Je. Murphyullrich et al., HEPARIN-BINDING PEPTIDES FROM THROMBOSPONDIN-1 AND THROMBOSPONDIN-2 CONTAIN FOCAL ADHESION-LABILIZING ACTIVITY, The Journal of biological chemistry, 268(35), 1993, pp. 26784-26789
The cell adhesion regulating extracellular matrix glycoprotein, thromb
ospondin (TSP), causes a loss of focal adhesion plaques from spread en
dothelial cells and fibroblasts. To localize the site on TSP that has
focal adhesion-labilizing activity, we initially tested proteolytic fr
agments of TSP for activity. The heparin-binding fragment has signific
ant focal adhesion-labilizing activity, whereas the nonheparin-binding
140-kDa fragment had no significant activity. These results were cons
istent with previous data that showed that both a monoclonal antibody
to the heparin-binding domain of TSP (A2.5) and heparin neutralized TS
P activity. Peptides from putative heparin binding sequences of the am
ino-terminal heparin-binding domain of TSP were synthesized and tested
for their ability to cause loss of focal adhesions. The hep I peptide
(amino acids 17-35) caused maximal loss of focal adhesions and was ac
tive at 0.1 muM, whereas peptide hep II (74-95) and peptide hep III (1
70-189) were inactive. The activity of the hep I peptide was neutraliz
ed by the addition of heparin and heparan sulfate but not by chondroit
in sulfate. The basic amino acids in the hep I sequence appear to be r
equired for focal adhesion-labilizing activity, because modification o
f the lysine residues at amino acids 24 and 32 rendered the peptide co
mpletely inactive. In addition, a peptide from the analogous sequence
of mouse TSP 2, in which basic residues are conserved, was nearly as a
ctive as hep I from TSP1. These data show that the anti-adhesive activ
ity of TSP is conserved in both TSP1 and TSP2 and that the active site
is located in a 19-amino acid sequence in the heparin-binding domain
of TSPs.