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[Role of proteins of the macroglobulin family in regulation of tumor growth].

Proteins of the macroglobulin family are an ancient and evolutionarily conservative link of the immune system, which is actively involved in both inhibition of tumor growth cells and proliferation of tumor cells. Two basically different binding sites and a great conformational plasticity of all representatives of the macroglobulin family, as well as the presence of two to four representatives of the family in the blood of most species allow them to transport diverse substances and exert various regulatory influences on both the tumor and the entire organism. For example, the capacity of macroglobulins for binding hydrolases makes it possible to inhibit enzyme mediated tumor invasion. At the same time, an excess of macroglobulin/hydrolase complexes can activate apoptosis. The tumor is able of using macroglobulins, especially pregnancy-associated proteins, for its own protection. Specifically, pregnancy-associated alpha2-glycoprotein, which is actively produced by human tumor cells, blocks the histocompatibility complex antigens. On the contrary, the capacity of binding zinc stimulates the thymulin-dependent activation of natural killer cells. Nevertheless, the actively growing tumor expresses many receptors to macroglobulins, which are the main carriers of some cytokines and growth factors essential for proliferation.

Animals↗

A Waldenström's macroglobulin with anti-G3m(b1) antibody activity.

A human monoclonal macroglobulin (IgM, K) from a patient (KI) with Waldenström's macroglobulinemia was shown to have antibody activity against a human IgG (Gm) allotype. In hemagglutination tests, only one anti-D serum with G3m(b0b1) reacted with macroglobulin KI. Antiglobulin specificity of macroglobulin KI was determined to be an anti-G3m(b1) antibody by hemagglutination inhibition tests. Fab fragments from macroglobulin KI could react with human IgG3 protein possessing G3m(b1), but Fc fragments could not react. Gm phenotype in IgG isolated from serum KI was determined to be Gm(a,z,g,b0,s,t,u). This is the first report of a Waldenström's macroglobulin with antiglobulin specificity against a Gm allotype.

Aged↗

Basis for the potent inhibition of influenza virus infection by equine and guinea pig alpha 2-macroglobulin.

The unique properties of equine and guinea pig sera which make them potent inhibitors of influenza virus adsorption and infection have been investigated. The inhibitory activities of both sera are found to reside entirely in their respective alpha 2-macroglobulins, high molecular weight glycoproteins which bind to viral hemagglutinins via sialic acids of their N-linked carbohydrate groups. Structure analysis has shown that both proteins contain 4-O-acetyl-N-acetylneuraminic acid (4-O-Ac-NeuAc) (Hanaoka, K., Pritchett, T. J., Takasaki, S., Kochibe, N., Sabesan, S., Paulson, J.C., and Kobata, A. (1989) J. Biol. Chem. 264, 9842-9849). These 4-O-acetylated sialic acids have been found in few species, making their coincidence with high inhibitory potency in equine and guinea pig alpha 2-macroglobulin striking. However, 4-O-Ac-NeuAc does not appear to increase the avidity of interaction with influenza virus since isolated oligosaccharides of equine alpha 2-macroglobulin are no more potent inhibitors of adsorption than isolated oligosaccharides of human alpha 2-macroglobulin, which is a relatively poor inhibitor and contains only NeuAc. Since 4-O-Ac-NeuAc is resistant to cleavage by viral sialidase it may serve to protect the inhibitor from inactivation. These and supporting results suggest that the key property of equine and guinea pig alpha 2-macroglobulin which make them high potency inhibitors is a spatial arrangement of sialic acid containing oligosaccharide groups which allows optimal interaction with multiple hemagglutinins. The implications of these results for the design of low molecular weight inhibitors of influenza virus infection are discussed.

Animals↗

[alpha 2-Macroglobulin in serum and plasma: reference values with carbobenzoxy-valyl-glycyl-arginine-p-nitroanilide, a chromogenic substrate].

alpha 2-Macroglobulin can be determined using the amidolytic activity of the alpha 2-macroglobulin-trypsin-complex. Reference values are reported using a commercially available kit for continuous assay at 25 degrees C. Reference ranges obtained in a population of 235 children and adults aged between 1 day and 61 years were found to be age-dependent. Reference ranges established for the different age groups of children were grouped together in order to obtain useful guide values. The following values were obtained for serum: 1 to 10 days: 5-11 kU/l 11 days to 18 years: 8-16 kU/l The reference values (5th to 95th percentile) determined for juveniles and adults were as follows: 19 to 25 years: 4.1-12.2 kU/l (serum); 4.0-11.2 kU/l (citrated plasma) greater than or equal to 25 to 61 years: 4.1-8.1 kU/l (serum); 3.7-7.4 kU/l (citrated plasma) No variation according to sex was observed, nor was any difference noted between preprandial and postprandial catalytic concentrations, or between values in smokers and non-smokers or between values in the group of women taking hormonal contraceptives and the group not taking such drugs. If the dilution of the blood sample with citrate solution is taken into account when calculating the catalytic concentrations in citrated plasma, the values in plasma are higher than those in serum (median 11%). Measurement of the alpha 2-macroglobulin-trypsin complex in plasma is assumed to give a more realistic reflection of the in vivo activity than measurement in serum. Comparison with two immunological methods (radial immunodiffusion and kinetic nephelometry) revealed that there is a linear relationship between the measurement of the catalytic concentration of the alpha 2-macroglobulin-trypsin complex and the immunological concentration of alpha 2-macroglobulin. Therefore mutual conversion is possible for the described concentration range.

Adolescent↗

The comparative evaluation of alpha-2-macroglobulin and immunoglobulin levels in sera of patients with bronchial asthma.

Alpha-2-macroglobulin is the major serum antiprotease capable of inhibiting proteolytic enzymes. Protease-antiprotease homeostasis within the respiratory tract can be altered in bronchial asthma. On the other hand, in their disorder marked changes in immunoglobulin levels are observed and are related to the clinical stage of the disease. Consequently, we hypothesized, that a correlation between the levels of alpha-2-macroglobulin and immunoglobulins might have been existing. In order to evaluate this concept, the comparative analysis of serum alpha-2-macroglobulin and immunoglobulin levels in patients with extrinsic (atopic) and intrinsic (non-atopic) bronchial asthma, and in patients with bronchospastic and emphysema dominated components, was carried out by radial immunodiffusion assays. The positive linear correlation between alpha-2-macroglobulin and IgM levels was observed in patients of all tested groups. These results were discussed in view of the known role of the natural inhibitor of proteolytic enzymes, i.e. alpha-2-macroglobulin, in the protection of asthmatic lung.

Adolescent↗

Primary structure of human alpha 2-macroglobulin. I. Isolation of the 26 CNBr fragments, amino acid sequence of 13 small CNBr fragments, amino acid sequence of methionine-containing peptides, and alignment of all CNBr fragments.

The isolation of the 26 CNBr fragments from the identical Mr = 180,000 subunits of human alpha 2-macroglobulin is described. The fragments have been purified by combinations of gel chromatography, ion-exchange chromatography, high voltage paper electrophoresis, paper chromatography, and high performance liquid chromatography. The complete amino acid sequences of 13 small CNBr fragments have been determined. These fragments include CB1 (residues 1-9), CB3 (residues 79-98), CB4 (residues 99-128), CB9 (residues 442-477), CB10 (residues 478-497), CB13 (residues 644-650), CB14 (residues 651-665), CB15 (residues 666-674), CB16 (residues 675-690), CB19 (residues 937-945), CB20 (residues 946-954), CB24 (residues 1356-1362), and CB25 (residues 1363-1375). The fragments determined account for 200 of the 1451 residues of the subunits of alpha 2-macroglobulin. Most likely, Cys-6 of CB9 is bound to the corresponding residue in CB9 from another subunit, thus forming an interchain disulfide bridge in alpha 2-macroglobulin. Cys-1 of CB15 is bound to Cys-35 of CB12. CB15 contains a pair of Gln residues that can react covalently with amines in a factor XIIIa-catalyzed process (Gln-5 and Gln-6). CB16 contains the primary cleavage sites for proteinases in the bait region of alpha 2-macroglobulin (-Arg7-Val-Gly-Phe-Tyr-Glu-). CB20 contains the residues which in native alpha 2-macroglobulin presumably form an internal reactive beta-cysteinyl-gamma-glutamyl thiol ester (Cys-4 and Glx-7). Partial NH2- and COOH-terminal sequence data are given for the 13 large CNBr fragments. Complete or partial sequence determination of 19 methionine-containing peptides or variants thereof allow the alignment of all the CNBr fragments.

Amino Acid Sequence↗

Primary structure of human alpha 2-macroglobulin. V. The complete structure.

The primary structure of the tetrameric plasma glycoprotein human alpha 2-macroglobulin has been determined. The identical subunits contain 1451 amino acid residues. Glucosamine-based oligosaccharide groups are attached to asparagine residues 32, 47, 224, 373, 387, 846, 968, and 1401. Eleven intrachain disulfide bridges have been placed (Cys25-Cys63, Cys228-Cys276, Cys246-Cys264, Cys255-Cys408, Cys572-Cys748, Cys619-Cys666, Cys798-Cys826, Cys824-Cys860, Cys898-Cys1298, Cys1056-Cys1104, and Cys1329-Cys1444). Cys-447 probably forms an interchain bridge with Cys-447 from another subunit. The beta-SH group of Cys-949 is thiol esterified to the gamma-carbonyl group of Glx-952, thus forming an activatable reactive site which can mediate covalent binding of nucleophiles. A putative transglutaminase cross-linking site is constituted by Gln-670 and Gln-671. The primary sites of proteolytic cleavage in the activation cleavage area (the "bait" region) are located in the sequence: -Arg681-Val-Gly-Phe-Tyr-Glu-. The molecular weight of the unmodified alpha 2-macroglobulin subunit is 160,837 and approximately 179,000, including the carbohydrate groups. The presence of possible internal homologies within the alpha 2-macroglobulin subunit is discussed. A comparison of stretches of sequences from alpha 2-macroglobulin with partial sequence data for complement components C3 and C4 indicates that these proteins are evolutionary related. The properties of alpha 2-macroglobulin are discussed within the context of proteolytically regulated systems with particular reference to the complement components C3 and C4.

Amino Acid Sequence↗

Alpha 2-macroglobulin is present in and synthesized by the cornea.

PURPOSE: The purposes of this study were to determine whether the proteinase inhibitor alpha 2-macroglobulin is present in the cornea, and, if so, where it is located, and whether it is synthesized by the cornea, and, if so, where it is being synthesized. METHODS: alpha 2-Macroglobulin was immunolocalized using a double antibody technique and quantified by immunodot blot assays, and its identity was confirmed by Western blot analysis. Corneal synthesis of this inhibitor was determined by immunoprecipitation of extracts from corneas incubated in organ culture with 35S-methionine. mRNA was localized by in situ hybridization of 3H-labeled cDNA to the inhibitor. RESULTS: alpha 2-Macroglobulin was localized in the epithelial, endothelial, and stromal cells. It was also found in the stromal extracellular matrix. When extracts of the epithelium, stroma, and Descemet's membrane-endothelium were analyzed by Western blot, an immunoreactive band for this inhibitor was detected in all extracts. This band comigrated with the alpha 2-macroglobulin form isolated from plasma. Metabolically labeled inhibitor was immunoprecipitated from the stromal layer but not from the epithelial or endothelial layer. However, when examined by in situ hybridization, mRNA was localized to epithelial and endothelial cells in addition to stromal keratocytes. CONCLUSIONS: Because alpha 2-macroglobulin has the ability to inhibit a wide range of proteinases, it is probable that this inhibitor plays an important role in protecting the cornea from damage caused by proteinases. This includes proteinases synthesized by the cornea and those released from inflammatory cells and invading organisms.

Adolescent↗

Studies on the effect of plasminogen activator on the interaction between alpha 2-macroglobulin and plasmin.

Six different plasmins were prepared by incubating human plasminogen with various amounts of streptokinase or urokinase. It was confirmed that the six different plasmins possessed similar caseinolytic activities, and the inhibitory effects of a alpha 1-antitrypsin on caseinolytic activities of the six different plasmins were all the same. On the other hand, interactions between the six different plasmins and alpha 2-macroglobulin were complicated. Plasmins activated by cleavage of plasminogen were almost immediately or effectively inhibited by alpha 2-macroglobulin. However, plasmin activated by complex formation of plasminogen with streptokinase was not so immediately or effectively inhibited by alpha 2-macroglobulin. It was supposed that the difference between these two results on the interaction between plasmin and alpha 2-macroglobulin might be due to the difference in molecular form of plasmin. In the present study, it was also confirmed that streptokinase or urokinase, in free form in the reaction mixture, interfered with the interaction between plasmin and alpha 2-macroglobulin. The cause for such interference was discussed.

Fibrinolysin↗

[The effect of plasmin and its complexes with alpha 2-antiplasmin and alpha 2-macroglobulin on secretion of proteinase and plasminogen inhibitors from peripheral mononuclear cells].

Secretion of proteinase inhibitors alpha2-macroglobulin, alpha1-proteinase inhibitor, inter-alpha-trypsin inhibitor and plasminogen was distinctly altered in mononuclear cell from peripheric blood of volunteers if these cells were preincubated simultaneously with alpha2-macroglobulin and alpha2-antiplasmin modified by plasmin. The complex plasmin-alpha2-macroglobulin was shown to stimulate most effectively the alpha2-macroglobulin and alpha1-proteinase inhibitor secretion. Besides, plasmin-alpha2-macroglobulin complex inhibited the inter-alpha-trypsin inhibitor secretion, whereas the complex plasmin-alpha2-antiplasmin--stimulated. Both these complexes augmented the rate of plasminogen synthesis in the mononuclear cells.

Antifibrinolytic Agents↗

Prostate specific antigen-alpha 2-macroglobulin complexes in prostate cancer patient sera.

Quantitative immunoblotting of prostate cancer patient sera revealed that most prostate specific antigen was in complexes with alpha 1-antichymotrypsin or alpha 2-macroglobulin with little of it being free antigen. Complexes of prostate specific antigen with these protease inhibitors in patient sera comigrated during electrophoresis with the respective purified complexes. Each complex was selectively removed from patient sera by absorption with specific antibodies. When prostate specific antigen was added to normal plasma, complexes with alpha 2-macroglobulin appeared first and after 1 hr, the distribution was approximately 40% free antigen, approximately 40% complexes with alpha 2-macroglobulin, and approximately 20% complexes with alpha 1-antichymotrypsin. These data show that prostate specific antigen reacts more readily with alpha 2-macroglobulin than with any other protease inhibitor in plasma and that the antigen complexes with alpha 2-macroglobulin in vivo in cancer patients.

Biomarkers, Tumor↗

Different binding kinetics of Serratia 56K protease with plasma alpha 2-macroglobulin and chicken egg white ovomacroglobulin.

We recently reported that Serratia 56K protease is inhibited by plasma alpha 2 macroglobulin (alpha 2M) temporarily and by chicken egg white ovomacroglobulin (ovoM) continuously (Molla, A. et al. (1986) Infect. Immun. 53, 522-529). The inhibition of this protease is almost complete with ovoM whereas it is incomplete with alpha 2M, although these two macroglobulins show homology and many similarities. In the present study we determined the apparent numbers of binding sites and binding constants for the two macroglobulins by means of the fluorescence polarization method using FITC-labeled 56K protease. The time courses of complex formation of 56K protease with alpha 2M and ovoM were different; with ovoM it was complete within 5 min while with alpha 2M 150 min was required. Their apparent molecular volumes were also different; the fluorescence polarization value of the E/I complex was 18.7% larger with ovoM than with alpha 2M. The association constants obtained on Scatchard plot analysis with 56K protease and alpha 2M or ovoM were 0.33 X 10(7) M-1 and 1.09 X 10(7) M-1, respectively. One molecule of each of these macroglobulins binds 1.13 and 1.35 molecules of 56K protease, respectively. Upon E/I complex formation, an increase in amino groups due to proteolysis was noted in both cases, but more progressive proteolysis was observed in the case of alpha 2M. Furthermore, when the 56K protease was inactivated through the depletion of Zn atoms, complex formation did not occur.

Amino Acids↗

alpha-Macroglobulin domain structure studied by specific limited proteolysis.

Limited proteolysis was used to probe the domain structures of rat alpha 1-inhibitor 3, human pregnancy zone protein, and rat alpha 1-macroglobulin representing monomeric, dimeric, and tetrameric members, respectively, of the alpha-macroglobulin family. Specific limited digestion with trypsin, chymotrypsin, elastase, subtilisin, or Staphylococcus aureus V8 proteinase produced well-defined fragments as monitored by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The fragments were electroblotted to polyvinylidene difluoride membranes and subjected to NH2-terminal sequence analysis to locate the cleavage sites by comparison with the known primary structures of these proteins. Based on these results and the sizes of the generated fragments the 180-kDa alpha-macroglobulin subunit is proposed to contain seven relatively large domains resistant to proteolytic digestion, constituted by approximate residues 1-200 (I), 290-400 (II), 415-660 (III), 710-860 (IVa), 920-1160 (IVb), 1203-1305 (Va), and 1314-1451 (Vb) (human alpha 2-macroglobulin numbering). The overall domain organization is similar to that recently proposed by D. S. Rubenstein, J. J. Enghild, and S. V. Pizzo (1991, J. Biol. Chem. 266, 11252-11261) from studies of rat alpha 1-inhibitor 3, but the present results suggest that the large domains IV and V proposed by these authors are each composed of two domains. The present study emphasizes that domain Vb contains the determinants necessary for receptor recognition.

Amino Acid Sequence↗

alpha2-macroglobulin and C1-inactivator are plasma inhibitors of human glandular kallikrein.

Human glandular kallikrein (hK2) is a possible new marker for prostate cancer that is homologous to prostate specific antigen. Purified hK2 added to serum or plasma reacted with endogenous protease inhibitors to form complexes of >350, 135, and 80 kDa, and some hK2 remained free, as judged by immunoblotting. The former two complexes could be removed by specific antibodies to alpha2-macroglobulin and to C1- inactivator, respectively, and they comigrated on SDS-PAGE with complexes formed between hK2 and purified alpha2-macroglobulin or C1-inactivator. hK2 complexes of 80 kDa could not be completely removed with any anti-serpin antibody used. Thus, these may consist of more than one type of hK2 complex. In contrast, essentially all hK2 complexes were removed from seminal plasma by antibody to protein C inhibitor, demonstrating that protein C inhibitor is the only significant inhibitor of hK2 in semen. hK2 reacted more rapidly with alpha2-macroglobulin than with any other inhibitor in plasma or serum. Divalent metal ions and heparin did not appreciably affect the rate of formation of any of the hK2 complexes in serum or plasma or with purified alpha2-macroglobulin or C1-inactivator. Measurement of one or more of the hK2 forms identified here may have diagnostic or prognostic potential for prostate cancer.

Biomarkers, Tumor↗

Identification of four genes coding for isoforms of murinoglobulin, the monomeric mouse alpha 2-macroglobulin: characterization of the exons coding for the bait region.

Murinoglobulins are the single chain members of the alpha 2-macroglobulin family of proteinase inhibitors in the mouse. DNA clones representing the genes coding for four different murinoglobulins were isolated from three independent mouse genomic DNA libraries. Sequence analysis demonstrated that in each gene two exons are coding for the bait region. This is the specific protein sequence in each alpha-macroglobulin, which is functionally important since it is extremely sensitive to cleavage by different proteinases. The molecular data established the existence of at least four different murinoglobulin genes. Three of these corresponded to the three cDNA clones previously identified. Sequencing of intron-exon boundaries and intron sizing allowed us to construct physical maps of the region from exon 15 to exon 25 (numbered in comparison to mouse alpha 2-macroglobulin) in each murinoglobulin gene. Southern blotting of genomic DNA from five different mouse strains confirmed this analysis and even suggested the possible existence of a fifth murinoglobulin gene. These data indicate that the mouse presents a genetic repertoire of the alpha 2-macroglobulin family much more complex than originally anticipated. The bait region exon sequences showed a considerably higher degree of divergence (72 to 88% sequence identity) than that of the flanking exon sequences coding for adjacent, structural domains of the murinoglobulin proteinase inhibitors (91 to 96%). Even more surprising was that adjacent intron sequences are conserved as faithfully as the nonbait region coding exons (90 to 96%). These data demonstrate a unique property of the bait region coding sequences, as they apparently are allowed to mutate considerably. This divergency must then confer divergent proteinase inhibitory properties to the resulting proteins.

3T3 Cells↗

Alpha 2-macroglobulin is the major neutralizing inhibitor of influenza A virus in pig serum.

Horse, pig, and rabbit sera contain distinct glycoprotein inhibitors of influenza A viruses that inhibit hemagglutinating activity and neutralize viral infectivity. Although alpha 2-macroglobulin has been identified as the inhibitor in horse serum, the inhibitors in pig and rabbit sera have not been identified. As an initial step in elucidating the structural differences among inhibitor molecules, we sought to isolate the inhibitor in pig serum. The purified inhibitor decreased the hemagglutinating activity of influenza A virus, A/Los Angeles/2/87 (H3N2), and represented the majority of the virus-neutralizing activity in pig serum. The inhibitor corresponded in size to alpha 2-macroglobulin and cross-reacted antigenically with human alpha 2-macroglobulin. Characterization of the inhibitor's oligosaccharide moiety using linkage-specific lectins revealed the presence of N-acetylneuraminic acid-alpha 2,6-galactose but not N-acetylneuraminic acid-alpha 2,3-galactose. These data indicate that alpha 2-macroglobulin is the major neutralizing inhibitor of influenza A virus in pig serum.

Animals↗

Endotoxin abolishes the induction of alpha 2-macroglobulin synthesis in cultured human monocytes indicating inhibition of the terminal monocyte maturation into macrophages.

Monocytes were prepared from human blood and allowed to differentiate into macrophages in vitro. Synthesis and secretion of the potent proteinase inhibitor alpha 2-macroglobulin is strongly induced during maturation of monocytes into macrophages establishing alpha 2-macroglobulin as a differentiation marker protein. When the monocytes were incubated with endotoxin (S. typhi) in a concentration as low as 100 ng/ml, induction of alpha 2-macroglobulin synthesis was completely abolished indicating impaired monocyte/macrophage differentiation by lipopolysaccharides (LPS). In contrast to alpha 2-macroglobulin, synthesis of alpha 1-proteinase inhibitor in mononuclear phagocytes was stimulated by endotoxin.

Cell Differentiation↗

Binding of monofluorophosphate to alpha2-macroglobulin and C3.

After administering an oral dose of monofluorophosphate (MFP) to human beings or rats, a fraction of the drug appears in plasma that is bound to proteins, establishing a previously undetected compartment of nondiffusible fluoride. This article documents experiments performed in vitro, describing the binding of MFP to two plasma globulins: alpha2-macroglobulin and C3 (a beta-globulin). MFP binds irreversibly to these proteins through a stable bond. MFP binds to purified alpha2-macroglobulin or to C3 with a molar ratio MFP: protein close to unity. MFP binding reduces significantly the biological activity of these proteins, which share in common a macrocyclic 4-residue ring thiolactone (Cys-Gly-Glu-Glu). The binding site of MFP is as yet unknown. Protein-bound MFP appeared in the plasma of volunteers during the 5-7 hours following intake. Peak concentration of protein-bound MFP and maximal reduction of alpha2-macroglobulin activity was observed 2 hours after intake. Clearance of protein-bound MFP coincided with the return of alpha2-macroglobulin to basal levels.

Animals↗