Abstract
Previously we have shown that THP-1 cells synthesize matrix metalloproteinase-9 (MMP-9) where a fraction of the enzyme is strongly linked to a proteoglycan (PG) core protein. In the present work we show that these pro-MMP-9.PG heteromers have different biochemical properties compared with the monomeric form of pro-MMP-9. In these heteromers, the fibronectin II-like domain in the catalytic site of the enzyme is hidden, and the fibronectin II-like-mediated binding to gelatin and collagen is prevented. However, a fraction of the pro-MMP-9.PG heteromers interacted with gelatin and collagen. This interaction was not through the chondroitin sulfate (CS) part of the PG molecule but, rather, through a region in the PG core protein, a new site induced by the interaction of pro-MMP-9 and the PG core protein, or a non-CS glycosaminoglycan part of the PG molecule. The interaction between pro-MMP-9.PG heteromers and gelatin was weaker than the interaction between pro-MMP-9 and gelatin. In contrast, collagen I bound to pro-MMP-9.PG heteromers and pro-MMP-9 with approximately the same affinity. Removal of CS chains from the PG part of the heteromers did not affect the binding to gelatin and collagen. Although the identity of the PG core protein is not known, this does not have any impact on the described biochemical properties of the heteromer or its pro-MMP-9 component. It is also shown that a small fraction of the PG, which is not a part of the pro-MMP-9.PG heteromer, can bind gelatin. As for the pro-MMP-9.PG heteromers, this was independent of the CS chains. The structure that mediates the binding of free PG to gelatin is different from the corresponding structure in the pro-MMP-9.PG heteromer, because they were eluted from gelatin-Sepharose columns under totally different conditions. Although only a small amount of pro-MMP-9.PG heteromer is formed, the heteromer may have fundamental physiological importance, because only catalytic amounts of the enzyme are required to digest physiological targets.
Highlights
A large number of genetically unrelated proteins are known to contain highly negatively charged glycosaminoglycan (GAG)2
Isolation of PG and Pro-matrix metalloproteinases (MMPs)-91⁄7PG Heteromer—The conditioned serum-free media from phorbol 12-myristate 13-acetate (PMA)-treated THP-1 cells incubated for 72 h in the presence or absence of [35S]sulfate was applied to Q-Sepharose chromatography as described previously [34, 41]
To determine which types of GAG chains were synthesized by the THP-1 cells, [35S]PG was treated with chondroitin ABC lyase (cABC), which degrades chondroitin sulfate (CS) but not heparan sulfate (HS) chains
Summary
Trichloroacetic acid, formaldehyde, Tris, urea, and sodium acetate were from Merck. Acrylamide, Coomassie Brilliant Blue G-250, and Triton X-100 were from BDH. S-2255), cetylpyridinium chloride, phorbol 12-myristate 13-acetate (PMA), DMSO, Hepes, Brij-35, silver nitrate, alkaline phosphatase-conjugated antibodies, chondroitin sulfate C, EDTA, gelatin, calf skin collagen, and alkaline phosphatase-conjugated antibody were purchased from Sigma. Proteinase-free chondroitin ABC lyase (cABC) was from Seikagaku Kogyo Co. Human recombinant TIMP-1, mouse monoclonal antibodies against human MMP-9 and TIMP-1 were from Calbiochem. Gelatin-Sepharose, Q-Sepharose, Superose 6 (HR 10/30), Sephadex G-50 (fine), Amplify, 14C-labeled RainbowTM protein molecular weight standards, and [35S]sulfate were obtained from Amersham Biosciences. Unlabeled molecular weight standards were from BioRad. Cronex 4 medical x-ray film was obtained from Sterling Diagnostic Imaging. Ultima Gold XR was from PerkinElmer Life Sciences, and CDP-StarTM chemiluminescent substrate was from New England Biolabs
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
