Rat alpha2 acute-phase macroglobulin. Isolation and physicochemical properties.
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ABSTRACT:
SUBMITTER: Gauthier F
PROVIDER: S-EPMC1164166 | biostudies-other | 1976 Dec
REPOSITORIES: biostudies-other
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Project description:Alpha2-M (alpha2-macroglobulin) was purified from human plasma by two different procedures. As well as having no detectable impurities by the usual criteria for testing the homogeneity of protein preparations, these alpha2M preparations showed a single component, after reduction in urea, of 185000 daltons by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. The molecular weight of the alpha2M was found to be 718000 by sedimentation equilibrium experiments using the gravimetrically determined -v of 0.731 ml/g. The interaction of several proteinases with alpha2M was studied by using a novel discontinuous polyacrylamide-gel system, which showed clear separation of the enzyme-complexed alpha2M from the free alpha2M. These studies indicated that urokinase, as well as trypsin, chymotrypsin, plasmin and thrombin forms complexes with alphaM. The cleavage of the 185000-dalton subunit to a 85000-dalton species on interaction of trypsin with alpha2M was demonstrated by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis after reduction of the alpha2M-trypsin complex in urea. The amino acid composition, carbohydrate content, absorption coefficient at 280 nm, the specific refractive increment and the sedimentation coefficient for these alpha2M preparations were measured. The stability of the trypsin-binding activity of the alpha2M preparations was also studied under several storage situations.
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Project description:The relationship between various amyloidoses and chaperones is gathering attention. In patients with dialysis-related amyloidosis, ?(2)-macroglobulin (?2M), an extracellular chaperone, forms a complex with ?(2)-microglobulin (?2-m), a major component of amyloid fibrils, but the molecular mechanisms and biological implications of the complex formation remain unclear. Here, we found that ?2M substoichiometrically inhibited the ?2-m fibril formation at a neutral pH in the presence of SDS, a model for anionic lipids. Binding analysis showed that the binding affinity between ?2M and ?2-m in the presence of SDS was higher than that in the absence of SDS. Importantly, SDS dissociated tetrameric ?2M into dimers with increased surface hydrophobicity. Western blot analysis revealed that both tetrameric and dimeric ?2M interacted with SDS-denatured ?2-m. At a physiologically relevant acidic pH and in the presence of heparin, ?2M was also dissociated into dimers, and both tetrameric and dimeric ?2M interacted with ?2-m, resulting in the inhibition of fibril growth reaction. These results suggest that under conditions where native ?2-m is denatured, tetrameric ?2M is also converted to dimeric form with exposed hydrophobic surfaces to favor the hydrophobic interaction with denatured ?2-m, thus dimeric ?2M as well as tetrameric ?2M may play an important role in controlling ?2-m amyloid fibril formation.
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Project description:BackgroundAlpha 2 Macroglobulin family members have been studied extensively with respect to their roles in physiology and human disease including innate immunity and Alzheimer's disease, but little is known about a possible role in liver development loss-of-function in model systems.Principal findingsWe report the isolation of the zebrafish alpha2 macroglobulin-like (A2ML) gene and its specific expression in the liver during differentiation. Morpholino-based knock-down of A2ML did not block the initial formation of the liver primordium, but inhibited liver growth and differentiation.SignificanceThis report on A2ML function in zebrafish development provides the first evidence for a specific role of an A2M family gene in liver formation during early embryogenesis in a vertebrate.
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Project description:Surfactant protein D (SP-D) is an innate immune collectin that recognizes microbes via its carbohydrate recognition domains, agglutinates bacteria, and forms immune complexes. During microbial infections, proteases, such as elastases, cleave the carbohydrate recognition domains and can inactivate the innate immune functions of SP-D. Host responses to counterbalance the reduction of SP-D-mediated innate immune response under these conditions are not clearly understood. We have unexpectedly identified that SP-D could interact with protein fractions containing ovomucin and ovomacroglobulin. Here, we show that SP-D interacts with human alpha(2)-macroglobulin (A2M), a protease inhibitor present in the lungs and serum. Using enzyme-linked immunosorbent assays, surface plasmon resonance, and carbohydrate competition assays, we show that SP-D interacts with A2M both in solid phase (K(D) of 7.33 nM) and in solution via lectin-carbohydrate interactions under physiological calcium conditions. Bacterial agglutination assays further show that SP-D x A2M complexes increase the ability of SP-D to agglutinate bacteria. Western blot analyses show that SP-D, but not A2M, avidly binds bacteria. Interestingly, intact and activated A2M also protect SP-D against elastase-mediated degradation, and the cleaved A2M still interacts with SP-D and is able to enhance its agglutination abilities. We also found that SP-D and A2M can interact with each other in the airway-lining fluid. Therefore, we propose that SP-D utilizes a novel mechanism in which the collectin interacts with protease inhibitor A2M to decrease its degradation and to concurrently increase its innate immune function. These interactions particularly enhance bacterial agglutination and immune complex formation.
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Project description:The three-dimensional structure of the N-terminal domain (residues 18-112) of alpha2-macroglobulin receptor-associated protein (RAP) has been determined by NMR spectroscopy. The structure consists of three helices composed of residues 23-34, 39-65, and 73-88. The three helices are arranged in an up-down-up antiparallel topology. The C-terminal 20 residues were shown not to be in a well defined conformation. A structural model for the binding of RAP to the family of low-density lipoprotein receptors is proposed. It defines a role in binding for both the unordered C terminus and the structural scaffold of the core structure. Pathogenic epitopes for the rat disease Heymann nephritis, an experimental model of human membranous glomerulonephritis, have been identified in RAP and in the large endocytic receptor gp330/megalin. Here we provide the three-dimensional structure of the pathogenic epitope in RAP. The amino acid residues known to form the epitope are in a helix-loop-helix conformation, and from the structure it is possible to rationalize the published results obtained from studies of fragments of the N-terminal domain.
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Project description:High resolution images of rat acute-phase alpha 2-macroglobulin (AP alpha 2M) have been obtained by using dark-field electron microscopy. No staining or artifact-inducing procedures were used. Analysis of unfiltered electron microscope plates, exposed to minimal electron beam radiation, revealed highly contrasted particles of variable morphology with dimensions of approx. 19 nm X 14 nm. An electron-dense core with four to six projections could be seen. Two-fold symmetry was evident in selected images, supporting the four-subunit composition of the protein. Image processing and filtering confirmed the presence and configuration of the projections by demonstrating exact molecular dimensions of 16 nm X 9.5 nm and a shape with six projections like that of the Russian letter zh. SDS/polyacrylamide-gel electrophoresis revealed that this molecule was in the proteinase-bound form. C.d. data revealed a surprisingly low content of alpha-helical secondary structure (12%) and an atypically large content of beta-form structure (33%). Comparison of the amino acid compositions of AP alpha 2M and human alpha 2-macroglobulin indicated a high degree of homology between the two molecules. It is concluded that the conformation of rat AP alpha 2M, both at the molecular and secondary structural levels, is strikingly similar to that of human alpha 2-macroglobulin.
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Project description:1. A method is described for the isolation of certain of the alpha(1)-globulins of rat plasma that are known to increase in concentration after tissue damage (acute-phase globulins). 2. Although apparently homogeneous when examined by disc electrophoresis at pH9, these proteins could be subdivided further by isoelectric fractionation. 3. Treatment with neuraminidase removed approx. 60% of the sialic acid originally present in these proteins and gave almost completely homogeneous material of decreased mobility when examined by disc electrophoresis in polyacrylamide gel. When subjected to immunoelectrophoresis this material gave a single arc. 4. The homogeneity of the isolated materials was examined by ultracentrifugation. The single peak thus found is consistent with molecular weights of 45000-46000. 5. The isolated materials were shown to be glycoproteins containing approx. 15% of carbohydrate, and to have isoelectric points in the range pH4.4-4.8.
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Project description:Many benthic marine invertebrates, like barnacles, have a planktonic larval stage whose primary purpose is dispersal. How these species colonize suitable substrata is fundamental to understanding their evolution, population biology, and wider community dynamics. Unlike larval dispersal, settlement occurs on a relatively small spatial scale and involves larval behavior in response to physical and chemical characteristics of the substratum. Biogenic chemical cues have been implicated in this process. Their identification, however, has proven challenging, no more so than for the chemical basis of barnacle gregariousness, which was first described >50 years ago. We now report that a biological cue to gregarious settlement, the settlement-inducing protein complex (SIPC), of the major fouling barnacle Balanus amphitrite is a previously undescribed glycoprotein. The SIPC shares a 30% sequence homology with the thioester-containing family of proteins that includes the alpha(2)-macroglobulins. The cDNA (5.2 kb) of the SIPC encodes a protein precursor comprising 1,547 aa with a 17-residue signal peptide region. A number of structural characteristics and the absence of a thioester bond in the SIPC suggest that this molecule is a previously undescribed protein that may have evolved by duplication from an ancestral alpha(2)-macroglobulin gene. Although the SIPC is regarded as an adult cue that is recognized by the cyprid at settlement, it is also expressed in the juvenile and in larvae, where it may function in larva-larva settlement interactions.
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Project description:Human alpha2M (alpha2-macroglobulin) and the complement components C3 and C4 are thiol ester-containing proteins that evolved from the same ancestral gene. The recent structure determination of human C3 has allowed a detailed prediction of the location of domains within human alpha2M to be made. We describe here the expression and characterization of three alpha(2)M domains predicted to be involved in the stabilization of the thiol ester in native alpha2M and in its activation upon bait region proteolysis. The three newly expressed domains are MG2 (macroglobulin domain 2), TED (thiol ester-containing domain) and CUB (complement protein subcomponents C1r/C1s, urchin embryonic growth factor and bone morphogenetic protein 1) domain. Together with the previously characterized RBD (receptor-binding domain), they represent approx. 42% of the alpha2M polypeptide. Their expression as folded domains strongly supports the predicted domain organization of alpha2M. An X-ray crystal structure of MG2 shows it to have a fibronectin type-3 fold analogous to MG1-MG8 of C3. TED is, as predicted, an alpha-helical domain. CUB is a spliced domain composed of two stretches of polypeptide that flank TED in the primary structure. In intact C3 TED interacts with RBD, where it is in direct contact with the thiol ester, and with MG2 and CUB on opposite, flanking sides. In contrast, these alpha2M domains, as isolated species, show negligible interaction with one another, suggesting that the native conformation of alpha2M, and the consequent thiol ester-stabilizing domain-domain interactions, result from additional restraints imposed by the physical linkage of these domains or by additional domains in the protein.
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