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PubMed1995

Identification of the serum factor required for liposome-primed activation of mouse peritoneal macrophages. Modified alpha 2-macroglobulin enhances Fc gamma receptor-mediated phagocytosis of opsonized sheep red blood cells.

Culture supernatant, prepared by incubating fetal calf serum and liposome-treated B cells, augments mouse peritoneal macrophage Fc gamma receptor-mediated phagocytosis of IgG-opsonized sheep red blood cells. The activation process was hypothesized to be as follows. B-cell membranous glycosidases stimulated by liposomes, convert serum factor to a macrophage-stimulating active serum factor. As the active serum factor loses its activation potential by the addition of mannose or by digestion with alpha-mannosidase, mannose residues at the terminal present in the active serum factor are hypothesized to contribute to macrophage activation. The active serum factor was purified on a concanavalin A-Sepharose 4B column, and identified as a modified form of alpha 2-macroglobulin by immunochemical analysises. On non-denaturing polyacrylamide gel electrophoresis, modified alpha 2-macroglobulin showed a slow form, while alpha 2-macroglobulin-trypsin and alpha 2-macroglobulin-methylamine complexes, which bind specifically to alpha 2-macroglobulin receptors, showed a fast form and did not activate macropahges. These findings demonstrate that alpha 2-macroglobulin is the essential serum factor in liposome-primed macrophage activation, and that modified alpha 2-macroglobulin with mannose residues at the terminal sugar chain binds to macrophage mannose receptors, but not alpha 2-macroglobulin receptors, and increases Fc gamma receptor-mediated phagocytosis of IgG-opsonized sheep red blood cells.

Animals↗

PubMed1995

Expression of mouse alpha-macroglobulins, lipoprotein receptor-related protein, LDL receptor, apolipoprotein E, and lipoprotein lipase in pregnancy.

The expression of the proteinase inhibitors of the alpha-macroglobulin family and of their clearance receptor was analyzed in the mouse during pregnancy, embryonal development, and adolescence. In total we studied seven partners of a complicated network of interactions in proteolysis and lipid metabolism:alpha-2-macroglobulin, murinoglobulin, the alpha-2-macroglobulin receptor/lipoprotein receptor related protein, the murine equivalent of the receptor associated protein or the 44 kDa heparin binding protein, the low density lipoprotein receptor, apolipoprotein E, and lipoprotein lipase. The data demonstrate that: i) the regulation of expression of mouse tetrameric alpha-2-macroglobulin results in very constant levels, similar to alpha-2-macroglobulin in humans; ii) single chain murinoglobulin, not alpha-2-macroglobulin, is subject to regulation of expression during pregnancy, around birth, and in adolescence; iii) an important role seems implicated for the alpha-2-macroglobulin receptor in placental lipid metabolism, probably making it the most important lipoprotein receptor to supply the fetus; iv) the massive increase in apolipoprotein E synthesis in uterus and placenta accentuate the changed lipid metabolism during pregnancy to an apolipoprotein E-based uptake by the alpha-2-macroglobulin receptor/lipoprotein receptor related protein; v) the increased expression of lipoprotein lipase underlines its role in the generation of free fatty acids in uterus and placenta as another mechanism of supply, next to receptor mediated endocytosis of lipoproteins.

Age Factors↗

PubMed1996

Inhibition of transforming growth factor-beta-induced cataractous changes in lens explants by ocular media and alpha 2-macroglobulin.

PURPOSE: To investigate the ocular media for the presence of inhibitors of transforming growth factor-beta (TGF beta) using a lens epithelial explant system in which TGF beta induces cataractous changes. The effect of alpha 2-macroglobulin, an inhibitor of TGF beta in other systems, also was assessed. METHODS: Explants prepared from 21-day-old rats were cultured with TGF beta 2 with and without 50% bovine aqueous or vitreous or alpha 2-macroglobulin. alpha 2-macroglobulin was added to an aqueous concentrate, shown to contain endogenous TGF beta activity by blocking with anti-TGF beta. Explants were monitored by phase-contrast microscopy for 5 days and assessed in terms of capsule wrinkling, spindle-cell formation, blebbing, and cell loss. alpha 2-macroglobulin in the ocular media was assessed by enzyme-linked immunosorbent assay and Western blot analysis. RESULTS: At 50% strength, neither aqueous nor vitreous demonstrated TGF beta-like activity; however, aqueous partially and vitreous completely prevented cataractous changes induced by 25 and 100 pg/ml TGF beta 2, respectively. alpha 2-macroglobulin (50 to 200 micrograms/ml) also protected against these changes, with complete inhibition of TGF beta 2 or aqueous-derived TGF beta activity at the highest concentration. A threefold higher concentration of alpha 2-macroglobulin was detected in vitreous than aqueous. CONCLUSIONS: Both aqueous and vitreous contain molecule(s) that inhibit TGF beta 2 activity. alpha 2-macroglobulin has been identified in the ocular media and shown to block cataractous changes induced by TGF beta. Maintaining appropriate levels of alpha 2-macroglobulin or similar molecules in the ocular media may protect lens cells from the damaging effects of TGF beta, and reduced levels may predispose to cataract.

Adult↗

PubMed1983

Synthesis of alpha 2-macroglobulin in rat hepatocytes and in a cell-free system.

The biosynthesis and secretion of alpha 2-macroglobulin was studied in rat hepatocyte primary cultures. After immunoprecipitation of alpha 2-macroglobulin from a cell homogenate and the hepatocyte medium, two forms of alpha 2-macroglobulin with app. Mr of 176000 and 182000, respectively, were identified. A precursor-product relationship for the two alpha 2-macroglobulin forms was demonstrated by a pulse-chase experiment. The cellular form of alpha 2-macroglobulin could be deglycosylated by endoglucosaminidase H, whereas the medium form of alpha 2-macroglobulin remained unaffected. On the other hand, only the medium form of alpha 2-macroglobulin was found to be susceptible to neuraminidase. In vitro translation of rat liver poly(A)+ RNA resulted in a translation product of an app. Mr of 162000.

Animals↗

PubMed1985

Fluoresceinated alpha 2-macroglobulin as a probe for studying macrophages.

The potential value of fluorescein-conjugated human alpha 2-macroglobulin as a probe for studying macrophages in murine peritoneal exudate and spleen cell suspensions has been investigated using a fluorescence-activated cell sorter. These studies revealed that the number of alpha 2-macroglobulin-positive cells in the mixtures examined correlated closely with their macrophage content as determined morphologically. Furthermore cells separated on the FACS on the basis of their strong alpha 2-macroglobulin binding exhibited macrophage morphology and expressed Fc receptors on their surface. Conversely the alpha 2-macroglobulin-negative population contained few macrophages or Fc rosette-forming cells. Extensive blocking and comparative binding studies with a number of purified human alpha 2-macroglobulin preparations and derivatives thereof, and a variety of purified proteins, confirmed that the binding of fluorescein-conjugated alpha 2-macroglobulin to peritoneal exudate cells was specific. Furthermore the binding occurs in a highly reproducible manner. These observations suggest that fluorescein-conjugated alpha 2-macroglobulin in conjugation with flow cytometry is a sensitive and reliable method for both identifying and isolating subpopulations of macrophages.

Animals↗

PubMed1982

Radioimmunoassay of rat acute-phase alpha 2-macroglobulin.

A double-antibody radioimmunoassay (RIA) to acute-phase alpha 2-macroglobulin was developed for the quantitation of this large macromolecule in physiological fluids. The primary receptor for the RIA was a monospecific antiserum to purified acute-phase alpha 2-macroglobulin which produced a high titre (7.5 . 10(6)) antibody with a strong affinity for rat acute-phase alpha 2-macroglobulin (Ka = 1.24 . 10(11)) as measured by Scatchard analysis. The validity of the assay was confirmed by specificity for rat alpha 2-macroglobulin measured in various physiological fluids as assessed by parallel dose-response curves; and accuracy, measured by the analytical recovery of alpha 2-macroglobulin by the RIA in serum (104 +/- 7%) and buffer (103 +/- 7%), and the correlation (R = 0.999) of measurements of acute-phase alpha 2-macroglobulin-containing samples measured in serum and buffer. Reference acute-phase serum measured by this RIA and by rocket immunoelectrophoresis were 98.6% in agreement. Radioimmunoassay sensitivity was estimated at less than 1.0 ng alpha 2-macroglobulin/ml, measured over a range of 0-160 ng. Precision was assessed by intraassay (2.99 +/- 0.97%) and interassay (8.76 +/- 2.64%) variation. Evaluation confirmed that quantitation of rat acute-phase alpha 2-macroglobulin by this RIA met the criteria of sensitivity, validity and precision.

Animals↗

PubMed1996

Preparation and characterization of a C-terminal fragment of pregnancy zone protein corresponding to the receptor-binding peptide from human alpha 2-macroglobulin.

Digestion of the pregnancy zone protein with papain at pH 4.5 yields an 18 kDa C-terminal fragment. This fragment consists of the 145 C-terminal amino-acid residues cleaved at Asn-1288 Ile and is homologous to the C-terminal receptor binding fragment of human alpha 2-macroglobulin obtained by cleavage with papain. The fragment contains an intrachain disulfide bond between 1308Cys and 1423Cys corresponding to that between 1304Cys and 1419Cys in alpha 2-macroglobulin. An oligosaccharide chain, is present in the C-terminal fragment of pregnancy zone protein as in human alpha 2-macroglobulin. The PZP C-terminal fragment was demonstrated to bind to the LRP/alpha 2M-receptor. Both the pregnancy zone protein and alpha 2-macroglobulin fragments bind three mAb's (alpha 1:1, R35, and 7H11D6) generated against alpha 2-macroglobulin. The mAb 7H11D6 was generated against the alpha 2-macroglobulin-proteinase complex (Isaacs, I.J., Steiner, J.P., Roche, P.A., Pizzo, S.V. and Strickland, D.K. (1988) J. Biol. Chem. 263, 6709-6714) and the binding of this to the C-terminal fragments of both pregnancy zone protein and alpha 2-macroglobulin indicates that both proteins use the same receptor recognition site for binding to the LRP/alpha 2M-receptor.

Amino Acid Sequence↗

PubMed1992

Rat Sertoli cell clusterin, alpha 2-macroglobulin, and testins: biosynthesis and differential regulation by germ cells.

Clusterin, alpha 2-macroglobulin and testins are three novel Sertoli cell proteins whose physiological functions may be related to cell-cell interactions in the seminiferous epithelium of the testis. We have demonstrated the biosynthesis of clusterin, alpha 2-macroglobulin, and testins by Sertoli cells in vitro using pulse-chase labeling analysis. For clusterin, two precursors with an apparent molecular weight (M(r)) of 72,000 (PH) and 66,000 (PL) were detected in the Sertoli cell cytosol in addition to the alpha (M(r) 43,000) and beta (M(r) 35,000) subunits of the mature protein. However, the precursors were not secreted into the medium since only the alpha and beta subunits of clusterin were detected. For alpha 2-macroglobulin and testins, no precursor molecules were detected either in the Sertoli cell cytosol or culture medium. The polarized secretory pattern of these proteins and their regulation by follicle stimulating hormone (FSH) and testosterone (T) were examined using a bicameral culture chamber that mimics the in vivo physiological conditions. Clusterin was secreted almost exclusively into the apical chamber of the bicameral culture unit with an apical:basal ratio of 30:1. In contrast, alpha 2-macroglobulin and testins had an apical:basal ratio of 1:1 and 1.5:1, respectively. Thus, the polarized secretory pattern for clusterin is different from alpha 2-macroglobulin and testins. It was noted that FSH and T, the known Sertoli cell regulators, did not affect the secretion of either clusterin or alpha 2-macroglobulin. Due to the morphological intimacy between Sertoli cells and germ cells in the adluminal compartment of the testis, the effects of germ cell-conditioned medium were investigated. Addition of germ cell-conditioned medium (1-30 micrograms protein) to the apical chamber of the bicameral culture unit caused a dose-dependent inhibition of clusterin and testins apical secretion and a slight but statistically significant stimulation of their basal secretion. In contrast, the secretion of alpha 2-macroglobulin by Sertoli cells was stimulated both apically and basally. These observations suggest that germ cell-conditioned medium contains a biological factor(s) that differentially regulates the bidirectional secretion of Sertoli cell proteins. These studies therefore reveal the complicated regulatory processes involved in cell-cell interactions in the seminiferous epithelium.

Animals↗

PubMed1975

Uptake of proteinase-alpha-macroglobulin complexes by macrophages.

Complexes of labelled proteinases (subtilopeptidase A, trypsin) with serum alpha 1-macroglobulin or alpha 2-macroglobulin are rapidly taken up in vitro by rabbit alveolar macrophages and peritoneal macrophages but not by mixed rabbit peripheral blood leukocytes. Enzyme, not bound to alpha 1- or alpha 2-macroglobulin, does not become associated with alveolar macrophages. Chemically inactivated subtilopeptidase A does not bind to alpha 1- or alpha 2-macroglobulin; chemically inactivated subtilopeptidase A in mixtures with alpha 1 - or alpha 2-microglobulin, does not interact with alveolar macrophages. Blocking experiments confirmed that the interaction of proteinase with alveolar macrophages is complex specific; uptake of labelled complex was prevented by the simultaneous addition of macroglobulin complexes formed with non-labelled subtilopeptidase A, subtilopeptidase B, trypsin or chymotrypsin but not by macroglobulin alone. The findings demonstrate a complex-specific interaction between proteinase-alpha-macroglobulin complexes and macrophages.

Animals↗

PubMed1983

Alpha 2-macroglobulin 'fast' forms inhibit superoxide production by activated macrophages.

Mouse peritoneal macrophages activated by bacillus Calmette-Guerin (BCG) were incubated with human alpha 2-macroglobulin converted to its 'fast' form with either trypsin or methylamine before being stimulated with phorbol myrystate acetate. Both alpha 2-macroglobulin-trypsin and alpha 2-macroglobulin-methylamine inhibited macrophage production of superoxide anion (O2-) while native alpha 2-macroglobulin had little effect except at high concentration. The alpha 2-macroglobulin 'fast' forms, which bind with a Kd of about 8 nM, inhibited 50% generation of O2- (ID50) at a concentration of 7 nM while alpha 2-macroglobulin inhibited O2- production with an ID50 of 141 nM. The 'fast' forms of alpha 2-macroglobulin may play a role in the feedback regulation of inflammatory reactions.

Animals↗

PubMed1986

Evidence for binding of human pregnancy zone protein-proteinase complex to alpha 2-macroglobulin receptors.

125I-labelled human pregnancy zone protein complexed with chymotrypsin was removed from the circulation with a half-time of 2.3 min after intravenous injection in rats. After 6 min about 67% of the label was present in the liver and about 3% was in the spleen, both in male and in female pregnant rats. The half-time of removal was more than 30 min for native pregnancy zone protein. Uptake into other organs, including placentae and feti, was negligible. 30 pM labelled pregnancy zone protein X chymotrypsin was specifically bound to rat hepatocytes and adipocytes and to human fibroblasts and monocyte-derived macrophages at 4 degrees C. Binding was almost completely abolished by a saturating concentration of unlabelled alpha 2-macroglobulin X trypsin. Binding of 15 pM labelled macroglobulin complex was completely abolished by a saturating concentration of pregnancy zone protein X chymotrypsin. In rat hepatocytes, binding of pregnancy zone protein complex was lower than that of alpha 2-macroglobulin complex at low ligand concentrations. Half-maximal receptor occupancy was obtained with about 300 pM pregnancy zone protein complex. Unlabelled alpha 2-macroglobulin or pregnancy zone protein complex failed to accelerate dissociation of the labelled pregnancy zone protein complex under conditions where dissociation of alpha 2-macroglobulin was markedly enhanced. It is concluded that pregnancy zone protein and alpha 2-macroglobulin complexes bind to the same receptors. The quantitative differences may be related to the fact that alpha 2-macroglobulin is a tetramer whereas the functional unit of pregnancy zone protein is probably a dimer.

Animals↗

PubMed1994

Ineffectiveness of the inhibition of the main haemorrhagic metalloproteinase from Bothrops jararaca venom by its only plasma inhibitor, alpha 2-macroglobulin.

Observations that a haemorrhagic metalloproteinase (jararhagin) from Bothrops jararaca venom had less effect on platelets suspended in plasma than in washed platelet suspensions, suggested that plasma contains naturally occurring inhibitor(s) of this enzyme. By using radiolabelled jararhagin and crossed immunoelectrophoresis, we have demonstrated the binding of this enzyme to alpha 2-macroglobulin in plasma. SDS-PAGE analysis of this binding revealed the presence of radioactivity in four bands with relative molecular masses of 640, 570, 520 and 410 kDa; in addition a small amount of 47 kDa free enzyme was demonstrable. Reduced samples showed an additional non-complexed 90 kDa fragment of alpha 2-macroglobulin generated by jararhagin. These results are compatible with a model in which, upon multiple cleavages of alpha 2-macroglobulin, the enzyme becomes covalently bound to the inhibitor, and the two halves of the inhibitor become crosslinked. However, jararhagin activity was not completely inhibited even after long incubation (60 min) with a large (10-fold) molar excess of alpha 2-macroglobulin either in plasma or a purified alpha 2-macroglobulin preparation. Kinetic studies showed that inhibition was comparatively slow, although jararhagin readily cleaved alpha 2-macroglobulin in the bait region. Therefore, the ineffectiveness of the inhibition could have resulted from a low tendency of this proteinase to form covalent complexes with the inhibitor. We conclude that the pronounced haemorrhagic activity of jararhagin can be attributed to prolonged access of this enzyme to high molecular weight substrates, even in the presence of a large molar excess of alpha 2-macroglobulin.

Animals↗

PubMed1998

Alpha2-macroglobulin does not function as a C3 homologue in the plasma hemolytic system of the American horseshoe crab, Limulus.

A major problem of comparative immunology is the characterization of the internal defense systems that lyse foreign cells, such as bacteria and other microbial pathogens that have gained entry into the body. The plasma cytolytic system of the American horseshoe crab, Limulus polyphemus, is sensitive to treatment with methylamine, which inactivates the abundant plasma defense protein alpha2-macroglobulin. This has been interpreted to mean that alpha2-macroglobulin plays an important role in hemolysis, analogous to the role of complement component C3 of the mammalian complement system (Enghild et al., 1990). Sensitivity to methylamine has been suggested to reflect an evolutionary homology with the plasma cytolytic system of mammals, in which the complement system is inactivated by the reaction of methylamine with complement components C3 and C4. C3, C4 and alpha2-macroglobulin contain an internal thiol ester bond linking cysteinyl and glutamic acid residues and methylamine inactivates all three proteins by reaction with the thiol-esterified glutamic acid. However, we have recently shown that the principal effector of hemolysis in Limulus is the plasma lectin, limulin (Armstrong et al., 1996). In this article we show that native, unreacted alpha2-macroglobulin is not involved directly in hemolysis but instead that methylamine-reacted alpha2-macroglobulin inhibits the hemolytic activity of limulin. Thus the thiol ester proteins alpha2-macroglobulin and C3 operate very differently in the hemolytic systems of Limulus and mammals and are not functionally homologous. Limulus alpha2-macroglobulin functions indirectly in hemolysis: its inactivation yields an inhibitory molecule for limulin-mediated hemolysis.

Animals↗

PubMed1984

Mechanism of alpha 2-macroglobulin-proteinase interactions. Studies with trypsin and plasmin.

The time course of the interactions of alpha 2-macroglobulin with trypsin and with plasmin was studied by measuring the generation of thiol groups, the concentration of alpha 2-macroglobulin subunits cleaved at the bait regions, and the change in intrinsic protein fluorescence of alpha 2-macroglobulin-enzyme reaction mixtures as functions of time. The interaction of alpha 2-macroglobulin with trypsin was found to be very fast but could be studied in the presence of benzamidine, a rather strong competitive inhibitor of trypsin. The results obtained indicate that alpha 2-macroglobulin-proteinase reactions, known to involve specific limited proteolysis of the bait regions, gross conformational changes, and cleavage of the internal beta-cysteinyl-gamma-glutamyl thiol esters of native alpha 2-macroglobulin, may proceed via at least two different reaction pathways determined by the nature of or the concentration of the reacting proteinase. After initial cleavage of one bait region at high proteinase activity the next step presumably is a fast cleavage of a second bait region before any substantial rearrangements leading to generation of thiol groups and the final incorporation of the proteinase occur. At low proteinase activity no further bait region cleavages occur and only the two thiol groups of half of the alpha 2-macroglobulin molecule are generated in the final 1:1 complex.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1985

Complexes of rat alpha 1-macroglobulin and subtilisin are endocytosed by parenchymal liver cells.

Rat alpha 1-macroglobulin was isolated from plasma. Gel electrophoresis of the denatured and reduced protein showed two bands, with Mr values of 163 000 and 37 000. The large subunit contained an autolytic site. This subunit was also split after reaction of the macroglobulin with trypsin. Electron microscopy showed that the macroglobulin changed towards a more compact conformation after reaction with this proteinase. Subtilisin, or alpha 1-macroglobulin, was labelled with a sucrose-containing radio-iodinated group that stays in lysosomes after endocytosis and breakdown of the tagged protein. After intravenous injection into rats, alpha 1-macroglobulin was cleared from plasma with first-order kinetics, showing a half-life of about 9 h, whereas complexes of alpha 1-macroglobulin and subtilisin were cleared with half-lives of only 3 min. Liver contained about 60% of the label at 30 min after injection of complexes. About 90% of the liver radioactivity was found in parenchymal cells isolated after perfusion of the liver with a collagenase solution. Subcellular fractionation indicated a lysosomal localization of the complexes. We conclude that endocytosis by parenchymal liver cells is the major cause of the rapid clearance of alpha 1-macroglobulin-proteinase complexes from plasma.

Animals↗

PubMed1977

Cultured human monocytes synthesize and secrete alpha2-macroglobulin.

alpha2-Macroglobulin levels in the supernates of cultures of different subpopulations of human peripheral blood mononuclear leukocytes were assayed by a radioimmunoassay. Unfractionated mononuclear leukocytes produced greater amounts of the macroglobulin (4.0 vs. 0.8 ng/10(6) cells) than did subpopulations enriched in T or B+T lymphocytes, by passage through nylon wool or cotton wool columns, respectively. Still higher concentrations of alpha2-macroglobulin (40 ng/10(6) cells) were measured in the supernates of glass-adherent mononuclear leukocyte cultures. These results suggest that cells of monocyte-macrophage lineage are mainly, if not exclusively, responsible for the appearance of alpha2- macroglobulin in the supernate of human peripheral blood leukocyte cultures. The de novo synthesis and release of alpha2-macroglobulin by cultured monocytes was demonstrated by immunoprecipitation of radioactivity from supernates of 32S-methionine-labeled glass-adherent cells. Antiserum against purified alpha2-macroglobulin was used in both Ouchterlony double diffusion and double antibody precipitation tests. SDS-polyacrylamide gel electrophoresis of immunoprecipitates showed that most of the radioactivity comigrated with authentic alpha2-macroglobulin subunit at about 160,000 daltons.

B-Lymphocytes↗

PubMed1990

Clearance of desialylated mouse alpha-macroglobulin and murinoglobulin in the mouse.

Mouse alpha-macroglobulin and murinoglobulin were labeled with 125I and utilized for plasma clearance studies performed with mice. Desialylated murinoglobulin was rapidly cleared from the circulation with a half-life of about 5 min. On the other hand, desialylated alpha-macroglobulin showed a biphasic curve: about half was cleared at a rate similar to that of the intact molecule while the remaining half had a shorter half-life of about 20 min which was prolonged by a simultaneous injection of a 200-fold excess of unlabeled asialoorosomucoid. Virtually no cross competition was observed between these asialoglobulins and formaldehyde-treated bovine serum albumin or trypsin-bound alpha-macroglobulin. These results suggest that the intravascular elimination of desialylated alpha-macroglobulin and murinoglobulin is independent of the clearance systems responsible for formaldehyde-modified proteins or proteinase-bound alpha-macroglobulins, and that the structure or spatial arrangement, or both, of oligosaccharide units of alpha-macroglobulin is somewhat different from that of murinoglobulin, resulting in a difference of avidity of interaction with the asialoglycoprotein receptor. The desialylated alpha-macroglobulin and murinoglobulin accumulated principally in the liver.

Animals↗

PubMed1994

Lysosomal enzyme replacement using alpha 2-macroglobulin as a transport vehicle.

Improvement of the delivery of exogenous enzymes is essential to achieve effective enzyme replacement therapy in lysosomal storage diseases. To test whether alpha 2-macroglobulin, an endogenous plasma protein, could serve as a transport vehicle of therapeutic agents to cells, alpha 2-macroglobulin and acid alpha-glucosidase or alpha-galactosidase A were coupled using two heterobifunctional cross-linking reagents. The alpha-glucosidase-alpha 2-macroglobulin conjugate was internalized and transported into lysosomes of acid alpha-glucosidase-deficient fibroblasts. The enzyme activity was stable after being taken up by the cells. Uptake of the conjugate resulted in the degradation of glycogen accumulated in lysosomes. The alpha-galactosidase A-alpha 2-macroglobulin conjugate was also internalized into the lysosomes of alpha-galactosidase A-deficient fibroblasts. Internalized alpha-galactosidase A-conjugate degraded globotriaosylceramide accumulated in lysosomes. The endocytosis of both conjugate was inhibited by alpha 2-macroglobulin-trypsin complex, indicating that the conjugates were endocytosed by an alpha 2-macroglobulin receptor system. These results showed the usefulness of alpha 2-macroglobulin as a transport vehicle of lysosomal enzymes for effective enzyme replacement.

Animals↗