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In the current concept of bacterial infections, pathogen-associated molecular patterns (PAMPs) derived from pathogens and damage-associated molecular patterns (DAMPs) released from damaged/necrotic host cells are crucial factors in induction of innate immune responses. However, the implication of DAMPs in apical and marginal periodontitis is unknown. Serum amyloid A (SAA) is a DAMP that is involved in the development of various chronic inflammatory diseases, such as rheumatoid arthritis. In the present study, we tested whether SAA is involved in the pathogenesis of periapical lesions, using human periapical surgical specimens and mice deficient in SAA and Toll-like receptors (TLR). SAA1/2 was locally expressed in human periapical lesions at the mRNA and protein levels. The level of SAA protein appeared to be positively associated with the inflammatory status of the lesions. In the development of mouse periapical inflammation, SAA1.1/2.1 was elevated locally and systemically in wild-type (WT) mice. Although SAA1.1/2.1 double-knockout and SAA3 knockout mice had redundant attenuation of the extent of periapical lesions, these animals showed strikingly improved inflammatory cell infiltration versus WT. Recombinant human SAA1 (rhSAA1) directly induced chemotaxis of WT neutrophils in a dose-dependent manner in vitro. In addition, rhSAA1 stimulation significantly prolonged the survival of WT neutrophils as compared with nonstimulated neutrophils. Furthermore, rhSAA1 activated the NF-κB pathway and subsequent IL-1α production in macrophages in a dose-dependent manner. However, TLR2/TLR4 double deficiency substantially diminished these SAA-mediated proinflammatory responses. Taken together, the SAA-TLR axis plays an important role in the chronicity of periapical inflammation via induction of inflammatory cell infiltration and prolonged cell survival. The interactions of PAMPs and DAMPs require further investigation in dental/oral inflammation.

Background and Aims: Serum amyloid A (SAA) predicts cardiovascular events. Overexpression of SAA increases atherosclerosis development; however, deficiency of two of the murine acute phase isoforms, SAA1.1 and SAA2.1, has no effect on atherosclerosis. SAA3 is a pseudogene in humans, but is an expressed acute phase isoform in mice. The goal of this study was to determine if SAA3 affects atherosclerosis in mice., Methods: ApoE -/- mice were used as the model for all studies. SAA3 was overexpressed by an adeno-associated virus or suppressed using an anti-sense oligonucleotide approach., Results: Over-expression of SAA3 led to a 4-fold increase in atherosclerosis lesion area compared to control mice (p = 0.01). Suppression of SAA3 decreased atherosclerosis in mice genetically deficient in SAA1.1 and SAA2.1 (p < 0.0001)., Conclusions: SAA3 augments atherosclerosis in mice. Our results resolve a previous paradox in the literature and support extensive epidemiological data that SAA is pro-atherogenic., (Published by Elsevier B.V.)

The serum amyloid A (SAA) family of proteins consists of inducible acute-phase members and a constitutive member that are minor apolipoproteins of normal high density lipoprotein (HDL). During inflammation, HDL cholesterol and apolipoprotein A-I (apoA-I) protein are decreased, and these changes are thought to be partly related to the increase in acute-phase SAA proteins that associate with the HDL particle to become the major apolipoprotein species. To determine the specific role of SAA in the alteration of HDL in the absence of a generalized acute-phase response, acute-phase Saa1.1 transgene expression was directed via an inducible mouse metallothionein promoter. Elevated levels of SAA1.1 (28+/-9 mg/dL) comparable to a moderate acute-phase response were achieved over a 5-day period. SAA association with the HDL particles at this concentration did not significantly alter the apoA-I or HDL cholesterol levels or change the lipoprotein profiles in the transgenic mice compared with wild-type mice. In addition, we used adenoviral vectors to increase the SAA expression to levels seen in a major acute-phase response. Injection of adenovirus expressing the mouse SAA1.1 protein resulted in high-level expression (72+/-8 mg/dL) but did not alter apoA-I levels. However, the SAA associated with the HDL particle gave rise to significantly larger HDL particles ( approximately 10%). Adenoviral expression of the constitutive SAA4 protein resulted in an increase in HDL size ( approximately 10%) and an increase in very low density lipoprotein levels (20-fold) and triglyceride levels (1.7-fold). These studies suggest that increases in acute-phase SAA proteins alone are insufficient to alter HDL cholesterol or apoA-I levels during inflammation. A role for constitutive SAA4 in HDL-very low density lipoprotein interactions should be considered.

Serum amyloid A (SAA) is an acute phase reactant that can become the predominant apolipoprotein of high density lipoprotein (HDL) during severe inflammatory states. However, the function of SAA is unknown. To study the ability of SAA to form HDL in the absence of apolipoprotein A-I, we expressed the mouse SAA pI 6.15 (CE/J) isoform in apolipoprotein A-I knock-out (apoA-I (-/-)) mice using a recombinant adenovirus. As a control, apoA-I (-/-) mice were injected with an adenovirus expressing human apoA-I. High level expression of plasma SAA was obtained in the absence of any endogenous acute phase SAA production. SAA expression increased plasma HDL cholesterol levels about 2-fold, but to a lesser extent than the expression of apoA-I (about 10-fold). The HDL particles isolated by density ultracentrifugation from SAA-expressing mice were heterogeneous in size and composition and rich in free cholesterol as well as apoE and apoA-IV. Of the SAA expressed in the plasma, only a small fraction (4%) was associated with HDL particles in contrast to expressed apoA-I, of which 62% was associated with HDL. We conclude that SAA is unable to substitute for apoA-I in HDL particle formation.

Site-directed mutagenesis of the acute-phase human serum amyloid A (SAA1 alpha) protein was used to evaluate the importance of the N-terminal amino acid residues, namely RSFFSFLGEAF The full-length cDNA clone of SAA1 alpha (pA1.mod.) was used to create two mutations, namely Gly-8 to Asp-8 and an 11 amino acid truncation between Arg-1 and Phe-11 respectively. Wild-type and mutant cDNAs were expressed in Chinese hamster ovary (CHO) cells under the control of the human cytomegalovirus promoter, which resulted in the secretion of the processed proteins into the culture media. Wild-type recombinant human SAA (rSAA) protein was shown to have pI values of 6.0 and 6.4, similar to the human SAA isoform SAA1 alpha and SAA1 alpha desArg found in acute-phase plasma. N-terminal sequencing of 56 residues confirmed its identity with human SAA1 alpha. The total yield of wild-type rSAA measured by ELISA was between 3.5 and 30 mg/l. The two mutations resulted in reduced expression levels of the mutant SAA proteins (3-10 mg/l). Further measurements of rSAA concentration in lipid fractions of culture medium collected at a density of 1.21 g/ml (high-density liporotein; HDL) and 1.063-1.18 g/ml (very-low-density lipoprotein/low-density lipoprotein; VLDL/LDL) showed that 76% of the wild-type protein was found in the HDL fraction and the remaining 24% in the infranatant non-lipid fraction. In contrast the relative concentration of mutant rSAA in HDL and infranatant fractions was reversed. This is consistent with the previously proposed involvement of the 11 amino acid peptide in anchoring. SAA protein on to HDL3 [Turnell, Sarra, Glover, Baum, Caspi, Baltz and Pepys (1986) Mol. Biol. Med. 3, 387-407]. Wild-type rSAA protein was shown to from amyloid fibrils in vitro under acidic conditions as shown by electron microscopy, and stained positive with Congo Red and exhibited apple-green birefringence when viewed under polarized light. Under the same conditions mutSAA(G8D) and mutSAA delta 1-11 did not form amyloid fibrils. In conclusion, replacement of Gly-8 by Asp-8 or deletion of the first 11 amino acid residues at the N-terminus of rSAA diminishes its capacity to bind to HDL and decreases amyloid fibril formation.

The serum amyloid A (SAA) proteins are a polymorphic family of apolipoproteins associated with high-density lipoproteins (HDL). Three distinct subfamilies have been identified: (i) a cytokine-induced acute phase subfamily that is hepatically produced and can become the major apolipoprotein on HDL (SAA1, SAA2); (ii) a peripherally produced acute phase SAA3 that is only a minor HDL apolipoprotein; and (iii) a constitutive subfamily (SAA4) that is a minor normal HDL apolipoprotein comprising more than 90% of the SAA during homeostasis. Here we define the structure of the Saa4 gene. Similar to other Saa family members, it has four exons and three introns. It is 4588 bp long from the transcription start site to the end of the 3'-untranslated region and is approximately 20% larger than other Saa genes. We have located Saa4 11 kb upstream from Saa3 and 5 kb downstream from Saa1, with the pseudogene approximately 1 kb from the 5' end of Saa4. Saa4 has the same orientation as most other Saa family members, with only Saa2 having an opposing orientation. These data promote our understanding of the evolution of the Saa family. They enhance our ability to develop the mouse as a transgenic and gene deletion model to advance the understanding of the function of these apolipoproteins.

Background: Experimental amyloid A protein (AA) amyloidosis in mice is the most rapid type of amyloid formed, thus providing a valuable model to study amyloid formation. Recent studies have suggested the importance of apolipoprotein E (apoE) in Alzheimer's disease and systemic amyloidoses. To help understand the role of apoE in amyloidoses, we examined amyloid tissue for the presence of apoE in mouse AA amyloid., Experimental Design: Mice were injected with amyloid-enhancing factor and silver nitrate to induce amyloid deposition. Spleens were examined for Congo red staining and serum amyloid A, serum amyloid P component, and apoE immunostaining. In addition, RNA analysis was performed to measure the expression of apoE in various tissues after amyloid induction., Results: We have found that apoE is associated with mouse amyloid. Ab to apoE consistently detected the presence of increased levels of apoE in amyloid tissue. Immunohistochemical analyses confirmed that the apoE immunoreactivity co-associated with AA and serum amyloid P in the amyloid fibrils. Northern blot analysis of amyloid tissue showed an increase in apoE messenger RNA compared with control tissue., Conclusions: This is the first demonstration of apoE in mouse amyloid tissue. The data presented here, along with previous studies, suggest that apoE may be involved in amyloidogenesis. These studies validate the mouse model for studying the role of apoE in amyloid fibrillogenesis. The use of transgenic and gene-inactivated mice will help to elucidate the role and mechanism of apoE in amyloid formation.

The acute-phase proteins serum amyloid A protein (SAA) and secretory phospholipase A2 (sPLA2) are simultaneously expressed during inflammatory conditions. SAA associates with high-density lipoprotein (HDL) altering its physicochemical composition. We found that purified acute-phase SAA, but not the constitutive form, markedly enhances the lipolytic activity of sPLA2 in a dose-related manner with phosphatidylcholine/lysophosphatidylcholine or phosphatidylethanolamine/lysophosphatidylethanolamine liposomal substrates. Normal HDL was found to reduce activity of sPLA2 in a dose-dependent manner, but when acute-phase HDL containing 27% SAA was tested, it enhanced sPLA2 activity. Immunopurified monospecific antibodies against SAA completely abolished the enhancing activity of SAA and acute-phase HDL. Given the central role of HDL in lipoprotein metabolism, the interaction between HDL, SAA and sPLA2 may account for changes detected in lipoprotein metabolism during the acute phase.

Normal high density lipoprotein (N-HDL) is remodeled during acute phase (AP) reactions by the association of serum amyloid A (SAA) and the depletion of apolipoprotein (apo) A-I. To determine the impact of this remodeling on HDL function, the capacities of N-HDL and AP-HDL to associate with and promote cholesterol efflux from human monocytic THP-1 cells were compared. THP-1 cells preferentially bound AP-HDL compared with N-HDL. Examination of the AP-HDL particles bound to THP-1 cells revealed a disproportionate association of an apoSAA-enriched, apoA-I-depleted subpopulation compared with the composition of the starting material. However, N-HDL and AP-HDL promoted cholesterol efflux from THP-1 cells equally efficiently and in a dose-dependent manner. When N-HDL was experimentally remodeled with apoSAA to achieve an apoprotein composition similar to that of the preferentially bound particles, cellular cholesterol efflux was reduced by 30%. The remodelling of HDL with apoSAA during the acute phase reaction alters cholesterol efflux only when apoSAA constitutes more than 50% of the HDL protein.

Serum amyloid A proteins (SAAs), a family of homologous molecules, are apolipoproteins of high density lipoprotein (HDL). They can be divided into two groups. The first group comprises the well-characterized acute phase SAAs that associate with HDL during inflammation, thereby remodeling the HDL particle by displacing apolipoprotein (apo)A-I. The second group consists of the recently discovered constitutive SAAs, mouse SAA5 and human SAA4. They exist as minor apolipoproteins on HDL but constitute more than 90% of the total SAA during homeostasis. We have characterized human SAA4 as an apolipoprotein. During homeostasis, SAA4 is synthesized only in the liver. Purification of SAA4 has been described and its plasma concentration has been established at 55 +/- 13 micrograms/ml in 26 healthy individuals. It was present on all HDL density classes and very low density lipoprotein (VLDL) but was absent from low density lipoprotein (LDL). Using two-dimensional electrophoresis and phosphorimaging, SAA4 was found to be associated with a specific subpopulation of only three HDL particles, not involved in the initial cholesterol transfer from cells.

During inflammation, serum amyloid A (SAA) protein increases up to 1000-fold and can become a major component of high-density lipoprotein (HDL). We have identified a new apolipoprotein molecule (SAA5) as a distinct member of the SAA family. It differs from the inflammatory isotypes (SAA1, SAA2, and SAA3) not only in structure but in the fact that it is constitutive on normal HDL where it accounts for more than 90% of total SAA. Whereas all members of the SAA family, including SAA5, responded to endotoxin administration, SAA5 contrasted with other SAAs in its resistance to induction either by a high-fat, high-cholesterol atherogenic diet or the injection of mildly oxidized LDL (MM-LDL). These data provide further evidence that the induction of inflammatory molecules by oxidized lipids is selective. In atherosclerosis-susceptible C57BL/6 mice and atherosclerosis-resistant C3H/HeJ mice, the inflammatory SAA isotypes (SAA1, SAA2, and SAA3) responded in a strain-specific manner to oxidized lipids either generated with feeding of the atherogenic diet or introduced by MM-LDL injection. We hypothesize that the constitutive SAA5 molecules on normal HDL may contribute to its normal physiological role and that the dramatic induction of the inflammatory SAA subfamily equips the particle for an altered yet related functional role appropriate to the inflammatory state.

A novel member of the mouse serum amyloid A protein family, SAA5, has been identified as a normal apolipoprotein component of non-acute-phase high density lipoprotein (HDL). The structure of SAA5 was derived from a clone isolated from a normal Balb/c liver cDNA library. The clone predicts a pre-SAA5 molecule of 130 residues from which an 18-residue leader peptide is cleaved. The mature molecule has an octapeptide insert spanning from position 70 to 77. Similar inserts are found in human C-SAA and, paradoxically, in acute-phase SAA molecules of a number of other species. There is 48% amino acid identity between apo-SAA5 and the other mouse SAA proteins and 57% identity between the human C-SAA and apo-SAA5. The SAA5 mRNA is three times larger than previously identified SAA mRNAs. Although SAA5 is constitutively expressed in the liver, it has a rapid albeit muted response to inflammatory stimuli. The increase of SAA5 mRNA is due to increased transcription rather than mRNA stabilization. Plasma SAA5 levels during the acute phase are biphasic, either because of translational control or displacement from HDL and rapid clearance. We propose that constitutive SAAs (SAA5) on normal HDL contribute to its normal physiological role, whereas the dramatically inducible family members (SAA1, SAA2, SAA3) equip this particle for an altered functional role during inflammation.

Inbred CE/J mice have been identified as extremely resistant to azocasein-induced amyloidosis relative to five commonly used inbred strains, A/J, CBA/J, C57BL/6J, C3H/HeN, and SJL/J. The enhanced amyloid resistance in CE/J mice seems to derive from the novel structure of the SAA gene family in CE/J mice, as determined by Southern blot hybridization analysis of SAA gene structure and isoelectric focusing analysis of acute phase SAA proteins in the six inbred strains. In CE/J mice, a single, novel SAA isoform of pI 6.15 is present, whereas in the other strains the amyloidogenic SAA2 isoform (pI 6.3) is codominantly expressed with SAA1 (pI 6.45). Two other inbred strains, PERU and IS/CAM, share common SAA specific HindIII DNA fragments with CE/J mice. Wild-derived Mus musculus mice differ from all of the inbred strains studied, both in SAA gene structure and in the pattern of SAA isoform production; two isoforms, one pI 6.15 and the other pI 6.3 (corresponding to SAA2), were codominantly expressed. Only the pI 6.15 isoform, not SAA1 and 2, was produced by CE/J mice in response to lipopolysaccharide, casein, silver nitrate, interleukin-1, or tumor necrosis factor; tumor necrosis factor was a weaker stimulus than interleukin-1 for the pI 6.15 isoform as it is for SAA1 and 2 production in the other inbred strains. This study provides a new line of evidence supporting the role of precursor structure as a determining factor in murine amyloid A amyloidosis and provides a valuable model for studies of amyloidogenesis.

Most studies of experimental amyloid A protein (AA) amyloidosis in mice have been performed in type A mice with BALB/c as the prototype. In these mice the products of two genes, SAA1 and SAA2, are the major apo-SAA isoforms on high density lipoprotein (HDL). Of these two isoforms, that differ at nine amino acids, only apo-SAA2 is rapidly cleared and deposited as amyloid fibrils. No mouse strain has ever been shown to be completely resistant to amyloid induction. We have found the CE/J mouse strain to be exceedingly resistant to amyloidogenesis. Data indicate that this resistance is not due to a lack of apo-SAA synthesis but rather resides in the unique apo-SAA isoform in this strain. CE/J mice have a single major apo-SAA isoform (pI 6.15) the product of a single gene. This is a hybrid molecule with features of both apo-SAA1 and apo-SAA2, differing from the latter at only six amino acids. When CD studies were performed to explore the structural relationship of this isoform to apo-SAA1 and apo-SAA2, we found that when bound to heparan sulfate proteoglycan the CE/J pI 6.15 isoform fails to undergo the beta-sheet folding typical for apo-SAA2. This evidence suggests that the folding effect of heparan sulfate proteoglycan on apo-SAA2 is important in amyloid formation.

Amyloid A (AA) amyloidosis is widespread throughout the animal kingdom. Several factors including: 1) precursor production; 2) precursor structure; 3) precursor degradation; and 4) precursor/product interaction with the pentraxin serum amyloid P have been implicated in amyloidogenesis, but the exact sequence of events leading to AA fibril formation and deposition remains unclear. Most models of experimental amyloidosis, including golden Syrian hamsters (Mesocricetus auratus), involve massive and repeated inflammatory stimulation; however, the model of spontaneous amyloidosis with aging in female, but not male, Syrian hamsters permits analysis of amyloidogenic factors in the absence of inflammation. Another genus, the Armenian hamster (Cricetulus migratorius), differs from Syrian hamsters both in gender-specific serum amyloid P expression and susceptibility to AA amyloidosis. In this study, we describe novel SAA molecules in the Syrian hamster in the presence and absence of inflammation. We demonstrate that, based on isoelectric separation, the Syrian hamster SAA proteins can be separated into two broad subfamilies. Plasma SAA concentration in female Syrian hamsters increases spontaneously with age, and fragments of a basic SAA isotype expressed both hepatically and extrahepatically are selectively deposited as AA fibrils. After inflammatory stimulation, the patterns of SAA gene expression in Syrian and Armenian hamsters differ. In Syrian hamsters, both hepatic SAA mRNA and the high density lipoprotein apoSAA content increase approximately 1000-fold; in Armenian hamsters, hepatic SAA mRNA is limited in quantity and different in structure; and although plasma SAA proteins increase three- to fivefold, apoSAA is not detectable in high density lipoprotein. The results suggest that regulation and site of precursor production as well as precursor structure influence AA amyloidogenesis in these two hamster genera.

Four serum amyloid A protein (SAA) genes and two gene products, apo-SAA1 and apo-SAA2 were identified in BALB/c mice (type A). SJL/J mice (type B) are thought to be defective in apo-SAA2 expression. A unique variant of mouse apo-SAA was identified in SJL/J mice by isoelectric-focusing analysis of high-density lipoprotein from endotoxin-treated mice. Complete amino-acid-sequence analysis of this quantitatively major form of SJL/J apo-SAA (pI 5.9) showed it to be identical with the apo-SAA2 isoform from BALB/c mice, except for the substitution of aspartic acid for alanine at position 101. Isoform-specific analysis of mRNA from liver of BALB/c and SJL/J mice and their F1 hybrid progeny (CSJLF1/J) mice revealed further differences in the 3' untranslated regions of the genes, not only encoding apo-SAA2 and apo-SAA pI 5.9, but also apo-SAA1. The SAA genes of SJL/J mice thus differ from BALB/c in exon 4. Additional minor isoforms corresponding to apo-SAA2 (pI 6.3) in SJL/J mice and apo-SAA (pI 5.9) in BALB/c mice were identified. We propose that, when analysing a multigene family such as SAA, thorough analysis at the protein level should complement molecular-biological approaches where the use of a too-limited repertoire of probes can obscure complexities.

Serum amyloid A protein (SAA), an acute-phase reactant and apolipoprotein of high-density lipoprotein, is a polymorphic protein with six reported isoforms. These are the products of three genes, i.e., cDNA pA1, cDNA pSAA82 and genomic DNA SAAg9, the last two being allelic variants at a single locus. We have identified an individual with additional novel SAA isoforms on isoelectric-focusing analysis. By using 3-bromo-3-methyl-2-(2'-nitrophenylsulphenyl)-indolenine (BNPS-skatole) cleavage of the protein at tryptophan residues we obtained the complete amino acid sequence of a novel isoform. Additional cleavage by endoproteinase Asp-N allowed verification of the tryptophan residues and complete amino acid sequence of both isoforms. The suitability of this approach to the rapid sequencing of SAA was demonstrated. Sequence analysis and quantification suggest that these isoforms are the result of the first confirmed allelic variation at the SAA1 locus. We designate the protein products of this allele SAA1 beta (pI 6.1) and SAA1 beta des-Arg (pI 5.6).

A novel serum amyloid A protein (SAA) has been identified as a normal apolipoprotein component of non-acute phase high density lipoprotein. This novel SAA has been designated "constitutive" SAA (C-SAA) to distinguish it from "acute phase" SAA (A-SAA). C-SAA was partially sequenced, and immunochemical analyses indicated that it constitutes a distinct subclass of apolipoproteins within the SAA superfamily. A C-SAA cDNA clone was isolated from a human liver library and sequenced. The clone predicts a pre-C-SAA molecule of 130 residues from which an 18-residue leader peptide is cleaved. The 112-residue mature molecule is 8 residues longer than human A-SAA; the size difference is due to the presence of an octapeptide between positions 70 and 77 that is not found in the corresponding region of human A-SAA. Paradoxically, octapeptides of similar composition are found at similar positions in the A-SAAs of a number of other species. The C-SAA octapeptide specifies the first two residues of a NSS tripeptide, the only potential N-linked glycosylation site in the molecule. Studies indicate that approximately 50% of these sites are glycosylated, thereby giving rise to two size classes, 14 and 19 kDa, of C-SAA in vivo. Human acute phase liver contains little C-SAA mRNA relative to the levels of A-SAA mRNA, and the treatment of PLC/PRF/5 hepatoma cells with monocyte-conditioned medium does not induce C-SAA mRNA concentrations to detectable levels, in contrast to the massive induction of A-SAA mRNA observed. C-SAA is therefore not a major acute phase reactant.

Four serum amyloid A protein (SAA) genes and two SAA gene products, SAA1 and SAA2, were identified in BALB/c mice. Using analytical isoelectric focusing we have identified a quantitatively significant new member of the SAA family and designated it 'SAA5'. This protein has characteristics never before described for any SAA molecule. In the highly conserved region between amino acids 33 and 44, identical in all SAAs from all species examined, SAA5 had four amino acid substitutions. In addition, the induction of SAA5 by lipopolysaccharide had different kinetics from that of the other mouse SAAs. Our data suggest that the mouse SAA gene family is more complex in composition and regulation than previously surmised.

Serum amyloid A protein (apo-SAA) is an acute-phase reactant and an apolipoprotein of high density lipoproteins (HDL). Six major isoforms of apo-SAA occur in humans (pI 6.0, 6.4, 7.0, 7.4, 7.5, 8.0). In this report we have rationalized the phenotypic expression of apo-SAA isoforms with published apo-SAA structures predicted from apo-SAA cDNA's pA1 and pSAA82 and the genomic DNA SAAg9. The six apo-SAA isoforms fall into three pairs, pI 6.0/6.4, 7.0/7.5, and 7.4/8.0, which are products of cDNA pA1, cDNA pSAA82, and genomic DNA SAAg9, respectively. The second of each isoform pair (i.e. pI 6.4, 7.5, and 8.0) is the "primary" product: a 104-residue peptide with the NH2-terminal sequence Arg-Ser-Phe-Phe. Each primary product is processed either to a major 103-residue peptide with the NH2-terminal sequence Ser-Phe-Phe or processed to a minor 102-residue product which results from the loss of both an Arg and a Ser residue from the NH2 termini. These "secondary" products have the lower pI values of 6.0, 7.0, and 7.4, respectively. The isoelectric points of the SAAg9 products were confirmed by expression of SAAg9 in transfected mouse L-cells. Both the pI 8.0 and 7.4 isoforms were present in cellular extracts, suggesting that post-translational modification of apo-SAA may occur intracellularly. However, the greater relative abundance of the pI 7.4 isoform extracellularly suggests that the major conversion may occur after secretion. Whereas the gene corresponding to the pA1 cDNA sequence does not show allelic variation, the segregation characteristics of the pI 7.0/7.5 and 7.4/8.0 isoform pairs amongst individuals suggests that these isoforms are the products of genes (with sequences corresponding to pSAA82 and SAAg9, respectively) which are allelic variants at a single locus distinct from that for the pI 6.0/6.4 isoform pair.

Monokine-induced hepatic secretion of serum amyloid A protein (apo-SAA), an acute-phase reactant, is followed by rapid association with high-density lipoprotein (HDL) in plasma. Plasma clearance of apo-SAA is more rapid than any of the other HDL apolipoproteins. It has been shown that, of the acute-phase HDL3 apolipoproteins, apo-SAA preferentially associates with neutrophil membranes. HDL apolipoproteins have been shown to activate protein kinase C in endothelial cells. We therefore investigated potential phosphorylation of HDL3 apolipoproteins by protein kinase C. Apo-SAA was the only apolipoprotein phosphorylated (Km = 12 mM). Phosphorylation of the apo-SAA-containing HDL3 particle was selective for the more basic isoforms of apo-SAA (pI 7.0, 7.4, 7.5 and 8.0), with more acidic isoforms being phosphorylated when delipidated acute-phase apolipoproteins were used as substrate. However, phosphorylation was not in itself responsible for the establishment of the apo-SAA isoforms.