A transient COS-7 cell expression system was used to
investigate the functional domain arrangement of tissue
inhibitor of metalloproteinases-3 (TIMP-3), specifically
to assess the contribution of the amino- and carboxylterminal
domains of the molecule to its matrix metalloproteinase
(MMP) inhibitory and extracellular matrix
(ECM) binding properties. Wild type TIMP-3 was entirely
localized to the ECM in both its glycosylated (27
kDa) and unglycosylated (24 kDa) forms. A COOH-terminally
truncated TIMP-3 molecule was found to be a non-
ECM bound MMP inhibitor, whereas a chimeric TIMP
molecule, consisting of the NH2-terminal domain of
TIMP-2 fused to the COOH-terminal domain of TIMP-3,
displayed ECM binding, albeit with a lower affinity than
the wild type TIMP-3 molecule. Thus the functional domain
arrangement of TIMP-3 is analogous to that seen in
TIMP-1 and -2, namely that the NH2-terminal domain is
responsible for MMP inhibition whereas the COOH-terminal
domain is most important in mediating the specific
functions of the molecule. A mutant TIMP-3 in
which serine 181 was changed to a cysteine, found in
Sorsby’s fundus dystrophy, a hereditary macular degenerative
disease, was also expressed in COS-7 cells. This
gave rise to an additional 48-kDa species (possibly a
TIMP-3 dimer) that retained its ability to inhibit MMPs
and localize to the ECM. These data favor the hypothesis
that the TIMP-3 mutations seen in Sorsby’s fundus dystrophy
contribute to disease progression by accumulation
of mutant protein rather than by the loss of functional
TIMP-3
