A critical crosslink region in human-bone-derived collagen type I. Specific cleavage site at residue Leu95.
Collagen was extracted from human adult bone by limited pepsin digestion and collagen types were purified by consecutive salt precipitation first under neutral and then under acid conditions. In SDS/PAGE, all collagen type I preparations showed a protein band [alpha 1s(I)] migrating between alpha 1(I) and alpha 2(I) as well as a band [alpha 2s(I)] migrating in front of alpha 2(I). The collagenous nature of the pepsin-stable alpha 1s(I) protein was clearly demonstrated by digestion with human-leucocyte-derived collagenase, immunoblotting with antibodies against collagen type I and amino acid analysis. Partial amino acid sequencing of alpha 1(I) and alpha 1s(I) identified alpha 1s(I) as a shortened alpha 1(I) chain due to a specific cleavage site between residues Leu95 and Asp96 which is in close vicinity to the hydroxylysine-derived crosslink at position 87. In circular dichroism, the proportion of thermally labile collagen molecules was proportional to the amount of shortened alpha 1(I) and alpha 2(I) chains, respectively. The melting temperature was found to be 36 +/- 0.5 degrees C as judged from circular dichroism and susceptibility to proteolysis. Our data provide clear evidence that a shortened alpha 1-derived collagen chain can be extracted from human adult bone whereas it is hardly found in human skin. The unique cleavage site might provide important information about the collagen I molecule embedded in the calcified matrix of human bone.
Bätge
B
B
Institut für Medizinische Molekularbiologie, Medizinische Universität zu Lübeck, Federal Republic of Germany.
Notbohm
H
H
Diebold
J
J
Lehmann
H
H
Bodo
M
M
Deutzmann
R
R
Müller
P K
PK
D000328:Adult; D000375:Aging / Q000378:metabolism; D000595:Amino Acid Sequence; D001842:Bone and Bones / Q000032:analysis*; D002146:Bony Callus / Q000032:analysis / Q000378:metabolism; D003094:Collagen / Q000302:isolation & purification / Q000378:metabolism*; D003432:Cross-Linking Reagents / Q000378:metabolism; D004586:Electrophoresis; D006358:Hot Temperature; D006801:Humans; D007930:Leucine / Q000378:metabolism; D003012:Microbial Collagenase / Q000378:metabolism; D008969:Molecular Sequence Data; D010013:Osteogenesis Imperfecta / Q000378:metabolism; D010434:Pepsin A / Q000378:metabolism; D010455:Peptides / Q000302:isolation & purification / Q000378:metabolism; D012867:Skin / Q000032:analysis
D003432:Cross-Linking Reagents; D010455:Peptides; D003094:Collagen; D010434:Pepsin A; D003012:Microbial Collagenase; D007930:Leucine