Sequence identity between the alpha 2-macroglobulin receptor and low density lipoprotein receptor-related protein suggests that this molecule is a multifunctional receptor.

Ten peptides, derived from human alpha 2-macroglobulin (alpha 2M) receptor by chemical or proteolytic digestion, were sequenced. Comparative analysis revealed that all of the resulting sequences were present within the cDNA-deduced structure of low density lipoprotein receptor-related protein (LRP) (Herz, J., Hamann, U., Rogne, S., Myklebost, O., Gausepohl, H., and Stanley, K. K. (1988) EMBO J. 7, 4119-4127). The findings provide evidence that the alpha 2M receptor and LRP are the same molecule. Further evidence comes from immunoprecipitation experiments using a monoclonal antibody specific for the alpha 2M receptor that show this molecule, like LRP, to contain two polypeptides of approximately 420 and 85 kDa that are noncovalently associated. An additional component of this receptor system is a 39-kDa polypeptide that co-purifies with the alpha 2M receptor during affinity chromatography. Solid phase binding studies reveal that the 39-kDa polypeptide binds with high affinity (Kd = 18 nM) to the 420-kDa component of the alpha 2M receptor. The apparent identity of LRP and the alpha 2M receptor suggests that this molecule is a multifunctional receptor with the capacity to bind diverse biological ligands and highlights a possible relationship between two previously unrelated biological processes, lipid metabolism and proteinase regulation.     

Reaction of alpha 2-macroglobulin with plasmin increases binding of transforming growth factors-beta 1 and beta 2.

The binding of 125I-transforming growth factors-beta 1 and beta 2 (TGF-beta 1 and TGF-beta 2) to alpha 2-macroglobulin (alpha 2M) was studied before and after reaction with plasmin, thrombin, trypsin, or methylamine. Complex formation between TGF-beta and native or reacted forms of alpha 2M was demonstrated by non-denaturing polyacrylamide gel electrophoresis and autoradiography. Reaction of native alpha 2M with plasmin or methylamine markedly increased the binding of 125I-TGF-beta 1 and 125I-TGF-beta 2 to alpha 2M. The alpha 2M-plasmin/TGF-beta complexes were minimally dissociated by heparin. Reaction of alpha 2M with thrombin or trypsin reduced the binding of 125I-TGF-beta 1 and 125I-TGF-beta 2; the resulting complexes were readily dissociated by heparin. Complexes between TGF-beta 2 and native or reacted forms of alpha 2M were less dissociable by heparin than the equivalent complexes with TGF-beta 1. These studies demonstrate that the TGF-beta-binding activity of alpha 2M is significantly affected by plasmin, thrombin, trypsin and methylamine. Observations that alpha 2M-plasmin preferentially binds TGFs-beta suggest a mechanism by which alpha 2M may regulate availability of TGFs-beta to target cells in vivo.     

Comparative studies of pregnancy associated plasma protein-A and alpha 2-macroglobulin using metal chelate chromatography

Pregnancy Associated Plasma Protein-A (PAPP-A), shares many physicochemical and functional similarities with alpha-2-macroglobulin (alpha 2M). Comparative studies between these two proteins and various metal chelate Sepharoses have shown that both PAPP-A and alpha 2M bind to the copper and zinc, but not to the calcium, manganese or magnesium chelate gels. In addition to PAPP-A and alpha 2M, fibronectin and Pregnancy Zone Protein (PZP) were also found to bind to the copper and zinc chelate gels. Analysis of proteins bound to the zinc chelate Sepharose showed alpha 2M to have the highest affinity for the matrix, followed by PAPP-A and fibronectin, which coeluted, and then PZP. Following the zinc chelate chromatography minor qualitative changes were detected only in PAPP-A, but the eluted proteins retained in-vitro functional activity. By atomic absorption, PAPP-A, alpha 2M and fibronectin were found to contain zinc.     

The conformation-dependent interaction of alpha 2-macroglobulin with vascular endothelial growth factor. A novel mechanism of alpha 2-macroglobulin/growth factor binding

alpha(2)-Macroglobulin (alpha(2)M) is a highly conserved proteinase inhibitor present in human plasma at high concentration (2-4 mg/ml). alpha(2)M exists in two conformations, a native form and an activated, receptor-recognized form. While alpha(2)M binds to numerous cytokines and growth factors, in most cases, the nature of the alpha(2)M interaction with these factors is poorly understood. We examined in detail the interaction between alpha(2)M and vascular endothelial growth factor (VEGF) and found a novel and unexpected mechanism of interaction as demonstrated by the following observations: 1) the binding of VEGF to alpha(2)M occurs at a site distinct from the recently characterized growth factor binding site; 2) VEGF binds different forms of alpha(2)M with distinct spatial arrangement, namely to the interior of methylamine or ammonia-treated alpha(2)M and to the exterior of native and proteinase-converted alpha(2)M; and 3) VEGF (molecular mass approximately 40 kDa) can access the interior of receptor-recognized alpha(2)M in the absence of a proteinase trapped within the molecule. VEGF bound to receptor-recognized forms of alpha(2)M is internalized and degraded by macrophages via the alpha(2)M receptor, the low density lipoprotein receptor-related protein. Oxidation of both native and receptor-recognized alpha(2)M results in significant inhibition of VEGF binding. We also examined the biological significance of this interaction by studying the effect of alpha(2)M on VEGF-induced cell proliferation and VEGF-induced up-regulation of intracellular Ca(2+) levels. We demonstrate that under physiological conditions, alpha(2)M does not impact the ability of VEGF to induce cell proliferation or up-regulate Ca(2+).     

A chemically modified preparation of alpha2-macroglobulin binds beta-amyloid peptide with increased affinity and inhibits Abeta cytotoxicity

Macromolecules that bind beta-amyloid peptide (Abeta) and neutralize Abeta cytotoxicity offer a promising new approach for treating Alzheimer's disease. When the plasma protein, alpha2-macroglobulin (alpha2M), is treated with methylamine (alpha2M-MA), it undergoes conformational change and acquires Abeta-binding activity. In this study, we demonstrate that a chemically stabilized preparation of human alpha2M conformational intermediates (alpha2M-cis-Pt/MA) binds Abeta with greatly increased affinity, compared with alpha2M-MA. alpha2M-cis-Pt/MA was generated by reacting alpha2M with the protein cross-linking reagent, cis-Pt, followed by methylamine. Increased Abeta-binding to alpha2M-cis-Pt/MA was demonstrated by co-migration of radio-iodinated proteins in non-denaturing PAGE, chemical cross-linking, and co-immunoprecipitation. The apparent K(D) for Abeta-binding to alpha2M-cis-Pt/MA was decreased 10-fold, compared with alpha2M-MA, to 29 nm. Native alpha2M demonstrated negligible Abeta-binding, as anticipated. alpha2M-cis-Pt/MA markedly counteracted Abeta-induced C6 cell apoptosis. Essentially complete inhibition of apoptosis was observed even when the Abeta was present at fourfold molar excess to alpha2M-cis-Pt/MA. Under equivalent conditions, alpha2M-MA inhibited apoptosis by 25 +/- 6%. When Abeta and alpha2M-cis-Pt/MA were added to human plasma in vitro, significant binding was detected. No binding was observed when an equivalent concentration of native alpha2M or alpha2M-MA was added to plasma. We propose that alpha2M-cis-Pt/MA is a novel alternative to Abeta-specific antibodies, for studying the efficacy of Abeta-binding agents in vitro and in vivo.     

Isolation of a novel integrin receptor mediating Arg-Gly-Asp-directed cell adhesion to fibronectin and type I collagen from human neuroblastoma cells. Association of a novel beta 1-related subunit with alpha v

We report the isolation from two human neuroblastoma cell lines of an Arg-Gly-Asp-dependent integrin complex capable of binding to vitronectin, fibronectin, and type I collagen. The two neuroblastoma cell lines, SK-N-SH and IMR-32, exhibit specific attachment to fibronectin and type I collagen. SK-N-SH cells exhibit a much stronger attachment to vitronectin than the IMR-32 cells, which attach poorly to this substrate. Affinity chromatography of octylglucoside extracts of 125I surface-labeled cells on GRGDSPK-Sepharose columns resulted in the specific binding and elution with GRGDSP of three radiolabeled polypeptides with relative molecular masses of 135, 115, and 90 kD when analyzed by SDS-PAGE under nonreducing conditions. In the SK-N-SH cells the 135- and 90-kD polypeptides were more abundant whereas in the IMR-32 cells the 135- and 115-kD polypeptides were more highly expressed. Liposomes prepared from fractions containing all three polypeptides bound to vitronectin, fibronectin, and type I collagen, whereas liposomes prepared from the 135- and 115-kD polypeptides bound only to fibronectin and type I collagen. Polyclonal antibodies against the alpha/beta complexes of both the vitronectin receptor and the fibronectin receptor immunoprecipitated all three polypeptides. A monoclonal antibody against beta 1 immunoprecipitated only the 135- and the 115-kD polypeptides, whereas a monoclonal antibody against beta 3 subunit immunoprecipitated the 135- and 90-kD polypeptides. Although, the 115-kD polypeptide could be recognized by an anti-beta 1 antibody, a comparison of peptide maps generated by V8 protease digestion of the 115-kD polypeptide and beta 1 subunit immunoprecipitated from GRGDSPK-Sepharose flow-through material indicated that these two polypeptides are distinct. Depletion of the 90-kD polypeptide with an anti-beta 3 monoclonal antibody did not effect the ability of the 115- and 135-kD polypeptides to bind to GRGDSPK-Sepharose. These data indicate that the SK-N-SH and IMR-32 neuroblastoma cells express a novel "beta 1-like" integrin subunit that can associate with alpha v and can bind to RGD. We propose to name this beta 1-like subunit beta n. The data reported here thus demonstrate that in these two cell lines alpha v associates with two beta subunits, beta n and beta 3, forming two heterodimers. The alpha v beta n complex mediates binding to fibronectin and type I collagen, whereas the alpha v beta 3 complex mediates binding to vitronectin.     

Modulation of mouse peritoneal macrophage Ia and human peritoneal macrophage HLA-DR expression by alpha 2-macroglobulin "fast" forms

alpha 2-Macroglobulin (alpha 2M) is converted from its native form into electrophoretically "fast" forms by reaction with proteinases or with methylamine. The "fast" forms both bind to specific receptors on macrophages (MP). We have previously shown that alpha 2M "fast" forms modulate effector functions of murine peritoneal MP. In the present study, alpha 2M "fast" forms antagonized the increase in MP HLA-DR and Ia expression induced in vitro by interferon-gamma (IFN-gamma). This effect was observed with human peritoneal MP, as well as MP from peptone-injected and bacillus Calmette-Guérin-infected mice of three strains. alpha 2M-trypsin, which had been reacted with aprotinin and alpha 2M-methylamine, both of which lack proteolytic activity, also antagonized interferon-induced Ia expression. alpha 2M "fast" forms also reduced the ability of MP to serve as accessory cells for lectin-induced lymphocyte proliferation. alpha 2M "fast" form is an immune modulator of human and murine MP function, probably through a specific receptor-mediated mechanism.     

The low density lipoprotein receptor-related protein/alpha2-macroglobulin receptor is a receptor for connective tissue growth factor

Connective tissue growth factor (CTGF) expression is regulated by transforming growth factor-beta (TGF-beta) and strong up-regulation occurs during wound healing; in situ hybridization data indicate that there are high levels of CTGF expression in fibrotic lesions. Recently the binding parameters of CTGF to both high and lower affinity cell surface binding components have been characterized. Affinity cross-linking and SDS-polyacrylamide gel electrophoresis analysis demonstrated the binding of CTGF to a cell surface protein with a mass of approximately 620 kDa. We report here the purification of this protein by affinity chromatography on CTGF coupled to Sepharose and sequence information obtained by mass spectroscopy. The binding protein was identified as the multiligand receptor, low density lipoprotein receptor-related protein/alpha2-macroglobulin receptor (LRP). The identification of LRP as a receptor for CTGF was validated by several studies: 1) binding competition with many ligands that bind to LRP, including receptor-associated protein; 2) immunoprecipitation of CTGF-receptor complex with LRP antibodies; and 3) cells that are genetically deficient for LRP were unable to bind CTGF. Last, CTGF is rapidly internalized and degraded and this process is LRP-dependent. In summary, our data indicate that LRP is a receptor for CTGF, and may play an important role in mediating CTGF biology.     

Primary structure of alpha 2-macroglobulin receptor-associated protein. Human homologue of a Heymann nephritis antigen.

The alpha 2-macroglobulin (alpha 2M) receptor complex as purified by affinity chromatography contains three polypeptides: a 515-kDa heavy chain, an 85-kDa light chain, and a 39-kDa associated protein. Previous studies have established that the 515/85-kDa components are derived from a 600-kDa precursor whose complete sequence has been determined by cDNA cloning (Herz, J., Hamann, U., Rogne, S., Myklebost, O., Gassepohl, H., and Stanley, K. (1988) EMBO J. 7,4119-4127). We have now determined the primary structure of the human 39-kDa polypeptide, termed alpha 2M receptor-associated protein, by cDNA cloning. The deduced amino acid sequence contains a putative signal sequence that precedes the 323-residue mature protein. Comparative sequence analysis revealed that alpha 2M receptor-associated protein has 73% identity with a rat protein reported to be a pathogenic domain of Heymann nephritis antigen gp 330 and 77% identity to a mouse heparin-binding protein termed HBP-44. The high overall identity suggests that these molecules are interspecies homologues and indicates that the pathogenic domain, previously thought to be a portion of gp 330, is in fact a distinct protein. Further, the 120-residue carboxyl-terminal region of alpha 2M receptor-associated protein has 26% identity with a region of apolipoprotein E containing the low density lipoprotein receptor binding domain. Pulse-chase experiments revealed that the newly formed alpha 2M receptor-associated protein remains cell-associated, while surface labeling experiments followed by immunoprecipitation suggest that this protein is present on the cell surface forming a complex with the alpha 2M receptor heavy and light chains.     

Purification and characterization of a "half-molecule" alpha 2-macroglobulin from the southern grass frog: absence of binding to the mammalian alpha 2-macroglobulin receptor

An alpha-macroglobulin (alpha 2M), which is a dimer consisting of two non-disulfide-bonded subunits, was identified and purified from frog plasma by Ni2+ chelate affinity chromatography. This frog "half-molecule" alpha-macroglobulin migrated as an alpha 2-globulin in cellulose-acetate electrophoresis rather than as the previously described frog alpha 1M, which exists as a tetramer formed by the noncovalent association of disulfide-bonded pairs. A molecular weight of approximately 380 000 was obtained by gel-filtration high-pressure liquid chromatography, and in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) the protein migrated as a single band of Mr approximately 180 000 before and after reduction. No evidence was obtained for association of this protein to a higher molecular weight species. After the preparation was heated, additional bands were obtained in SDS-PAGE with Mr approximately 60 000 and 12 000. The additional bands were not obtained after heating methylamine-treated preparations. The circular dichroic spectrum of frog alpha 2M exhibits negative ellipticity over the region 205-250 nm with a minimum at 216 nm. After reaction with proteinase, a decrease in the absolute mean residue rotation was obtained. Amino acid analysis demonstrated that frog alpha 2M and alpha 1M are similar in composition to avian and mammalian alpha-macroglobulins; however, there are sufficient differences in the composition of these two amphibian alpha-macroglobulins to support the conclusion that they are distinct proteins. Frog alpha 2M bound approximately 0.5 mol of trypsin/mol of inhibitor. This binding was abolished by pretreatment with methylamine.(ABSTRACT TRUNCATED AT 250 WORDS)     

Differences in hydrophobic properties for human alpha 2-macroglobulin and pregnancy zone protein as studied by affinity phase partitioning

Human alpha 2-macroglobulin and pregnancy zone protein are related with regard to primary structure, physicochemical properties, and quarternary structure. Both proteins undergo conformational changes when they form complexes with proteinases or react with primary amines. The surface properties of the native, chymotrypsin-treated and methylamine-treated forms of alpha 2-macroglobulin and pregnancy zone protein were studied by partitioning in aqueous two-phase systems composed of 7.5% dextran T70 and 5% poly(ethylene glycol) 8000. All proteins and their derivatives had a high potential for hydrophobic interaction as analyzed in terms of affinity for poly(ethylene glycol) esters of fatty acids included in the phase systems. Treatment of alpha 2-macroglobulin with methylamine or chymotrypsin increased the surface hydrophobicity significantly compared to that of the native protein. No difference in hydrophobic interaction was found for native and methylamine-treated pregnancy zone protein, but the chymotrypsin-treated protein showed a marked increase in binding to the hydrophobic ligand. The changes in surface hydrophobicity parallel changes in receptor binding properties of the derivatized forms of alpha 2-macroglobulin and could be a signal for binding to cell-surface receptors, followed by internalization.     

Isolation and characterization of horse alpha 2-macroglobulin protease inhibitor

Several publications have described in the past properties of partly purified horse alpha 2-macroglobulin (alpha 2M) which are strikingly different from the human alpha 2M. Horse alpha 2M was therefore isolated to purity by classical procedures, i.e. affinity chromatography, ion exchange chromatography and gel filtration, and its properties are compared with those of its human counterpart. The molecular weight of the native protein and its subunits, the isoelectrofocusing pattern and the change in electrophoretic mobility caused by interaction with protease were similar to those of human alpha 2M. Horse alpha 2M had a broad enzyme specificity and inhibited enzymatic action on macromolecules but not on small molecular weight synthetic substrates. In addition the horse and human alpha 2M were found to be immunochemically related when examined by specific antisera to human as well as to horse alpha 2-macroglobulin.     

Characterization of thrombin binding to alpha 2-macroglobulin

The formation and structural characteristics of the human alpha 2-macroglobulin (alpha 2M)-thrombin complex were studied by intrinsic protein fluorescence, sulfhydryl group titration, electrophoresis in denaturing and nondenaturing polyacrylamide gel systems, and in macromolecular inhibitor assays. The interaction between alpha 2M and thrombin was also assessed by comparison of sodium dodecyl sulfate-gel electrophoretic patterns of peptides produced by Staphylococcus aureus V-8 proteinase digests of denatured alpha 2M-125I-thrombin and alpha 2M-125I-trypsin complexes. In experiments measuring fluorescence changes and sulfhydryl group exposure caused by methylamine, we found that thrombin produced its maximum effects at a mole ratio of approximately 1.3:1 (thrombin:alpha 2M). Measurements of the ability of alpha 2M to bind trypsin after prior reaction with thrombin indicated that thrombin binds rapidly at one site on alpha 2M, but occupies the second site with some difficulty. Intrinsic fluorescence studies of trypsin binding to alpha 2M at pH 5.0, 6.5, and 8.0 not only revealed striking differences in trypsin's behavior over this pH range, but also some similarities between the behavior of thrombin and trypsin not heretofore recognized. Structural studies, using sodium dodecyl sulfate-polyacrylamide gel electrophoresis to measure alpha 2M-125I-thrombin covalent complex formation, indicated that covalency reached a maximum at a mole ratio of approximately 1.5:1. At this ratio, only 1 mol of thrombin is bound covalently per mol of alpha 2M. These gel studies and those of proteolytic digests of denatured alpha 2M-125I-trypsin and alpha 2M-125I-thrombin complexes suggest that proteinases form covalent bonds with uncleaved alpha 2M subunits. The sum of our results is consistent with a mechanism of proteinase binding to alpha 2M in which the affinity of the proteinase for alpha 2M during an initial reversible interaction determines its binding ratio to the inhibitor.     

PDGF-stimulated fibroblast proliferation is enhanced synergistically by receptor-recognized alpha 2-macroglobulin.

alpha-Macroglobulins derived from plasma or secreted by macrophages are platelet-derived growth factor (PDGF) binding proteins that compete with cell-surface receptors on fibroblasts for PDGF binding. alpha 2-Macroglobulin (alpha 2M) derived from bovine plasma was tested for its ability to modulate the PDGF-induced proliferation of primary passage rat lung fibroblasts (RLFs) and a human skin fibroblast cell line (CRL 1508). Fibroblasts were grown in 10% fetal bovine serum (FBS) for 24 hr, then washed with serum-free medium before adding serum-free defined medium (SFDM) containing insulin and transferrin. To this medium were added varying concentrations of human plasma-derived AB-PDGF and alpha 2 M, alone or in combination. Receptor-recognized alpha 2M was prepared by treatment with methylamine. Both native alpha 2M and the alpha 2M-methylamine (alpha 2M-MA) were tested for growth promoting activity in the absence or presence of PDGF. After 3 days, a concentration-dependent growth curve of fibroblast proliferation was demonstrated for PDGF alone, with near maximal stimulation reached at 15-20 ng/ml PDGF. alpha 2M and alpha 2M-MA alone had no effect on cell proliferation. However, alpha 2M-MA concentrations above 32 micrograms/ml synergistically enhanced PDGF-stimulated proliferation greater than 100% in the presence of 15 ng/ml PDGF. Native alpha 2M enhanced PDGF-stimulated growth 80-100% above PDGF controls only at low concentrations (32-64 micrograms/ml alpha 2M). High concentrations of native alpha 2M (128-256 micrograms/ml) either had no effect on growth or were inhibitory to PDGF-stimulated growth, depending on the cell type tested. Rat lung fibroblasts were shown to secrete a factor(s) that inhibited the trypsin-binding capacity of native alpha 2M. We further demonstrated that early passage RLFs possess specific cell-surface receptors for [125I]-PDGF and [125I]-alpha 2M-MA, and preincubation of RLFs with alpha 2M-MA increased the specific binding of [125I]-PDGF to the cell surface of these fibroblasts. Considered together, these data support the view that receptor-recognized alpha 2M synergistically enhances the proliferative capacity of PDGF. We postulate that receptor-recognized alpha Ms enhance PDGF-stimulated growth by increasing the local concentration of PDGF at the cell surface, where the PDGF could be released in close proximity to its own receptors.     

The properties of rabbit alpha1-macroglobulin upon activation are distinct from those of rabbit and human alpha2-macroglobulins

We have characterized native and activated forms of rabbit alpha1M and compared them to rabbit and human alpha2M. Similar to human alpha2M, rabbit alpha1M is a tetramer associated via disulfide bonds and non-covalent interactions that exhibits autolysis into two fragments when heated. Like human alpha2M, rabbit alpha1M is cleaved by trypsin at one site; however, rabbit alpha1M shares characteristics with rabbit alpha2M that are different from the properties of human alpha2M. Amine or trypsin treatment of rabbit alpha-macroglobulins does not result in a significant conformational change or cleavage of four thiolester bonds. Full thiolester cleavage is only observed for rabbit alpha1M after exposure to both trypsin and a small amine. Additionally, amine-treated rabbit alpha-macroglobulins retain trypsin inhibitory potential and do not fully shield bound proteinases. Methylamine and trypsin treatment of rabbit alpha1M results in two dissimilar conformations that display differing exposure of the receptor-recognition site. While ammonia- and methylamine-modified rabbit alpha1M bind to macrophages with similar affinity to that of human alpha2M, trypsin-treated rabbit alpha1M exhibits dramatically lower affinity. This suggests that rabbit alpha1M may not play the same proteinase-inhibiting physiological role as human alpha2M.     

Expression of human alpha 2-macroglobulin cDNA in baby hamster kidney fibroblasts: secretion of high levels of active alpha 2-macroglobulin.

Human alpha 2-macroglobulin (alpha 2M) is a unique 720-kDa proteinase inhibitor with a broad specificity. Unlike most other proteinase inhibitors, it does not inhibit proteolytic activity by blocking the active site of the proteinase. During complex formation with a proteinase, alpha 2M entraps the proteinase molecule in a reaction that involves large conformational changes in alpha 2M. We describe the molecular cloning of alpha 2M cDNA from the human hepatoblastoma cell line HepG2. The cDNA was subcloned under control of the adenovirus major late promoter in a mammalian expression vector and introduced into the baby hamster kidney (BHK) cell line. Transformed clones were isolated and tested for production of human alpha 2M with a specific enzyme-linked immunosorbent assay. Human recombinant alpha 2M (r alpha 2M), secreted and purified from isolated transfected BHK cell lines, was structurally and functionally compared to alpha 2M purified from human serum. The results show that r alpha 2M was secreted from the BHK cells as an active proteinase-binding tetramer with functional thiol esters. Cleavage reactions of r alpha 2M with methylamine and trypsin showed that the recombinant product, which was correctly processed at the N-terminus, exhibited molecular characteristics similar to those of the human serum derived reference. Moreover, r alpha 2M-trypsin complex bound to purified human placental alpha 2M receptor with an affinity indistinguishable from that of a complex formed from serum-derived alpha 2M and trypsin.     

Preparation and initial characterization of an intermediate, half-cleaved form of human alpha 2-macroglobulin

A form of human alpha 2-macroglobulin (alpha 2M) has been prepared that has properties intermediate to those of native alpha 2-macroglobulin and 2:1 protease-alpha 2 M ternary complex by using Sepharose-linked chymotrypsin. The intermediate form has mobility on native polyacrylamide gels between the fast and slow forms of alpha 2M and migrates as a diffuse band. Two bait regions and two thiol esters per alpha 2M tetramer are cleaved, although no chymotrypsin is detectable in the modified alpha 2-macroglobulin species. The remaining bait regions and thiol esters can be cleaved by further reaction with other proteases. Intermediate-form alpha 2M can trap 1.18 mol of chymotrypsin, 0.85 mol of trypsin, and 0.65 mol of thrombin. Although both thrombin and methylamine react with intermediate-form alpha 2M at rates not distinguishable within experimental error from those of their reactions with native alpha 2M, chymotrypsin-Sepharose reacts much more slowly with the intermediate form than with native alpha 2 M, indicating a nonequivalence of the two reactive sites on alpha 2M. This nonequivalence may be present initially or be induced by reaction at the first site. Comparison of ESR results obtained from spin-labeling methylamine-treated or protease-reacted alpha 2M with those from spin-labeling of the free SH groups in intermediate-form alpha 2M shows that trapped protease influences the mobility of the attached nitroxide either through direct contact or by producing a different conformation from that present in methylamine-treated or intermediate-form alpha 2M.     

Electron microscopy of alpha 2-macroglobulin with a thiol ester bound ligand.

In order to covalently bind the hydrolyzed thiol ester groups of the human alpha 2-macroglobulin (alpha 2M) transformed by methylamine, the phospholipase A2 (PLA2), a small enzyme (M(r) = 13,000) from Naja nigricollis snake venom was activated by succinimidyl 4-(maleimidomethyl)cyclohexane-1-carboxylate (SMCC). Average images determined from electron micrographs of the methylamine-transformed alpha 2M, with and without activated PLA2, were determined by image processing and compared. A localization of the PLA2 was achieved by subtracting the average image of alpha 2M transformed by methylamine from that containing PLA2. The results are consistent with previous work showing the central localization of chymotrypsin trapped in alpha 2M. They also suggest that the four thiol esters are located near the center of the alpha 2M molecule.     

Immunochemical detection and characterization of pregnancy-associated endometrial alpha 1- and alpha 2-globulins secreted by human endometrium and decidua

Antisera raised against the soluble antigens of the endometrium of early pregnancy detected two antigenic proteins of alpha 1 and alpha 2 mobility in extracts of this tissue and were termed antigens A and B. Neither antigen was detected in pregnancy sera or extracts of proliferative endometrium, but antigen B was detected in extracts of secretory endometrium and both were present in amniotic fluid and medium from in-vitro incubations of pregnancy endometrium. Fractionation of radiolabelled medium on ion-exchange chromatography demonstrated that antigens A and B co-eluted with the proteins from which EP14 and EP15 were derived and which were the major secretory polypeptides of pregnancy endometrium in vitro. Further biochemical purification revealed that EP14 (Mr 32 000) was derived from a protein of native molecular weight 36 000 which existed in two forms, whereas EP15 (Mr 28 000) was derived from a dimeric glycoprotein of native molecular weight 56 000. Immunochemical studies demonstrated that antigens A and B are identical to these two secretory proteins and have been termed pregnancy-associated endometrial alpha 1- and alpha 2-globulins (alpha 1- and alpha 2-PEG).     

The 39-kDa receptor-associated protein interacts with two members of the low density lipoprotein receptor family, alpha 2-macroglobulin receptor and glycoprotein 330.

The 39-kDa receptor-associated protein (RAP) binds to the alpha 2-macroglobulin receptor/low density lipoprotein receptor-related protein (alpha 2MR/LRP) and inhibits binding of ligands to this receptor. The in vivo function of RAP may be to regulate ligand binding and/or assist in the correct biosynthetic processing or trafficking of the alpha 2MR/LRP. Here we show that RAP binds another putative receptor, the kidney glycoprotein 330 (gp330). Gp330 is a high molecular weight glycoprotein that is structurally similar to both the alpha 2MR/LRP and low density lipoprotein receptor. The ability of RAP to bind to gp330 was demonstrated by ligand blotting and solid phase binding assays, which showed that RAP binds to gp330 with high affinity (Kd = 8 nM). Exploiting the interaction of gp330 and RAP, we purified gp330 by affinity chromatography with a column of RAP coupled to Sepharose. Gp330 preparations obtained by this procedure were notably more homogeneous than those obtained by conventional methods. Immunocytochemical staining of human kidney sections localized RAP to the brush-border epithelium of proximal tubules. The fact that gp330 is also primarily expressed by proximal tubule epithelial cells strengthens the likelihood that the interaction between gp330 and RAP occurs in vivo. The functional significance of RAP binding to gp330 may be to antagonize ligand binding as has been demonstrated for the alpha 2MR/LRP or to assist in the biosynthetic processing and/or trafficking of this receptor.