Abstract
Type X collagen is a short chain collagen expressed in the hypertrophic zone of calcifying cartilage during skeletal development and bone growth. The alpha1(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 alpha1(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.
Highlights
Type X collagen is a short chain collagen expressed in the hypertrophic zone of calcifying cartilage during skeletal development and bone growth
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
While no molecular weight change was observed for the Y598D amino acid substitution (Fig. 2, lane 4), both the single base deletion of cytosine 1952 (NC1⌬C) and the 10-base pair deletion (NC1⌬10) of the NC1 domain resulted in shortened pre␣1(X) chains (Fig. 2, lanes 2 and 3) when compared with the normal pre␣1(X) chain of 680 amino acids (Fig. 2, lane 1)
Summary
Construction of a Full-length Human Type X Collagen cDNA Containing the 3Ј-Untranslated Region—The production of a full-length ␣1(X) cDNA construct, pTM1-h10, in a T7-driven expression vector suitable both for in vitro transcription-translation and transient expression in cells using the vaccinia/T7 phage system [19] has been described previously [9]. The digested plasmid was purified by agarose electrophoresis, recovered by electroelution, the resultant overhang sequences were filled in with Klenow, and blunt ends were ligated to produce the plasmid pTM1-helix⌬. This procedure removed the amino acid residues 72–354,3 a total of 283 amino acids within the amino-terminal region of the triple helical domain of human type X collagen (Fig. 1). The plasmid pTM1-h10wt (5 ng) was used as a template for the primary rounds of PCR with primer pairs pTM1-1 (sense)/NC2⌬B (antisense) and NC2⌬A (sense)/ HX10 (antisense) (Table I) to generate the two independent fragments with overlapping sequences. Sequence assignment numbers for the type X collagen primers are taken from Reichenberger et al [31] and for pTMl from Fuerst et al [22]
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