Abstract
Serum amyloid A1 (SAA1) is a prototypic acute phase protein, induced to extremely high levels by physical insults, including inflammation and infection. Human SAA and its NH2-terminal part have been studied extensively in the context of amyloidosis. By contrast, little is known about COOH-terminal fragments of SAA. Intact SAA1 chemoattracts leukocytes via the G protein-coupled receptor formyl peptide receptor like 1/formyl peptide receptor 2 (FPR2). In addition to direct leukocyte activation, SAA1 induces chemokine production by signaling through toll-like receptor 2. We recently discovered that these induced chemokines synergize with intact SAA1 to chemoattract leukocytes in vitro and in vivo. Gelatinase B or matrix metalloproteinase-9 (MMP-9) is also induced by SAA1 during infection and inflammation and processes many substrates in the immune system. We demonstrate here that MMP-9 rapidly cleaves SAA1 at a known consensus sequence that is also present in gelatins. Processing of SAA1 by MMP-9 at an accessible loop between two alpha helices yielded predominantly three COOH-terminal fragments: SAA1(52–104), SAA1(57–104), and SAA1(58–104), with a relative molecular mass of 5,884.4, 5,327.3, and 5,256.3, respectively. To investigate the effect of proteolytic processing on the biological activity of SAA1, we chemically synthesized the COOH-terminal SAA fragments SAA1(52–104) and SAA1(58–104) and the complementary NH2-terminal peptide SAA1(1–51). In contrast to intact SAA1, the synthesized SAA1 peptides did not induce interleukin-8/CXCL8 in monocytes or fibroblasts. Moreover, these fragments possessed no direct chemotactic activity for neutrophils, as observed for intact SAA1. However, comparable to intact SAA1, SAA1(58–104) cooperated with CXCL8 in neutrophil activation and migration, whereas SAA1(1–51) lacked this potentiating activity. This cooperative interaction between the COOH-terminal SAA1 fragment and CXCL8 in neutrophil chemotaxis was mediated by FPR2. Hence, proteolytic cleavage of SAA1 by MMP-9 fine tunes the inflammatory capacity of this acute phase protein in that only the synergistic interactions with chemokines remain to prolong the duration of inflammation.
Highlights
Serum amyloid A (SAA) is an acute phase protein, mainly produced in the liver under inflammatory conditions (1), but extrahepatic production of SAA has been reported (2, 3)
To determine whether serum amyloid A1 (SAA1) is cleaved by gelatinase B/matrix metalloproteinases (MMPs)-9, recombinant intact SAA1 (11.8 kDa) was incubated at 37°C with the enzyme at a 1:50 enzyme:substrate ratio and analyzed by SDS–PAGE
The cleavage products were already visible after 0.25 h, and the intensity of the SAA1 cleavage products increased over time
Summary
Serum amyloid A (SAA) is an acute phase protein, mainly produced in the liver under inflammatory conditions (1), but extrahepatic production of SAA has been reported (2, 3). At low concentrations (10–500 ng/ml), SAA induces cytokines, chemokines, and matrix metalloproteinases (MMPs) (14–17). One of the most important biological activities of SAA, exerted at low concentrations, is its direct and indirect chemotactic activity for monocytes, neutrophils, immature dendritic cells, and regulatory T cells (17–21). The direct chemotactic activity of SAA is mediated by the G proteincoupled receptor (GPCR) formyl peptide receptor 2 (FPR2) (22). SAA is indirectly chemotactic for leukocytes via induction of chemokines through binding to toll-like receptor 2 (TLR2) (17, 21). Following induction by serum amyloid A1 (SAA1), these chemokines synergize with each other, and with SAA, to enhance leukocyte migration to the inflammatory site (17). Chemokines are small chemotactic cytokines, directing leukocyte migration under homeostatic and inflammatory conditions through binding to GPCRs (26). Posttranslational truncation of chemokines can increase, decrease, or even completely block their chemotactic capacity (27, 28)
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