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J Gliemann

Publications and source records attributed to J Gliemann.

At least 55 records · Page 3Linked to original sources
PubMed1995

Evidence that epithelial glycoprotein 330/megalin mediates uptake of polybasic drugs.

Glycoprotein 330 (gp330) is an endocytic receptor expressed in the renal proximal tubules and some other absorptive epithelia, e.g., in the inner ear. The present study shows that the antifibrinolytic polypeptide, aprotinin, and the nephro- and ototoxic antibiotics, aminoglycosides, and polymyxin B compete for binding of 125I-urokinase-plasminogen activator inhibitor type-1 complexes to purified rabbit gp330. Half maximal inhibition was measured at 4 microM for aprotinin, 50 microM for gentamicin, and 0.5 microM for polymyxin B. Drug binding to gp330 was validated by equilibrium dialysis of [3H] gentamicin-gp330 incubations and binding/uptake studies in rat proximal tubules and gp330-expressing L2 carcinoma cells. Analyses of mutant aprotinins expressed in Saccharomyces cerevisiae revealed that basic residues are essential for the binding to gp330 and renal uptake. The polybasic drugs also antagonized ligand binding to the human alpha 2-macroglobulin receptor. However, the rapid glomerular filtration of the drugs suggests kidney gp330 to be the quantitatively most important target. In conclusion, a novel role of gp330 as a drug receptor is demonstrated. The new insight into the mechanism of epithelial uptake of polybasic drugs might provide a basis for future design of drugs with reduced toxicity.

Animals↗
PubMed1994

A carboxyl-terminal fragment of lipoprotein lipase binds to the low density lipoprotein receptor-related protein and inhibits lipase-mediated uptake of lipoprotein in cells.

It has previously been shown that lipoprotein lipase can mediate uptake of remnant lipoprotein particles via binding to the low density lipoprotein receptor-related protein/alpha 2-macroglobulin receptor (LRP). Binding of lipoprotein lipase, and of triglyceride-rich lipoproteins associated with the lipase, to LRP depends on an intact carboxyl-terminal folding domain of the lipase (Nykjaer, A., Bengtsson-Olivecrona, G., Lookene, A., Moestrup, S. K., Petersen, C. M., Weber, W., Beisiegel, W., and Gliemann, J. (1993) J. Biol. Chem. 268, 15048-15055). Here we show that the site for binding to the receptor is within residues 380-425 of the bovine and residues 378-423 of the human lipoprotein lipase. We demonstrate that a carboxyl-terminal fragment of human lipoprotein lipase (residues 378-448), expressed as fusion protein in Escherichia coli, binds to purified and cellular LRP but not to lipoproteins. Binding of the fragment to purified LRP was blocked by heparin. In addition, the fragment inhibited the binding of lipase and the lipase-mediated binding of lipoproteins to the purified receptor. The fragment exhibited reduced binding to proteoglycan-deficient cells. Moreover, the fragment inhibited the uptake of lipoproteins in cells mediated by the lipase via binding to heparan sulfate proteoglycans and LRP. We conclude that the fragment contains the site for binding to LRP and a candidate site for interaction with heparan sulfate proteoglycans, whereas binding to lipoproteins is inefficient. The fragment can therefore inhibit the lipase-mediated lipoprotein uptake, a process that may promote the development of atherosclerosis when occurring in cells of the arterial wall.

Amino Acid Sequence↗
PubMed1994

Regions involved in binding of urokinase-type-1 inhibitor complex and pro-urokinase to the endocytic alpha 2-macroglobulin receptor/low density lipoprotein receptor-related protein. Evidence that the urokinase receptor protects pro-urokinase against binding to the endocytic receptor.

The alpha 2-macroglobulin receptor/low density lipoprotein receptor-related protein (alpha 2MR/LRP) binds several ligands, including complex between the two chain urokinase-type plasminogen activator (uPA) and type-1 plasminogen activator inhibitor (PAI-1), and the single chain zymogen pro-urokinase (pro-uPA). We have used truncated variants of uPA and PAI-1 as well as Fab fragments of monoclonal antibodies with known epitopes to identify regions in the uPA.PAI-1 complex and in pro-uPA involved in binding to alpha 2MR/LRP.uPA.PAI-1 complex bound with high affinity (EC50 about 0.4 nM) via contacts in the PAI-1 moiety as well as the uPA serine proteinase domain and the uPAA chain. Pro-uPA bound with lower affinity (EC50 about 10 nM), and efficient binding to alpha 2MR/LRP was dependent on contact with both the A chain and the serine proteinase domain. We analyzed the effect of complex formation with the urokinase receptor since this is the primary target for binding of uPA.PAI-1 and pro-uPA at the cell surface, and since it has been demonstrated that urokinase receptor-bound uPA.PAI-1 complex is internalized following interaction with alpha 2 MR/LRP (Nykjaer, A., Petersen, C. M., Møller, B., Jensen, P.H., Moestrup, S.K., Holtet, T.L., Etzerodt M., Thøgersen, H.C., Munch, M., Andreasen, P.A., and Gliemann, J. (1992) J. Biol. Chem. 267, 14543-14546). Soluble recombinant urokinase receptor blocked the binding of pro-uPA to alpha 2MR/LRP but caused only a slight reduction in the affinity for binding of uPA.PAI-1. Moreover, pro-uPA bound to the urokinase receptor at the cell surface was not internalized and degraded unless activated to uPA and complexed with PAI-1. We conclude that pro-uPA is protected against degradation via alpha 2MR/LRP when bound to uPAR due to shielding of a binding contact in the A chain, whereas the affinity of uPAR-bound uPA.PAI-1 complex for binding to alpha 2MR/LRP remains sufficient to allow rapid internalization and degradation.

3T3 Cells↗
PubMed1994

Receptor-binding domain of human alpha 2-macroglobulin. Expression, folding and biochemical characterization of a high-affinity recombinant derivative.

A recombinant version of the receptor binding domain (RBDv) of human alpha 2-macroglobulin (alpha 2M) has been expressed in E. coli and refolded using a novel iterative procedure. RBDv (Val1299-Ala1451) is extended by 15 residues at the N-terminal side of the Lys1313-Glu papain cleavage site in human alpha 2M. RBDv contains the intra-chain bridge Cys1329-Cys1444 and is soluble and monomeric. Competition experiments with 125I-labelled methylamine-treated alpha 2M reveal that RBDv binds to the placental receptor for transformed alpha 2M with a Kd of 8 nM, i.e. the binding affinity of RBDv is of the same order of magnitude as the intrinsic affinity for binding of one domain in transformed alpha 2M to one receptor molecule.

Amino Acid Sequence↗
PubMed1994

Receptor-mediated endocytosis of plasminogen activators and activator/inhibitor complexes.

Recent findings have elucidated the mechanism for clearance from the extracellular space of the two types of plasminogen activators, urokinase-type plasminogen activator (u-PA) and tissue-type plasminogen activator (t-PA), and their type-1 inhibitor (PAI-1). Activator/PAI-1 complexes and uncomplexed t-PA bind to the multi-ligand receptors alpha 2-macroglobulin receptor/low density lipoprotein receptor-related protein (alpha 2MR) and epithelial glycoprotein 330 (gp330). These receptors mediate endocytosis and degradation of u-PA/PAI-1 complex bound to the glycosyl phosphatidyl inositol-anchored urokinase receptor (u-PAR) on cell surfaces, and participate, in cooperation with other receptors, in hepatic clearance of activator/PAI-1 complexes and uncomplexed t-PA from blood plasma. The alpha 2MR- and gp330-mediated endocytosis of a ligand (u-PA/PAI-1 complex) initially bound to another receptor (u-PAR) is a novel kind of interaction between membrane receptors. Binding to alpha 2MR and gp330 is a novel kind of molecular recognition of serine proteinases and serpins.

Amino Acid Sequence↗
PubMed1994

Identification of glutamine and lysine residues in Alzheimer amyloid beta A4 peptide responsible for transglutaminase-catalysed homopolymerization and cross-linking to alpha 2M receptor.

The beta-amyloid peptide (beta A4), derived from a larger amyloid precursor protein, is the principal component of senile plaques in Alzheimer's disease. Here we report that the full-length (1-40) synthetic beta A4 peptide, containing one glutamine and two lysine residues, is able to form homopolymers in a transglutaminase-mediated reaction. Moreover, transglutaminase catalysed the formation of heteropolymers in reactions of beta A4 with alpha 2M receptor, a constituent of amyloid plaques, and with extracellular matrix proteins. Incorporation of site-specific probes followed by enzymatic digestion and sequencing of tracer-containing fractions demonstrated that both Lys16, Lys28 and Gln15 in beta A4 were susceptible to cross-linking by transglutaminase.

Alzheimer Disease↗
PubMed1994

Urokinase receptor. An activation antigen in human T lymphocytes.

The ability of activated T lymphocytes to extravasate and reach inflammatory and malignant foci in the tissues is a basic function of cellular immunity. Recent evidence strongly suggests that the urokinase receptor (uPAR) holds a central position in the development of human two-chain urokinase-mediated pericellular proteolysis and matrix degradation, an important element in cell migration. In this report we establish uPAR as a pan T cell activation Ag. As determined by FACS analysis, CD3+ lymphocytes from healthy donors exhibited no significant uPAR expression. In contrast, patients (e.g., HIV-positive donors) showed distinct uPAR expression, confined to HLA-DR+ cells, in up to 80% of all T cells. In vitro activation by PMA caused a rapid up-regulation of membrane uPAR in all healthy donor T cells and was accompanied by enhanced receptor synthesis and elevated uPAR mRNA levels. A similar induction resulted from activation via the TCR/CD3 complex using mitogens (PHA, and Con A), anti-CD3 antibodies, and alloantigen. Receptor expression at single cell level was also modulated by a number of cytokines. IL-2, IL-4 and IL-7 increased uPAR presentation on 20 to 50% of the T cell population, and combined stimulation of bulk cultures demonstrated an additive effect of IL-2 and IL-7, whereas the response to each of the two was inhibited by IL-4. In addition, TGF-beta 1 substantially reduced the uPAR expression in T cell cultures responding to PHA, IL-2, and IL-7. Irrespective of the activating reagent, the T cells appeared to produce the same molecular uPAR species, but the affinity of uPAR expressed in PMA blasts was decreased, presumably because of a differential location at the cell surface. All activated cultures showed co-expression of uPAR and CD25. The finding that the urokinase receptor is an activation Ag may suggest that cell-associated plasminogen activation is involved in extravasation and migration of activated T cells.

Base Sequence↗
PubMed1994

Cleaved intracellular plasminogen activator inhibitor 2 in human myeloleukaemia cells is a marker of apoptosis.

The proteolytic modification of plasminogen activator inhibitor 2 (PAI-2) was studied during apoptosis in the human promyelocytic leukaemic NB4 cell line during treatment with the phosphatase inhibitors okadaic acid and calyculin A as well as the protein synthesis inhibitor cycloheximide. The apoptic type of cell death was ascertained by morphological and biochemical criteria. In cell homogenates PAI-2 was probed by [125I]urokinase plasminogen activator (uPA) and detected as a sodium dodecyl sulphate-stable M(r) 80,000 complex after reducing sodium dodecyl sulphate-polyacrylamide gel electrophoresis and autoradiography. During apoptosis a smaller (M(r) 70,000) uPA-PAI-2 complex was consistently detected. The modification was in the PAI-2 moiety, as the [125I]uPA tracer could be extracted in its intact form from the complex. Thus the cleaved PAI-2 isoform is a biochemical marker of apoptosis in the promyelocytic NB4 cell line. The modified PAI-2 isoform was also detected in homogenates made from purified human mononuclear leukaemic cells aspirated from the bone marrow of patients suffering from acute and chronic myeloid leukaemia.

Apoptosis↗
PubMed1994

Expression of alpha 2-macroglobulin receptor/low density lipoprotein receptor-related protein and scavenger receptor in human atherosclerotic lesions.

Macrophage- and smooth muscle cell (SMC)-derived foam cells are typical constituents of human atherosclerotic lesions. At least three receptor systems have been characterized that could be involved in the development of foam cells: alpha 2-macroglobulin receptor/LDL receptor-related protein (alpha 2 MR/LRP), scavenger receptor, and LDL receptor. We studied the expression of these receptors in human atherosclerotic lesions with in situ hybridization and immunocytochemistry. An abundant expression of alpha 2MR/LRP mRNA and protein was found in SMC and macrophages in both early and advanced lesions in human aortas. alpha 2MR/LRP was also present in SMC in normal aortas. Scavenger receptor mRNA and protein were expressed in lesion macrophages but no expression was found in lesion SMC. LDL receptor was absent from the lesion area but was expressed in some aortas in medial SMC located near the adventitial border. The results demonstrate that (a) alpha 2MR/LRP is, so far, the only lipoprotein receptor expressed in lesions SMC in vivo; (b) scavenger receptors are expressed only in lesion macrophages; and (c) both receptors may play important roles in the development of human atherosclerotic lesions.

Adult↗
PubMed1993

Epithelial glycoprotein-330 mediates endocytosis of plasminogen activator-plasminogen activator inhibitor type-1 complexes.

Epithelial glycoprotein 330 (gp330) is structurally similar to the multifunctional alpha 2-macroglobulin receptor/low density lipoprotein receptor-related protein (alpha 2MR/LRP), gp330 and alpha 2MR/LRP bind Ca2+ with high affinity, and both receptors bind and mediate endocytosis of alpha 2MR-associated protein (RAP). In the present report, we describe that affinity-purified gp330 from rabbit renal cortex binds plasminogen activator inhibitor type-1 (PAI-1) complexed with urokinase-type plasminogen activator (uPA). alpha 2M-methylamine, which binds with high affinity to alpha 2MR/LRP, did not bind to gp330. The apparent Kd for binding of uPA.PAI-1 complexes was about 0.8 nM at 4 degrees C. The binding was calcium-dependent and inhibited by recombinant RAP (rRAP) and tissue type plasminogen activator-PAI-1 complexes. Thin sections of rabbit renal proximal tubules bound 125I-labeled uPA.PAI-1 and rRAP in the apical part of proximal tubules corresponding to the localization of gp330. The binding of 125I-uPA.PAI-1 complexes in tubules was abolished by excess unlabeled rRAP, and a rRAP-inhibitable endocytosis and degradation of labeled uPA.PAI-1 complexes was demonstrated by perfusion of isolated rabbit proximal tubules. The results establish an endocytotic function of gp330 and suggest that gp330 is an important component of the fibrinolytic system in gp330-containing epithelial as found in, for example, kidney and lung.

Amino Acid Sequence↗
PubMed1993

The alpha 2-macroglobulin receptor/low density lipoprotein receptor-related protein binds lipoprotein lipase and beta-migrating very low density lipoprotein associated with the lipase.

Lipoprotein lipase (LPL) causes a marked increase in the cellular binding of beta-migrating very low density lipoprotein (beta-VLDL) to a large receptor compatible with the alpha 2-macroglobulin receptor (alpha 2MR)/low density lipoprotein receptor-related protein (LRP) (Beisiegel, U., Weber, W., and Bengtsson-Olivecrona, G. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 8342-8346). Here we demonstrate that LPL binds to the alpha-chain of purified alpha 2MR/LRP immobilized on microtiter plates. The binding, apparently to multiple sites, was blocked by heparin and inhibited by the alpha 2MR-associated protein (alpha 2MRAP) and by EDTA. Immobilized LPL bound alpha 2MR/LRP in solution as well as beta-VLDL prepared from cholesterol-fed rabbits. Both binding reactions were dependent on an intact carboxyl-terminal folding domain of LPL, but were independent of its dimeric structure and intact catalytical function. Dimeric LPL could mediate binding of beta-VLDL to immobilized alpha 2MR/LRP and to cells, e.g. monocytes. In contrast, LPL monomers were not able to mediate binding to immobilized alpha 2MR/LRP, presumably because of cross-inhibition due to close relation between the binding regions for the lipoprotein and for the receptor in the carboxyl-terminal domain of the LPL monomer. Heparin, but not alpha 2MRAP, inhibited cellular binding of 125I-LPL or 125I-beta-VLDL supplemented with LPL. However, alpha 2MRAP inhibited degradation of the two ligands by about 90% and 40-50%, respectively. The results show that LPL is a ligand for alpha 2MR/LRP and, because of its affinity for lipoprotein particles, dimeric LPL can mediate or strengthen binding of beta-VLDL to this receptor. It is proposed that LPL binds primarily to cell surface heparan sulfate in monocytes and is presented for endocytosis and degradation by alpha 2MR/LRP. Moreover, beta-VLDL may be further supplemented with LPL at the cell surface and achieve affinity for alpha 2MR/LRP.

Animals↗
PubMed1993

Alpha 2-macroglobulin-proteinase complexes, plasminogen activator inhibitor type-1-plasminogen activator complexes, and receptor-associated protein bind to a region of the alpha 2-macroglobulin receptor containing a cluster of eight complement-type repeats.

A region containing sites for ligand binding was localized in the 4525-amino acid residue alpha 2-macroglobulin receptor/low density lipoprotein receptor-related protein (alpha 2MR/LRP) by ligand- and immunoblotting of proteinase and CNBr digests of the purified human placental protein. 125I-Labeled rat alpha 1-macroglobulin light chain, urokinase-plasminogen activator inhibitor type-1 complex, and alpha 2MR-associated protein all bound to a 75-kDa CNBr-generated fragment (68 kDa after deglycosylation). In addition to the three ligands, the fragment bound a novel monoclonal antibody reacting in the region defined by amino acid residues 1165-1246 as determined by binding to recombinant fragments of alpha 2MR/LRP. The positions of methionine residues in alpha 2MR/LRP suggested that the ligand-binding CNBr fragment contained three disulfide-linked peptides comprising the residues 776-1399. This origin was confirmed by partial amino acid sequencing of the electroblotted fragment and polypeptides generated by reduction of the fragment. The identified region represents 13.6% of the molecular mass (nonglycosylated) of alpha 2MR/LRP and contains one of three large clusters of complement-type repeats.

Amino Acid Sequence↗