[CITATION][C] Characterization of the Interaction between Integrins and Recombinant Human Osteopontina

DD Hu, JR Hoyer, JW Smith - Annals of the New York Academy …, 1995 - Wiley Online Library
DD Hu, JR Hoyer, JW Smith
Annals of the New York Academy of Sciences, 1995Wiley Online Library
Osteopontin (OPN) is a multifunctional extracellular matrix protein that supports osteoclast
adhesion to the bone by binding to integrin avp3.'We characterized the interaction between
OPN and integrin a, p3 with recombinant human osteopontin (rOPN) and the urinary form of
human OPN, uropontin (UP). The cDNA of human OPN was modified and engineered into
an expression vector pGEX-Sx-1 (Invitrogen). The recombinant osteopontin was expressed
in E. coli (BL26, Novagen) as a fusion protein with glutathione S-transferase (GST) and …
Osteopontin (OPN) is a multifunctional extracellular matrix protein that supports osteoclast adhesion to the bone by binding to integrin avp3.'We characterized the interaction between OPN and integrin a, p3 with recombinant human osteopontin (rOPN) and the urinary form of human OPN, uropontin (UP). The cDNA of human OPN was modified and engineered into an expression vector pGEX-Sx-1 (Invitrogen). The recombinant osteopontin was expressed in E. coli (BL26, Novagen) as a fusion protein with glutathione S-transferase (GST) and cleaved from GST with factor Xa. The protein was purified by DEAE chromatography and HPLC. rOPN migrated at 60 kD on a 10% SDS-PAGE, but mass spectroscopic measurement showed its mass to be 27,046 daltons. The amino acid sequencing analysis reported this form of OPN has an intact amino terminus. Therefore rOPN is truncated, presumably by bacterial proteases, at arginine residue 228, 70 amino acids short of the native C-terminus. Thus it is termed rOP27. Human UP was purified under nondenaturing conditions by monoclonal antibody affinity chromatography.
UP and rOP27 are functionally identical in their ability to support cell adhesion and to bind purified integrin a, &. Both events are RGD dependent. In addition, an aVp3-specific antibody, Fab9, 3 abolished cell adhesion to rOP27, proving that the adhesion is mediated by a, p3. Further study showed that neither rOP27 nor UP binds platelet integrin~~ nb P 3 with detectable affinity. Thus, osteopontin is the first naturally occurring RGD-containing protein found to have a substantially greater affinity for the a, integrin than for the (YIlb integrin. The major finding from this study is that divalent cations regulate the interaction between integrins and rOP27. Cell adhesion was most efficient in Mn2+ and Mg2+. Moreover, cell adhesion to rOPN supported by either Mn2+ or MgZf is blocked
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