## 3 Discussion
Such as the humans' SAP, OnSAP also has calcium-binding sites; each monomer could bind 2 Ca\({}^{2+}\), and the amino acid of these binding sites are the same except for one. In humans, SAP shared 51% sequence homology with CRP, which is also short pentraxin and is the main acute phase protein in humans [28; 29; 30; 31]. Based on this information, we compared the tridimensional structure predicted by Alphafold 2.0 between them, and the result demonstrates that they were highly similar (Figure 3H). Moreover, the PTX domain of long-pentraxin OnPTX3 is also the same as OnSAP. Structure analysis told us these above-mentioned pentraxins might share similar functions, and therefore, due to the extra domain sequence named N-terminal region of PTX3, different functions would be found between OnPTX3 and OnSAP (data unpublished). Pentraxin is a kind of protein that is formed with five of the same monomers via non-covalent bonds. Moreover, recombinant protein demonstrated abilities in bacteria combination and agglutination in the presence of Ca\({}^{2+}\), which strongly demonstrated that PTX domain is the critical part of pathogen identification [6]. The function of SAP regulating phagocytosis is a typical phenomenon among many species, from teleost to mammals. For example, SAP-opsonizing zymosans could be phagocytosed by human macrophages through FcyRIIa [5]. Moreover, SAP plays an important role in assisting phagocytosis towards the _Streptococcus pneumoniae_ through the classical pathway of complement system instead of interacting with FcyR directly [9; 23]. In teleost fish, SAP-treated _Pseudomonas fluorescens_ enhanced the phagocytosis of half-smooth tongue sole [2]. In this study, we demonstrated that OnSAP was able to promote the MO/Mo phagocytosis of _S. agalactiae_ and _A. hydrophila_. Based on the above result, OnSAP acts as a PRRs in early vertebrate: OnSAP could bind bacteria and then interact with the cell surface phagocytosis receptor, perhaps Fc Receptor, which might be important for early vertebrate resistance to foreign invasion. In this study, we demonstrated the function of the OnSAP regulating complement system. CRBCs were employed for the hemolysis assay (Figure 4H). (r)OnSAP could significantly improve the lysis rate. SAP, which has the ability to interact with C1q, MBL and C4 BP, is a regulator that assists different complement molecules [11; 14]. On the one hand, the classical pathway could be activated through SAP binding antigen, resulting in recruiting C1 complexes to the target surface [9; 28]. On the other hand, in humans, MBL combines with SAP with its CLR domain and formed a complex that could amplify complement activation; this process may circumvent MSAP (MBL-associated serine pro tease) [6]. However, such studies are still rare in bony fish that are necessary to explore the relationship among OnSAP, OnMBL and OnMASP or OnSAP and OnC1q. In conclusion, the functional characterization of SAP in Nile tilapia was explored and identified in this study. First, the transcript of OnSAP was found to express widely in many tissues and its expression was up-regulated significantly after in vivo stimulation with bacterial pathogen. Further, recombinant OnSAP possessed the capabilities to bind and agglutinate bacteria, and even promoted phagocytosis bacteria cells by monocytes and macrophages. Moreover, the OnSAP could enhance complement-mediated hemolysis. To sum up, our result enriched the function of SAP in a primary vertebrate, which could help us to better understand the biological functions of the pentrazin family.