COOH-terminal SAA1 peptides fail to induce chemokines but synergize with CXCL8 and CCL3 to recruit leukocytes via FPR2.

A natural leukocyte chemoattractant was isolated from bovine serum by an established 4-step purification procedure. Based on its relative molecular mass of 7287 and NH2-terminal sequence, the protein was identified as a carboxy-terminal peptide of the acute phase protein serum amyloid A1 (SAA1). This SAA1(46-112) fragment and its human equivalent SAA1(47-104) were chemically synthesized. Unlike intact SAA1α, these SAA fragments failed to directly chemoattract neutrophils and monocytes, to induce chemokines, and to stimulate downstream extracellular signal-regulated kinase signaling in monocytes. However, the SAA fragments potently synergized with CCL3 to induce monocyte migration and with CXCL8 to stimulate neutrophil shape changes and chemotaxis. Unlike intact SAA1α, SAA1(46-112) did not induce CXCL6 ex vivo but provoked a cooperative intraperitoneal neutrophil recruitment in mice when coinjected with CXCL6 into the peritoneal cavity. Moreover, SAA1(47-104) desensitized the synergy between intact SAA1α and CXCL8 in neutrophil chemotaxis, suggesting that this peptide binds formyl peptide receptor 2 (FPR2). This was evidenced by a complete blockade of synergy between the COOH-terminal SAA1 fragments and CXCL8 or CCL3 in neutrophil and monocyte chemotaxis, respectively, by the FPR2 antagonist WRW4 Thus, SAA1 is degraded into fragments lacking chemokine-inducing capacity, while keeping synergy with cytokine-induced chemokines to sustain limited inflammation.

Electrostatic Interactions at N- and C-Termini Determine Fibril Polymorphism in Serum Amyloid A Fragments.

Amyloid polymorphism presents a challenge to physical theories of amyloid formation and stability. The amyloidogenic protein serum amyloid A (SAA) exhibits complex and unexplained structural polymorphism in its N-terminal fragments: the N-terminal 11-residue peptide (SAA1-11) forms left-handed helical fibrils, while extension by one residue (SAA1-12) produces a rare right-handed amyloid. In this study, we use a combination of vibrational spectroscopy and ultramicroscopy to examine fibrils of these peptides and their terminally acetylated and amidated variants, in an effort to uncover the physical basis for this effect. Raman spectroscopy and atomic force microscopy provide evidence that SAA1-12 forms a ß-helical fibril architecture, while SAA1-11 forms more typical stacked ß-sheets. Importantly, N-terminal acetylation blocks fibril formation by SAA1-12 with no effect on SAA1-11, while C-terminal amidation has nearly the opposite effect. Together, these data suggest distinct electrostatic interactions at the N- and C-termini stabilize the two fibril structures; we propose model fibril structures in which C-terminal extension changes the favored intermolecular interaction between peptide monomers from an Arg1-C-terminus charge pair to an N-terminus-C-terminus charge pair. This model suggests a general mechanism for charge-mediated amyloid polymorphism and may inform strategies for design of peptide-based nanomaterials stabilized by engineered intermolecular contacts.

Evaluation of colostrum-derived human mammary-associated serum amyloid A3 (M-SAA3) protein and peptide derivatives for the prevention of enteric infection: in vitro and in murine models of intestinal disease.

In vitro experiments confirmed that a 10-mer peptide derived from human mammary-associated serum amyloid A3 (M-SAA3) protected intestinal epithelial cells from enteropathogenic Escherichia coli (EPEC) adherence. The entire 42-mer human M-SAA3 protein was even more effective, reducing EPEC binding by 72% relative to untreated cells (P<0.05), compared with 25% and 57% reductions for the human 10-mer and Lactobacillus GG, respectively. However, none of the M-SAA3 peptides reduced Salmonella invasion in vitro (P>0.05). Each of the M-SAA3 10-mer peptides and the 42-mer was then administered orally to mice at 500 mug day(-1) for 4 days before deliberate infection with either Citrobacter rodentium (mouse model of EPEC) or Salmonella Typhimurium. None of the peptides protected against Salmonella infection and the 42-mer may even increase infection, as there was a trend towards increased Salmonella counts in the liver and small intestine in 42-mer-treated mice compared with those in sodium acetate-treated control mice. Citrobacter counts were reduced in the caecum of mice administered the 42-mer relative to a scrambled 10-mer (P<0.05), but not compared with the sodium acetate control and no reductions were observed in the faeces or colon. Overall, although promising anti-infective activity was demonstrated in vitro, neither the 42-mer M-SAA3 protein nor a 10-mer peptide derivative prevented enteric infection in the animal models tested.