**3. Production and characterization of SAA fragments**

In AA amyloidosis, the insoluble AA amyloid protein is derived from the proteolytic cleavage of SAA, generating an N-terminal fragment of SAA. In some cases, this AA amyloid protein lacks amino acids at both N- and C-terminus compared to the full-length SAA. One reported study found that the AA fibril protein purified from rheumatoid arthritis patients with secondary amyloidosis contained 2 fragments with residues 1–50 and 1–45 [37]. However, a SAA fragment with residues 1–76 (or 2–76) was most commonly found in amyloid fibrils, such as those from the livers and spleens of patients with familial Mediterranean fever (FMF), tuberculosis, Hodgkin's diseases and bronchiectasis [24, 38].

In patients with rheumatoid arthritis, higher serum levels of metalloproteinases (MMPs)-1, -2, -3 and -9 were detected compared to healthy controls [39, 40], and

the production of these enzymes could be stimulated by SAA [41, 42]. Besides, these MMPs were shown to cleave SAA and AA amyloid protein *in vitro* to produce various sizes of SAA fragments (see **Table 1**). In addition to generating the AA fragments commonly identified in secondary amyloidosis, MMP-1, -2 and -3 cleaved SAA into fragments with residues 1–57, 1–51 and 8–55, respectively [43]. The spanning region (residues 51–57) contains sites that may be cleaved by all three MMPs. In addition, MMP-2 and MMP-3 can also cleave at other residues including residues 7–8 (MMP-2 and -3), 16–17 (MMP-3) and 23–24 (MMP-3). In other species studied, MMP-1 and -3 are able to cleave rabbit SAA3 at residues 50–57, showing conservation between the rabbit SAA3 and human SAA1 [44]. Therefore it was suggested that these MMPs might contribute to the pathogenesis of AA amyloidosis by generating SAA fragments.

In addition to their roles in AA amyloidosis, SAA-derived fragments may have other biological functions. A recent study demonstrated that MMP-9 could rapidly cleave human SAA1 within 30 minutes *in vitro* to produce COOH-terminal fragments, SAA1 (58–104), SAA1 (52–104) and SAA1 (57–104) [46]. These fragments account for 50, 30 and 20% of the total cleaved fragments by MMP-9, respectively. The synthetic peptides of these fragments failed to induce CXCL8 production in human monocytes and diploid fibroblasts, as well as neutrophil chemotaxis; however they potentiated CXCL8-induced neutrophil chemotaxis in a dosedependent manner via FPR2 [46]. The authors of this report suggested that intact SAA first initiates the inflammatory response and induces the release of MMP-9, which cleaves SAA and modulates the response of SAA by potentiating activities of selected chemokines to prolong the inflammation process. In addition to MMPs,


*The table lists known SAA fragments and synthetic peptides that have been identified. References are provided on the column to the right.*

**115**

*Serum Amyloid A and Immunomodulation DOI: http://dx.doi.org/10.5772/intechopen.81617*

amyloid protein [35, 49, 50].

region of SAA1 in LPS binding [53].

**4. The cytokine-like activities of recombinant SAA**

different from the known structural properties of chemokines [57].

cathepsins, endosomal and lysosomal proteases, were also shown to cleave SAA and might also be involved in AA amyloidosis. Cathepsin B was shown to cleave SAA at residues 76–77 to produce the most common form of AA found in amyloidosis [49]. Another study also reported that both cathepsin B and L completely cleaved SAA, and cathepsin B could produce 9 AA amyloid-like proteins; however, cathepsin L produced no fragments resembling AA amyloid proteins by cleaving within the N-terminus [45]. All amyloid-like SAA fragments described to date have either an intact N-terminus or one that only lacks 1–2 amino acids. Elastase and cathepsin D that cleave SAA further along the N-terminus can prevent the formation of AA

Accumulating evidence suggests that some of the observed biological functions of SAA, other than those related to amyloidosis, may be attributed to SAA-derived fragments rather than the intact protein. In some of these studies, synthetic peptides based on SAA protein sequence were prepared to verify or identify the potential functions. SAA-derived peptides with IFNγ-inducing capability were found in human rheumatic synovial fluid [51]. An SAA2-derived peptide with chemotactic activity for B lymphocytes was found in cow milk [52]. In a recent study, a fragment of SAA1 (46–112) was found in bovine serum and is equivalent to human SAA1 (47–104). The synthetic peptides of this fragment failed to directly induce chemotaxis and chemokine production (CXCL8 and CCL3) in human neutrophils and monocytes, but it synergized with CXCL8 or CCL3 to induce chemotaxis via FPR2 [47]. Studies were also conducted to examine potential functions of SAA and its peptides in LPS-induced inflammatory response. SAA-derived fragments lacking both N- and C-terminal residues were expressed as recombinant proteins and texted for their activities *in vitro*. Fragments such as one with amino acids 11–58 of human SAA1 exhibited minimal proinflammatory activity but enhanced ability to induce IL-10 expression and to counteract LPS-induced inflammation and lung injury [48]. In a recent study, a peptide consisting amino acids 32–47 of human SAA1 was found to disrupt the binding of SAA1 to LPS, suggesting the involvement of this

Recombinant SAA was used in an early study that identified the SAA protein as a chemoattractant for phagocytes [54]. Xu et al. reported that SAA also induced the migration and adhesion of lymphocytes [55]. These studies were among the first to identify leukocyte-activating activities of the recombinant SAA protein. SAA differs from chemokines as it lacks the characteristic cysteine residues that form disulfide bonds for structural stabilization. It was not until 2014 when the crystal structures of two SAA proteins were solved [32, 56]. The 4-helix bundle structure of the SAA monomers and the propensity of forming multimers [32, 56] are strikingly

Studies conducted by Patel et al. [58] and Fulaneto et al. [59] revealed cytokinelike activities of SAA for its induction of IL-1β, TNFα, IL-1RA and IL-8. Of note, the study conducted by Patel and coworkers used both the recombinant human SAA (rhSAA) and purified SAA-HDL complex, although they found that the cytokine-inducing activity of the SAA-HDL complex was much lower than that of rhSAA. These studies were followed by reports that SAA in neutrophils could induce IL-8 expression through one of the chemoattractant receptors [60] that also mediates anti-inflammatory activities when stimulated by the eicosanoid lipoxin A4 [61, 62]. In addition to proinflammatory cytokines, rhSAA was found to stimulate monocyte expression of tissue factor [63]. Injection of rhSAA to mice increased G-CSF
