Purification and characterization of native type XIV collagen.
A new molecule, type XIV collagen, with domains homologous to type IX and XII collagens has been recently discovered in pepsin extracts of fetal bovine tissues (Dublet, B., and van der Rest, M. (1991) J. Biol. Chem. 266, 6853-6858). In the present study, we describe the purification and the characterization of the intact native form of this newly discovered collagen. By using only two chromatographic steps we were able to obtain pure type XIV collagen. Furthermore, minor modifications of the protocol allowed us to perform the simultaneous large scale purification of type XII and type XIV collagens from the same tissue. Intact type XIV collagen migrates on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) as two bands of 220 and 290 kDa (reducing conditions). After collagenase treatment, a single band of 190 kDa is observed, which represents the large non-collagenous domain of the molecule (NC3). Rotary shadowing electron micrographs of intact type XIV collagen show a cross-shaped structure formed by a thin tail attached through a central globule to three identical "fingers." These properties are similar to those previously described for intact chicken type XII collagen (Dublet, B., Oh, S., Sugrue, S. P., Gordon, M. K., Gerecke, D. R., Olsen, B. R., and van der Rest, M. (1989) J. Biol. Chem. 264, 13150-13156), but the two molecules are different gene products and have charge and glycosylation differences. Finally, we show that the three chains of purified type XIV collagen have an apparent molecular mass of approximately 220 kDa and are not cross-linked to each other by bonds other than disulfide bridges. The same observation was made for type XII collagen. In both cases, the 290-kDa migrating band in SDS-PAGE is due to incomplete denaturation in electrophoresis sample buffer in the absence of urea.
Aubert-Foucher
E
E
Institute of Biology and Chemistry of Proteins (CNRS-UPR 412), Lyon, France.
Font
B
B
Eichenberger
D
D
Goldschmidt
D
D
Lethias
C
C
van der Rest
M
M
D000818:Animals; D002642:Chick Embryo; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D003094:Collagen / Q000737:chemistry / Q000302:isolation & purification* / Q000648:ultrastructure; D004591:Electrophoresis, Polyacrylamide Gel; D015151:Immunoblotting; D003012:Microbial Collagenase; D008854:Microscopy, Electron; D008970:Molecular Weight; D010434:Pepsin A; D010446:Peptide Fragments / Q000302:isolation & purification; D012867:Skin / Q000737:chemistry*; D013710:Tendons / Q000737:chemistry*; D014508:Urea
D010446:Peptide Fragments; D014508:Urea; D003094:Collagen; D010434:Pepsin A; D003012:Microbial Collagenase