IMedical Hypotheses
Serum Amyloid A (SAA), a Protein without a Function: Some Suggestions with Reference to Cholesterol Metabolism R. KISILEVSKY Department of Pathology, Queen’s University, and the Syi and Molly Apps Research Center Kingston General Hospital, Kingston, Ontario, Canada K7L 3N6
Abstract - Serum amyloid A, as an apolipoprotein, is present on high density lipoprotein only during inflammatory states. When viewed from HDL’s established function as a mechanism for reverse cholesterol transportation, it is postulated that serum amyloid A represents a signal to redirect HDL to sites of tissue destruction where cholesterol is being collected by macrophages. The object is to direct the reverse cholesterol transporter to sites of cholesterol accumulation for the subsequent removal of these cholesterol stores. The hypothesis has relevance to the process of atheroma formation.
Introduction
Not uncommonly, investigation into the structure of an altered molecule, which has been identified in a specific disease, has provided clues not only to the pathogenesis of the disease concerned, but also to the natural function of the component under consideration. This has yet to take place with the serum amyloid A (SAA) protein. This protein is known by virtue of its association with a relatively rare form of amyloidosis, the form of amyloid which is seen as a complication of persistent acute infiammation. The rarity of this form of amyloid and its associated circulating component have probably been responsible for the general lack of interest in SAA. Yet, if the speculations to be put forth in this article prove to be correct, understanding the natural function of SAA
may well provide some new perspectives on cholesterol metabolism, SAA’s possible protective role in the development of atherosclerosis, and potentially modes of interference in atheroma formation. Serum amyloki A In the late, 196Os,
techniques were developed for the isolation of amyloid fibrils, which upon denaturation and protein fractionation provided peptides which could be sequenced with great accuracy (1). Once these peptides and their pimary structure were elucidated, attempts were made to raise antisera to the peptide for further study. Several laboratori~ were successful and used such antisera to examine serum for cross-reactive material (2-5). SAA was discov-
Date naivcd ._24_October 1990 ____ Date mxeptcd 3 Jammy
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338 ered approximately 15 years ago in the course of studies examining the serum for potential precursors to the inflammation-associated AA form of amyloid. Following the amino acid sequencing of SAA, it became apparent that the AA peptide responsible for the inllammation-associated amyloid fibril represented a fragment (usually the amino terminal 2/3) of the SAA protein (6, 7). The antisera also provided a tool to investigate the regulation of SAA in various physiological conditions, and where SAA was synthesized. These studies have provided a great deal of information but have yet to show where SAA exerts its function and, for that matter, what this natural function might be. SAA- molecular biology Knowledge of the amino acid sequence of SAA eventually led to the preparation of cDNAs, their cloning, and the identiIication of genes possessing the information for this protein (6,8). It became apparent that SAA was not a single protein, but rather a family of several related proteins, data supporting information which had already been obtained from an analysis of SAA in serum. Two serum forms were identified in the mouse, SAAt and SAA2 (9). Analogous forms were identified in the human (10). Four murine genes have now been identified; one is a pseudogene, and the remaining three are each known to be responsible for a protein product (8, 11, 12). In the mouse, only one of these actually serves as the precursor to amyloid, and is designated as SAA2 (9). Much work has now been done showing that during an inflammatory reaction the cytokines interleukin-1, interleukin6, and tumor necrosis factor, are responsible for regulating the transcription of the SAA gene in liver (13, 14). Substantial quantities of messenger RNA appear in this tissue within hours (4-8h) of the onset of an inflammatory reaction (15). In fact, 25% of total liver protein synthesis can be devoted to the synthesis of SAA during an acute inflammatory process, clearly an indicator of an important function (16). Shortly thereafter (12-15h), the protein appears in the circulation with concentrations 5Of&lOOO-foldgreater than in non-intIammatory states (15). SAA concentrations increase from I-5 pg/ml to 500-1000 pg/ml. It has been shown that tissues other than liver and the gastrointestinal tract can express SAA3 mRNA (12). But, the liver and gastrointestinal tract seem to be the major sources of SAAt and SAA2 (17, 18). These are the proteins which occur in the circulation during an inllammatory reaction. The organization of the linear structure of the SAA genes is very similar to other apolipoproteins. SAA contains four exons spaced by
introns in an analogous fashion to that seen in other apolipoproteins (19). SAA- the protein In its native state, SAA, a molecule of 104 amino acids and 12-14 000 daltons, exists as a complex bound primarily (90% or more) to high density lipoproteins (I-IDL) (20,21,22). The association of SAA and HDL likely occurs in the serum following the secretion of SAA (23,24). SAA has all the features of an apolipoprotein including the amphipathic properties (7,25). However, it is produced and forms a substantial part of HDL only during inflammation. SAA can displace apoA-I from HDL but apparently does not alter HDL lipid composition (26). Conversely, apoA-I and apoA-II can displace SAA (27). Preliminary evidence indicates that SAA and apoA-I are handled physiologically in an independent fashion with apoA-I having a half-life of approximately lOh, while that of SAA is only 90 min (28). During inflammation, the half-life of apoA-I decreases to 3-3.511, but SAA retains its half-life of approximately 90 min (28). The very fact that SAA’s serum concentration has increased by several orders of magnitude during inflammation indicates that during inlbunmation there is an enormous capacity to clear SAA from the circulation. Preliminary evidence indicates that the mechanism of SAA clearance is not saturable even with concentrations as high as 500-1000 pg/ml(28). Hypothesis SAA-potential
functions
Relatively little work has been done examining SAA’s fundamental function. Several studies have suggested that SAA may influence lymphocytic responses to antigens, suppressing this type of activity (29, 30). The rationale for such studies stems from the old concept that amyloid deposits generally arise in some way from an immunological disturbance. Such a concept is no longer tenable, as there are clearly many forms of amyloid and amyloid peptides which have no known association with an immune process. More recent studies have suggested that SAA has signiiicant influence on lecithin cholesterol acyl transferase activity (LCAT) associated with the high density lips proteins (31). This work arises from the known structural association of SAA, HDL and LCAT on the same physical particle. These findings may be closer to the truth. Functional considerations suggest, however, a fundamentally different role for SAA. Our suggestions
sAAANDcH-
OL MErABoLIsM
are based on three well established observations. The tirst is that SAA is present in the circulation in substantial quantities only during inflammation. The second is that 9096 or more of SAA is associated with high density lipoproteins, and the third is HDL’swell established function in reverse cholesterol transport (32). Whatever role one can postulate for SAA should tie these observations together, Teleologically the association of SAA with HDL could occur for at least one of several masons: 1) SAA has a specific function to play while on HDL particles; 2) SAA uses HDL as a vehicle to be transported to sites where HDL normally exercises its function; or 3) SAA is a signal to HDL to go to sites which HDL does not normally visit. With reference to the tirst possiblity, as indicated earlier, SAA may influence LCAT activity on HDL. This begs the question as to why it should bc necessary to alter LCAT activity during inflammation. Are there particular aspects of cholesterol metabolism that are altered during inflammation? No doubt there are. This question will be addressed more deeply below for it involves more than a consideration of the simple association of SAA and HDL and LCAT activity. Secondly, if SAA simply associates with HDL and uses HDL only as a carrier to reach sites which HDL normally visits, one is left with having to understand why SAA is produced during inflammation to go to such sites and what possible role it could be playing there. This possibility simply shifts our lack of understanding of SAA function from the HDL particle to a variety of both known and unknown tissue sites without giving us any additional insights. A more exciting possibility is the third consideration, namely that SAA serves as a signal to HDL, redirecting HDL to specific anatomic sites or cells. In the context of inflammation, such sites may represent the areas of tissue destruction. Here HDL could effect its usual function of cholesterol scavenging, and altered LCAT activity could be part of this function. The rationale for considering SAA as a new signal to HDL to redimct it to sites of tissue destruction is as follows. At sites of tissue injury and during the development of the inflammatory response large numbers of cells are being destroyed with the release of many lipid, protein, and nucleic acid components. With some of these cellular components the local release of lytic enzymes, such as nucleases or proteases, may be sufficient to digest the material in situ, either intracelhilarly or extracellularly.In the case of lipid material, lipase attack on triglyceride or phospholipids would release free fatty acids provoking a further intense inflammatory response. Lipids are
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more likely to be picked up in large quantities by the inflammatoty cells, and as in the case of cholesterol packaged within cells such as macrophages, genemting the so called foam cells, a lipid laden microphage. Locally, the cholesterol may be reutilized and made available to proliferating cells in the area of repair. or cholesterol may be removed from such cells to sites of ultimate cholesterol excretion utilizing the HDL reverse cholesterol transport mechanism. To effect the latter process a communications system is necessary to inform various body parts that inflammation and tissue destruction are in progress. At the same time such signals should elicit a process to direct both cellular and circulatory scavenging mechanism to such inflammatory sites to handle the liberated cellular molecules. The cellular aspects of this process have been understood for some time (i.e. inflammation proper). Involvement of serum macromolecular complexes such as HDL in such a process has not previously been considered. The above mentioned scenario is fulfilled in the generation of cytokines by the inflammatory cells at the site of tissue destruction. These serve as signals to a variety of cells and organs. In the case of the liver, the cytokines induce the generation of acute phase proteins, such as SAA. We postulate that SAA in turn has evolved in such a way to associate with high density lipoproteins, and serves to redirect HDL to lipid containing cells such as the macrophage. This process would redirect the reverse cholesterol transporter to a site of cholesterol uptake and storage. This process would not necessarily depend on the appearance of a new receptor on such cells for HDL, although such an event is not excluded. Rather, such a receptor might already exist on macrophages. Its ligand, SAA would appear only when necessary, addressing HDL to such cells at the time of greatest need, i.e. inflammation. While each HDL particle might not necessarily carry any more cholesterol, an increased number of vehicles (HDL) appearing at sites of cholesterol storage (foam cells) could have a significant impact on the quantity of cholesterol removed from these sites. These considerations make several predictions: 1) the association of SAA with HDL should change the affinity of HDL for such cell types; and 2) an SAA receptor should be present on macrophages. It is precisely these considerations which prompted recent work where we undertook binding studies between HDL, or HDLBAA, with macrophages from animals in different physiologic states. It became immediately apparent that SAA on an HDL particle increases the affinity of HDL for such a cell by a factor of 2-3, even though the apoA-I content of HDL decreases by
340 50% (Subrahmanyan and Kisilevsky, manuscript in preparation). In addition, competitive binding experiments have suggested there is a specillc SAA receptar on these inflammatory cells (Subrahmanyan and Kisilevsky, manuscript in preparation). The above considerations may have importance not only in reference to HDL/SAA function during period8 of inllammation, but may also play a role in the process of atherogenesis. It has been known for many years that individuals with long-standing inflammation, and individuals with debilitating tumours, when examined at autopsy, have relatively little atherosclerosis when matched with others of comparable age. The glib explanation has been that these individuals are poorly nourished because of their long-standing disease and therefore have less atherosclerosis. The mechanism is not, however, explained. Interestingly, in both these clinical situations SAA levels tend to be high (33-37). In light of our hypothesis our postulated mechanism may explain the removal of cholesterol from foam cells at sites of atherogenesis in the above mentioned clinical states. This model of SAA/HDL function can by analogy also be used to consider the function of other acute phase proteins, such as SAP. SAP has recently been shown to play a role in maintaining fragments of chromatin released from dead cells in a soluble state (38). Such solubilixed chromatin would also be more easily removed from inflammatory sites.
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Summary Our hypothesis suggests that SAA is a signal to redirect HDL to inflammatory cells such as macrophages for the purpose of cholesterol removal. Supporting evidence exists in the form of afIinity and competitive binding data, and anecdotal clinical reports. This hypothesis can be extended to other acute phase proteins, which may be considered as a grouping of proteins which not only limit the activity of enzymes released during an acute inflammatoryreaction to the site of inflammation, but also play a direct role in the removal of specific constituents from sites of tissue destruction. Acknowledgements This wotk was suppotted by grants frcm the Medical Research Council of Canada MT-3153 and the Heatt and Sttoke Foundation of Canada. ‘lhe author is indebted to Mrs ti Northcottfor her able admiistrative assistance and Mn B Latiier and Ma K Wowk for their able secmmrial assistance.
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