Cartilage oligomeric matrix protein (COMP), a large pentameric
glycoprotein and member of the
thrombospondin (TSP) group of extracellular
proteins, is found in the territorial matrix surrounding chondrocytes. More than 50 unique COMP mutations have been identified as causing two skeletal dysplasias:
pseudoachondroplasia (
PSACH); and
multiple epiphyseal dysplasia (EDM1). Recent studies suggest that
calcium-binding and
calcium-induced protein folding differ between wild type and
mutant proteins, and abnormal processing of the mutant COMP
protein contributes to the characteristic enlarged lamellar appearing rER cisternae in
PSACH and EDMI chondrocytes in vivo and in vitro. Towards the goal of delineating the pathogenesis of
PSACH and EDM1, in-vivo
PSACH growth plate and in-vitro
PSACH chondrocytes cultured in
alginate beads were examined to identify and localize the chaperone
proteins participating in the processing of the retained
extracellular matrix proteins in the
PSACH rER.
Aggrecan was localized to both the rER cisternae and matrix while COMP and
type IX collagen were only found in the rER.
Type II collagen was solely found in the ECM suggesting that it is processed and transported differently from other retained ECM
proteins. Five chaperone
proteins: BiP (
Grp78);
calreticulin (CRT);
protein disulfide (PDI);
ERp72; and
Grp94, demonstrated immunoreactivity in the enlarged
PSACH cisternae and the short rER channels of chondrocytes from both in-vivo and in-vitro samples. The chaperone
proteins cluster around the electron dense material within the enlarged rER cisternae. CRT, PDI and
GRP94 AB-
gold particles appear to be closely associated with COMP. Immunoprecipitation and Western blot, and Fluorescence Resonance Energy Transfer (FRET) analyses indicate that CRT, PDI and
GRP94 are in close proximity to normal and mutant COMP and BiP to mutant COMP. These results suggest that these
proteins play a role in the processing and transport of wild type COMP in normal chondrocytes and in the retention of mutant COMP in
PSACH chondrocytes.