Sequence determination and analysis of the 3' region of chicken pro-alpha 1(I) and pro-alpha 2(I) collagen messenger ribonucleic acids including the carboxy-terminal propeptide sequences

Three pro-alpha 1 collagen cDNA clones, pCg1, pCg26, and pCg54, and two pro-alpha 2 collagen cDNA clones, pCg 13 and pCg45, were subjected to extensive DNA sequence determination. The combined sequences specified the amino acid sequences for chicken pro-alpha 1 and pro-alpha 2 type I collagens starting at residue 814 in the collagen triple-helical region and continuing to the procollagen C-termini as determined by the first in-phase termination codon. Thus, the sequences of 272 pro-alpha 1 C-terminal, 260 pro-alpha 2 C-terminal, 201 pro-alpha 1 helical, and 201 pro-alpha 2 helical amino acids were established. In addition, the sequences of several hundred nucleotides corresponding to noncoding regions of both procollagen mRNAs were determined. In total, 1589 pro-alpha 1 base pairs and 1691 pro-alpha 2 base pairs were sequenced, corresponding to approximately one-third of the total length of each mRNA. Both procollagen mRNA sequences have a high G+C content. The pro-alpha 1 mRNA is 75% G+C in the helical coding region sequenced and 61% G&C in the C-terminal coding region while the pro-alpha 2 mRNA is 60% and 48% G+C, respectively, in these regions. The dinucleotide sequence pCG occurs at a higher frequence in both sequences than is normally found in vertebrate DNAs and is approximately 5 times more frequent in the pro-alpha 1 sequence than in the pro-alpha 2 sequence. Nucleotide homology in the helical coding regions is very limited given that these sequences code for the repeating Gly-X-Y tripeptide in a region where X and Y residues are 50% conserved. These differences are clearly reflected in the preferred codon usages of the two mRNAs.     

Proteasomal degradation of unassembled mutant type I collagen pro-alpha1(I) chains.

We have previously shown that type I procollagen pro-alpha1(I) chains from an osteogenesis imperfecta patient (OI26) with a frameshift mutation resulting in a truncated C-propeptide, have impaired assembly, and are degraded by an endoplasmic reticulum-associated pathway (Lamandé, S. R., Chessler, S. D., Golub, S. B., Byers, P. H., Chan, D., Cole, W. G., Sillence, D. O. and Bateman, J. F. (1995) J. Biol. Chem. 270, 8642-8649). To further explore the degradation of procollagen chains with mutant C-propeptides, mouse Mov13 cells, which produce no endogenous pro-alpha1(I), were stably transfected with a pro-alpha1(I) expression construct containing a frameshift mutation that predicts the synthesis of a protein 85 residues longer than normal. Despite high levels of mutant mRNA in transfected Mov13 cells, only minute amounts of mutant pro-alpha1(I) could be detected indicating that the majority of the mutant pro-alpha1(I) chains synthesized are targeted for rapid intracellular degradation. Degradation was not prevented by brefeldin A, monensin, or NH(4)Cl, agents that interfere with intracellular transport or lysosomal function. However, mutant pro-alpha1(I) chains in both transfected Mov13 cells and OI26 cells were protected from proteolysis by specific proteasome inhibitors. Together these data demonstrate for the first time that procollagen chains containing C-propeptide mutations that impair assembly are degraded by the cytoplasmic proteasome complex, and that the previously identified endoplasmic reticulum-associated degradation of mutant pro-alpha1(I) in OI26 is mediated by proteasomes.     

Construction of a full-length cDNA encoding human pro-alpha 2(I) collagen and its expression in pro-alpha 2(I)-deficient W8 rat cells

We have constructed a cDNA encoding the entire human pro-alpha 2(I) collagen molecule. Sequence determination for 2196 base pairs at the 5' end of the cDNA clone, and comparison with previously characterized human alpha 2(I) sequences, identified a number of nucleotide and amino acid polymorphisms. Functionality of the cDNA clone, under control of the long terminal repeat of Rous sarcoma virus, was demonstrated by its introduction into the W8 cell line. The W8 line, a chemically transformed variant of K16 rat liver epithelial cells, has been previously shown to lack detectable levels of alpha 2(I) RNA, but to secrete alpha 1(I) homotrimers. Introduction of the human cDNA into W8 cells, resulted in secretion of chimeric type I collagen comprised of rat alpha 1(I) and human alpha 2(I) chains. Availability of a functional full-length clone of human alpha 2(I) cDNA, combined with the W8 cell line as expression system, will allow detailed analysis, through site-directed mutagenesis, of domains on the pro-alpha 2(I) molecule involved in assembly, transport, secretion, and fibrillogenesis.     

High levels of expression of a minigene version of the human pro alpha 1 (I) collagen gene in stably transfected mouse fibroblasts. Effects of deleting putative regulatory sequences in the first intron.

A minigene version of the human gene for the pro alpha(I) chain of type I procollagen (COL1A1) was prepared that contained -2.3 kilobases of the 5'-flanking sequence, the first 5 exons and introns, the last 6 exons and introns, and about 2 kilobases of the 3'-flanking sequence. The gene was then used for stable transfection experiments with mouse NIH 3T3 fibroblasts. Because the products of the minigene were shorter, it was possible to compare expression of the minigene with expression of the endogenous pro alpha 1 (I) gene by Northern and Western blot analyses. The results demonstrated that the construct contained enough of the gene to obtain high levels of expression in many of the stably transfected cells. Since previous observations suggested that the first intron of the pro alpha 1 (I) gene contained important cis-regulatory elements, two versions of the minigene were prepared in which most of the first intron was deleted. Comparison of expression of the minigene with expression of two deleted versions of the same gene established that 85% of the total sequences in the first intron are not essential for high levels of expression of the gene in stably transfected mouse fibroblasts.