Assembly of the type 1 procollagen molecule: selectivity of the interactions between the alpha 1(I)- and alpha 2(I)-carboxyl propeptides.
Assembly of the heterotrimeric procollagen I molecule is initiated by interactions between the carboxyl propeptide domains of the completed nascent pro alpha chains. The [pro alpha 1(I)]2[pro alpha 2(I)] heterotrimer is the predominant molecule, with much smaller amounts of stable [pro alpha 1(I)]3 homotrimer also being formed. However, the [pro alpha 2(1)]3 homotrimer has not been detected, raising questions as to the mechanism of chain assembly and why [pro alpha2(1)]3 homotrimers are not formed. These questions have been examined here by expressing the intact and amino- or carboxyl-terminal truncated C-propeptides of the pro alpha chains recombinantly in bacteria and in a coupled transcription/translation reticulocyte lysate system. Their interactions were studied in vitro by binding analyses and in vivo by using the yeast two-hybrid system. The C-pro alpha 1(I) interacted with itself, and with C-pro alpha 2(I), as expected. Surprisingly, the C-pro alpha 2(I) also interacted with itself, both in vitro and in vivo. While the interaction of C-pro alpha 2(I) with itself and C-pro alpha 1(I) in vitro was strong, these interactions were weaker in vivo. Deletion of 36 amino acids from the C-terminal domain of C-pro alpha 1 had no effect on its binding to intact self or intact C-pro alpha 2, but the same deletion in C-pro alpha 2 completely abolished its binding to intact C-pro alpha 2 and to C-pro alpha 1. Comparable N-terminal deletions in C-pro alpha 1 or C-pro alpha 2 diminished, but did not abolish, their binding to intact C-pro alpha 1 and C-pro alpha 2. In the yeast two-hybrid system, C-pro alpha 2 interacted with itself more weakly than with C-pro alpha 1. Molecular modeling and circular dichroism analyses showed that C-pro alpha 1 and C-pro alpha 2 have different folded structures and stability. Studies with antibodies specific to the C-pro alpha1 and alpha2 peptides showed them to precipitate different, specific, and distinct cell proteins from fibroblast lysates. The C-pro alpha 2(I) antibody complexed with more cell proteins. We hypothesize that the lack of pro alpha 2(I) homotrimers is not due to the inability of the C-pro alpha 2(I) to interact with itself, but rather to the competing presence of other cell proteins. The specificity of these interactions may reside in conformational differences in N- and C-terminal sequences of the two propeptides or in their different folding patterns.
Alvares
K
K
Division of Oral Biology, Northwestern University Dental School, Chicago, Illinois 60611, USA.
Siddiqui
F
F
Malone
J
J
Veis
A
A
D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D002460:Cell Line; D002474:Cell-Free System; D002942:Circular Dichroism; D004220:Disulfides / Q000737:chemistry; D004926:Escherichia coli / Q000235:genetics; D005822:Genetic Vectors / Q000737:chemistry; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011233:Precipitin Tests; D011347:Procollagen / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017510:Protein Folding; D011499:Protein Processing, Post-Translational; D011817:Rabbits; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D012441:Saccharomyces cerevisiae
D004220:Disulfides; D010446:Peptide Fragments; D011347:Procollagen; D011994:Recombinant Proteins; C042813:procollagen type I carboxy terminal peptide