Interaction of Pro-matrix Metalloproteinase-9/Proteoglycan Heteromer with Gelatin and Collagen

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-9⅐PG 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-9⅐PG 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-9⅐PG heteromers and gelatin was weaker than the interaction between pro-MMP-9 and gelatin. In contrast, collagen I bound to pro-MMP-9⅐PG 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-9⅐PG heteromer, can bind gelatin. As for the pro-MMP-9⅐PG 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-9⅐PG heteromer, because they were eluted from gelatin-Sepharose columns under totally different conditions. Although only a small amount of pro-MMP-9⅐PG heteromer is formed, the heteromer may have fundamental physiological importance, because only catalytic amounts of the enzyme are required to digest physiological targets.A large number of genetically unrelated proteins are known to contain highly negatively charged glycosaminoglycan (GAG) 2 chains. Such core proteins, substituted with GAG chains, constitute an entity of glycoproteins called proteoglycans (PGs). There are several types of GAG chains, where chondroitin sulfate (CS) and heparin/heparan sulfate (HS) are two major types (1). All GAG chains are unbranched, and they contain a variable number of negatively charged sulfate groups that are important for their function (2). Some PGs are associated with cells, whereas others are secreted and are a part of the extracellular matrix (ECM). Almost all mammalian cells synthesize PGs. Monocytes and macrophages synthesize PGs, which are mainly substituted with CS chains (CSPG) and only a minor proportion of HS (3, 4). In resting monocytes most of the CSPG is not released but sorted to the endocytic pathway and degraded (4). However, when monocytes are stimulated and differentiated to macrophages, both the biosynthesis and the secretion of CSPG are i...

Gene expression profiling of DEHP-treated cardiomyocytes reveals potential causes of phthalate arrhythmogenicity

“…Upregulation of endothelin receptors results in remodeling of MMPs and downregulation of connexin-43 through a common pathway (Peng, et al 2010). These MMPs are termed “gelatinases” and are known to degrade basement membrane proteins (Ahmed, et al 2006, Malla, et al 2008). An upregulation in MMP mRNA expression was observed following treatment with DEHP.…”

The effect of stromelysin-1 (MMP-3) on non-collagenous extracellular matrix proteins of demineralized dentin and the adhesive properties of restorative resins

“…This is not documented in dentin, but we cannot exclude that the cleavage of the heteromer by MMP-3 may release activated MMP-9. Even in very small amount MMP-9 may digest physiological targets [27,28].…”

Gelatin In Situ Zymography on Fixed, Paraffin-embedded Tissue: Zinc and Ethanol Fixation Preserve Enzyme Activity

“…We recently showed that DMSO and Triton X-100 have different abilities to detach gelatinase complexes from gelatin-Sepharose columns (Malla et al 2008a). Therefore, DMSO was also used to extract gelatin-degrading enzymes in the present study.…”

“…Gelatin Zymography SDS-PAGE substrate zymography was carried out as described previously (Malla et al 2008a) with gels (7.5 3 8.5 cm 3 0.75 mm) containing 0.1% (w/v) gelatin and 4% and 7.5% (w/v) polyacrylamide in the stacking and separating gels, respectively. Eight ml of extract was mixed with 2 ml of loading buffer (333 mm TrisHCl, pH 6.8, 10% SDS, 0.03% bromophenol blue, and 50% glycerol).…”