Hyaluronic acid associated with the surfaces of cultured fibroblasts is linked to a serum-derived 85-kDa protein.
Hyaluronic acid (HA) was extracted from the cell layer of cultured mouse dermal fibroblasts with 6 M guanidine HCl in the presence of 8% (w/v) Zwittergent. HA could be separated from the bulk of extracted proteins by consecutive isopycnic centrifugation and gel and ion-exchange chromatography under dissociative conditions. The final preparation was the complex of HA (viscosity average molecular weight approximately 2 x 10(6)) and a protein of Mr approximately 85,000 in a molar ratio of 1:1. Since the extraction procedure employed has been shown to break most noncovalent bonds between HA and proteins, they would appear to be covalently linked. However, the HA-binding protein remained unlabeled even after long incubation of the cells in the presence of a highly radioactive amino acid mixture, suggesting that it is an exogenous protein derived from the fetal calf serum added to culture medium. The presence of a HA-binding 85-kDa protein could in fact be demonstrated in fetal calf serum as well as sera from various other sources. This protein cross-reacted with antibodies raised against the HA-protein complex purified from cultured mouse dermal cells and was retained on octyl-Sepharose. Like the cell-derived 85-kDa protein, the serum 85-kDa protein, once bound to HA, could not be released from the complex by various dissociative procedures. These results, taken together, suggest that the hydrophobic serum protein can be intercalated into cell surface membranes, thereby mediating the binding of HA to the cell surface.
Institute of Molecular Science of Medicine, Aichi Medical University, Japan.
M
M
Yoneda
S
S
Suzuki
K
K
Kimata
D000818:Animals; D000831:Animals, Newborn; D002352:Carrier Proteins / Q000097:blood / Q000302:isolation & purification / Q000378:metabolism*; D002460:Cell Line; D002478:Cells, Cultured; D002499:Centrifugation, Density Gradient; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D005347:Fibroblasts / Q000378:metabolism; D018960:Hyaluronan Receptors; D006820:Hyaluronic Acid / Q000302:isolation & purification / Q000378:metabolism*; D015151:Immunoblotting; D051379:Mice; D008815:Mice, Inbred Strains; D008970:Molecular Weight; D012867:Skin / Q000378:metabolism
D002352:Carrier Proteins; D018960:Hyaluronan Receptors; D006820:Hyaluronic Acid