Changes of matrilin forms during endochondral ossification. Molecular basis of oligomeric assembly

To understand the molecular properties of matrilin-3, a newly discovered member of the novel extracellular matrix protein family, we cloned a MAT-3 cDNA from developing chicken sterna. Real time quantitative reverse-transcription polymerase chain reaction indicates that MAT-3 mRNA is mainly expressed in the proliferation zone of a growth plate. It is also expressed in the maturation zone, overlapping with that of the mature chondrocyte-abundant matrilin-1 mRNA. This suggests that matrilin-3 may self-assemble in the proliferation zone, in addition to its co-assembly with matrilin-1 during endochondral ossification. Transfection of a MAT-3 cDNA into COS-7 cells shows that MAT-3 predominantly forms a homotetramer but also a trimer and a dimer. Co-transfection of both MAT-3 and MAT-1 cDNAs results in three major matrilins as follows: (MAT-1)(3), (MAT-3)(4), and (MAT-1)(2)(MAT-3)(2). Thus matrilin-3 may assemble into both homotypic and heterotypic oligomers. Our analysis shows that the assembly of MAT-3 does not depend on the number of epidermal growth factor repeats within the molecule, but the presence of Cys(412) and Cys(414) within the coiled-coil domain, which form covalent disulfide linkage responsible for both homo-oligomerization of MAT-3 and hetero-oligomerization of MAT-3 and MAT-1. Our data suggest that the varying synthetic levels of matrilins in different zones of a growth plate may result in a change of matrilin oligomeric forms during endochondral ossification.