Altered proteoglycan synthesis by micromelial limbs induced by 6-aminonicotinamide. Appearance of abnormal forms of cartilage-characteristic proteoglycan (PG-H)

Abstract

Since administration of 6-aminonicotinamide (10 micrograms) to day-4 chick embryos in ovo was shown to induce micromelial limbs, biosynthesis of cartilage-characteristic proteoglycan-H (PG-H) as an important index of limb chondrogenesis was examined in day-7 normal and micromelial hind limbs by biochemical and immunological methods. (1) Metabolic labelling of the micromelial limbs with [6-3H]glucosamine and either [35S]sulphate or [35S]methionine, followed by analyses of labelled PG-H by glycerol density-gradient centrifugation under dissociative conditions, showed a marked reduction in the PG-H synthesis. (2) PG-H synthesized by the micromelial limbs was much lower than that synthesized by the normal limbs in the biosynthetic ratio of chondroitin sulphate to keratan sulphate and glycoprotein-type oligosaccharide, although no significant difference was observed in the immunological properties of these proteoglycans. (3) The degree of sulphation of chondroitin sulphates of PG-H was lowered in the micromelial limbs as judged by the increase of unsulphated disaccharide (delta Di-OS) released by chrondroitinase ABC digestion, although there were no significant differences between the normal and the micromelial limbs in the average molecular size (Mr = 38,000) of labelled chondroitin sulphates of PG-H. (4) Addition of beta-D-xyloside, an artificial initiator for chondroitin sulphate synthesis, to the micromelial limbs in culture recovered the incorporation of labelled glucosamine into chondroitin sulphate to that comparable with the normal control with beta-D-xyloside, although the incorporation of [35S]sulphate was lower in the micromelia than in the control with beta-D-xyloside. These results suggest that the reduction in the biosynthesis of the PG-H as well as the production of altered forms of PG-H induced by 6-aminonicotinamide during a critical period of limb morphogenesis may be an important factor for the micromelia.