Interactions between collagen-binding stress protein HSP47 and collagen. Analysis of kinetic parameters by surface plasmon resonance biosensor.
A 47-kDa heat shock protein (HSP47) is a collagen-binding stress protein which is localized in the endoplasmic reticulum (ER) of collagen-secreting cells. Recent studies have shown that HSP47 transiently binds to newly synthesized procollagens and that conformationally abnormal procollagen is also bound by HSP47 for a much longer time in the ER (Nakai, A., Satoh, M., Hirayoshi, K., and Nagata, K. (1992) J. Cell Biol. 117, 903-914). HSP47 is thus suggested to have a collagen-specific molecular chaperone-like function. In this report, we analyzed the interaction of HSP47 and types I to V collagen using BIAcore system, an optical biosensor based on the principles of surface plasmon resonance. Types I-V collagen were purified from porcine skin, porcine articular cartilage, bovine lens capsule, and porcine placenta and immobilized on sensorchips of the BIAcore system at a surface concentration of 10-15 ng/mm2. Purified recombinant mouse HSP47 (rmHSP47) expressed in Escherichia coli was passed over the sensorchips at a flow rate of 2 microliters/min and binding curves of rmHSP47 to collagens were monitored. Using this approach, accurate association and dissociation rate constants were determined in addition to dissociation constants. rmHSP47 was found to bind to types I-V collagen with similar dissociation constants of the order of 10(-7) M. This relatively low dissociation constant resulted from the rapid dissociation rate constant (kdiss > 10(-2) s-1) and considerably high (kass approximately 2 x 10(4) M-1 s-1) association rate constant. These kinetic parameters may reflect a transient interaction between HSP47 and procollagen in vivo.
Natsume
T
T
Research & Development Center, Nippon Meat Packers Inc., Ibaraki, Japan.
Koide
T
T
Yokota
S
S
Hirayoshi
K
K
Nagata
K
K
D000595:Amino Acid Sequence; D000818:Animals; D015374:Biosensing Techniques; D002478:Cells, Cultured; D002642:Chick Embryo; D003094:Collagen / Q000378:metabolism*; D006360:Heat-Shock Proteins / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D011485:Protein Binding
D006360:Heat-Shock Proteins; D003094:Collagen