Site-directed mutagenesis of human type X collagen. Expression of α1(X) NC1, NC2, and helical mutations in vitro and in transfected cells

Abstract

Type X collagen is a short chain collagen expressed in the hypertrophic zone of calcifying cartilage during skeletal development and bone growth. The α1(X) homotrimer consists of three protein domains, a short triple helix (COL1) flanked by nonhelical amino-terminal (NC2) and carboxyl-terminal (NC1) domains. While mutations of the NC1 domain result in Schmid metaphyseal chondrodysplasia, which suggests a critical role for this protein domain, little biochemical detail is known about type X collagen synthesis, secretion, and the mechanisms of molecular assembly. To study these processes, a range of mutations were produced in human α1 (X) cDNA and the biochemical consequences determined by in vitro expression, using T7-driven coupled transcription and translation, and by transient transfection of cells. Three NC1 mutants, which were designed to be analogous to Schmid mutations (1952delC, 1963del10, and Y598D), were unable to assemble into type X collagen homotrimers in vitro, but the mutant chains did not associate with, or interfere with, the efficiency of normal chain assembly in co- translations with a normal construct. Expression in transiently transfected cells confirmed that mutant type X collagen assembly was also compromised in vivo. The mutant chains were not secreted from the cells but did not accumulate intracellularly, suggesting that the unassociated mutant chains were rapidly degraded. In-frame deletions within the helix (amino acid residues 72-354) and the NC2 domain (amino acid residues 21-54) were also produced. In contrast to the NC1 mutations, these mutations did not prevent assembly. Mutant homotrimers and mutant-normal heterotrimers were formed in vitro, and the mutant homotrimers formed in transiently transfected cells had assembled into pepsin-stable triple helical molecules which were secreted.