The intronic deletion polymorphism of the Alpha2- macroglobulin gene modulates the severity and extent of atherosclerosis in the circle of Willis.
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Aged↗
Biomedical subjects
G Birkenmeier
Publications and source records attributed to G Birkenmeier.
Plasma levels of transforming growth factor-1beta and alpha2-macroglobulin before and after radical prostatectomy: association to clinicopathological parameters.
BACKGROUND: To study the levels of transforming growth factor-1beta (TGF-beta1) and of alpha2-macroglobulin (alpha2-M), a high affinity binding protein of TGF-beta1, in comparison to prostate-specific antigen (PSA) in prostate cancer (PCa) patients before and up to 12 months after prostatectomy, and to correlate the results with clinicopathological parameters. METHODS: Eighty-one patients who underwent radical prostatectomy for PCa were included in this study. Pre- and postoperatively, plasma levels of TGF-beta1, alpha2-M and PSA were measured in the same samples by ELISA, and were correlated with pathological parameters and clinical outcomes. RESULTS: The preoperative TGF-beta1 levels were significantly elevated as compared to the controls; they showed a positive correlation with the Gleason score. Patients with initial androgen-deprivation therapy had lower TGF-beta1 levels than untreated patients. Elevated concentrations of TGF-beta1 levelled off 12 months after prostatectomy approaching values of healthy individuals. Decreased plasma levels of total and transformed alpha2-M (proteinase-complexed form) were observed in PCa. Preoperative levels of TGF-beta1 but not of alpha2-M seem to be influenced by the body mass index (BMI). CONCLUSIONS: Elevated TGF-beta1 and decreased alpha2-M were consistently found in patients with PCa, and may be considered as risk factors for tumor development and progression. In comparison to PSA, the TGF-beta1 levels displayed a slow decline after radical prostatectomy; this indicates that TGF-beta1 is mainly produced outside the prostatic tissue. Since TGF-beta1 levels are influenced by the BMI, this indicates that PCa might be sensitive to diet.
Aged↗
Human alpha2-macroglobulin: genotype-phenotype relation.
A pentanucleotide deletion polymorphism in the gene of alpha2-macrolgobulin (alpha2-M) is suggested to be associated with late-onset Alzheimer's disease (AD), though controversial results have been reported. The underlying assumption is that the intronic pentanucleotide deletion may affect the biological function and quantity of the inhibitor and thus contribute to the AD pathology. In the present study we have analyzed the distribution of the deletion polymorphism within a group of 227 healthy Caucasians. In parallel studies, we determined the plasma concentrations of total and transformed alpha2-M. A strong correlation of the total concentration of alpha2-M with age was ascertained (r(s) = -0.54, P < 0.001). However, no significant correlation between age and the genotypes (P = 0.68) was detected, and no statistically significant effect of the genotype on the concentrations of total and transformed alpha2-M was found (P = 0.49 and 0.96, respectively). A significant correlation was observed between total and transformed alpha2-M in the genotype groups Ins/Ins (r(s) = 0.56, P < 0.001) and Ins/Del (r(s) = 0.35, P < 0.004). Furthermore, in the entire data set, a significantly elevated concentration of total alpha2-M was found in females as compared to males (P = 0.003). There was a slight but nonsignificant difference in the genotype distributions between males and females (P = 0.14). To test the proposed existence of genotype-specific alterations of functional properties of alpha2-M, we isolated alpha2-M from the plasma of carriers with different genetic background and analyzed the alpha2-M subunit structure as well as the binding of the inhibitor to growth factors/cytokines, to amyloid-beta and to the receptor. The experiments failed to reveal any genotype-specific functional alterations of the alpha2-M. The absence of abnormalities in alpha2-M mRNA and protein suggests that the alpha2-M deletion polymorphism is probably not associated with functional deficiencies important in AD pathology. However, it can be speculated that the observed general age-related alpha2-M deficiency may lead to accelerated accumulation of amyloid-beta, which might be relevant to AD pathology.
Adult↗
Alpha 2-macroglobulin-mediated degradation of amyloid beta 1--42: a mechanism to enhance amyloid beta catabolism.
Peptides derived from proteolytic degradation of the amyloid precursor protein, e.g., amyloid beta (A beta), are considered to be central to the pathology of Alzheimer's disease (AD). Soluble A beta is present in measurable concentrations in cerebrospinal fluid and blood. There are indications that soluble A beta present in circulation can cross the blood-brain barrier via transcytosis mediated by brain capillary endothelial cells. It implies that A beta originating from circulation may contribute to vascular and parenchymal A beta deposition in AD. Enhancing of A beta catabolism mediated by proteolytic degradation or receptor-mediated endocytosis could be a key mechanism to maintain low concentrations of soluble A beta. To launch A beta clearance we have exploited the A beta-degrading activity of diverse alpha 2-macroglobulin (alpha 2-M)-proteinase complexes. Complexes with trypsin, alpha-chymotrypsin, and bromelain strongly degrade (125)I-A beta 1--42 whereas complexes with endogenous proteinases, e.g., plasmin and prostate-specific antigen, were not effective. A beta degradation by the complexes was not inhibited by alpha 1-antichymotrypsin and soybean trypsin inhibitor which normally would inactivate the free serine proteinases. A prerequisite for A beta degradation is its binding to specific binding sites in alpha 2-M that may direct A beta to the active site of the caged proteinase. Ex vivo, enhanced degradation of (125)I-A beta 1--42 in blood could be achieved upon oral administration of high doses of proteinases to volunteers. These results suggest that up-regulation of A beta catabolism could probably reduce the risk of developing AD by preventing A beta accumulation in brain and vasculature.
Alzheimer Disease↗
Modulation of growth factor binding properties of alpha2-macroglobulin by enzyme therapy.
PURPOSE: To investigate the binding of transforming growth factor-beta (TGF-beta) to human alpha2-macroglobulin upon oral treatment of patients with proteases. METHODS: Volunteers were given a cocktail of active proteinases (Phlogenzym) composed of trypsin, bromelain and the additive rutoside orally over a period of 7 days at low dose followed by a bolus application. Before and after medication plasma was immediately withdrawn and binding of 125I-TGF-beta to the proteinase inhibitor alpha2-macroglobulin was determined by electrophoresis and gamma-counting. Cell culture experiments were performed to study the effect of transformed alpha2-macroglobulin on TGF-beta-stimulated proliferation of skin fibroblasts. RESULTS: Ingestion of proteinases was found to trigger the formation of intermediate forms of alpha2-macroglobulin displaying high affinity to TGF-beta. Maximum binding of TGF-beta was observed 1-2 h after bolus ingestion, and steadily levelled off with time. In vitro experiments demonstrated that complex formation of diverse proteinases (trypsin, alpha-chymotrypsin, bromelain and plasmin) with alpha2-macroglobulin conferred binding of 125I-TGF-beta, alpha2-Macroglobulin transformed by methylamine or proteinases was found to abolish the TGF-beta effect on fibroblasts in cell culture. CONCLUSIONS: Intestinal absorption of proteinases triggers the formation of TGF-beta binding species of alpha2-macroglobulin in blood. Mediated by this process high concentrations of TGF-beta might be reduced via enhanced clearance of alpha2-macroglobulin-TGF-beta complexes. Thus, proteinase therapy may have beneficial effects in treatment of fibrosis and certain cancers accompanied by excessively high TGF-beta concentrations.
Administration, Oral↗
Localization of alpha(2)-macroglobulin receptor/low-density lipoprotein receptor in third-trimester human placentas: a preliminary immunohistochemical study.
The alpha(2)-macroglobulin receptor/low-density lipoprotein receptor-related protein (alpha(2)-M-R/LRP) is involved in a variety of transcellular transportations, e.g. of apolipoprotein E, and represents an inhibitor of proteinases. After creation of monoclonal antibodies of the alpha- and beta-subunits of the alpha(2)-M-R/LRP and its receptor-associated protein, we have tested their distribution in five 3rd-trimester placentas, using cryosections, immunohistochemically and by immunofluorescence. All three monoclonal antibodies showed positive staining in villous and extravillous cytotrophoblasts and intravillous vessels, but not in syncytiotrophoblasts. Thus, alpha(2)-M-R/LRP plays a possible role in the transplacental nutritional transportation process. It is necessary to search the distribution of alpha(2)-M-R/LRP under pathological conditions, like fetal growth retardation and preeclampsia.
Female↗
Protease administration decreases enhanced transforming growth factor-beta 1 content in isolated glomeruli of diabetic rats.
Overproduction of transforming growth factor (TGF)-beta 1 messenger RNA is of fundamental importance in the pathogenesis of diabetic nephropathy. In vitro studies have recently shown that the serine protease trypsin diminishes the enhanced TGF-beta 1-expression induced by advanced glycation end products. Moreover, proteolytic enzymes may accelerate the removal of TGF-beta 1 from renal tissue via a protease-induced activation of alpha 2-macroglobulin (alpha 2M). This activation results in the binding of numerous cytokines, including TGF-beta 1 and is followed by enhanced plasma clearance of the protease alpha 2M-cytokine complex. In the present study in streptozotocin-diabetic rats we investigated whether the administration of Phlogenzym, a fixed combination of the proteases trypsin and bromelain combined with the antioxidant rutosid, modulates renal hypertrophy and the formation of TGF-beta 1 in isolated glomeruli. Three weeks after induction of diabetes, renal hypertrophy developed with an enhanced kidney/body weight ratio. When compared with normal rats, an elevated content of intraglomerular TGF-beta 1 (44.25 +/- 21.9 vs. 71.1 +/- 23.4 ng/microgram DNA, p < 0.05) as well as fibronectin (2.62 +/- 0.49 vs. 3.42 +/- 0.62 ng/microgram DNA, p < 0.05) was observed. In the diabetic rats, treatment with intraperitoneal proteases prevented the rise of intraglomerular TGF-beta 1 content (34.9 +/- 22.2 ng/microgram DNA, p < 0.01) and attenuated the rise of fibronectin (3.03 +/- 1.12 ng/microgram DNA NS). Furthermore, a decrease in the kidney/body weight ratio (p < 0.01) was achieved. Protease administration did not affect blood glucose concentration and was without visible adverse effects.
Animals↗
Clearance mechanism of prostate specific antigen and its complexes with alpha2-macroglobulin and alpha1-antichymotrypsin.
PURPOSE: To investigate the rate of elimination of prostate specific antigen (PSA) and its complexes with human alpha2-macroglobulin (alpha2-M) and alpha1-antichymotrypsin (ACT) and to elucidate the role of the alpha2-macroglobulin-receptor/low density lipoprotein receptor-related protein (alpha2-M-R/LRP) in the clearance mechanism. MATERIALS AND METHODS: PSA and complexes of PSA with alpha2-M and ACT were prepared and radiolabeled with [125I]Na (Amersham, Braunschweig, Germany). Radiolabeled proteins were injected into rats and the elimination of radioactivity from circulation was measured by gamma-counting of 20 microL aliquots over time. After 30 minutes different organs were removed and the total radioactivity was counted. The elimination rate and distribution of PSA and PSA-complexes was studied in the absence and presence of an excess of transformed alpha2-M. RESULTS: Radiolabeled PSA is rapidly eliminated from circulation with an initial half-life of 6.4+/-2.1 minutes mainly due to extraction by the liver and kidney. The clearance is slightly inhibited by transformed alpha2-M. PSA-alpha2-M is solely eliminated by the liver with a half-life of 6.7+/-1 minutes. Uptake by the liver is competitively inhibited by transformed alpha2-M. PSA-ACT is eliminated by the liver and kidney with an initial half-life of 3.51+/-1.1 minutes. Transformed alpha2-M failed to inhibit the clearance of PSA-ACT. CONCLUSIONS: Free PSA and PSA-inhibitor complexes are removed from the circulation by different clearance mechanisms. The sites of metabolism of the different forms of PSA are different but include liver and kidney as main organs for uptake. There are indications that alpha2-M-R/LRP is involved in PSA elimination. Thus, factors which modulate the receptor function and expression as well as the concentration of its natural ligands may interfere with the steady state concentrations of different PSA forms in blood.
Animals↗
Prostate-specific antigen triggers transformation of seminal alpha2-macroglobulin (alpha2-M) and its binding to alpha2-macroglobulin receptor/low-density lipoprotein receptor-related protein (alpha2-M-R/LRP) on human spermatozoa.
BACKGROUND: The aim of this study was to identify the proteolytic activity which triggers the transformation of human alpha2-macroglobulin (alpha2-M) in seminal fluid and its binding to its receptor. METHODS: Measurement of the concentrations of total and transformed alpha2-M in seminal fluid was accomplished by ELISA. Zymography of seminal plasma was performed in SDS-polyacrylamide gels containing casein as proteolytic substrate. Rate electrophoresis, SDS-PAGE, and Western blotting were applied to study the complex formation of prostate-specific antigen (PSA) with alpha-M. Ligand-binding analysis of sperm cells was performed using [125I] labeled proteins. Detection of receptor on sperm cells was achieved by immunofluorescence. RESULTS: The mean concentration of total alpha2-M in a random collection of seminal plasma was 4.6 microg/ml. On average, between 33-98% of the inhibitor was found to be transformed. Zymography of seminal plasma revealed a proteolytic activity which is associated with a 33-kDa protein identified as PSA. Its proteolytic activity could be inhibited by 0.2-M. Both purified PSA and seminal plasma were capable of transforming native alpha2-M. Binding of PSA to alpha2-M triggers the exposition of receptor binding sites in the inhibitor molecule, which causes binding of the complex to alpha2-M-R/LRP identified on spermatozoa. CONCLUSIONS: PSA, the main proteinase in seminal fluid, is responsible for the transformation of alpha2-M and for its binding to alpha2-M-R/LRP present on spermatozoa. The binding of alpha2-M-PSA complexes to the spermatozoa receptor may exert an impact on normal sperm-cell functions.
Binding Sites↗
Different expression of the alpha2-macroglobulin receptor/low-density lipoprotein receptor-related protein in human keratinocytes and fibroblasts.
We compared, using a combination of different immunological methods and by competitive PCR, the expression of the alpha2-macroglobulin receptor/low-density lipoprotein receptor-related protein (alpha2-M-R/LRP) in human keratinocytes and fibroblasts. This receptor has previously been found in skin only in dermal cells associated with fibroblasts and dendritic cells. For immunodetection we used mouse monoclonal antibodies against the two subunits of the receptor and against the receptor-associated protein (RAP), known as the regulatory protein of the receptor activity. The alpha2-M-R/LRP was found to be predominantly located intracellularly in keratinocytes whereas a distinct labelling of the outer membrane surface was found in fibroblasts. RAP is abundant in fibroblasts but is less expressed in keratinocytes. In frozen skin sections receptor immunoreactivity was detected in the epidermis with increased reactivity of basal keratinocytes, as well as in the dermis in association with dermal fibroblasts. By immunoprecipitation of biotinylated cell extracts, polypeptides were identified corresponding to the alpha-subunit and beta-subunit of the receptor as well as to the coprecipitating RAP. Competitive PCR revealed the presence of 67.9 and 2049.7 ag of alpha2-M-R/LRP mRNA per cell in keratinocytes and fibroblasts, respectively. The results demonstrate that both cell types express alpha-M-R/LRP mRNA and contain receptor protein as well as RAP but in different quantities and subcellular localizations.
Animals↗
Prostate-specific antigen forms complexes with human alpha 2-macroglobulin and binds to the alpha 2-macroglobulin receptor/LDL receptor-related protein.
PURPOSE: To investigate the binding of the prostate-specific antigen (PSA) to human alpha 2-macroglobulin (alpha 2-M) and to alpha 1-antichymotrypsin (ACT). MATERIALS AND METHODS: Binding analysis was evaluated by electrophoresis, Western-blotting, enzyme-linked immunosorption assay (ELISA) and size exclusion chromatography. Quantification of PSA and of different forms of alpha 2-M was performed using commercial test kits. The cleavage site of PSA in alpha 2-M was analyzed by SDS-PAGE and microsequencing. RESULTS: Binding of PSA to alpha 2-M is initiated by the cleavage of the peptide bond between amino acids Tyr 686 and Glu 687 of the bait region indicating a chymotrypsin-like activity of the PSA. The PSA's proteolytic cleavage triggers the transformation of alpha 2-M as detected by conformation-specific monoclonal antibodies. Kinetic analysis revealed faster binding of PSA to alpha 2-M than to ACT. The PSA bound to alpha 2-M is caged by the inhibitor and thus escapes detection by antibodies. This results in an incorrect calculation of the level of PSA when released from prostate into the blood. Complexes of PSA-alpha 2-M and PSA-ACT were found to bind to the alpha 2-macroglobulin receptor/LDL receptor-related protein (alpha 2-M-R/LRP) which may be the clearance receptor for PSA. CONCLUSIONS: Quantifying free PSA and PSA-ACT complexes, as routinely done in managing prostate-associated diseases, does not represent the total secretion capacity of the prostate. The proteinase inhibitor alpha 2-M has to be considered as a main contributor to PSA complex formation in the blood.
Amino Acid Sequence↗
Human leptin forms complexes with alpha 2-macroglobulin which are recognized by the alpha 2-macroglobulin receptor/low density lipoprotein receptor-related protein.
OBJECTIVE: To identify binding proteins of leptin in human plasma. METHODS: Binding was evaluated by electrophoresis, size exclusion chromatography (SEC), Western blotting, and radioisotope labeling. Quantification of leptin and the different forms of alpha2-macroglobulin (alpha2-M) was performed by ELISA. RESULTS: Leptin interacts with the proteinase inhibitor, alpha2-M. 125I-labeled leptin specifically binds to the transformed inhibitor, which arises by reaction with proteinases or with reactive primary amines. No leptin binding was observed to the native alpha2-M, which abundantly occurs in plasma. The complex formation between leptin and alpha2-M was found to proceed within minutes and was stable, as it resisted separation by SEC and electrophoresis. The Kd of the complex was 2.14 +/- 0.78 micromol/l. Complex formation with transformed alpha2-M did not interfere with the immunological determination of leptin in plasma. The leptin-alpha2-M complex was found to be recognized by the alpha2-M receptor/low density lipoprotein receptor-related protein. By computer analysis, a simple model is presented showing that the degree of transformation of alpha2-M may significantly influence the leptin concentration in blood. CONCLUSIONS: The proteinase inhibitor, alpha2-M, may act as a leptin-binding protein in human plasma. Binding of leptin to transformed alpha2-M and its rapid clearance by the alpha2-M receptor may significantly influence the bioavailability of leptin in human plasma.
Adult↗
Separation of paraproteins from human plasma by membrane chromatography.
Membrane chromatography using a commercially available blotting membrane was performed in a dead-end filtration mode to separate paraproteins from plasma of patients suffering from paraproteinemia. The affinity membrane was found to display distinct specificity to monoclonal IgG1. A dissociation constant (Kd) of 3.2 microM and a maximum binding capacity of 1.43 mg/cm2 IgG1 paraprotein were obtained from the adsorption isotherm of the affinity membrane. The membrane was found to absorb immunoglobulins species-dependently because no binding of immunoglobulins from mouse, rat and rabbit could be observed.
Adsorption↗
The subunits of alpha2-macroglobulin receptor/low density lipoprotein receptor-related protein, native and transformed alpha2-macroglobulin and interleukin 6 in Alzheimer's disease.
To explore the role of alpha2-macroglobulin receptor/low density lipoprotein receptor-related protein (alpha2M-R/LRP) and its ligands in the pathogenesis of Alzheimer's disease (AD), antibodies were raised against its alpha- and beta-subunits and their expression pattern in the CNS in AD and control cases was correlated with that of native and transformed alpha2-macroglobulin (alpha2M) and interleukin 6 (IL-6). The transmembranous beta-subunit of alpha2M-R/LRP and transformed alpha2M were found in plaque cores in AD. Extramembranous alpha-subunit and native alpha2M immunoreactivities were localized in activated plaque-associated astrocytes and extracellularly in plaques. IL-6 immunostaining was associated with neurofibrillary changes, and was also found extracellularly in the center of plaques and in microglial cells. Our finding that plaque cores contain a second transmembranous protein fragment, the beta-subunit of alpha2 M-R/LRP, suggests ongoing membrane-protein degradation. By altering clearance and scavenger-like functions, fragmentation and breakdown of alpha2M-R/LRP may have an important role in extracellular amyloid deposition and the formation of neurofibrillary tangles in AD.
Alzheimer Disease↗
Epitope mapping by screening of phage display libraries of a monoclonal antibody directed against the receptor binding domain of human alpha2-macroglobulin.
The human proteinase inhibitor, alpha2-macroglobulin (a2-M), inhibits a large number of proteinases. Alpha2-M-proteinase complexes are rapidly cleared from the circulation by binding to a cellular receptor (alpha2-M-R/LRP) via the receptor binding domain (RBD) which is made up of a 20 kDa C-terminal stretch of the 180 kDa monomer of the inhibitor. A monoclonal antibody (mab alpha-1) has been described which reacts with the receptor-recognizable form of the inhibitor, the so called transformed alpha2-M (a2-Mt). By screening of a phage display library an epitope in the RBD of the inhibitor was identified that reacts with mab alpha-1. Out of 25 phage clones a heptapeptide sequence (S-x1-x2-D-x3-x4-K) was obtained containing identical amino acids in three positions. A consensus peptide (S-R-S-D-P-P-K) was synthesized and found to displace alpha2-Mt from binding to mab alpha-1 and to receptor. The specificity of competition was demonstrated by a reversed peptide and a control antibody. By structural comparison it was found that the consensus heptapeptide mimics a discontinuous conformationally constrained epitope present in the RBD of the inhibitor. This is the first report describing the detection of discontinuous epitopes by phage display using a short linear peptide.
Amino Acid Sequence↗
[Mucinous adenocarcinomas of the large intestine in Crohn disease].
In two patients with Crohn's disease of long duration after more than 17 and 12 years respectively a mucinous adenocarcinoma of the ascending colon and a perianal mucinous carcinoma deriving from the anal ducts developed. The possible pathogenetic connections between chronic inflammatory bowel disease and tumour origin are considered. In particular, both the histologically identifiable dysplasia of the mucosa and the fistulae originating in the anal ducts are discussed.
Adenocarcinoma, Mucinous↗
Immunocytochemical demonstration of alpha 2-M-R/LRP on Müller (glial) cells isolated from rabbit and human retina.
The alpha 2-macroglobulin receptor/low-density lipoprotein receptor-related protein (alpha 2-M-R/LRP) is a multifunctional receptor which has been implicated in lipoprotein metabolism, clearance of proteinase-proteinase inhibitor complexes and regulation of growth factor/cytokine metabolism. This receptor is abundantly present in numerous tissues and organs such as liver, lung, placenta and brain. In brain it is expressed in neurones but not in normal macroglia. Using immunocytochemistry and monoclonal antibodies against the large extracellular receptor subunit we have detected alpha 2-M-R/LRP on enzymatically isolated retinal Müller (glial) cells. This receptor may be involved in vital functions of Müller cells.
Animals↗
Gene transfer mediated by alpha2-macroglobulin.
alpha2-Macroglobulin covalently linked to poly(L)-lysine can be used as a vehicle for receptor-mediated gene transfer. This modified alpha2-macroglobulin maintains its ability to bind to the alpha2-macroglobulin receptor, and was shown to introduce a luciferase reporter gene plasmid into HepG2 human hepatoma cells in vitro. The alpha2-macroglobulin receptor is a very large and multifunctional cell surface receptor, whose rapid and efficient internalization rate makes it attractive for gene therapy, e.g. for hepatic gene targeting via injection into the portal vein.
Gene Transfer Techniques↗