**2. SAA and its role in amyloidosis**

SAA is the general name of a family of proteins with high sequence homology but encoded by distinct genes [16]. Both humans and mice have 4 SAA genes, but in human the *SAA3* is a pseudogene that does not express [17]. SAA4 is constitutively expressed in both humans and mice. In contrast, the expression of SAA1, SAA2 and in mice, SAA3, is highly inducible [18]. These SAA proteins are therefore termed acute-phase SAAs based on their induced expression during the acute-phase response [18, 19]. The human SAA genes are located on chromosome 11 while the mouse SAA genes are found in a cluster on chromosome 7 [20, 21].

At the primary sequence level, the human and mouse SAA proteins share high sequence homology (**Figure 1**), suggesting that these proteins may have similar functions although their modes of expression vary. Of note, although mouse SAA3 has an expression profile different from that of SAA1 and SAA2, its sequence is as homologous to human SAA1 as mouse SAA1 and SAA2 (**Figure 1**). The sequence homology suggests that the functions of SAA3, expressed upon induction by inflammatory cues in various mouse tissues, may be similar to those of human SAA1 and SAA2.

Human SAA1 has been widely studied for its functions. SAA1 was first identified as a serum component recognized by antibodies raised against the amyloid fibril protein known as amyloid A (AA). In one of the studies, antisera were prepared against the major nonimmunoglobulin component of secondary amyloidosis. The antisera were able to detect a serum component that was present at much higher levels in more than half of the pathological samples collected from

#### **Figure 1.**

*Comparison of the amino acid sequence of human and mouse inducible SAA proteins. The amino acid sequences of mature SAA protein (without signal peptides) are shown, and identical amino acids are marked with asterisks (\*). Inset shows the percent of sequence homology between the 3 inducible mouse SAA proteins and human SAA1.*

**113**

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

product of SAA.

event [31].

sess intrinsic amyloidogenicity.

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

sis, Hodgkin's diseases and bronchiectasis [24, 38].

patients compared to only 7% of normal controls [1]. Husby and Natvig found that the serum component detected by the antisera against AA was larger and its circulation level was increased with age and during pregnancy [2]. The protein immuoprecipitated by the antisera was of low molecular weight with similar but not identical amino acid composition of the AA fibrils [22]. It was thought that AA could be a subunit of the SAA protein [22], which was identified as a cleavage

Amyloidosis develops when insoluble amyloid fibrils accumulate in the extracellular space of the tissues and organs in the body. Patients with chronic inflammatory diseases may develop AA amyloidosis, also termed secondary amyloidosis [23, 24]. SAA as an amyloid protein has the propensity of fibril formation.

However, how SAA forms fibril is not fully understood. A number of observations suggest that SAA produced in inflammatory tissues is endocytosed into macrophages [25], where the acidic environment of lysosome promotes fibril formation [26]. The small amount of fibril formed is then exocytosed to the cell surface, prompting a nucleation-dependent incorporation of additional SAA into fibrils [27]. More recent studies have shown that SAA forms stable oligomers at pH of 3.5–4.5, that are resistant to proteolysis and undergo α-helix to β-sheet conversion. The SAA accumulated in lysosomes eventually escape from the cells [28]. Based on these studies, AA fibril formation is a biphasic process [27, 29] that involves an intracellular phase and an extracellular phase. Proteolysis is involved probably in both phases [27, 30]. In the second phase, additional SAA proteins may be recruited with nucleation of AA fibrils, and cleavage of SAA may be a post-fibrillogenic

Recent delineation of the crystal structure of human SAA1 provides a structural basis for AA amyloidosis [32]. Despite high levels of sequence homology, different SAA isoforms have different propensity in forming AA fibrils. Human SAA1.1 has a high tendency of amyloidogenicity, whereas SAA2.2 found in the CE/J mice did not form amyloid fibrils [33, 34] despite sequence homology as high as 94% with SAA1.1. It was found that the structural determinants for amyloidogenicity reside in the first 10–15 residues of mature SAA protein [35]. In a more recent study, SAA2.2 was found to form small fibrils within a few hours, in contrast to the long lag time of SAA1.1 that was characteristically oligomer-rich [36]. These fibrils exhibited different morphology and the fibrils of SAA1.1 were found to be pathogenic. The results of this study suggest that fibrillation kinetics and prefibrillar oligomers of different SAA isoforms may determine their pathogenicity even though they all pos-

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), tuberculo-

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

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

*Amyloid Diseases*

SAA1 and SAA2.

proteins. For other functions of SAA, the interested reader is referred to several

SAA is the general name of a family of proteins with high sequence homology but encoded by distinct genes [16]. Both humans and mice have 4 SAA genes, but in human the *SAA3* is a pseudogene that does not express [17]. SAA4 is constitutively expressed in both humans and mice. In contrast, the expression of SAA1, SAA2 and in mice, SAA3, is highly inducible [18]. These SAA proteins are therefore termed acute-phase SAAs based on their induced expression during the acute-phase response [18, 19]. The human SAA genes are located on chromosome 11 while the

At the primary sequence level, the human and mouse SAA proteins share high sequence homology (**Figure 1**), suggesting that these proteins may have similar functions although their modes of expression vary. Of note, although mouse SAA3 has an expression profile different from that of SAA1 and SAA2, its sequence is as homologous to human SAA1 as mouse SAA1 and SAA2 (**Figure 1**). The sequence homology suggests that the functions of SAA3, expressed upon induction by inflammatory cues in various mouse tissues, may be similar to those of human

Human SAA1 has been widely studied for its functions. SAA1 was first identified as a serum component recognized by antibodies raised against the amyloid fibril protein known as amyloid A (AA). In one of the studies, antisera were prepared against the major nonimmunoglobulin component of secondary amyloidosis. The antisera were able to detect a serum component that was present at much higher levels in more than half of the pathological samples collected from

*Comparison of the amino acid sequence of human and mouse inducible SAA proteins. The amino acid sequences of mature SAA protein (without signal peptides) are shown, and identical amino acids are marked with asterisks (\*). Inset shows the percent of sequence homology between the 3 inducible mouse SAA proteins* 

excellent reviews that have been published recently [9, 11–15].

mouse SAA genes are found in a cluster on chromosome 7 [20, 21].

**2. SAA and its role in amyloidosis**

**112**

**Figure 1.**

*and human SAA1.*

patients compared to only 7% of normal controls [1]. Husby and Natvig found that the serum component detected by the antisera against AA was larger and its circulation level was increased with age and during pregnancy [2]. The protein immuoprecipitated by the antisera was of low molecular weight with similar but not identical amino acid composition of the AA fibrils [22]. It was thought that AA could be a subunit of the SAA protein [22], which was identified as a cleavage product of SAA.

Amyloidosis develops when insoluble amyloid fibrils accumulate in the extracellular space of the tissues and organs in the body. Patients with chronic inflammatory diseases may develop AA amyloidosis, also termed secondary amyloidosis [23, 24]. SAA as an amyloid protein has the propensity of fibril formation. However, how SAA forms fibril is not fully understood. A number of observations suggest that SAA produced in inflammatory tissues is endocytosed into macrophages [25], where the acidic environment of lysosome promotes fibril formation [26]. The small amount of fibril formed is then exocytosed to the cell surface, prompting a nucleation-dependent incorporation of additional SAA into fibrils [27]. More recent studies have shown that SAA forms stable oligomers at pH of 3.5–4.5, that are resistant to proteolysis and undergo α-helix to β-sheet conversion. The SAA accumulated in lysosomes eventually escape from the cells [28]. Based on these studies, AA fibril formation is a biphasic process [27, 29] that involves an intracellular phase and an extracellular phase. Proteolysis is involved probably in both phases [27, 30]. In the second phase, additional SAA proteins may be recruited with nucleation of AA fibrils, and cleavage of SAA may be a post-fibrillogenic event [31].

Recent delineation of the crystal structure of human SAA1 provides a structural basis for AA amyloidosis [32]. Despite high levels of sequence homology, different SAA isoforms have different propensity in forming AA fibrils. Human SAA1.1 has a high tendency of amyloidogenicity, whereas SAA2.2 found in the CE/J mice did not form amyloid fibrils [33, 34] despite sequence homology as high as 94% with SAA1.1. It was found that the structural determinants for amyloidogenicity reside in the first 10–15 residues of mature SAA protein [35]. In a more recent study, SAA2.2 was found to form small fibrils within a few hours, in contrast to the long lag time of SAA1.1 that was characteristically oligomer-rich [36]. These fibrils exhibited different morphology and the fibrils of SAA1.1 were found to be pathogenic. The results of this study suggest that fibrillation kinetics and prefibrillar oligomers of different SAA isoforms may determine their pathogenicity even though they all possess intrinsic amyloidogenicity.
