Stimulation of peripheral blood mononuclear cells with lipopolysaccharide induces expression of the plasma protein alpha2-macroglobulin

The human alpha(2)-macroglobulin gene is approximately 48 kb in size and consists of 36 exons, which encode the 180 kDa subunit of this large tetrameric protein. In this investigation, a procedure of sequencing human alpha(2)-macroglobulin mRNA, using mRNA from lipopolysaccharide-stimulated peripheral blood mononuclear cells as template in RT-PCR, was developed. Incubation of peripheral blood mononuclear cell populations with lipopolysaccharide induced alpha(2)-macroglobulin mRNA expression reaching levels detectable by RT-PCR. Extracted human alpha(2)-macroglobulin mRNA was used to determine the nucleotide sequence of a 500 bp DNA segment encoding the most C-terminal, receptor-binding part of the protein, using alpha(2)-macroglobulin specific primers. The sequence obtained matched the earlier published sequence of human alpha(2)-macroglobulin, except for three point mutations, i.e., cytosine for guanine, cytosine for thymidine and thymidine for adenine substitutions at positions 4369, 4423, and 4511, respectively. None of these alterations, however, affect the amino acid sequence of the protein. In conclusion, we demonstrate a new, improved, approach to sequence human alpha(2)-macroglobulin mRNA by overexpressing the protein in peripheral blood mononuclear cells. This procedure may be useful in the search for mutations in alpha(2)-macroglobulin, examining its role in the pathogenesis of human diseases.     

Seasonal rhythms of "acute phase proteins" in humans

"Acute phase proteins" comprise a group of proteins whose concentrations increase or decrease by at least 25% after a damaging stimulus (burn, trauma, tissue damage, etc.) or during inflammation. We investigated the seasonal variation in the concentrations of several acute phase proteins--alpha1-antichymotrypsin (ACT), alpha1-acid glycoprotein (AGP), transferrin (Tf), alpha2-macroglobulin (alpha2-M), ceruloplasmin (Cp), antitrypsin (AT), and haptoglobin (Hp). Blood samples were collected from 15 healthy volunteers, who were subjected to the seasonal changes in illumination, were drawn at 08:00 h every 3 months (August, November, January/February, March/April, June/July). With the exception of Hp, all acute phase proteins showed an annual rhythm (ANOVA; p < 0.01). Lowest concentrations occurred in the winter months (November through February), with the exception of Tf, which was oppositely phased.     

Plasma levels of immunoreactive ceruloplasmin and other acute phase proteins during lactation

Lactation elevates plasma copper as well as oxidase activity levels of the copper-containing, acute phase protein ceruloplasmin (Cp). The present study provides an initial inquiry into the mechanisms behind these changes. Plasma obtained from 12 lactating women, 1 month postpartum, displayed a greater percentage increase in immunoreactive Cp levels (mean increase = 89%) than in plasma copper (mean = 66%) or Cp oxidase activity (mean = 42%). Lactation did not increase plasma content of C-reactive protein or alpha 1-antitrypsin but significantly elevated haptoglobin concentrations. Plasma alpha 2-macroglobulin contents correlated with immunoreactive Cp levels in lactating women but not in controls. These results strengthen the hypothesis that plasma content of individual acute phase proteins is regulated by both overlapping and individualized processes. In addition, the present findings raise the possibility that lactation increases both Cp synthesis and plasma turnover time of Cp-bound copper.     

Retarded protein folding of deficient human alpha 1-antitrypsin D256V and L41P variants

alpha(1)-Antitrypsin is the most abundant protease inhibitor in plasma and is the archetype of the serine protease inhibitor superfamily. Genetic variants of human alpha(1)-antitrypsin are associated with early-onset emphysema and liver cirrhosis. However, the detailed molecular mechanism for the pathogenicity of most variant alpha(1)-antitrypsin molecules is not known. Here we examined the structural basis of a dozen deficient alpha(1)-antitrypsin variants. Unlike most alpha(1)-antitrypsin variants, which were unstable, D256V and L41P variants exhibited extremely retarded protein folding as compared with the wild-type molecule. Once folded, however, the stability and inhibitory activity of these variant proteins were comparable to those of the wild-type molecule. Retarded protein folding may promote protein aggregation by allowing the accumulation of aggregation-prone folding intermediates. Repeated observations of retarded protein folding indicate that it is an important mechanism causing alpha(1)-antitrypsin deficiency by variant molecules, which have to fold into the metastable native form to be functional.     

Characterization, size estimation and solubilization of α-macroglobulin complex receptors in liver membranes

Receptors for α₂-macroglobulin-proteinase complexes have been characterized in rat and human liver membranes. The affinity for binding of ¹²⁵I-labelled α₂-macroglobulin · trypsin to rat liver membranes was markedly pH-dependent in the physiological range with maximum binding at pH 7.8–9.0. The half-time for association was about 5 min at 37°C in contrast to about 5 h at 4°C. The half-saturation constant was about 100 pM at 4°C and 1 nM at 37°C (pH 7.8). The binding capacity was approx. 300 pmol per g protein for rat liver membranes and about 100 pmol per g for human membranes. Radiation inactivation studies showed a target size of 466 ± 71 kDa (S.D., n = 7) for α₂-macroglobulin · trypsin binding activity. Affinity cross-linking to rat and human membranes of ¹²⁵I-labelled rat α₁-inhibitor-3 · chymotrypsin, a 210 kDa analogue which binds to the α₂-macroglobulin receptors in hepatocytes (Gliemann, J. and Sottrup-Jensen, L. (1987) FEBS Lett. 221, 55–60), followed by SDS-polyacrylamide gel electrophoresis, revealed radioactivity in a band not distinguishable from that of cross-linked α₂-macroglobulin (720 kDa). This radioactivity was absent when membranes with bound ¹²⁵I-α₁-inhibitor-3 complex were treated with EDTA before cross-linking and when incubation and cross-linking were carried out in the presence of a saturating concentration of unlabelled complex. The saturable binding activity was maintained when membranes were solubilized in the detergent 3-[(3-cholamidopropyl)dimethylammonio]profane sulfonate (CHAPS) and the size of the receptor as estimated by cross-linking experiments was shown to be similar to that determined in the membranes. It is concluded that liver membranes contain high concentrations of an approx. 400–500 kDa α₂-macroglobulin receptor soluble in CHAPS. The soluble preparation should provide a suitable material for purification and further characterization of the receptor.     

Recombinant human interleukin-6 (IL-6/BSF-2/HSF) regulates the synthesis of acute phase proteins in human hepatocytes

Recombinant human IL-6 (rhIL-6) is a potent inducer of the synthesis of acute phase proteins in adult human hepatocytes. A wide spectrum of acute phase proteins is regulated by this mediator. After labeling of rhIL-6 stimulated human hepatocytes with [35S]methionine acute phase protein synthesis was measured by immunoprecipitation. Serum amyloid A, C-reactive protein, haptoglobin, alpha 1-antichymotrypsin and fibrinogen were strongly induced (26-, 23-, 8.6-, 4.6- and 3.8-fold increases, respectively). Moderate increases were found for alpha 1-antitrypsin (2.7-fold) and alpha 1-acid glycoprotein (2.7-fold). RhIL-6 had no effect on alpha 2-macroglobulin, whereas fibronectin, albumin and transferrin decreased to 64, 56 and 55% of controls. In the cases of serum amyloid A, haptoglobin, alpha 1-antichymotrypsin, alpha 1-antitrypsin and alpha 1-acid glycoprotein, dexamethasone enhanced the action of rhIL-6. We conclude that rhIL-6 controls the acute phase response in human liver cells.     

A novel serine protease complexed with α2-macroglobulin from skeletal muscle of lizard fish (Saurida undosquamis)

A novel fish muscle serine protease named muscle soluble serine protease (MSSP) was purified from the soluble fraction of lizard fish (Saurida undosquamis: Synodontidae) muscle by ammonium sulfate fractionation followed by four steps of column chromatographies. In native-PAGE, the purified enzyme appeared as a single band with an estimated mol. mass of approximately 380 kDa by gel filtration. In SDS-PAGE under reducing conditions, the purified enzyme migrated as two protein bands at 110 and 100 kDa, named subunits A and B, respectively. The 20 residues of N-terminal amino acid sequence of subunit B showed 70% of homology to β-chain of carp α₂-macroglobulin-1. Moreover, both subunits A and B showed immunoreactivity with anti carp α₂-macroglobulin antibody. Purified MSSP was inactivated by Pefabloc SC, aprotinin, benzamidine and TLCK, but not by α₁-antitrypsin. After acid treatment (pH 2, 24 h), however, the enzyme activity eluted at 14 kDa from Sephacryl S-200 carried out under acidic conditions was inhibited by α₁-antitrypsin. Lizard fish MSSP most rapidly hydrolyzed Boc-Val-Pro-Arg-MCA and Boc-Gln-Arg-Arg-MCA, but did not hydrolyzed Suc-Leu-Leu-Val-Tyr-MCA and Suc-Ala-Ala-Pro-Phe-MCA, and was not suppressed either by E-64, pepstatin A and ethylenediaminetetraacetic acid (EDTA). These results indicate that the purified MSSP is a serine protease complexed with α₂-macroglobulin, and the entrapped protease was dissociated by the acid treatment. Purified and free MSSPs were most active at pH 10.0 and 9.0, respectively. Purified MSSP degraded myofibrillar proteins and casein but time courses of degradation of these substrates by the enzyme differed.     

Interleukin-6 is the major regulator of acute phase protein synthesis in adult human hepatocytes

The three monokines interleukin-1 beta (IL-1 beta), tumor necrosis factor alpha (TNF alpha), and interleukin-6 (IL-6) modulate acute phase plasma protein synthesis in adult human hepatocytes. Only IL-6 stimulates the synthesis of the full spectrum of acute phase proteins as seen in inflammatory states in humans, i.e. synthesis and secretion of C-reactive protein, serum amyloid A, fibrinogen, alpha 1-antitrypsin, alpha 1-antichymotrypsin and haptoglobin are increased while albumin, transferrin and fibronectin are decreased. IL-1 beta as well as TNF alpha, although having a moderate effect on the positive acute phase proteins and inhibiting the synthesis of fibrinogen, albumin and transferrin, fail to induce serum amyloid A and C-reactive protein. These data suggest that IL-6 plays the key role in the regulation of acute phase protein synthesis in human hepatocytes.     

Inhibition of human factor Xa by various plasma protease inhibitors

The inhibitory effects of the plasma protease inhibitors antithrombin III, alpha 2-macroglobulin and alpha 1-antitrypsin on the activity of human factor Xa have been studied using purified proteins. The rate of inhibition was determined by measuring the residual factor Xa activity at timed intervals utilizing the synthetic peptide susbtrate Bz-Ile-Glu(piperidyl)-Gly-Arg-pNA. Kinetic analysis with varying molar concentrations of inhibitors demonstrated that the inhibition of factor Xa by antithromin III, alpha 2-macroglobulin and alpha 1-antitrypsin followed second-order kinetics. Calculated values of the rate constants for the inhibition of factor Xa by antithrombin III, alpha 2-macroglobulin and alpha 1-antitrypsin were 5.8 . 10(4), 4.00 . 10(4) and 1.36 . 10(4) M -1 . min -1, respectively. The plasma concentrations of the inhibitors can be used to assess their potential relative effectiveness against factor Xa. In plasma this was found as alpha 1-antitrypsin greater than antithrombin III greater than alpha 2-macroglobulin in the ratio 4.64: 2.08: 1.0. Cephalin was shown to inhibit the rate of reaction between factor Xa and antithrombin III.     

Regulation of rabbit acute phase protein biosynthesis by monokines.

We defined the acute phase behaviour of a number of rabbit plasma proteins in studies (in vivo) and studied the effects of monokine preparations on their synthesis by rabbit primary hepatocyte cultures. Following turpentine injection, increased serum levels of C-reactive protein, serum amyloid A protein, haptoglobin, ceruloplasmin, and decreased concentrations of albumin were observed. In contrast to what is observed in man, concentrations of alpha 2-macroglobulin and transferrin were increased. Co-culture of primary hepatocyte cultures with lipopolysaccharide-activated human peripheral blood monocytes or incubation with conditioned medium prepared from lipopolysaccharide-activated human or rabbit monocytes resulted in dose-dependent induction of serum amyloid A, haptoglobin, ceruloplasmin and transferrin and depression of albumin synthesis, while C-reactive protein synthesis and mRNA levels remained unchanged. A variety of interleukin-1 preparations induced dose-dependent increases in the synthesis and secretion of serum amyloid A, haptoglobin, ceruloplasmin and transferrin and decreased albumin synthesis. Human recombinant tumour necrosis factor (cachectin) induced a dose-dependent increase in synthesis of haptoglobin and ceruloplasmin. In general, human interleukin-1 was more potent than mouse interleukin-1 and tumour necrosis factor. None of the monokines we studied had an effect on C-reactive protein synthesis or mRNA levels. These data confirm that C-reactive protein, serum amyloid A, haptoglobin and ceruloplasmin display acute phase behaviour in the rabbit, and demonstrate that, in contrast to their behaviour in man, alpha 2M and transferrin are positive acute phase proteins in this species. While both interleukin-1 and tumour necrosis factor regulate biosynthesis of a number of these acute phase proteins in rabbit primary hepatocyte cultures, neither of these monokines induced C-reactive protein synthesis. Comparison of these findings with those in human hepatoma cell lines, in which interleukin-1 does not induce serum amyloid A synthesis, suggests that the effect of interleukin-1 on serum amyloid A synthesis may be indirect.     

Modifications of serum glycoproteins the days following a prolonged physical exercise and the influence of physical training.

Eight male subjects (mean age 24.1 +/- 2.6 years) performed at intervals of 2 weeks successively a 3 h and two 2 h runs of different running speed. The days following the running there were moderate elevations of C-reactive protein, haptoglobin, alpha-1-acid glycoprotein, coeruloplasmin, transferrin, alpha-1-antitrypsin and plasminogen. There were small or no changes of albumin, alpha-2-macroglobulin and hemopexin. The elevations of the "acute phase reactants" were examined in three male subjects following a 2 h run before and after an endurance training period of 9 weeks. This demonstrated a decreased acute phase response after training as illustrated by the changes of C-reactive protein, haptoglobin and alpha-1-acid glycoprotein in spite of higher posttraining running speeds. Well-trained athletes have elevated levels of the serum protease inhibitors alpha-1-antitrypsin, alpha-2-macroglobulin and C1-inhibitor. These antiproteolytic glycoproteins might limit exercise-induced inflammatory reactions.     

Cloning and expression of human haptoglobin subunits in Escherichia coli: delineation of a major antioxidant domain

Human plasma haptoglobin (Hp) comprises alpha and beta subunits. The alpha subunit is heterogeneous in size, therefore isolation of Hp and its subunits is particularly difficult. Using Escherichia coli, we show that alpha1, alpha2, beta, and alpha2beta chain was abundantly expressed and primarily present in the inclusion bodies consisting of about 30% of the cell-lysate proteins. Each cloned subunit retained its immunoreactivity as confirmed using antibodies specific to alpha or beta chain. By circular dichroism, the structure of each expressed subunit was disordered as compared to the native Hp. The antioxidant activity was found to be associated with both alpha and beta chains when assessed by Cu(2+)-induced oxidation of low density lipoprotein (LDL). Of remarkable interest, the antioxidant activity of beta chain was extremely potent and markedly greater than that of native Hp (3.5x), alpha chain (10x) and probucol (15x). The latter is a clinically proved potent compound used for antioxidant therapy. The "unrestricted" structure of beta subunit may therefore render its availability for free-radical scavenge, which provides a utility for the future design of a "mini-Hp" in antioxidant therapy. It may also provide a new insight in understanding the mechanism involved in the antioxidant nature of Hp.     

Circadian rhythm of acute phase proteins under the influence of bright/dim light during the daytime

We investigated the influence of two different light intensities, dim (100 lx) and bright (5000 lx), during the daytime on the circadian rhythms of selected acute phase proteins of C-reactive protein (CRP), alpha1-acid glycoprotein (AGP), alpha1-antichymotrypsin (ACT), transfferin (TF), alpha2-macroglobulin (alpha2-m), haptoglobin (HP), and ceruloplasmin (CP). Serum samples were collected from 7 healthy volunteers at 4 h intervals during two separate single 24 h spans during which they were exposed to the respective light intensity conditions. A circadian rhythm was detected only in ACT concentration in the bright light condition. The concentration of ACT, a positive acute phase protein (APP), increased (significantly significant differences in the ACT concentration were detected at 14:00 and 22:00 h) and AGP showed a tendency to be higher under the daytime bright compared to dim light conditions. There were no significant differences between the time point means under daytime dim and bright light conditions for alpha2-M, AGP, Tf, Cp, or Hp. The findings suggest that some, but not all, APP may be influenced by the environmental light intensity.     

The in vitro interactions of rat pancreatic elastase and normal and inflammatory ray serum.

The partition of labelled rat pancreatic elastase (EC 3.4.21.11) between the different protease inhibitors of rat plasma was studied at different levels of saturation of the inhibitors of rat plasma was studied at different levels of saturation of the inhibitor capacity of plasma with the enzyme. The reaction mixtures were analysed by immunoelectrophoretic methods utilizing specific antisera against the different inhibitors and by gel filtration on Sephadex G-200. Rat serum was shown to contain four elastase binding proteins. alpha 1-antitrypsin, alpha 1-macroglobulin and alpha 2-acute phase protein and alpha 1-inhibitor 3 which exhibits immunologic cross-reaction with human inter-alpha-trypsin inhibitor and is of similar molecular weight. With minute amounts of labelled elastase the partition among the binding protein was alpha 1-macroglobulin 60%, alpha 1-antitrypsin 24% and alpha 1-I3 16%. The 60% value of alpha 1-M bound radioactivity in normal serum corresponds to the sum of alpha 1-M and alpha 2-AP labelling in inflammatory serum.     

Effect of protein deficiency on endogenous pyrogen-mediated acute phase protein responses

Endogenous pyrogen (EP) is known to trigger a rise in the plasma concentrations of various acute phase reactant proteins. This study describes the effects of chronic protein deficiency in rabbits on EP-mediated changes in the plasma concentrations of fibrinogen, albumin, and alpha 2-macroglobulin. Injection (i.v.) of EP from healthy donors into protein-deprived rabbits produced a smaller rise in plasma fibrinogen and alpha 2-macroglobulin, and a smaller fall in plasma albumin than injection of EP into controls. Injection of EP, obtained from malnourished donors, into healthy rabbits also resulted in an attenuation of the acute phase protein response. These data are consistent with the hypothesis that EP activity is influenced by the nutritional status of both the donor and recipient of EP.     

Molecular cloning and expression analysis of alpha 2-macroglobulin in the kuruma shrimp, Marsupenaeus japonicus

The cDNA encoding the kuruma shrimp, Marsupenaeus japonicus alpha(2)-macroglobulin (alpha(2)M) was obtained by screening a haemocyte cDNA library and 5' RACE PCR amplification. The full length cDNA of 4748 bp contains an open reading frame of 4518 nucleotides that translates into a 1505-amino acid putative peptide, with a 5'untranslated region (UTR) of 59 bp and a 3'UTR of 171 bp. The open reading frame encodes an N-terminal signal sequence of 17 residues and a mature protein of 1488 residues. The entire amino acid sequence is similar to the alpha(2)M sequences of arthropods (30-31% identity), mammals (26-27% identity) and fish (25-28% identity). The M. japonicus alpha(2)M sequence contains putative functional domains including a bait region, an internal thiol ester site, and a receptor-binding domain, which are present in mammalian alpha(2)Ms. In a healthy shrimp, the mRNA of alpha(2)M was mainly expressed in haemocytes. In addition, the expression level of alpha(2)M mRNA was dramatically increased by through time upon oral administration of peptidoglycan (PG), which is an immune stimulant. The highest expression of alpha(2)M mRNA was observed 7 days after feeding with PG. These results suggest that the shrimp alpha(2)M is an important molecule in immune system.     

EFFECT OF ULCERATIVE COLITIS ACTIVITY ON PLASMA CONCENTRATION OF TRANSFORMING GROWTH FACTOR β1

Enhanced expression of transforming growth factor-β₁(TGF-β₁) demonstrated in human colonic mucosa of patients with ulcerative colitis (UC), indicates its possible significance in the pathogenesis of this disease. The aim of this study was to evaluate plasma TGF-β₁concentration in patients with different degrees of colonic mucosal injury, as a possible indicator of ulcerative colitis activity. TGF-β₁concentration was measured with an enzyme immunoassay (EIA) in plasma of 45 patients with endoscopically confirmed UC. Values observed in UC patients (40.5±15.9 ng/ml) were significantly higher than in healthy people (18.3±11.6 ng/ml) and higher than in patients with irritable colon syndrome (ICS), (20.5±13.6 ng/ml). The highest plasma TGF-β₁(58.6±112.1 ng/ml) was in patients with the severe UC course. TGF-β₁level analysed in all UC patients revealed significant positive correlation with scored degree of mucosal injury (r=0.396;P<0.01). Among other possible laboratory markers of the disease activity, only C-reactive protein concentration demonstrated significant correlation. Enhanced production of TGF-β₁can be related to inflammation activity. Measurement of plasma TGF-β₁may be considered as a biomarker of the disease activity.     

The identification of distinctive forms of human alpha 2-macroglobulin by using the numerical relationship between trypsin binding in alpha- and beta-modes.

Interactions between the serine proteinase trypsin and the protein proteinase inhibitors in human blood were expressed in terms of a coupled set of non-linear differential equations, which has been solved for each of 110 samples of serum obtained from colleagues and from a variety of hospital sources. Optimization of nine unknown theoretical parameters and 21 experimental rate measurements of the hydrolytic activity of trypsin in free and bound states after admixture with various amounts of a given serum was achieved by an iterative procedure using initial estimates of the parameters derived from the "four-straight-line" model described in the preceding paper [Topping & Seilman (1979) Biochem. J. 177, 493--499.] Such a procedure yielded the following information for each sample of serum examined: (a) the concentrations of alpha 1-antitrypsin and alpha 2-macroglobulin; (b) the unequivocal assignment of alpha 2-macroglobulin into one of seven categories on the basis of trypsin binding in two kinetically differentiated modes (alpha and beta); (c) the hydrolytic activities of trypsin (versus Bz-Arg-OEt) when bound to alpha 1-antitrypsin, and to alpha 2-macroglobulin in the alpha- and beta-modes. Molecular interpretations of the binding of trypsin to alpha 2-macroglobulin are discussed and the potential clinical value of recognizing the nature of such binding is reported.     

Acute phase proteins, body temperature and urinary melatonin under the influence of bright and dim light intensities during the daytime

Concentrations of five acute phase proteins: C-reactive protein (CRP), alpha 1-antichymotrypsin (ACT), transferin (Tf), alpha 2-macroglobulin (alpha 2-M) and haptoglobin (Hp) as well as glycosylation profiles of alpha 1-antichymotrypsin (ACT) were studied in sera samples with 7 healthy volunteers under the influence of two different light intensities during the daytime dim (100 lx) and bright (3000 lx) light. Concentration of transferin (negative proteins) under the influence of bright light during the daytime decreased significantly. Other proteins have the tendency to increase (positive proteins) under the influence of daytime bright light. The microheterogeneity of ACT did not change under the influence of different light intensities. Melatonin and rectal temperature were also measured simultaneously. Rectal temperature decreased to be lower during the first half of the night and urinary melatonin secretion rate increased to be higher during the night when the subjects spent time under the bright light during the day. Thus, it is concluded that the diurnal bright light exposure may activate some parameters of acute phase proteins, increase nocturnal melatonin secretion and accelerate a fall of rectal temperature during first half period of night sleep.