Prospects and limitations of the rational engineering of fibrillar collagens.
Recombinant collagens are attractive proteins for a number of biomedical applications. To date, significant progress was made in the large-scale production of nonmodified recombinant collagens; however, engineering of novel collagen-like proteins according to customized specifications has not been addressed. Herein we investigated the possibility of rational engineering of collagen-like proteins with specifically assigned characteristics. We have genetically engineered two DNA constructs encoding multi-D4 collagens defined as collagen-like proteins, consisting primarily of a tandem of the collagen II D4 periods that correspond to the biologically active region. We have also attempted to decrease enzymatic degradation of novel collagen by mutating a matrix metalloproteinase 1 cleavage site present in the D4 period. We demonstrated that the recombinant collagen alpha-chains consisting predominantly of the D4 period but lacking most of the other D periods found in native collagen fold into a typical collagen triple helix, and the novel procollagens are correctly processed by procollagen N-proteinase and procollagen C-proteinase. The nonmutated multi-D4 collagen had a normal melting point of 41 degrees C and a similar carbohydrate content as that of control. In contrast, the mutant multi-D4 collagen had a markedly lower thermostability of 36 degrees C and a significantly higher carbohydrate content. Both collagens were cleaved at multiple sites by matrix metalloproteinase 1, but the rate of hydrolysis of the mutant multi-D4 collagen was lower. These results provide a basis for the rational engineering of collagenous proteins and identifying any undesirable consequences of altering the collagenous amino acid sequences.
Majsterek
Ireneusz
I
Department of Dermatology and Cutaneous Biology, Jefferson Medical College, Jefferson Institute of Molecular Medicine, Thomas Jefferson University, Philadelphia, Pennsylvania 19107, USA.
McAdams
Erin
E
Adachi
Eijiro
E
Dhume
Shirish T
ST
Fertala
Andrzej
A
D000596:Amino Acids / Q000737:chemistry; D001665:Binding Sites; D055395:Bone Morphogenetic Protein 1; D019485:Bone Morphogenetic Proteins / Q000737:chemistry; D002241:Carbohydrates / Q000737:chemistry; D045744:Cell Line, Tumor; D003094:Collagen / Q000737:chemistry; D004247:DNA / Q000737:chemistry; D005109:Extracellular Matrix / Q000378:metabolism; D024022:Fibrillar Collagens / Q000737:chemistry*; D006801:Humans; D006868:Hydrolysis; D020782:Matrix Metalloproteinases / Q000737:chemistry; D008666:Metalloendopeptidases / Q000737:chemistry; D008854:Microscopy, Electron; D011348:Procollagen N-Endopeptidase / Q000737:chemistry; D015202:Protein Engineering / Q000379:methods*; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry; D013696:Temperature
D000596:Amino Acids; D019485:Bone Morphogenetic Proteins; D002241:Carbohydrates; D024022:Fibrillar Collagens; D011994:Recombinant Proteins; D003094:Collagen; D004247:DNA; D020782:Matrix Metalloproteinases; D008666:Metalloendopeptidases; D011348:Procollagen N-Endopeptidase; C525705:BMP1 protein, human; D055395:Bone Morphogenetic Protein 1