Proteoglycan Lt from chicken embryo sternum identified as type IX collagen.

Proteoglycan Lt (PG-Lt), isolated from 17-day-old chicken embryo sterna, appeared to differ from its counterpart from tibia and femur (Noro, A., Kimata, K., Oike, Y., Shinomura, T., Maeda, N., Yano, S., Takahashi, N., and Suzuki, S. (1983) J. Biol. Chem. 258, 9323-9331). The intact disulfide-bonded molecule of approximately 300 kDa was separable into three chains of 115, 84, and 68 kDa on reduction, the molecular masses being relative to those of collagen standards on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). This is in contrast to tibial cartilage PG-Lt, from which there was no observed release of a 68-kDa chain (100 kDa relative to globular protein standards) after reduction. The 115-kDa chain of sternum PG-Lt consists of a core 68-kDa polypeptide to which the chondroitin sulfate chains are attached. The ratio of 4-sulfated to 6-sulfated disaccharides released after either chondroitinase ABC or AC digestion is 3:1. Identity of PG-Lt with type IX collagen was indicated by their similar elution profiles on DEAE-Trisacryl and by the presence in both proteins of co-migrating 84- and 68-kDa bands on SDS-PAGE. This identity was confirmed by immunoblotting PG-Lt after SDS-PAGE, with affinity-purified polyclonal antibodies specific for a triple helical domain (HMW) of type IX collagen. The nonreduced high molecular mass material and all three bands of the reduced PG-Lt were immunoreactive, giving immunostaining patterns similar to autoradiographs from the [14C]glycine-labeled protein.