Abstract
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.
Highlights
The C-propeptides of the pro␣ 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␣1(I), pC␣2(I), and pC␣1(III) chains, i.e. pro␣ 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 ␣foldon chains were expressed in Pichia pastoris cells that expressed the two types of subunit of human prolyl 4-hydroxylase; the foldon domain was subsequently removed by pepsin treatment, which digests non-triple helical collagen chains, whereas triple helical collagen molecules are resistant to it
Summary
The C-propeptides of the pro␣ chains of type I and type III procollagens are believed to be essential for correct chain recognition and chain assembly in these molecules. In recent years experiments have been performed using recombinant expression in cultured insect cells (9 –11), yeasts [12,13,14] and plants [15] These cells have been shown to assemble partially [13, 15] or fully (9 –12, 14) hydroxylated recombinant collagen chains into molecules with partially [13, 15] or fully (9 –12, 14) stable triple helices, provided that they co-express the two types of subunit of a recombinant prolyl 4-hydroxylase. In the present work we have studied whether fully hydroxylated recombinant human type I collagen ␣ chains lacking C-propeptides can assemble effectively into triple helical molecules in the yeast Pichia pastoris when it co-expresses the two types of subunits of a recombinant human prolyl 4-hydroxylase [12, 14]. No data are available to indicate whether foldon could be used to replace the C-propeptides of pro␣ chains in the assembly of stable triple helical collagen molecules
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