Studies of the structure and binding properties of hamster female protein.

We report here the characterization of hamster female protein (FP), a member of the pentraxin family of plasma proteins, as a molecule composed of glycosylated subunits of 25,655 MW containing a single intrachain disulphide bridge. In the presence of EDTA the subunits are non-covalently associated as pentamers of mass approximately 128,000 MW, and in the presence of calcium they aggregate further, probably to form decamers. This pentamer-decamer transition at physiological ionic strength has not been described in other pentraxins. As previously reported, FP shares the capacity of C-reactive protein (CRP) in other species to bind phosphocholine and we show here that it also resembles human CRP in binding only weakly to agarose, to human AA amyloid fibrils in vitro, and to mouse AA amyloid deposits in vivo. It thus differs markedly from human and mouse serum amyloid P component (SAP) but it is nevertheless deposited in hamster AA amyloid in vivo and clearly is the hamster counterpart of SAP in other species. These results illustrate the subtle diversity among members of the otherwise conserved pentraxin family of vertebrate plasma proteins.

Experimental amyloidosis in the hamster: correlation between hamster female protein levels and amyloid deposition.

Reactive systemic AA amyloidosis was induced in female, male and castrated male hamsters either by repeated injection of casein or by injection of amyloid enhancing factor (AEF) followed by casein. The circulating concentrations of serum amyloid A protein (SAA), the putative precursor of the AA amyloid fibril protein, and of female protein (FP), the pentraxin homologue of serum amyloid P component (SAP) of other species, were measured and correlated with the speed and extent of amyloid deposition. The SAA responses of the three groups of hamsters were indistinguishable in both experiments but, in confirmation of previous reports, castrated males had FP levels higher than those of control males though still lower than in females. No differences were seen between groups in amyloid induction by casein injection alone. However, in the accelerated model using AEF, amyloid deposition occurred sooner and was more extensive in both females and castrated males than in unoperated males. These results strengthen the association between SAP, of which FP is the hamster counterpart, and the pathogenesis of amyloidosis.

Serum amyloid A protein (SAA) in horses: objective measurement of the acute phase response.

A sensitive and precise immunoassay for equine serum amyloid A protein (SAA) was established and used to determine, for the first time, the circulating concentration of this protein in health and disease. As in other species, equine SAA was present only at trace levels in healthy animals but behaved as an extremely sensitive and rapidly responding acute phase reactant following most forms of tissue injury, infection and inflammation, objectively reflecting the extent and activity of disease. Measurements of SAA should make a significant contribution to diagnosis and management of viral and bacterial infection in horses, and routine serial assays could provide an objective criterion for monitoring prospectively the general health of horses in training and racing.

Imaging of experimental amyloidosis with 131I-labeled serum amyloid P component.

131I-labeled human serum amyloid P component, which was injected into mice with experimentally induced systemic AA amyloidosis and into controls, became specifically localized and was retained in amyloidotic organs. In comparison, it was rapidly and completely eliminated from unaffected tissues and from control animals. Distinctive images of this amyloid-specific deposition of labeled serum amyloid P component were derived from whole body scanning, in vivo, of amyloidotic mice. These findings suggest that such imaging may have applications for the diagnosis and quantitation of amyloid deposits in humans.

C-reactive protein in dogs.

Serum concentrations of C-reactive protein (CRP) in dogs with various diseases or undergoing various procedures were measured by specific immunoassay. In 20 healthy dogs from various sources, values were all less than 5 mg/L, but in 22 healthy dogs from a single source, values ranged from less than 5 mg/L in 14 dogs and from 8 to 67 mg/L in 8 dogs. Increased concentrations of serum CRP were attained 24 hours after injection of casein (n = 9; median 188 mg/L), ovariohysterectomy (n = 11; median, 144 mg/L), or elective, nonacute orthopedic surgery (n = 10; median, 83 mg/L). After inoculation of Leptospira interrogans serovar canicola (n = 5), the behavior of serum CRP as an acute-phase reactant provided a sensitive and precise objective reflection of in vivo response. The CRP concentration in random single-serum samples from 73 dogs with other inflammatory and noninflammatory disorders ranged from normal (less than 5 mg/L) to 246 mg/L and generally correlated with the extent and activity of disease.

Isolation and characterisation of goat C-reactive protein.

A pentraxin was isolated from acute phase goat serum by its calcium-dependent affinity for agarose, and although it did not bind to phosphorylcholine immobilised on Sepharose, its binding to agarose was reversed by exposure to fluid phase phosphorylcholine. It was identified as goat C-reactive protein on the basis of its immunochemical cross-reactivity with human and bovine C-reactive protein. The molecule was composed of five identical, glycosylated, non-covalently associated subunits, each of molecular weight approx. 24,000. Acute phase serum levels in a small number of samples were not significantly different from normal levels (means 72 and 55 micrograms/ml, respectively), suggesting that goat C-reactive protein is not a major acute phase reactant. No other pentraxin was detected in goat serum.

Acute-phase high-density lipoprotein in the rat does not contain serum amyloid A protein.

Serum amyloid A protein (SAA) is an acute-phase apolipoprotein of high-density lipoprotein (HDL). Its N-terminal sequence is identical with that of amyloid A protein (AA), the subunit of AA amyloid fibrils. However, rats do not develop AA amyloidosis, and we report here that neither normal nor acute-phase rat HDL contains a protein corresponding to SAA of other species. mRNA coding for a sequence homologous with the C-terminal but not with the N-terminal part of human SAA is synthesized in greatly increased amounts in acute-phase rat liver. These observations indicate that the failure of rats to develop AA amyloid results from the absence of most of the AA-like part of their SAA-like protein, and that the N-terminal portion of SAA probably contains the lipid-binding sequences.

Circulating serum amyloid P component is the precursor of amyloid P component in tissue amyloid deposits.

Intravenous administration of 125I-labelled isolated mouse serum amyloid P component (SAP) to mice with systemic amyloidosis was followed by specific deposition of the labelled protein in amyloidotic organs. Although only a small proportion of the total injected dose became localized in this way, the amount correlated with the quantity of amyloid present in different organs and was greatest in the spleen. No such localization was detected in the organs of control, untreated mice or animals which had received inflammatory stimuli but did not have amyloidosis. The labelled SAP was found by autoradiography to be present in the same distribution within the tissues as the Congophilic amyloid deposits. These observations establish directly, for the first time, that circulating SAP is the precursor of the amyloid P component (AP) in systemic amyloidosis. They were confirmed by the further finding that intravenous injection into amyloidotic mice of human SAP, either in whole human serum or in isolated pure form, was followed by appearance of the human SAP in the mouse amyloid deposits. In addition to elucidating one aspect of the pathogenesis of amyloid deposition and strengthening the homology of functional behaviour between SAP of different species, the present results suggest a means for selective targeting of diagnostic tracers and/or effector agents to amyloid deposits in vivo.

Is the serum amyloid A protein in acute phase plasma high density lipoprotein the precursor of AA amyloid fibrils?

Serum amyloid A protein (SAA), an apolipoprotein of high density lipoprotein (HDL), is generally considered to be the precursor of AA protein, which forms the fibrils in reactive systemic amyloidosis in man and animals. This view is based on amino acid sequence identity between AA and the amino-terminal portion of SAA. However, in extensive and well-controlled studies of experimentally induced murine AA amyloidosis, we were unable to demonstrate a direct precursor-product relationship between SAA, in SAA-rich HDL preparations from acute phase or amyloidotic mouse or human serum, and AA protein in the amyloid deposits. This raises the possibility that SAA in its usual form, as an apolipoprotein of HDL synthesized during the acute phase response, may not be the major precursor of AA fibrils. The amyloidogenic forms of circulating SAA molecules may not be isolated during the preparation of HDL. Alternatively, particularly in the light of recent evidence that SAA mRNA is expressed in many different tissues throughout the body of appropriately stimulated animals, amyloidogenic SAA may be derived from sources other than the liver cells in which SAA-rich HDL is synthesized.

Secondary structure prediction of human SAA1. Presumptive identification of calcium and lipid binding sites.

Serum amyloid A protein (SAA), an apolipoprotein of high density lipoprotein (HDL), is an acute phase protein thought to be the precursor of amyloid fibrils in reactive systemic (AA) amyloidosis. A prediction of the secondary structure of the human serum amyloid protein SAA1(alpha) is presented. The prediction was based upon one-dimensional Fourier analysis of the amino acid sequence together with sequence matching to known structural motifs. The results were compared with those from prediction algorithms based upon statistical techniques. Our findings are consistent with available experimental data. They include the putative identification of the amino-terminal 11 residues as the functionally important lipid-binding site of SAA and of a likely, neutral, calcium-binding sequence: Gly48-Pro49-Gly50-Gly51. Sequence comparisons between SAA and protein tyrosine kinases, phospholipases A2 and delta-crystallin, all of which bind both calcium and phospholipid, revealed significant homologies that support our proposals concerning structure-function relationships in SAA.

X-ray scattering and diffraction by wet gels of AA amyloid fibrils.

Systemic amyloidosis is characterized by the extracellular accumulation of protein fibrils with typical ultrastructural morphology. The persistence in vivo of amyloid fibrils, which is responsible for their serious clinical effects, has been thought to reflect the particular, specific conformation of peptide chains constituting the fibrils. On the basis of earlier structural studies this conformation is generally considered to be almost exclusively anti-parallel beta-sheets. We have re-examined X-ray scattering by human amyloid A protein (AA) amyloid fibrils, with careful attention to the state of hydration of the preparations. We show that a stack of anti-parallel sheets is not consistent with the details of the X-ray pattern, which contains diffracted intensities that can be indexed on a 33A X 33A lattice. A structural model for the AA fibre consistent with the X-ray data is presented. The model takes account of the prediction of the secondary structure of the AA precursor SAA1(alpha) presented in our accompanying paper, and has the AA monomers arranged on a primitive lattice, with two unique molecules per unit cell.

Acute phase protein changes in antigen-induced mono-articular arthritis in rabbits and mice.

Acute phase protein levels have been measured during the induction and progression of antigen-induced mono-articular arthritis in rabbits and mice. In rabbits there was a short lived elevation in serum C-reactive protein (CRP) and erythrocyte sedimentation rate (ESR) immediately following intra-articular injection which returned to baseline levels 10-12 days after the injection. In BALB/c mice, serum amyloid P-component (SAP) and the third component of complement (C3) were elevated after intra-articular injection, returning towards baseline levels 6 weeks after the injection. The levels of CRP and SAP correlated with the inflammatory changes in the joints during the acute phase of the arthritic response (7 days after intra-articular injection). During the chronic phase the levels of these acute phase proteins bore no relationship to the degree of connective tissue destruction.

Studies of the in vivo synthesis and catabolism of serum amyloid P component (SAP) in the mouse.

The production of mouse serum amyloid P component (SAP), a major acute phase protein of liver origin, was studied immunocytochemically using the peroxidase staining technique. SAP was not detectable in the cytoplasm of hepatocytes from normal, unstimulated mice, nor was it observed before 24 h after an acute phase stimulus. 125I-labelled mouse SAP was cleared from the plasma in vivo with a half-life of 7.0-8.25 h in all animals studied including: normal mice of different strains with different genetically determined plasma SAP concentrations; mice undergoing acute phase responses with greatly elevated plasma SAP levels and mice with casein-induced amyloidosis. The circulating level of SAP is thus independent of its rate of clearance and catabolism and is determined by the rate of synthesis and/or secretion of SAP.

Human serum amyloid P component, a circulating lectin with specificity for the cyclic 4,6-pyruvate acetal of galactose. Interactions with various bacteria.

Serum amyloid P component (SAP), a normal plasma glycoprotein, has recently been shown to have Ca2+-dependent binding specificity for methyl 4,6-O-(1-carboxyethylidene)-beta-D-galactopyranoside (MO beta DG) [Hind, Collins, Renn, Cook, Caspi, Baltz & Pepys (1984) J. Exp. Med. 159, 1058-1069]. SAP was found to bind in vitro to Klebsiella rhinoscleromatis, the cell wall of which is known to contain this particular cyclic pyruvate acetal of galactose. SAP also bound in similar amounts (approx. 6000 molecules per organism) to group A Streptococcus pyogenes, but very much less was taken up on Xanthomonas campestris, which contains the 4,6-cyclic pyruvate acetal of mannose. No SAP bound to Escherichia coli, which contains the 4,6-cyclic pyruvate acetal of glucose, or to Streptococcus pneumoniae type 4, which contains the 2,3-cyclic pyruvate acetal of alpha- rather than beta-galactopyranoside, or to other organisms (Streptococcus agalactiae, Staphylococcus aureus and Staphylococcus epidermidis), the carbohydrate structures of which are less well characterized. Binding of SAP to those organisms which it did recognize was completely inhibited or reversed by millimolar concentrations of free MO beta DG. SAP, a human plasma protein, thus behaves as a lectin and may be a useful probe for its particular specific ligand in the cell walls of bacteria and other organisms.

In vivo turnover studies of C-reactive protein.

The in vivo plasma clearance rate of the acute phase reactant C-reactive protein (CRP) was studied in mice and rats. The clearance rate of 125I-human CRP in mice and 125I-rat CRP in rats showed a T1/2 of approximately 4 h. The T1/2 was independent of circulating levels of CRP and was not affected by the presence of C-polysaccharide (CPS), a ligand to which CRP binds. However, in mice receiving sufficient CPS, more radioactivity localized to the spleen compared to mice receiving 125I-CRP only. 125I-CPS was rapidly cleared at the same rate by normal mice and by mice undergoing an acute phase response while rats cleared 125I-CPS more slowly despite having high circulating CRP concentrations. These findings suggest that CRP does not provide a mechanism for extremely rapid clearance of its ligands from the circulation, although the handling and subsequent fate of these ligands may be affected.

In vivo turnover studies of C-reactive protein and lipoproteins in the rabbit.

Clearance from the plasma of 125I-labelled rabbit C-reactive protein (CRP) was significantly slower in animals undergoing an acute phase response (T1/2, mean +/- s.d., 6.48 +/- 1.4 h, n = 4) than in normal rabbits (3.17 +/- 0.4 h, n = 4), P less than 0.01. In contrast there was no difference between the rates of clearance of CRP in rabbits on a normal diet and hypercholesterolaemic rabbits, the plasma of which contained high levels of beta-VLDL with which CRP is known to form circulating complexes. Furthermore the apparent rates of clearance of 125I-labelled beta-VLDL, VLDL and LDL did not differ between normal rabbits and rabbits undergoing an acute phase response. The significance of these findings is discussed.

Isolation and characterization of C-reactive protein from the dog.

Using calcium-dependent affinity chromatography on Sepharose-bearing, covalently-coupled pneumococcal C-polysaccharide, a protein was isolated from the serum of dogs that had undergone general anaesthesia and major surgery. This protein was confirmed as the canine analogue of C-reactive protein (CRP) in other species by virtue of its electron microscopic appearance, subunit composition and behaviour as an acute phase reactant. Dog CRP had an apparent molecular weight of approximately 100,000 and was composed of five subunits of approximately 20,000 MW each. Two of the five subunits in each molecule were glycosylated. Negatively stained preparations had the typical cyclic pentameric disc-like structure of proteins of the pentraxin family, and in some preparations had a tendency to form stacks. Serum from normal healthy dogs of various strains usually contained less than 5 mg/l of CRP but, following the stimulus of major surgery, an increase in the CRP concentration was first detected at 4 hr.