Expression of wild-type and modified proalpha chains of human type I procollagen in insect cells leads to the formation of stable [alpha1(I)]2alpha2(I) collagen heterotrimers and [alpha1(I)]3 homotrimers but not [alpha2(I)]3 homotrimers.
Insect cells coinfected with a baculovirus coding for the proalpha1(I) chain of human type I procollagen and a double promoter virus coding for the alpha and beta subunits of human prolyl 4-hydroxylase produced homotrimeric [proalpha1(I)]3 procollagen molecules. The use of an additional virus coding for the proalpha2(I) chain led to the formation of a heterotrimeric molecule with the correct 2:1 ratio of proalpha1 to proalpha2 chains of type I procollagen (proalpha1(I) and proalpha2(I) chains, respectively), unless the proalpha1(I) chain was expressed in a relatively large excess. Replacement of the sequences coding for the signal peptide and the N propeptide of the proalpha1(I) chain with those of the proalpha1(III) chain increased level of expression of the proalpha1(I) chain, whereas no similar effect was found when the corresponding modification was made to the virus coding for the proalpha2(I) chain. Molecules containing such modified N propeptides were found to be processed at their N terminus more rapidly than those containing the wild-type propeptides. The Tm of the type I collagen homotrimer was similar to that of the heterotrimer, both values being about 42-43 degrees C when determined by circular dichroism. The wild-type proalpha2(I) chain formed no homotrimers. Replacement of the C propeptide of the proalpha2(I) chain with that of the proalpha1(I) chain or proalpha1 chain of type III procollagen (proalpha1(III) chain) led to the formation of homotrimers, but the alpha2(I) chains in such molecules were completely digested by pepsin in 1 h at 22 degrees C. The data thus suggest that, in addition to control at the level of the C propeptide, other restrictions may exist at the level of the collagen domain that prevent the formation of stable homotrimeric [proalpha2(I)]3 molecules in insect cells.
Myllyharju
J
J
Collagen Research Unit, Biocenter and Department of Medical Biochemistry, University of Oulu, FIN-90220 Oulu, Finland.
Lamberg
A
A
Notbohm
H
H
Fietzek
P P
PP
Pihlajaniemi
T
T
Kivirikko
K I
KI
D000818:Animals; D002942:Circular Dichroism; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D011347:Procollagen / Q000378:metabolism*; D011393:Procollagen-Proline Dioxygenase / Q000378:metabolism; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry; D015183:Restriction Mapping; D018411:Spodoptera
D011347:Procollagen; D011994:Recombinant Proteins; D011393:Procollagen-Proline Dioxygenase