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The C-propeptides of the pro alpha chains of type I and type III procollagens are believed to be essential for correct chain recognition and chain assembly in these molecules. We studied here whether the 30-kDa C-propeptides of the human pC alpha 1(I), pC alpha 2(I), and pC alpha 1(III) chains, i.e. pro alpha chains lacking their N-propeptides, can be replaced by foldon, a 29-amino acid sequence normally located at the C terminus of the polypeptide chains in the bacteriophage T4 fibritin. The alpha foldon chains were expressed in Pichia pastoris cells that also expressed the two types of subunit of human prolyl 4-hydroxylase; the foldon domain was subsequently removed by pepsin treatment, which also digests non-triple helical collagen chains, whereas triple helical collagen molecules are resistant to it. The foldon domain was found to be very effective in chain assembly, as expression of the alpha 1(I)foldon or alpha 1(III)foldon chains gave about 2.5-3-fold the amount of pepsin-resistant type I or type III collagen homotrimers relative to those obtained using the authentic C-propeptides. In contrast, expression of chains with no oligomerization domain led to very low levels of pepsin-resistant molecules. Expression of alpha 2(I)foldon chains gave no pepsin-resistant molecules at all, indicating that in addition to control at the level of the C-propeptide other restrictions at the level of the collagen domain exist that prevent the formation of stable [alpha 2(I)]3 molecules. Co-expression of alpha 1(I)foldon and alpha 2(I)foldon chains led to an efficient assembly of heterotrimeric molecules, their amounts being about 2-fold those obtained with the authentic C-propeptides and the alpha 1(I) to alpha 2(I) ratio being 1.91 +/- 0.31 (S.D.). As the foldon sequence contains no information for chain recognition, our data indicate that chain assembly is influenced not only by the C-terminal oligomerization domain but also by determinants present in the alpha chain domains.

Lysyl oxidase (EC 1.4.3.13), an extracellular copper amino oxidase, initiates the cross-linking of collagens and elastin by catalyzing oxidative deamination of the ⑀-amino group in certain lysine and hydroxylysine residues. We developed here a polymerase chain reaction (PCR) method for the quantification of lysyl oxidase mRNA in which a synthetic RNA is used as an internal standard for coamplification with the targeted mRNA. The amount of lysyl oxidase mRNA when studied by Northern blot analysis and the number of lysyl oxidase mRNA molecules when determined by the quantitative PCR method were found to be markedly low in various malignantly transformed cell lines relative to control cell lines, quantitative PCR indicating values of about 2-10% of those in the controls. No difference was found in the number of ␤-actin mRNA molecules between the transformed cells and the controls. Nuclear runoff experiments indicated that most if not all of the decrease in the number of lysyl oxidase mRNA molecules can be explained by diminished transcription of the respective gene.Lysyl oxidase (EC 1.4.3.13), an extracellular copper enzyme, initiates the cross-linking of collagens and elastin by catalyzing oxidative deamination of the ⑀-amino group in certain lysine and hydroxylysine residues of collagens and lysine residues of elastin (for reviews, see Refs. 1 and 2). Molecular cloning and complete cDNA-derived amino acid sequences have been reported for the rat (3, 4), human (5, 6), and chick (7) enzymes, which were found to be synthesized in precursor forms of 411, 417, and 420 amino acids, respectively. The human lysyl oxidase gene is located on chromosome 5 (5, 6) and the mouse gene on chromosome 18 (8 -10), both genes consisting of seven exons (11,12). Increased lysyl oxidase activity has been reported in fibrotic disorders (1), while a deficiency is found in two Xlinked, recessively inherited human disorders, the occipital horn syndrome and Menkes syndrome, and in the X-linked recessively inherited mottled series of allelic mutant mice (13-15). In these X-linked disorders, the low enzyme activity appears to be secondary to abnormalities in copper metabolism.Lysyl oxidase activity is markedly low in the culture medium of many malignantly transformed human cell lines (16). The cDNA-derived amino acid sequence of the mouse ras recision gene, rrg (17), has been found to match that of rat lysyl oxidase (18), suggesting that rrg and lysyl oxidase are identical. The levels of rrg mRNA (17) and lysyl oxidase activity (18) are markedly decreased in NIH 3T3 cells transformed by LTR-cHa-ras compared with those in nontransformed NIH 3T3 or in cells after reversion following prolonged treatment with interferon-␤ (17). Transfection of the revertants with antisense rrg constructs leads to a transformed morphology again, and the cells become tumorigenic in nude mice (17).The purpose of this work was to explore further the reasons for the low lysyl oxidase activity observed in the culture medium of malignantly transformed cells. For this pu...

In the original publication of this paper, the middle initial of Michael D. Ferrari's name was inadvertently left out. This has since been corrected online. The authors apologize for the error.