a’ or ‘c’) or gap zone (band ‘d’ or ‘e’) are shown to be KS-PGs or DS-PGs respectively. Four separate and specific PG binding sites on Type I collagen fibrils have thus been identified.3. Rabbit corneal KS and DS PGs each contain two kinds of PG (Gregory JD, Coster L & Damle SP (1982) J. Biol. Chem.257, 6965–6970). We propose that each ‘small” protein-rich PG is associated with a specific binding site on the collagen fibril.

‘Small’-proteoglycan: collagen interactions: Keratan sulphate proteoglycan associates with rabbit corneal collagen fibrils at the ‘a’ and ‘c’ bands

The histochemistry of connective tissue proteoglycans (PGs) poses two major requirements, 1. for the specific demonstration of a given PG and, 2. at the ultrastructural level, for the examination of the shape of the PG, and its interactions with other molecules and tissue elements. Techniques for the localisation and identification of PGs are discussed, according to the principles behind their application. The strengths and weaknesses of antibody stains are compared with those of mini-molecular reagents, based on resolution, sensitivity, stoichiometry and tissue penetration. The concept of specificity is analysed in this context. The polyanionic characteristics of PGs are exploited to allow very sensitive detection and localisation by cationic probes, both macro- and mini-molecular. Complex formation by heavy metal cations, basic dyestuffs and polycations with PGs is a simple ion-exchange process (basophilia), which can be manipulated to give considerable specificity, e.g. using the critical electrolyte concentration (CEC) concept. The fundamental physical chemical unity of the phenomena of basophilia, CEC and biochemical fractionations of tissue polyanions by anion-exchange is discussed. This unity allows direct quantitative and qualitative comparisons to be made between histochemical and biochemical results at all levels from the tissue sample to the single molecule of PG in situ. The great value of the partnership between microscopy and analytical biochemistry is emphasised. The expanded solution domain of PGs collapses during formation and processing of the 'stained' complex, obscuring molecular detail and possibly resulting in translocation of the PG. Approaches aimed at restoring the initial situation, and their application to electron microscopy of PGs in tissues are outlined. Complexes of PG with ambient polycations may form, either as artefacts during processing or as an integral part of in vivo ultrastructure. Uptake of stain may be partly or totally blocked, in consequence. Ways of investigating, avoiding, or making use of this phenomenon are described. Application of integrated approaches to the study of PG--collagen and PG--elastin interactions in tissues are discussed. Specific interactions and stoichiometric relationship, particularly of dermatan sulphate proteoglycans with type I collagen have been observed, in skin, tendon, and other non-calcifying tissue, but not in bone.

Proteoglycan Histochemistry — A Valuable Tool for Connective Tissue Biochemists

Publisher Summary This chapter discusses the general composition of cartilage, glycosaminoglycans, keratan sulfate, hyaluronic acid, and proteoglycans. Several types of connective tissue represented by ligaments, tendons, synovial membrane, and cartilage are found in diarthrodial joints. However, with the exception of cartilage, little is known about the ground substance of these tissues. Cartilage is an avascular tissue in which the cells are sparsely distributed in a stiff matrix. It is mechanically perhaps the most important tissue of diarthrodial joints and is well adapted to perform an exacting mechanical function, in having to withstand very high repetitive compressive loads that generate localized stresses. The water content of articular cartilage is about 65–75% and is highest near the joint surface of normal femoral condyles. The water content of patellar cartilage is similar. Although the content of water is high, it is precisely the presence of water in conjunction with proteoglycans and collagen that makes cartilage tough and resilient. Collagen accounts for about half the dry weight of human adult articular cartilage.

The Chemistry of the Ground Substance of Joint Cartilage

Hyaluronidase digestion and alkaline treatment of bovine tracheal cartilage proteoglycans. Isolation and characterisation of different keratan sulfate proteins.

Vittorio Pedrini

Papers

Electrophoretic Heterogeneity of Proteinpolysaccharides