The C Terminus of Mouse Monocyte Chemoattractant Protein 1 (MCP1) Mediates MCP1 Dimerization while Blocking Its Chemotactic Potency

Abstract

The extracellular protease plasmin cleaves mouse MCP1 (monocyte chemoattractant protein 1) at lysine 104, releasing a 50-amino acid C-terminal domain. The cleavage event increases the chemotactic activity of MCP1 and, by doing so, promotes the progression of excitotoxic injury in the central nervous system in pathological settings. The mechanism through which the cleavage event enhances MCP1-mediated chemoattraction is unknown; to investigate it, we use wild-type and mutant forms of recombinant MCP1. Full-length MCP1 (FL-MCP1) is secreted by cells as a dimer or multimer. We show that a mutant truncated at the C terminus, K104Stop-MCP1, does not dimerize, revealing that the C terminus mediates the interaction. Microglia, the immune-like cells normally present in the brain, derive from the bone marrow (1-5), enter the central nervous system (CNS) early during development, and reside in the parenchyma in a resting state characterized by ramified morphology. In the healthy brain, they continually extend and retract their processes to sense changes in the surrounding microenvironment (6). However, when there is an injury in the CNS, microglia switch to an activated state characterized by changes in gene expression, morphology, and proliferation (6 -16). The activated microglia then migrate to the site of injury and modify the injury outcome.The migration of microglia to the site of injury is stimulated by the local release of chemokines, a superfamily of structurally related small proteins that function as chemoattractants. A potent chemoattractant for monocytes/microglia (17), MCP1 (monocyte chemoattractant protein-1; also called CCL2), is upregulated in many types of CNS injury, including ischemia, hemorrhage, trauma, infection, hypoxia, and peripheral nerve axotomy (18 -24). The MCP1 protein is highly conserved in the N terminus among different species, whereas the C terminus is much more variable. The rodent MCP1 C terminus is decorated extensively with O-linked carbohydrates (25), which bind both soluble glycosaminoglycans (GAGs) and GAGs immobilized on the cell surface. Soluble GAGs inhibit the binding of MCP1 to its high affinity receptor CCR2 (30), which is expressed by microglia, astrocytes, and microvascular endothelial cells in the brain (26,27). Cell surface GAGs, on the other hand, have been reported to concentrate MCP1 locally, promote MCP1 oligomerization, and thus facilitate the binding of MCP1 to CCR2 (28 -31). Whether MCP1 functions as a monomer or homodimer, however, is still under debate. Although it is believed that MCP1 exerts its function as a homodimer (25,32), it has also been suggested that MCP1 can bind to CCR2 and induce downstream signaling as a monomer (33,34). It should be noted, however, that these experiments were conducted using human MCP1, not rodent MCP1, which contains a heavily glycosylated C-terminal extension.Previous studies in our laboratory have shown that 1) the highly glycosylated C-terminal extension of mouse MCP1 is removed by plasmin, the active protease of plasminogen,...

Truncation of monocyte chemoattractant protein 1 by plasmin promotes blood–brain barrier disruption

“…However, the effect was more potent in MCP1 -/-mice than in wild-type mice. It has been shown that MCP1 binds to and reduces membrane 1491 Truncated MCP1 disrupts the blood-brain barrier CCR2 levels (probably through endocytosis) (Jung et al, 2009;Yao and Tsirka, 2010). The dramatic change observed in MCP1 -/-mice could be due to higher membrane CCR2 density, as in the absence of MCP1 there would be no recycling of CCR2 off the plasma membrane.…”

“…These data suggest that upon MCP-1 binding to endothelial CCR2, a property that all MCP-1 mutants that retain the N-terminal portion of the protein exhibit, an increase in cortical actin is observed. However, the next step of the process, the internalization of CCR2 and re-arrangements of actin cytoskeleton to allow the cells to retract/reorganize the TJPs, is taking place only when the active FL-and K104Stop-MCP-1 proteins are present (Yao and Tsirka, 2010).…”

“…However, it is not known whether plasmin-mediated cleavage of MCP1 at K104, which enhances its chemotactic activity (Sheehan et al, 2007), affects MCP1-induced BBB compromise. To address this question, we used four recombinant MCP1 proteins, full-length MCP1 (FL), plasmin-noncleavable MCP1 (K104A), constitutively cleaved MCP1 (K104Stop) and MCP1 C-terminal extension (CT), that we previously characterized (Yao and Tsirka, 2010). First, we evaluated the cytotoxicity of these MCP1 mutants in mouse or human BMECs (HBMECs) using lactate dehydrogenase (LDH) cytotoxicity assays.…”

“…CT-MCP1 was subcloned into a pTYB1 vector with an N-terminal His 6 tag (Yao and Tsirka, 2010). BL21 cells were transformed with these constructs and expression of the target proteins was induced for 5 hours using isopropyl-b-D-thiogalactopyranoside.…”

“…The rodent MCP1 differs from the human protein at the C-terminus, with the rodent protein having a longer highly glycosylated Cterminus. We have previously shown that mouse MCP1 can be cleaved by plasmin at K104, and the truncated MCP1 is more potent than the full-length MCP1 in inducing microglial migration (Sheehan et al, 2007;Yao and Tsirka, 2010) through its receptor CCR2.…”