Search

Objective To investigate the expression changes in transport receptor and catabolic enzymes of amyloid β-protein (Aβ) in the brain of aged rats after surgery. Methods One hundred healthy SD rats were randomly divided into 4 groups according to their ages: aged control group (n=10), aged surgery group (n=40), young control group (n=10), and young surgery group (n=40). Rats in surgery group underwent hepatic lobectomy under anesthesia with 2% sevoflurane, followed by a 2-hour continuous anesthesia after the surgery, and then sacrificed on the 1st, 3rd, 7th and 15th day after surgery to obtain specimens. The expression of low-density lipoprotein receptor-related protein 1 (LRP-1) and receptor for advanced glycation end products (RAGE) in the hippocampus, and the expression of insulin-degrading enzyme (IDE) and neprilysin (NEP) in the cerebral cortex were determined by immunohistochemistry. The mRNA expression of IDE and NEP in the hippocampus was determined with RT-PCR. Results Compared with aged control group, the expression of LRP-1, NEP and NEP mRNA decreased and the expression of RAGE increased at each time point, the expression of IDE decreased at 1st and 15th day after surgery, and the expression of IDE mRNA decreased at 3rd and 7th day and increased at 15th day after surgery in aged surgery group (P

RAP (receptor-associated protein) is a three domain 38 kDa ER (endoplasmic reticulum)-resident protein that is a chaperone for the LRP (low-density lipoprotein receptor-related protein). Whereas RAP is known to compete for binding of all known LRP ligands, neither the location, the number of binding sites on LRP, nor the domains of RAP involved in binding is known with certainty. We have systematically examined the binding of each of the three RAP domains (D1, D2 and D3) to tandem and triple CRs (complement-like repeats) that span the principal ligand-binding region, cluster II, of LRP. We found that D3 binds with low nanomolar affinity to all (CR)2 species examined. Addition of a third CR domain increases the affinity for D3 slightly. A pH change from 7.4 to 5.5 gave only a 6-fold increase in Kd for D3 at 37 degrees C, whereas temperature change from 22 degrees C to 37 degrees C has a similar small effect on affinity, raising questions about the recently proposed D3-destabilization mechanism of RAP release from LRP. Surprisingly, and in contrast to literature suggestions, D1 and D2 also bind to most (CR)2 and (CR)3 constructs with nanomolar affinity. Although this suggested that there might be three high-affinity binding sites in RAP for LRP, studies with intact RAP showed that only two binding sites are available in the intact chaperone. These findings suggest a new model for RAP to function as a folding chaperone and also for the involvement of YWTD domains in RAP release from LRP in the Golgi.

Previous experiments have shown that human neoplastic and embryonic glial cell lines synthesize and secrete in culture, alpha 2 macroglobulin (alpha 2M), a broad spectrum proteinase inhibitor present in serum and extracellular fluids. The present study was aimed to investigate the presence of alpha 2M receptors on glial cell membrane, since several non-neural cell types producing alpha 2M also express alpha 2M receptors. By flow cytometric analysis, immunofluorescence and immunoelectronmicroscopy techniques we demonstrate an alpha 2M receptor-related immunoreactivity on the plasma membrane of a human glioma cell line. Ultrastructural experiments reveal a close colocalization of immunoreactivities for alpha 2M and its receptor in clathrin-coated pits and vesicles, structures typically involved in receptor-mediated endocytic pathways.

The low density lipoprotein receptor-related protein (LRP) is the principal clearance receptor for serpins and serpin-proteinase complexes. The ligand binding regions of LRP consist of clusters of cysteine-rich approximately 40-residue complement-like repeats (CR), with cluster II being the principal ligand-binding region. To better understand the specificity of binding at different sites within the cluster and the ability of LRP to discriminate in vivo between uncomplexed and proteinase-complexed serpins, we have systematically examined the affinities of plasminogen activator inhibitor-1 (PAI-1) and proteinase nexin-1 (PN-1) in their native, cleaved, and proteinase-complexed states to (CR)(2) and (CR)(3) fragments of LRP cluster II. A consistent blue shift of the CR domain tryptophan fluorescence suggested a common mode of serpin binding, involving lysines on the serpin engaging the acidic region around the calcium binding site of the CR domain. High affinity binding of non-proteinase-complexed PAI-1 and PN-1 occurred to all fragments containing three CR domains (3-59 nm) and most that contain only two CR domains, although binding energies to different (CR)(3) fragments differed by up to 18% for PAI-1 and 9% for PN-1. No detectable difference in affinity was seen between native and cleaved serpin. However, the presence of proteinase in complex with the serpin enhanced affinity modestly and presumably nonspecifically. This may be sufficient to give preferential binding of such complexes in vivo at the relevant physiological concentrations.

A new, easier and efficient purification method, using Sephacryl and DEAE-Sephacel, of the C-terminal fragment of two alpha-macroglobulins, alpha(2)-M and PZP, is presented. Two larger peptides were identified for each protein as the C-terminal fragment, with molecular weights of approximately 30 kDa and the N-terminal sequences were determined to be SSTQDTV for alpha(2)-M and VALHLS for PZP. The smaller peptides with molecular weights of 18 kDa correspond to a shorter C-terminal sequence of these proteins, and they were determined to be EEFPFA for alpha(2)-M and ALKVQTV for PZP, with no interfering sequences detected. The results confirmed the discriminatory capacity of the purification procedure and the purity of the fragments. This new methodology facilitates biological studies of alpha-macroglobulins, and will enable elucidation of the role the C-terminal region may exert to eliminate alpha-macroglobulin-proteinases complexes from the circulation by the LRP/receptor.

OBJECTIVE - In vitro studies implicate that the low-density lipoprotein receptor (LDLR)-related protein (LRP) in macrophages has a pro-atherogenic potential. In the present study, we investigated the in vivo role of macrophage specific LRP in atherogenesis independent of its role in the uptake of lipoproteins. METHODS AND RESULTS - We generated macrophage-specific LRP-deficient mice on an apoE/LDLR double-deficient background. Macrophage LRP deletion did not affect plasma cholesterol and triglyceride levels, lipoprotein distribution, and blood monocyte counts. Nevertheless, macrophage LRP deficiency resulted in a 1.8-fold increase in total atherosclerotic lesion area in the aortic root of 18-week-old mice. Moreover, LRP deficiency also resulted in a relatively higher number of advanced lesions. Whereas macrophage and smooth muscle cell content did not differ between LRP-deficient mice and control littermates, a 1.7-fold increase in collagen content and 2.3-fold decrease in relative number of CD3+ T cells were observed in lesions from macrophage specific LRP-deficient mice. CONCLUSIONS - Our data demonstrate that independent of its role in lipoprotein uptake, absence of LRP in macrophages resulted in more advanced atherosclerosis and in lesions that contained more collagen and less CD3+ T cells. In contrast to previous in vitro studies, we conclude that macrophage LRP has an atheroprotective potential and may modulate the extracellular matrix in the atherosclerotic lesions. © 2006 American Heart Association, Inc. Chemicals / CAS: cholesterol, 57-88-5; collagen, 9007-34-5; Apolipoproteins E; Collagen, 9007-34-5; LDL-Receptor Related Protein 1; Lipoproteins; Receptors, LDL

OBJECTIVE - In vitro studies implicate that the low-density lipoprotein receptor (LDLR)-related protein (LRP) in macrophages has a pro-atherogenic potential. In the present study, we investigated the in vivo role of macrophage specific LRP in atherogenesis independent of its role in the uptake of lipoproteins. METHODS AND RESULTS - We generated macrophage-specific LRP-deficient mice on an apoE/LDLR double-deficient background. Macrophage LRP deletion did not affect plasma cholesterol and triglyceride levels, lipoprotein distribution, and blood monocyte counts. Nevertheless, macrophage LRP deficiency resulted in a 1.8-fold increase in total atherosclerotic lesion area in the aortic root of 18-week-old mice. Moreover, LRP deficiency also resulted in a relatively higher number of advanced lesions. Whereas macrophage and smooth muscle cell content did not differ between LRP-deficient mice and control littermates, a 1.7-fold increase in collagen content and 2.3-fold decrease in relative number of CD3+ T cells were observed in lesions from macrophage specific LRP-deficient mice. CONCLUSIONS - Our data demonstrate that independent of its role in lipoprotein uptake, absence of LRP in macrophages resulted in more advanced atherosclerosis and in lesions that contained more collagen and less CD3+ T cells. In contrast to previous in vitro studies, we conclude that macrophage LRP has an atheroprotective potential and may modulate the extracellular matrix in the atherosclerotic lesions. © 2006 American Heart Association, Inc. Chemicals / CAS: cholesterol, 57-88-5; collagen, 9007-34-5; Apolipoproteins E; Collagen, 9007-34-5; LDL-Receptor Related Protein 1; Lipoproteins; Receptors, LDL

Analysis of genetic susceptibility factors for Alzheimer's disease (AD) in populations with different genetic and environmental background may be useful to understand AD etiology. There are few genetic association studies of AD in Latin America. In the present work, we analyzed polymorphisms in 3 candidate genes; the LDL receptor related protein-1, the microtubule-associated protein Tau and the serotonin transporter genes in a sample of 106 Colombian AD patients and 97 control subjects. We did not find a significant allelic or genotypic association with any of the three polymorphisms analyzed using different statistical analysis, including a neural network model or different sample stratifications. To date, APOE polymorphisms are the only genetic risk factors identified for AD in the Colombian population. It may be factible that future combination of high-throughput genotyping platforms and multivariate analysis models may lead to the identification of other genetic susceptibility factors for AD in the Colombian population. © Springer-Verlag 2005.

Udgivelsesdato: 2004-Dec-24 The low density lipoprotein receptor-related protein (LRP) mediates the endocytic clearance of various proteinases and proteinase.inhibitor complexes, including thrombospondin (TSP)-dependent endocytosis of matrix metalloproteinase (MMP)-2 (or gelatinase A), a key effector of extracellular matrix remodeling and cancer progression. However, the zymogen of MMP-2 (pro-MMP-2) mostly occurs in tissues as a complex with the tissue inhibitor of MMPs (TIMP-2). Here we show that clearance of the pro-MMP-2.TIMP-2 complex is also mediated by LRP, because addition of receptor-associated protein (RAP), a natural LRP ligand antagonist, inhibited endocytosis and lysosomal degradation of (125)I-pro-MMP-2.TIMP-2. Both TIMP-2 and the pro-MMP-2 collagen-binding domain independently competed for endocytosis of (125)I-pro-MMP-2.TIMP-2 complex. Surface plasmon resonance studies indicated that pro-MMP-2, TIMP-2, and pro-MMP-2.TIMP-2 directly interact with LRP in the absence of TSP. LRP-mediated endocytic clearance of (125)I-pro-MMP-2 was inhibited by anti-TSP antibodies and accelerated upon complexing with TSP-1, but these treatments had no effect on (125)I-pro-MMP-2.TIMP-2 uptake. This implies that mechanisms of clearance by LRP of pro-MMP-2 and pro-MMP-2.TIMP-2 complex are different. Interestingly, RAP did not inhibit binding of (125)I-pro-MMP-2.TIMP-2 to the cell surface. We conclude that clearance of pro-MMP-2.TIMP-2 complex is a TSP-independent two-step process, involving (i) initial binding to the cell membrane in a RAP-insensitive manner and (ii) subsequent LRP-dependent (RAP-sensitive) internalization and degradation.

The low-density lipoprotein (LDL) receptor-related protein (LRP) has a well-established role in the hepatic removal of atherogenic apolipoprotein E (APOE)-rich remnant lipoproteins from plasma. In addition, LRP recognizes multiple distinct pro- and antiatherogenic ligands in vitro. Here, we investigated the role of hepatic LRP in atherogenesis independent of its role in removal of APOE-rich remnant lipoproteins. Mice that allow inducible inactivation of hepatic LRP were combined with LDL receptor and APOE double-deficient mice (MX1Cre+LRPflox/floxLDLR -/-APOE-/- On an LDLR-/-APOE-/- background, hepatic LRP deficiency resulted in decreased plasma cholesterol and triglycerides (cholesterol: 17.1 ± 5.2 vs 23.4 ± 6.3 mM, P = . 025; triglycerides: 1.1 ± 0.5 vs 2.2 ± 0.8 mM, P = .002, for MX1Cre+LRPflox/flox-LDLR-/-APOE-/- and control LRPflox/flox-LDLR-/-APOE-/- mice, respectively). Lower plasma cholesterol in MX1Cre+LRP flox/flox-LDLR-/-APOE-/- mice coincided with increased plasma lipoprotein lipase (71.2 ± 7.5 vs 19.1 ± 2.4 ng/ml, P = .002), coagulation factor VIII (4.4 ± 1.1 vs 1.9 ± 0.5 U/mL, P = .001), von Willebrand factor (2.8 ± 0.6 vs 1.4 ± 0.3 U/mL, P = .001), and tissue-type plasminogen activator (1.7 ± 0.7 vs 0.9 ± 0.5 ng/ml, P = .008) compared with controls. Strikingly, MX1Cre +-LRPflox/floxLDLR-/-APOE-/- mice showed a 2-fold higher atherosclerotic lesion area compared with controls (408. 5 ± 115.1 vs 219.1 ± 86.0 103μm2, P = .003). Our data indicate that hepatic LRP plays a clear protective role in atherogenesis independent of plasma cholesterol, possibly due to maintaining low levels of its proatherogenic ligands. © 2004 by The American Society of Hematology. Chemicals/CAS: blood clotting factor 8, 9001-27-8; cholesterol, 57-88-5; lipoprotein lipase, 83137-80-8, 9004-02-8; tissue plasminogen activator, 105913-11-9; von Willebrand factor, 109319-16-6; Apolipoproteins E; Blood Coagulation Factors; Cholesterol, 57-88-5; LDL-Receptor Related Protein 1; Lipids; Receptors, LDL

The low-density lipoprotein (LDL) receptor-related protein (LRP) has a well-established role in the hepatic removal of atherogenic apolipoprotein E (APOE)-rich remnant lipoproteins from plasma. In addition, LRP recognizes multiple distinct pro- and antiatherogenic ligands in vitro. Here, we investigated the role of hepatic LRP in atherogenesis independent of its role in removal of APOE-rich remnant lipoproteins. Mice that allow inducible inactivation of hepatic LRP were combined with LDL receptor and APOE double-deficient mice (MX1Cre+LRPflox/floxLDLR -/-APOE-/- On an LDLR-/-APOE-/- background, hepatic LRP deficiency resulted in decreased plasma cholesterol and triglycerides (cholesterol: 17.1 ± 5.2 vs 23.4 ± 6.3 mM, P = . 025; triglycerides: 1.1 ± 0.5 vs 2.2 ± 0.8 mM, P = .002, for MX1Cre+LRPflox/flox-LDLR-/-APOE-/- and control LRPflox/flox-LDLR-/-APOE-/- mice, respectively). Lower plasma cholesterol in MX1Cre+LRP flox/flox-LDLR-/-APOE-/- mice coincided with increased plasma lipoprotein lipase (71.2 ± 7.5 vs 19.1 ± 2.4 ng/ml, P = .002), coagulation factor VIII (4.4 ± 1.1 vs 1.9 ± 0.5 U/mL, P = .001), von Willebrand factor (2.8 ± 0.6 vs 1.4 ± 0.3 U/mL, P = .001), and tissue-type plasminogen activator (1.7 ± 0.7 vs 0.9 ± 0.5 ng/ml, P = .008) compared with controls. Strikingly, MX1Cre +-LRPflox/floxLDLR-/-APOE-/- mice showed a 2-fold higher atherosclerotic lesion area compared with controls (408. 5 ± 115.1 vs 219.1 ± 86.0 103μm2, P = .003). Our data indicate that hepatic LRP plays a clear protective role in atherogenesis independent of plasma cholesterol, possibly due to maintaining low levels of its proatherogenic ligands. © 2004 by The American Society of Hematology. Chemicals/CAS: blood clotting factor 8, 9001-27-8; cholesterol, 57-88-5; lipoprotein lipase, 83137-80-8, 9004-02-8; tissue plasminogen activator, 105913-11-9; von Willebrand factor, 109319-16-6; Apolipoproteins E; Blood Coagulation Factors; Cholesterol, 57-88-5; LDL-Receptor Related Protein 1; Lipids; Receptors, LDL

Background. Human mesothelial cells (HMCs) have an important role in maintaining an adequately functioning fibrinolytic system in the peritoneal cavity by secreting the fibrinolytic enzymes tissue-type and urokinase-type plasminogen activator (t-PA and u-PA), as well as a specific PA inhibitor, PA inhibitor type 1 (PAI-1). In this study, we investigated whether the fibrinolytic capacity of HMCs is further counterbalanced by rapid uptake of t-PA and u-PA from the medium. Methods. Cultured HMCs were used to study the uptake and degradation of radiolabeled t-PA and u-PA in the absence or presence of an inhibitor of cellular protein degradation, chloroquine, and of specific receptor antagonists. Northern blotting and ligand-blotting techniques were applied to demonstrate the presence of specific receptors for binding of t-PA and u-PA. Results. At 37°C, HMCs rapidly internalized and degraded 125I-t-PA and 125I-u-PA, which could be inhibited by an excess of unlabeled t-PA anti u-PA, respectively, and by the lysosomotropic agent chloroquine. Northern blot analysis showed the expression of low-density lipoprotein (LDL) receptor-related protein (LRP), very low density lipoprotein (VLDL) receptor, and u-PA receptor. The addition of recombinant 39 kDa receptor-associated protein (RAP; an inhibitor of LRP and VLDL receptor) almost completely blocked the degradation of t-PA and partly that of u-PA. RAP ligand blotting demonstrated predominantly the presence of LRP, suggesting a major role for the LRP in mediating uptake and degradation of t- PA in HMCs. Endocytosis of u-PA occurs via two different pathways. After binding to u-PA receptor, a RAP-inhibitable and a non-RAP-inhibitable route for u-PA degradation was demonstrated. Tumor necrosis factor α (TNFα) diminished the fibrinolytic activity of HMCs by decreasing t-PA and increasing PAI-1 synthesis. The fall in t-PA levels could be counteracted by inhibiting t-PA degradation by either RAP or chloroquine. Interestingly, chloroquine also quenched the TNFα-induced changes in t-PA and PAI-1 mRNA levels. Using TNFα mutants and agonistic or blocking monoclonal antibodies specific for the TNF receptors p55 and p75, we found evidence that chloroquine interfered with the activation of the TNF receptor p55 and/or its intracellular signaling route. Conclusions. Receptor-mediated endocytosis plays a crucial role in regulating the fibrinolytic capacity of HMCs by its participation in the degradation of t-PA and u-PA, and in the TNFα-induced decrease in t-PA and the increase in PAI-1 expression. Chemicals/CAS: Antibodies; Carrier Proteins; Chloroquine, 54-05-7; Culture Media, Conditioned; Glutathione Transferase, EC 2.5.1.18; GST-RAP protein, recombinant; Iodine Radioisotopes; LDL-Receptor Related Protein 1; Lectins, C-Type; mannose receptor; Mannose-Binding Lectins; Plasminogen Activator Inhibitor 1; plasminogen activator, urokinase receptors; Receptors, Cell Surface; Receptors, Immunologic; Receptors, LDL; Recombinant Fusion Proteins; RNA, Messenger; Tissue Plasminogen Activator, EC 3.4.21.68; Tumor Necrosis Factor-alpha; Urinary Plasminogen Activator, EC 3.4.21.73; VLDL receptor

Background. Human mesothelial cells (HMCs) have an important role in maintaining an adequately functioning fibrinolytic system in the peritoneal cavity by secreting the fibrinolytic enzymes tissue-type and urokinase-type plasminogen activator (t-PA and u-PA), as well as a specific PA inhibitor, PA inhibitor type 1 (PAI-1). In this study, we investigated whether the fibrinolytic capacity of HMCs is further counterbalanced by rapid uptake of t-PA and u-PA from the medium. Methods. Cultured HMCs were used to study the uptake and degradation of radiolabeled t-PA and u-PA in the absence or presence of an inhibitor of cellular protein degradation, chloroquine, and of specific receptor antagonists. Northern blotting and ligand-blotting techniques were applied to demonstrate the presence of specific receptors for binding of t-PA and u-PA. Results. At 37°C, HMCs rapidly internalized and degraded 125I-t-PA and 125I-u-PA, which could be inhibited by an excess of unlabeled t-PA anti u-PA, respectively, and by the lysosomotropic agent chloroquine. Northern blot analysis showed the expression of low-density lipoprotein (LDL) receptor-related protein (LRP), very low density lipoprotein (VLDL) receptor, and u-PA receptor. The addition of recombinant 39 kDa receptor-associated protein (RAP; an inhibitor of LRP and VLDL receptor) almost completely blocked the degradation of t-PA and partly that of u-PA. RAP ligand blotting demonstrated predominantly the presence of LRP, suggesting a major role for the LRP in mediating uptake and degradation of t- PA in HMCs. Endocytosis of u-PA occurs via two different pathways. After binding to u-PA receptor, a RAP-inhibitable and a non-RAP-inhibitable route for u-PA degradation was demonstrated. Tumor necrosis factor α (TNFα) diminished the fibrinolytic activity of HMCs by decreasing t-PA and increasing PAI-1 synthesis. The fall in t-PA levels could be counteracted by inhibiting t-PA degradation by either RAP or chloroquine. Interestingly, chloroquine also quenched the TNFα-induced changes in t-PA and PAI-1 mRNA levels. Using TNFα mutants and agonistic or blocking monoclonal antibodies specific for the TNF receptors p55 and p75, we found evidence that chloroquine interfered with the activation of the TNF receptor p55 and/or its intracellular signaling route. Conclusions. Receptor-mediated endocytosis plays a crucial role in regulating the fibrinolytic capacity of HMCs by its participation in the degradation of t-PA and u-PA, and in the TNFα-induced decrease in t-PA and the increase in PAI-1 expression. Chemicals/CAS: Antibodies; Carrier Proteins; Chloroquine, 54-05-7; Culture Media, Conditioned; Glutathione Transferase, EC 2.5.1.18; GST-RAP protein, recombinant; Iodine Radioisotopes; LDL-Receptor Related Protein 1; Lectins, C-Type; mannose receptor; Mannose-Binding Lectins; Plasminogen Activator Inhibitor 1; plasminogen activator, urokinase receptors; Receptors, Cell Surface; Receptors, Immunologic; Receptors, LDL; Recombinant Fusion Proteins; RNA, Messenger; Tissue Plasminogen Activator, EC 3.4.21.68; Tumor Necrosis Factor-alpha; Urinary Plasminogen Activator, EC 3.4.21.73; VLDL receptor

We have used adenovirus-mediated gene transfer in mice to investigate low density lipoprotein receptor (LDLR) and LDLR-related protein (LRP)- independent mechanisms that control the metabolism of chylomicron and very low density lipoprotein (VLDL) remnants in vivo. Overexpression of receptor- associated protein (RAP) in mice that lack both LRP and LDLR (MX1cre+LRP(flox/flox)LDLR(-/-)) in their livers elicited a marked hypertriglyceridemia in addition to the pre-existing hypercholesterolemia in these animals, resulting in a shift in the distribution of plasma lipids from LDL-sized lipoproteins to large VLDL-sized particles. This dramatic increase in plasma lipids was not due to a RAP-mediated inhibition of a unknown hepatic high affinity binding site involved in lipoprotein metabolism, because no RAP binding could be detected in livers of MX1cre+LRP(flox/flox)LDLR(-/-) mice using both membrane binding studies and ligand blotting experiments. Remarkably, RAP overexpression also resulted in a 7-fold increase (from 13.6 to 95.6 ng/ml) of circulating, but largely inactive, lipoprotein lipase (LPL). In contrast, plasma hepatic lipase levels and activity were unaffected. In vitro studies showed that RAP binds to LPL with high affinity (K(d) = 5 nM) but does not affect its catalytic activity, in vitro or in vivo. Our findings suggest that an extrahepatic RAP-sensitive process that is independent of the LDLR or LRP is involved in metabolism of triglyceride-rich lipoproteins. There, RAP may affect the functional maturation of LPL, thus causing the accumulation of triglyceride-rich lipoproteins in the circulation. Chemicals/CAS: Cholesterol, 57-88-5; Chylomicrons; Detergents; Heymann Nephritis Antigenic Complex; LDL-Receptor Related Protein 1; Lipase, EC 3.1.1.3; Lipoprotein Lipase, EC 3.1.1.34; Membrane Glycoproteins; Polyethylene Glycols; Receptors, Immunologic; Receptors, LDL; Triglycerides; tyloxapol, 25301-02-4

We have used adenovirus-mediated gene transfer in mice to investigate low density lipoprotein receptor (LDLR) and LDLR-related protein (LRP)- independent mechanisms that control the metabolism of chylomicron and very low density lipoprotein (VLDL) remnants in vivo. Overexpression of receptor- associated protein (RAP) in mice that lack both LRP and LDLR (MX1cre+LRP(flox/flox)LDLR(-/-)) in their livers elicited a marked hypertriglyceridemia in addition to the pre-existing hypercholesterolemia in these animals, resulting in a shift in the distribution of plasma lipids from LDL-sized lipoproteins to large VLDL-sized particles. This dramatic increase in plasma lipids was not due to a RAP-mediated inhibition of a unknown hepatic high affinity binding site involved in lipoprotein metabolism, because no RAP binding could be detected in livers of MX1cre+LRP(flox/flox)LDLR(-/-) mice using both membrane binding studies and ligand blotting experiments. Remarkably, RAP overexpression also resulted in a 7-fold increase (from 13.6 to 95.6 ng/ml) of circulating, but largely inactive, lipoprotein lipase (LPL). In contrast, plasma hepatic lipase levels and activity were unaffected. In vitro studies showed that RAP binds to LPL with high affinity (K(d) = 5 nM) but does not affect its catalytic activity, in vitro or in vivo. Our findings suggest that an extrahepatic RAP-sensitive process that is independent of the LDLR or LRP is involved in metabolism of triglyceride-rich lipoproteins. There, RAP may affect the functional maturation of LPL, thus causing the accumulation of triglyceride-rich lipoproteins in the circulation. Chemicals/CAS: Cholesterol, 57-88-5; Chylomicrons; Detergents; Heymann Nephritis Antigenic Complex; LDL-Receptor Related Protein 1; Lipase, EC 3.1.1.3; Lipoprotein Lipase, EC 3.1.1.34; Membrane Glycoproteins; Polyethylene Glycols; Receptors, Immunologic; Receptors, LDL; Triglycerides; tyloxapol, 25301-02-4

Apo E3-leiden transgenic mice express human dysfunctional apo E variant and develop hyperlipidemia and atherosclerosis on a high fat/high cholesterol diet. We characterized diet-induced atherosclerotic lesions in apo E3-leiden transgenic mice using immunocytochemical methods in order to examine foam cell formation and determine whether advanced atherosclerotic lesions develop in these animals. Special attention was given to the presence of oxidized lipoproteins and expression of lipoprotein receptors. Plasma cholesterol levels in apo E3-leiden mice on an atherogenic diet increased from 2 to 36 mmol/l in 4 months. At this time apo E3-leiden mice had developed lesions, which ranged from early fatty streaks in thoracic and abdominal aorta to advanced lesions in aortic arch. Early fatty streaks were entirely composed of macrophages which also expressed scavenger receptors. Epitopes characteristic of oxidized LDL were present in macrophage-rich foam cells. Advanced atherosclerotic lesions also developed in apo E3-leiden mice including smooth muscle cell cap formation and erosion of the media. Macrophages and epitopes characteristic of oxidized LDL were present in core and shoulder regions. Scavenger receptors were expressed in macrophages in advanced lesions, whereas LDL-receptor-related protein (LRP) was mainly expressed in smooth muscle cells. It is concluded that: (1) macrophages are the major cell type in both early and advanced atherosclerotic lesions; (2) scavenger receptors and oxidized lipoproteins are present in lesion macrophages; and (3) LRP is mostly expressed in smooth muscle cells. Thus, lesions in apo E3-leiden transgenic mice have features in common with human atherosclerosis. Since lesion macrophages also retain their ability to synthesize endogenous apo E, apo E3-leiden transgenic mouse may be a useful model for studies on the development and genetics of atherosclerosis.

Apo E3-leiden transgenic mice express human dysfunctional apo E variant and develop hyperlipidemia and atherosclerosis on a high fat/high cholesterol diet. We characterized diet-induced atherosclerotic lesions in apo E3-leiden transgenic mice using immunocytochemical methods in order to examine foam cell formation and determine whether advanced atherosclerotic lesions develop in these animals. Special attention was given to the presence of oxidized lipoproteins and expression of lipoprotein receptors. Plasma cholesterol levels in apo E3-leiden mice on an atherogenic diet increased from 2 to 36 mmol/l in 4 months. At this time apo E3-leiden mice had developed lesions, which ranged from early fatty streaks in thoracic and abdominal aorta to advanced lesions in aortic arch. Early fatty streaks were entirely composed of macrophages which also expressed scavenger receptors. Epitopes characteristic of oxidized LDL were present in macrophage-rich foam cells. Advanced atherosclerotic lesions also developed in apo E3-leiden mice including smooth muscle cell cap formation and erosion of the media. Macrophages and epitopes characteristic of oxidized LDL were present in core and shoulder regions. Scavenger receptors were expressed in macrophages in advanced lesions, whereas LDL-receptor-related protein (LRP) was mainly expressed in smooth muscle cells. It is concluded that: (1) macrophages are the major cell type in both early and advanced atherosclerotic lesions; (2) scavenger receptors and oxidized lipoproteins are present in lesion macrophages; and (3) LRP is mostly expressed in smooth muscle cells. Thus, lesions in apo E3-leiden transgenic mice have features in common with human atherosclerosis. Since lesion macrophages also retain their ability to synthesize endogenous apo E, apo E3-leiden transgenic mouse may be a useful model for studies on the development and genetics of atherosclerosis.

Udgivelsesdato: 1998-May-15 BACKGROUND: The large plasma proteinase inhibitors of the alpha 2-macroglobulin superfamily inhibit proteinases by capturing them within a central cavity of the inhibitor molecule. After reaction with the proteinase, the alpha-macroglobulin-proteinase complex binds to the alpha-macroglobulin receptor, present in the liver and other tissues, and becomes endocytosed and rapidly removed from the circulation. The complex binds to the receptor via recognition sites located on a separate domain of approximately 138 residues positioned at the C terminus of the alpha-macroglobulin subunit. RESULTS: The crystal structure of the receptor-binding domain of bovine alpha 2-macroglobulin (bRBD) has been determined at a resolution of 1.9 A. The domain primarily comprises a nine-strand beta structure with a jelly-roll topology, but also contains two small alpha helices. CONCLUSIONS: The surface patch responsible for receptor recognition is thought to involve residues located on one of the two alpha helices of the bRBD as well as residues in two of the beta strands. Located on this alpha helix are two lysine residues that are important for receptor binding. The structure of bRBD is very similar to the approximately 100-residue C-terminal domain of factor XIII, a transglutaminase from the blood coagulation system.

Urokinase-type plasminogen activator (u-PA) is used as a thrombolytic agent in the treatment of acute myocardial infarction. In vitro, recombinant single-chain u-PA (rscu-PA) expressed in E. coli is recognized by the Low-Density Lipoprotein Receptor-related Protein (LRP) on rat parenchymal liver cells. In this study we investigated the role of LRP in the liver uptake and plasma clearance of rscu-PA in rats. A preinjection of the LRP inhibitor GST-RAP reduced the maximal liver uptake of 125I-rscu-PA at 5 min after injection from 50 to 30% of the injected dose and decreased the clearance of rscu-PA from 2.37 ml/min to 1.58 ml/min. Parenchymal, Kupffer and endothelial cells were responsible for 40, 50 and 10% of the liver uptake, respectively. The reduction in liver uptake of rscu-PA by the preinjection of GST-RAP was caused by a 91% and 62% reduction in the uptake by parenchymal and Kupffer cells, respectively. In order to investigate the part of rscu-PA that accounted for the interaction with LRP, experiments were performed with a mutant of rscu-PA lacking residues 11-135 (= delta125-rscu-PA). Deletion of residues 11-135 resulted in a 80% reduction in liver uptake and a 2.4 times slower clearance (0.97 ml/min). The parenchymal, Kupffer and endothelial cells were responsible for respectively 60, 33 and 7% of the liver uptake of 125I-delta125-rscu-PA. Preinjection of GST-RAP completely reduced the liver uptake of delta125-rscu-PA and reduced its clearance to 0.79 ml/min. Treatment of isolated Kupffer cells with PI-PLC reduced the binding of rscu-PA by 40%, suggesting the involvement of the urokinase-type Plasminogen Activator Receptor (u-PAR) in the recognition of rscu-PA. Our results demonstrate that in vivo LRP is responsible for more than 90% of the parenchymal liver cell mediated uptake of rscu-PA and for 60% of the Kupffer cell interaction. It is also suggested that u-PAR is involved in the Kupffer cell recognition of rscu-PA. Copyright © 1997 Schattauer Verlag

We recently found that the proteins of the proteolytic system of plasminogen activation emerge in late stages of melanocytic tumor progression. A large body of evidence suggests a role for two proteins, the low-density lipoprotein receptor-related protein (LRP)/α2-macroglobulin receptor and its receptor-associated protein (RAP), in the internalization of components of the plasminogen activation system. Here, we present data on the presence of these two proteins in human melanoma cell lines which differ in metastatic capacity, their corresponding xenografts, and in cutaneous melanocytic lesions. With flow cytometry, we found surface expression of LRP to be restricted to urokinase plasminogen activator, producing highly metastatic cell lines. These cell lines also produce higher levels of LRP mRNA, whereas RAP mRNA and protein are expressed at equal levels in all cell lines and not expressed at the cell surface. Xenografts of cell lines producing high levels of LRP remarkably contain only a small fraction of LRP-positive tumor cells. Using immunohistochemistry on frozen sections of 107 human melanocytic lesions comprising the various stages of melanocytic tumor progression, we found that expression of both LRP and RAP decreased in tumor progression. Furthermore, we noted that LRP and RAP are coexpressed within the same lesion. Using immunofluorescence double staining, we found that LRP and RAP colocalize in the same cells in the lesions studied and in the same cell structures in the cell lines studied. In conclusion, our results indicate that LRP and RAP are coordinately expressed in a decreased fashion in melanocytic tumor progression. Based on the staining results in xenografts and in human melanocytic lesions, we conclude that a strong correlation between expression of LRP and urokinase-type plasminogen activator seems not to exist in in vivo melanomas. Chemicals/CAS: LDL-Receptor Related Protein 1; Neoplasm Proteins; Receptors, Immunologic; Receptors, LDL; RNA, Messenger

We recently found that the proteins of the proteolytic system of plasminogen activation emerge in late stages of melanocytic tumor progression. A large body of evidence suggests a role for two proteins, the low-density lipoprotein receptor-related protein (LRP)/α2-macroglobulin receptor and its receptor-associated protein (RAP), in the internalization of components of the plasminogen activation system. Here, we present data on the presence of these two proteins in human melanoma cell lines which differ in metastatic capacity, their corresponding xenografts, and in cutaneous melanocytic lesions. With flow cytometry, we found surface expression of LRP to be restricted to urokinase plasminogen activator, producing highly metastatic cell lines. These cell lines also produce higher levels of LRP mRNA, whereas RAP mRNA and protein are expressed at equal levels in all cell lines and not expressed at the cell surface. Xenografts of cell lines producing high levels of LRP remarkably contain only a small fraction of LRP-positive tumor cells. Using immunohistochemistry on frozen sections of 107 human melanocytic lesions comprising the various stages of melanocytic tumor progression, we found that expression of both LRP and RAP decreased in tumor progression. Furthermore, we noted that LRP and RAP are coexpressed within the same lesion. Using immunofluorescence double staining, we found that LRP and RAP colocalize in the same cells in the lesions studied and in the same cell structures in the cell lines studied. In conclusion, our results indicate that LRP and RAP are coordinately expressed in a decreased fashion in melanocytic tumor progression. Based on the staining results in xenografts and in human melanocytic lesions, we conclude that a strong correlation between expression of LRP and urokinase-type plasminogen activator seems not to exist in in vivo melanomas. Chemicals/CAS: LDL-Receptor Related Protein 1; Neoplasm Proteins; Receptors, Immunologic; Receptors, LDL; RNA, Messenger

The balance of tissue-type plasminogen activator (t-PA) production and degradation determines its concentration in blood and tissues. Disturbance of this balance may result in either increased or decreased proteolysis. In the present study, we identified the receptor systems involved in the degradation of t-PA by human monocytes/macrophages in culture. Monocytes were cultured and became macrophages within 2 days. At 4°C, 125I-t-PA bound to macrophages with high (apparent dissociation constant [kd], 1 to 5 nmol/L) and low affinity (kd > 350 nmol/L). At 37°C, the cells internalized and degraded t-PA via the high affinity binding sites, which were partially inhibited by mannan. The low affinity binding sites were 6-aminohexanoic acid-inhibitable and not involved in t-PA degradation. Degradation of t-PA was upregulated during differentiation of monocytes to macrophages. Dexamethasone further upregulated the mannan-inhibitable t-PA degradation. Lipopolysaccharide downregulated both mannan-inhibitable and non-mannan- inhibitable t-PA degradation. Non-mannan-inhibitable degradation was completely blocked by recombinant 39-kD receptor-associated protein (RAP, inhibitor of lipoprotein receptor-related protein [LRP]), whereas mannan- inhibitable degradation was blocked by the addition of a monoclonal antibody against the mannose receptor. No differences between the degradation of t-PA and functionally inactivated t-PA were observed. We conclude that human monocyte-derived macrophages are able to bind, internalize, and degrade t- PA. Degradation of t-PA does not require complex formation with plasminogen activator inhibitors. The macrophages use two independently regulated receptors, namely, the mannose receptor and LRP, for the uptake and degradation of t-PA. Chemicals/CAS: aminocaproic acid, 1319-82-0, 60-32-2; tissue plasminogen activator, 105913-11-9; Dexamethasone, 50-02-2; LDL-Receptor Related Protein 1; Lipopolysaccharides; mannose receptor; Receptors, Cell Surface; Receptors, Immunologic; Tissue Plasminogen Activator, EC 3.4.21.68

The balance of tissue-type plasminogen activator (t-PA) production and degradation determines its concentration in blood and tissues. Disturbance of this balance may result in either increased or decreased proteolysis. In the present study, we identified the receptor systems involved in the degradation of t-PA by human monocytes/macrophages in culture. Monocytes were cultured and became macrophages within 2 days. At 4°C, 125I-t-PA bound to macrophages with high (apparent dissociation constant [kd], 1 to 5 nmol/L) and low affinity (kd > 350 nmol/L). At 37°C, the cells internalized and degraded t-PA via the high affinity binding sites, which were partially inhibited by mannan. The low affinity binding sites were 6-aminohexanoic acid-inhibitable and not involved in t-PA degradation. Degradation of t-PA was upregulated during differentiation of monocytes to macrophages. Dexamethasone further upregulated the mannan-inhibitable t-PA degradation. Lipopolysaccharide downregulated both mannan-inhibitable and non-mannan- inhibitable t-PA degradation. Non-mannan-inhibitable degradation was completely blocked by recombinant 39-kD receptor-associated protein (RAP, inhibitor of lipoprotein receptor-related protein [LRP]), whereas mannan- inhibitable degradation was blocked by the addition of a monoclonal antibody against the mannose receptor. No differences between the degradation of t-PA and functionally inactivated t-PA were observed. We conclude that human monocyte-derived macrophages are able to bind, internalize, and degrade t- PA. Degradation of t-PA does not require complex formation with plasminogen activator inhibitors. The macrophages use two independently regulated receptors, namely, the mannose receptor and LRP, for the uptake and degradation of t-PA. Chemicals/CAS: aminocaproic acid, 1319-82-0, 60-32-2; tissue plasminogen activator, 105913-11-9; Dexamethasone, 50-02-2; LDL-Receptor Related Protein 1; Lipopolysaccharides; mannose receptor; Receptors, Cell Surface; Receptors, Immunologic; Tissue Plasminogen Activator, EC 3.4.21.68

Udgivelsesdato: 1992-Nov-23 We have expressed receptor-binding domains of human alpha 2-macroglobulin and rat alpha 1-macroglobulin in Escherichia coli. Expression levels of both recombinants were quite high, but the human one was insoluble, probably forming inclusion bodies. The rat domain, which lacks the human disulfide, was produced in a soluble form and readily purified by two simple chromatographic steps. Purified recombinant rat alpha 1-macroglobulin receptor-binding domain was fully functional in binding to the alpha-macroglobulin receptor on human fibroblasts. This 142 residue domain should serve as an excellent template for analyzing the structural requirements for alpha-macroglobulin receptor ligation and dissecting the varied biological functions resulting from such ligation.

Udgivelsesdato: 1990-Oct

Udgivelsesdato: 1989-Feb-7 Efforts to characterize the receptor recognition domain of alpha-macroglobulins have primarily focused on human alpha 2-macroglobulin (alpha 2M). In the present work, the structure and function of the alpha-macroglobulin receptor recognition site were investigated by amino acid sequence analysis, plasma clearance, and cell binding studies using several nonhuman alpha-macroglobulins: bovine alpha 2M, rat alpha 1-macroglobulin (alpha 1M), rat alpha 1-inhibitor 3 (alpha 1I3), and proteolytic fragments derived from these proteins. Each alpha-macroglobulin bound to the murine peritoneal macrophage alpha-macroglobulin receptor with comparable affinity (Kd approximately 1 nM). A carboxyl-terminal 20-kDa fragment was isolated from each of these proteins, and this fragment bound to alpha-macroglobulin receptors with Kd values ranging from 10 to 125 nM. The amino acid identity between the homologous carboxyl-terminal 20-kDa fragments of human and bovine alpha 2M was approximately 90%, while the overall sequence homology between all carboxyl-terminal fragments studied was 75%. The interchain disulfide bond present in the human alpha 2M carboxyl-terminal 20-kDa fragment was conserved in bovine alpha 2M and rat alpha 1I3, but not in rat alpha 1M. The clearance of each intact alpha-macroglobulin-proteinase complex was significantly retarded following treatment with cis-dichlorodiammineplatinum(II) (cis-DDP). cis-DDP treatment, however, did not affect receptor recognition of purified carboxyl-terminal 20-kDa fragments of these alpha-macroglobulins. A carboxyl-terminal 40-kDa subunit, which can be isolated from rat alpha 1M, bound to the murine alpha-macroglobulin receptor with a Kd of 5 nM.(ABSTRACT TRUNCATED AT 250 WORDS)

Udgivelsesdato: 1987-Oct-1 125I-labelled pregnancy zone protein complex was injected intravenously in rats and after 6 min uptake into cells of the liver and spleen was determined by electron microscopic autoradiography. The liver took up 68% of the injected radioactivity; 61% was in the hepatocytes and 7% was in the liver macrophages (Kupffer cells). The spleen took up 3-4% and nearly all the radioactivity was in the macrophages of the red pulp. The uptake per cell volume was several times higher in the macrophage than in the hepatocyte. The radioactivity associated with macrophages was largely in endocytotic vacuoles and lysosomes. Binding of labelled pregnancy zone protein complex to peritoneal macrophages at 4 degrees C was 2-3 times higher than binding of the homologous alpha 2-macroglobulin complex. The two ligands competed for binding to the same receptors and the difference was due to a higher affinity of the pregnancy zone protein complex (Kd approx. 60 pM). After binding to the receptor, this ligand was internalised within 2-3 min at 37 degrees C and radioactivity inside the cells largely represented intact pregnancy zone protein complex. Radioactivity was released from the cell as iodotyrosine after a lag time of about 10 min. It is concluded that pregnancy zone protein complex is bound with a high affinity to the alpha 2-macroglobulin receptors in rat macrophages followed by receptor-mediated endocytosis and degradation of the ligand in the lysosomes.

Udgivelsesdato: 1988-May-15 Digestion of human alpha 2-macroglobulin-methylamine (alpha 2M-CH3NH2) with papain prior to gel filtration resulted in the resolution of three distinct peaks. The material in peak I (Mr approximately 600,000) and peak II (Mr approximately 55,000) did not have any receptor binding ability as determined by in vivo clearance studies and in vitro competitive binding studies using mouse peritoneal macrophages. In contrast, the material in peak III (Mr approximately 20,000) bound to macrophage alpha 2-macroglobulin (alpha 2M) receptors with a Kd of 250 nM. This represents a 500-fold decrease in affinity relative to undigested alpha 2M-CH3NH2. Sequence analysis demonstrated that this material constituted the carboxyl-terminal fragment (COOH-terminal fragment) of alpha 2M. alpha 2M is known to possess a methionyl residue which is susceptible to modification by cis-dichlorodiammineplatinum (II) (cis-DDP) with the result being a loss of receptor binding ability by alpha 2M. For this reason, experiments were performed to determine if the platinum-reactive methionyl residue is located in the COOH-terminal receptor binding fragment of alpha 2M. The results of this investigation demonstrate that cis-DDP is not reactive with either the isolated COOH-terminal fragment or the COOH-terminal fragment isolated from alpha 2M-CH3NH2 which had been pretreated with cis-DDP. In addition, the COOH-terminal fragment did not bind to monoclonal antibody 7H11D6, a monoclonal antibody which binds to the platinum-reactive epitope of the alpha 2M-CH3NH2 receptor recognition site. In contrast, the 55-kDa fragment of alpha 2M bound approximately 1 mol platinum/mol of 55-kDa fragment and also bound to monoclonal antibody 7H11D6. Since the COOH-terminal fragment retains some receptor binding ability, the results of this investigation demonstrate that this fragment is not the complete receptor recognition site and suggest that a platinum-reactive methionyl residue located in the 55-kDa fragment of alpha 2M is another component of this site.

Udgivelsesdato: 1995-Sep-18 The receptor-binding domains (RBDs) of human and bovine alpha 2-macroglobulin (alpha 2M) have been isolated after limited proteolysis of methylamine-treated alpha 2M with papain. Single crystals of the RBDs have been grown by vapour diffusion. Crystals of human RBD are very thin plates unsuited for data collection. However, crystals of RBD from bovine alpha 2M give diffraction patterns suitable for X-ray analysis, and a complete dataset with a maximum resolution of 2.3 A has been collected with synchrotron radiation at cryogenic temperature. The crystals belong to spacegroup P3(1)21 or P3(2)21 with cell parameters a = b = 106.8 A, c = 72.2 A.