Koji Kimata

Appearance of distinct types of proteoglycan in a well-defined temporal and spatial pattern during early cartilage formation in the chick limb

Our recent studies have shown that chick embryo epiphyseal cartilage synthesizes three distinct species of proteoglycan (PG-H, PG-Lb, and PG-Lt) which are analogous in having glycosaminoglycan side chains of the chondroitin (dermatan) sulfate type but different from one another in regard to the structure of core protein. In the present report, the expression of PG-H and PG-Lb has been studied in developing chick hind limbs (stages 19-33), using antibodies specific for these substances in indirect immunofluorescence. At the onset of cartilage morphogenesis (stage 24), PG-H became recognizable in the cartilage primordia, whereas a parallel section stained for PG-Lb showed no reaction. The first evidence of PG-Lb appearance was seen in a stage 28 cartilage (e.g., tibia) in which the cells in the middiaphysis became elongated in a direction perpendicular to the long axis of the cartilage. The PG-Lb fluorescence was confined to the zone of these flattened, disc-like cells, whereas the fluorescence for PG-H was uniformly distributed throughout the cartilage. With further development of cartilage (stage 29 approximately), the zone of flattened cells spread proximally and distally, and simultaneously large hypertrophied cells appeared at the diaphyseal region. During these zonal changes of cell morphology, the PG-Lb fluorescence remained restricted to the zone of flattened cells. Parallel sections stained for PG-H, in contrast, showed an evenly distributed pattern of the PG-H fluorescence throughout the cartilage. The results indicate that the appearance of PG-Lb is closely associated with the zonal changes of cell shape and orientation along the proximal-distal axis of the developing limb cartilage, and further suggest that the flattened chondrocytes in this particular zone have undergone additional changes in gene expression to form an extracellular matrix of still another chemical property.

Selective removal of heparan sulfate chains from proteoheparan sulfate with a commercial preparation of heparitinase

Procedures employing the commercial preparation of heparitinase were developed for isolating a protein-enriched core molecule from proteoheparan sulfate by selective removal of the heparan sulfate chains. Treatment of proteoheparan sulfate with the enzyme preparation caused seriously extensive degradation owing to the presence of proteolytic activity in the enzyme preparation. This effect could be avoided by using a series of protease inhibitors which prevented proteolytic degradation with less significant effect on the heparitinase activity. Application of the procedures to a purified preparation from the Engelbreth-Holm-Swarm tumor yielded a single protein-enriched core fraction with a molecular weight of approximately 450,000, as ascertained by sodium dodceyl sulfate-gel electrophoresis.

The occurrence of low buoyant density proteoglycans in embryonic chick cartilage

Abstract Two proteoglycan fractions, PCS-H and PCS-L, have previously been isolated from 4 M guanidine HCl extract of embryonic chick cartilages. This communication reports further studies with PCS-L indicating that this fraction contains several different forms, of which one differs from hitherto known cartilage proteoglycans in 1) markedly lower buoyant density, 2) susceptibility to reduction with 2-mercaptoethanol, 3) aggregate-forming ability in 4 M guanidine HCl, and 4) presence of dermatan sulfate-chondroitin sulfate copolymer chains. Also isolated from the PCS-L fraction is a keratan sulfate-rich proteoglycan which represents the smallest molecular size species in cartilage proteoglycan populations.

Morphological and biochemical differentiation of limb bud cells cultured in chemically defined medium.

Abstract When chick limb buds were isolated from stage 22–23 embryos and cultured in chemically defined medium “BGJb,” the explants grew and, after about 9 days, showed good chondrogenesis of recognizable cartilage segments. Cartilage-type proteoglycan (termed PCS-H) was not synthesized during early days of culture, but by Day 9, it became a major proteoglycan constituent of the tissue. Freshly dissociated limb bud cells, when plated as monodispersed cultures at a density of 7 × 10 6 cells/ml of BGJb, did not undergo chondrogenic differentiation and, instead, assumed the appearance of unhealthy or degenerated cells. During 9 days of culture, even though proteoglycans were synthesized, they were nevertheless of much smaller molecular size than PCS-H. When limb bud cells were cultured as a pellet containing 7 × 10 6 cells in 1 ml of BGJb, a more tightly packed aggregate of about 2 × 10 6 cells appeared in an inner region of the pullet during the first 24 hr of culture, and by Day 12 the aggregate had differentiated into a cartilage nodule surrounded by a thin layer of what appear to be ectodermal cells. As the conversion of aggregate into cartilage nodule progressed, newly formed proteoglycans gradually became more like cartilage-type proteoglycans, and by Day 12 they had many chemical and physical characteristics similar to those of the proteoglycans isolated from fully differentiated cartilages. The results indicate that the initial association of limb bud cells is an important factor for the chondrogenesis in BGJb and further suggest that the tight binding of the cell surfaces to one another may directly or indirectly stimulate the mechanism of synthesis of cartilage-type proteoglycans.

Nascent mucopolysaccharides attached to the Golgi membrane of chrondrocytes

Abstract As observed radioautographically in the cartilage of embryonic chick, radio-sulfate was concentrated only in the Golgi apparatus within 10 min of its addition. The labeled material was obtained by cell fractionation as it exists in a bound form on the Golgi membrane. Comparison of this material to protein-polysaccharide isolated from the extracellular matrix supported the concept that biosynthesis of some chondroitin sulfate chain takes place prior to its conversion to the mature form, protein-polysaccharide.

An enzyme-linked immunoassay for the cartilage proteoglycan.

Abstract Proteoglycan was purified from a rat chondrosarcoma and antiserum prepared. An enzyme-linked immunoassay was designed using this serum. The assay detected rat and murine, but not chick, high-molecular-weight cartilage proteoglycan. It did not detect noncartilage proteoglycan nor the low-molecular-weight proteoglycans found in cartilage. As little as 100 ng/ml of rat cartilage proteoglycan could be detected.

Gene expression of high- (p140trk) and low-affinity nerve growth factor receptor (LNGFR) in the adult and aged human peripheral nervous system.

Steady-state mRNA levels and immunoreactive proteins for high- (p140trk) and low-affinity nerve growth factor receptor (LNGFR) in the adult and aged human peripheral nervous system (PNS) were examined in autopsied material. trk mRNA expression was observed only in the sympathetic and dorsal root ganglia, while LNGFR mRNA was expressed widely through the PNS as well as non-neural tissues. Immunoreactive trk proto-oncogene product (p140trk) and LNGFR occurred in the perikarya of the subset of the sympathetic and dorsal root ganglion neurons, but only LNGFR immunoreactivity also occurred in the perineurium and the outer layer of the vessels. The spatial patterns of the trk and LNGFR gene expression in the adult human PNS were similar to those observed in the rat, mouse and chick, and their expression was well preserved in the aged.

Abnormal collagen fibrillogenesis in epiphyseal cartilage of CMD (cartilage matrix deficiency) mouse.

Light and electron microscopic observations on the structure of epiphyseal cartilages in the cmd/cmd mice, which had genetically failed to synthesize cartilage-characteristic proteoglycan but were normal in type II collagen synthesis, showed apparent abnormalities of collagen fibrils: e.g. increase in the diameter, appearance of periodic banding patterns and bundle-formation of collagen fibrils. These findings suggest that cartilage-characteristic proteoglycan normally limits the lateral growth of collagen fibrils and affects collagen fibrillogenesis in vivo.

Presence of link protein in cartilage from cmd/cmd (cartilage matrix deficiency) mice

Immunohistochemical and biochemical evidence that the cartilage from cmd/cmd mice, who have an autosomal recessive lethal mutation causing cartilage matrix deficiency, synthesizes link protein nearly at a normal level is provided. Since cartilage-characteristic proteoglycan is not synthesized in this mutant mouse (K. Kimata, H-J. Barrach, K. S. Brown, and J. P. Pennypacker (1981) J. Biol. Chem. 256, 6961-6968), link proteins are apparently not in conventional proteoglycan aggregate. However, the link proteins are functional and able to interact with exogenous cartilage-characteristic proteoglycan monomer and hyaluronic acid to form aggregates.