Search

The kidney proximal tubule is a major target tissue of the renin-angiotensin system (RAS). Megalin is an endocytic multiligand receptor abundantly expressed in the proximal tubule where it drives reabsorption of peptides and proteins from the glomerular ultrafiltrate. All major RAS components are present in the kidney proximal tubules. Here, megalin drives endocytosis of angiotensinogen (AGT), prorenin, and renin, while angiotensin-converting enzyme is localised at the brush border of the proximal tubule cells. Intrarenal formation of the key RAS effector angiotensin II (ANG II) occurs, and liver-derived AGT appears to be the primary source. New studies further suggest that megalin-mediated reabsorption of liver-derived AGT contributes to renal ANG II levels and thereby may influence renal RAS activity. This mini-review presents the recent advances on RAS in the proximal tubule and the involvement of megalin in the uptake and regulation of local RAS and discusses the possibility that megalin is involved in blood pressure regulation.

Many autoimmune diseases are characterized by germinal center (GC) derived affinity-matured, class-switched autoantibodies. Strategies to block GC formation and progression are currently being explored clinically, however, extrafollicular responses may also contribute to early events in autoimmunity. To investigate the relative contribution of these two pathways in autoimmune disease development, we leveraged a transgenic strategy to genetically block the GC pathway. Surprisingly, this accelerated extrafollicular responses and failed to curb autoimmune progression in two lupus models.In vitro, loss of the GC transcription factor Bcl-6 prevented cellular expansion and accelerated plasma cell differentiation, suggesting thein vivophenotype was caused by rewiring of B cell intrinsic transcriptional programming. In a competitive scenarioin vivo, B cells harboring the genetic GC block contributed disproportionately to the plasma cell output. Taken together, this emphasizes the extrafollicular pathway as a key contributor to autoimmune progression, and suggests that strategies aimed at blocking GCs should simultaneously target this pathway to avoid rerouting the pathogenic response.Highlights-A genetic GC block fails to prevent autoimmune progression in two lupus models-An intrinsic GC block drives B cell differentiation into terminally differentiated plasma cellsin vitro-B cells harboring a GC block competitively contribute to the plasma cell compartment in an autoreactive settingin vivo-Lupus mice with a GC block display immune complex deposition in kidney glomeruli that is indistinguishable from their wild-type counterpartsSummaryAffinity-matured autoantibodies generated in germinal centers are a hallmark of autoimmune diseases. Voss et al. block germinal centers in two autoimmune models, but surprisingly find that disease progresses unimpeded. They identify the extrafollicular pathway as a ‘backdoor to autoimmunity’.

Carrying the ε4 allele of the APOE gene encoding apolipoprotein E (APOE4) markedly increases the risk for late-onset Alzheimer's disease (AD), in which APOE4 exacerbates the brain accumulation and subsequent deposition of amyloid-β (Aβ) peptides. While the LDL receptor-related protein 1 (LRP1) is a major apoE receptor in the brain, we found that its levels are associated with those of insoluble Aβ depending on APOE genotype status in postmortem AD brains. Thus, to determine the functional interaction of apoE4 and LRP1 in brain Aβ metabolism, we crossed neuronal LRP1-knockout mice with amyloid model APP/PS1 mice and APOE3-targeted replacement (APO3-TR) or APOE4-TR mice. Consistent with previous findings, mice expressing apoE4 had increased Aβ deposition and insoluble amounts of Aβ40 and Aβ42 in the hippocampus of APP/PS1 mice compared with those expressing apoE3. Intriguingly, such effects were reversed in the absence of neuronal LRP1. Neuronal LRP1 deficiency also increased detergent-soluble apoE4 levels, which may contribute to the inhibition of Aβ deposition. Together, our results suggest that apoE4 exacerbates Aβ pathology through a mechanism that depends on neuronal LRP1. A better understanding of apoE isoform-specific interaction with their metabolic receptor LRP1 on Aβ metabolism is crucial for defining APOE4-related risk for AD.

Proximal tubule (PT) cells express a single saturable albumin-binding site whose affinity matches the estimated tubular concentration of albumin; however, albumin uptake capacity is greatly increased under nephrotic conditions. Deciphering the individual contributions of megalin and cubilin to the uptake of normal and nephrotic levels of albumin is impossible in vivo, as knockout of megalin in mice globally disrupts PT endocytic uptake. We quantified concentration-dependent albumin uptake in an optimized opossum kidney cell culture model and fit the kinetic profiles to identify albumin-binding affinities and uptake capacities. Mathematical deconvolution fit best to a three-component model that included saturable high- and low-affinity uptake sites for albumin and underlying nonsaturable uptake consistent with passive uptake of albumin in the fluid phase. Knockdown of cubilin or its chaperone amnionless selectively reduced the binding capacity of the high-affinity site, whereas knockdown of megalin impacted the low-affinity site. Knockdown of disabled-2 decreased the capacities of both binding sites. Additionally, knockdown of megalin or disabled-2 profoundly inhibited the uptake of a fluid phase marker, with cubilin knockdown having a more modest effect. We propose a novel model for albumin retrieval along the PT in which cubilin and megalin receptors have different functions in recovering filtered albumin in proximal tubule cells. Cubilin binding to albumin is tuned to capture normally filtered levels of the protein. In contrast, megalin binding to albumin is of lower affinity, and its expression is also essential for enabling the recovery of high concentrations of albumin in the fluid phase.

The megalin/cubilin receptor complex is required for proximal tubular endocytosis and degradation of filtered albumin. An additional high-capacity retrieval pathway of intact albumin for the recovery of large amounts of filtered albumin has been proposed, possibly involving cooperation between megalin/cubilin and the neonatal Fc receptor. To clarify the potential role of such a pathway, we examined the effects of megalin/cubilin gene inactivation on tubular albumin uptake and plasma albumin levels in nephrotic, podocin knockout mice. Immunofluorescence microscopy of megalin/cubilin/podocin knockout mouse kidneys demonstrated abolishment of proximal tubule albumin uptake, in contrast to the excessive albumin accumulation observed in podocin knockout mice compared to controls. Correspondingly, urinary albumin excretion was increased 1.4 fold in megalin/cubilin/podocin compared to podocin knockout mice (albumin/creatinine: 226 vs. 157 mg/mg). However, no difference in plasma albumin levels was observed between megalin/cubilin/podocin and podocin knockout mice, as both were reduced to approximately 40% of controls. There were no differences in liver albumin synthesis by mRNA levels and protein abundance. Thus, megalin/cubilin knockout efficiently blocks proximal tubular albumin uptake in nephrotic mice but plasma albumin levels did not differ as a result of megalin/cubilin-deficiency, suggesting no significance of the megalin/cubilin-pathway for albumin homeostasis by retrieval of intact albumin.

There is a great interest in exploring the mechanisms of oedema in nephrotic syndrome (NS) as evidenced by 2 parallel studies published in Acta Physiologica (1, 2). This has generated an editorial comment (3) which we respond to by this rebuttal. The interest in the nephrotic edema reflects not only the significant negative impact this has on the quality of life, but also the difficulties associated with identifying optimal treatment due to the lack of pathophysiological insight.

AIM: Activation of sodium reabsorption by urinary proteases has been implicated in sodium retention associated with nephrotic syndrome. The study was designed to test the hypothesis that nephrotic proteinuria in mice after conditional deletion of podocin leads to urokinase dependent, amiloride-sensitive plasmin-mediated sodium and water retention.METHODS: Ten days after podocin knockout, urine and feces was collected for 10 days in metabolic cages and analysed for electrolytes, plasminogen, protease activity, and ability to activate γENaC by patch clamp and western blot. Mice were treated with amiloride (2.5 mg-kg-1 for 2 days and 10 mg-kg-1 for 2 days) or an anti-urokinase-type plasminogen activator (uPA) targeting antibody (120 mg-kg-1 /24h) and compared to controls.RESULTS: Twelve days after deletion, podocin-deficient mice developed significant protein-and albuminuria associated with increased body weight, ascites, sodium accumulation, and suppressed plasma renin. This was associated with increased urinary excretion of plasmin and plasminogen that correlated with albumin excretion, urine protease activity co-migrating with active plasmin, and ability of urine to induce an amiloride-sensitive inward current in M1 cells in vitro. Amiloride treatment in podocin-deficient mice resulted in weight loss, increased sodium excretion, normalization of sodium balance and prevention of the activation of plasminogen to plasmin in urine in a reversible way. Administration of uPA targeting antibody abolished urine activation of plasminogen, attenuated sodium accumulation and prevented cleavage of γENaC.CONCLUSIONS: Nephrotic range glomerular proteinuria leads to urokinase dependent intratubular plasminogen activation and γENaC cleavage which contributes to sodium accumulation. This article is protected by copyright. All rights reserved.

Urinary albumin excretion is an important diagnostic and prognostic marker of renal function. Both animal and human urine contain large amounts of albumin fragments, but whether these fragments originate from renal tubular degradation of filtered albumin is unknown. Here, we used mice with kidneys lacking megalin and cubilin, the coreceptors that mediate proximal tubular endocytosis of albumin, to determine whether proximal tubular degradation of albumin forms the detectable urinary albumin fragments. After intravenous administration of (125)I-labeled mouse albumin to knockout and control mice, we examined kidney uptake of albumin and urinary excretion of both intact albumin and its fragments using size exclusion chromatography. In control mice, all labeled albumin eluted as albumin fragments in the urine. In megalin/cubilin-deficient mice, we observed decreased uptake and degradation of albumin and increased urinary excretion of intact albumin; we did not, however, detect a decrease in the excretion of albumin fragments. These results show that the generation of urinary albumin fragments occurs independently of renal tubular uptake and degradation of albumin, suggesting that the pathophysiological implications of changes in urinary albumin fragments require reevaluation.

The gene PRG2, encoding the proform of eosinophil major basic protein (proMBP), is one of the most highly expressed genes during human pregnancy, and low proMBP levels predict Down syndrome and poor pregnancy outcome. Reminiscent of a magnet, the primary structure of proMBP is extremely charge polarized, consisting of an N-terminal acidic propiece followed by a highly basic MBP domain in the C-terminal. Many tissues synthesize and secrete full-length proMBP, but only distinct cell types of the immune system process and store mature MBP in intracellular granules. MBP is released upon degranulation of eosinophil leukocytes and is toxic to bacteria, parasites, and mammalian cells. In contrast, proMBP is apparently nontoxic and functions in the inhibition of proteolysis and prohormone conversion. Recent research has revealed the complexity of proMBP biology and shed light on the process of MBP generation. ProMBP specifically forms disulfide-mediated, covalent complexes with the metzincin metalloproteinase pregnancy-associated plasma protein A (PAPPA) and the prohormone angiotensinogen (AGT). In both processes, PAPPA and AGT have reduced biological activity in the resulting complexes. In addition, proMBP is a component of high-molecular-weight AGT and, therefore, is potentially involved in the development of preeclampsia and in pregnancy-induced hypertension.

Members of the pappalysin family of metzincin metalloproteinases, pregnancy-associated plasma protein-A (PAPP-A, pappalysin-1) and PAPP-A2 (pappalysin-2), regulate the bioavailability of insulin-like growth factors (IGFs) by specific proteolytic inactivation of IGF-binding proteins (IGFBPs). PAPP-A cleaves IGFBP-4 and IGFBP-5, whereas PAPP-A2 cleaves only IGFBP-5. The pappalysins contain three Lin12-Notch repeat (LNR1-3) modules, previously considered unique to the Notch receptor family in which they function to regulate receptor cleavage. In contrast to the Notch receptor where three LNR modules are tandemly arranged, LNR3 is separated by more than 1000 residues from LNR1-2 in the pappalysin sequence. Each of the three LNR modules of PAPP-A is required for proteolysis of IGFBP-4, but not IGFBP-5. However, we here find that a C-terminal truncated variant of PAPP-A, which lacks LNR3 and therefore activity against IGFBP-4, cleaves IGFBP-4 when co-expressed with a PAPP-A variant, which is mutated in the active site. This suggests that LNR3 from the inactive subunit interacts in trans with LNR1-2 of the truncated PAPP-A subunit to form a functional trimeric LNR unit. We also show that formation of such a functional LNR unit depends on dimerization, as dissociation of a mutated non-covalent PAPP-A dimer results in reduced activity against IGFBP-4, but not IGFBP-5. Using PAPP-A/PAPP-A2 chimeras, we demonstrate that PAPP-A2 LNR1-2, but not LNR3, are functionally conserved with respect to IGFBP proteolysis. Additionally, we find that a sequence stretch C-terminal to LNR3 and single residues (Asp1521, Arg1529, and Asp1530) within this are required for LNR functionality. Udgivelsesdato: 2007-Apr-13

The activities of insulin-like growth factor (IGF)-I and -II are regulated by IGF-binding proteins (IGFBPs). Cleavage of IGFBP-4 by the metalloproteinase pregnancy-associated plasma protein-A (PAPP-A) causes release of bound IGF and has been established in several biological systems including the human reproductive system. Using flow cytometry, we first demonstrate that PAPP-A reversibly binds to the cell surface of several cell types analyzed. Heparin and heparan sulfate, but not dermatan or chondroitin sulfate, effectively compete for PAPP-A surface binding, and because incubation of cells with heparinase abrogated PAPP-A adhesion, binding is probably mediated by a cell surface heparan sulfate proteoglycan. Furthermore, the proteolytic activity of PAPP-A is preserved while bound to cells, suggesting that adhesion functions to target its activity to the vicinity of the IGF receptor, decreasing the probability that released IGF is captured by another IGFBP molecule before receptor binding. This mechanism potentially functions in both autocrine and paracrine regulation, as PAPP-A need not be synthesized in a cell to which it adheres. A truncated PAPP-A variant without the five short consensus repeats in the C-terminal third of the 1547-residue PAPP-A subunit, lacked surface binding. We also show that PAPP-A2, a recently discovered IGFBP-5 proteinase with homology to PAPP-A, does not bind cells. This finding allowed further mapping of the PAPP-A adhesion site to short consensus repeat modules 3 and 4 by the expression and analysis of nine PAPP-A/PAPP-A2 chimeras. Interestingly, the proteolytically inactive, disulfide-bound complex of PAPP-A and the proform of eosinophil major basic protein (proMBP), PAPP-A·proMBP, shows only weak surface binding, probably because the adhesion site of PAPP-A is occupied by heparan sulfate, known to be covalently bound to proMBP. This hypothesis was further substantiated by demonstrating that heparinase treatment of PAPP-A·proMBP restores surface binding. We finally propose a model in which IGF bioactivity is regulated by reversible cell surface binding of PAPP-A, which in turn is regulated by proMBP.

Pregnancy-associated plasma protein A2 (PAPP-A2, also known as pappalysin-2) is a large metalloproteinase that is known to be required for normal postnatal growth and bone development in mice. We here report the detection of zebrafish papp-a2 mRNA in the chordamesoderm, notochord and lower jaw of zebrafish (Danio rerio) embryos, and that papp-a2-knockdown embryos display broadened axial mesoderm, notochord bends and severely reduced cranial cartilages. Genetic data link these phenotypes to insulin-like growth factor (Igf)-binding protein-3 (Igfbp-3) and bone morphogenetic protein (Bmp) signaling, and biochemical analysis show specific Igfbp-3 proteolysis by Papp-a2, implicating Papp-a2 in the modulation of Bmp signaling by Igfbp-3 proteolysis. Knockdown of papp-a2 additionally resulted in angiogenesis defects, strikingly similar to previous observations in embryos with mutations in components of the Notch system. Accordingly, we find that Notch signaling is modulated by Papp-a2 in vivo, and, furthermore, that human PAPP-A2 is capable of modulating Notch signaling independently of its proteolytic activity in cell culture. Based on these results, we conclude that Papp-a2 modulates Bmp and Notch signaling by independent mechanisms in zebrafish embryos. In conclusion, these data link pappalysin function in zebrafish to two different signaling pathways outside the IGF system.

Pregnancy-associated plasma protein A2 (PAPP-A2, also known as pappalysin-2) is a large metalloproteinase that is known to be required for normal postnatal growth and bone development in mice. We here report the detection of zebrafish papp-a2 mRNA in the chordamesoderm, notochord and lower jaw of zebrafish ( Danio rerio ) embryos, and that papp-a2 -knockdown embryos display broadened axial mesoderm, notochord bends and severely reduced cranial cartilages. Genetic data link these phenotypes to insulin-like growth factor (Igf)-binding protein-3 (Igfbp-3) and bone morphogenetic protein (Bmp) signaling, and biochemical analysis show specific Igfbp-3 proteolysis by Papp-a2, implicating Papp-a2 in the modulation of Bmp signaling by Igfbp-3 proteolysis. Knockdown of papp-a2 additionally resulted in angiogenesis defects, strikingly similar to previous observations in embryos with mutations in components of the Notch system. Accordingly, we find that Notch signaling is modulated by Papp-a2 in vivo , and, furthermore, that human PAPP-A2 is capable of modulating Notch signaling independently of its proteolytic activity in cell culture. Based on these results, we conclude that Papp-a2 modulates Bmp and Notch signaling by independent mechanisms in zebrafish embryos. In conclusion, these data link pappalysin function in zebrafish to two different signaling pathways outside the IGF system.

The bioavailability of insulin-like growth factor (IGF)-I and -II is controlled by six IGF-binding proteins (IGFBPs 1–6). Bound IGF is not active, but proteolytic cleavage of the binding protein causes release of IGF. Pregnancy-associated plasma protein-A (PAPP-A) has recently been found to cleave IGFBP-4 in an IGF-dependent manner. To experimentally support the hypothesis that PAPP-A belongs to the metzincin superfamily of metalloproteinases, all containing the elongated zinc-binding motif HEXXHXXGXXH (His-482–His-492 in PAPP-A), we expressed mutants of PAPP-A in mammalian cells. Substitution of Glu-483 with Ala causes a complete loss of activity, defining this motif as part of the active site of PAPP-A. Interestingly, a mutant with Glu-483 replaced by Gln shows residual activity. Known metzincin structures contain a so-called Met-turn, whose strictly conserved Met residue is thought to interact directly with residues of the active site. By further mutagenesis we provide experimental evidence that Met-556 of PAPP-A, 63 residues from the zinc-binding motif, is located in a Met-turn of PAPP-A. Our hypothesis is also supported by secondary-structure prediction, and the ability of a 55-residue deletion mutant (d[S498-Y552]) to express and retain antigenecity. However, because PAPP-A differs in the features defining the individual established metzincin families, we suggest that PAPP-A belongs to a separate family. We also found that PAPP-A can undergo autocleavage, and that autocleaved PAPP-A is inactive. A lack of unifying elements in the sequences around the found cleavage sites of PAPP-A and a variant suggests steric regulation of substrate specificity.

RNAi-based strategies provide a great therapeutic potential for treatment of various human diseases including kidney disorders, but face the challenge of in vivo delivery and specific targeting. The chitosan delivery system has previously been shown to target siRNA specifically to the kidneys in mice when administered intravenously. Here we confirm by 2D and 3D bioimaging that chitosan formulated siRNA is retained in the kidney for more than 48 hours where it accumulates in proximal tubule epithelial cells (PTECs), a process that was strongly dependent on the molecular weight of chitosan. Chitosan/siRNA nanoparticles, administered to chimeric mice with conditional knockout of the megalin gene, distributed almost exclusively in cells that expressed megalin, implying that the chitosan/siRNA particle uptake was mediated by a megalin-dependent endocytotic pathway. Knockdown of the water channel aquaporin 1 (AQP1) by up to 50% in PTECs was achieved utilizing the systemic i.v. delivery of chitosan/AQP1 siRNA in mice. In conclusion, specific targeting PTECs with the chitosan nanoparticle system may prove to be a useful strategy for knockdown of specific genes in PTECs, and provides a potential therapeutic strategy for treating various kidney diseases.

Focal segmental glomerulosclerosis (FSGS) is a prevalent cause of end-stage renal disease, but the mechanisms underlying progression are unresolved. Lysosomal protein accumulation in the proximal tubule, mediated by megalin and cubilin endocytosis of increased amounts of filtered protein, is thought to result in inflammation and fibrosis. Here we determine whether release of inflammatory and fibrotic mediators in response to protein overload in the proximal tubule is caused by lysosomal enzyme deficits and insufficient proteolysis. As a model of FSGS, we used inducible podocyte-specific podocin-knockout mice analyzed at different time points. The content of megalin and cubilin ligands increased in the lysosomes after onset of proteinuria; however, protein and mRNA levels of megalin and cubilin showed only minor changes. To determine if the elevated lysosomal ligand content was caused by deficiency of enzymes, we analyzed protein and mRNA levels of lysosomal enzymes and found increased endogenous synthesis. Injection of dye-quenched fluorescent and iodinated albumin showed that proteolytic turnover in lysosomes of knockout mice adapted to the increased protein load. Inflammatory and fibrotic signals were increased early in disease, although the majority of lysosomes degraded endocytosed proteins effectively. Thus, insufficient lysosomal degradation in FSGS is not the cause of the inflammation and fibrosis during kidney disease.Kidney International advance online publication, 12 June 2013; doi:10.1038/ki.2013.218.

(99m)Tc-labeled dimercaptosuccinic acid ((99m)Tc-DMSA) accumulates in the kidney cortex and is widely used for imaging of the renal parenchyma. Despite its extensive clinical use, the mechanism for renal targeting of the tracer is unresolved. Megalin and cubilin are cooperating receptors essential to the proximal tubule endocytic uptake of proteins from the glomerular ultrafiltrate. We have used megalin/cubilin-deficient mice produced by gene knockout to determine whether receptor-mediated endocytosis is responsible for the renal uptake of (99m)Tc-DMSA. METHODS: Control or megalin/cubilin-deficient mice were injected intravenously with 0.5 MBq of (99m)Tc-DMSA or (99m)Tc-mercaptoacetyltriglycine (MAG3). Whole-body scintigrams and the activity in plasma, urine, and the kidneys were examined 6 h after injection. The size and identity of (99m)Tc-DMSA-bound proteins in urine were analyzed by fractionation by centrifugation and separation by sodium dodecyl sulfate polyacrylamide gel electrophoresis, followed by autoradiography and mass spectrometry. RESULTS: No renal accumulation of (99m)Tc-DMSA was identified in scintigrams of megalin/cubilin-deficient mice. The renal accumulated activity of the tracer was reduced to 11.4% (±2.5%, n = 7) of the normal uptake in control mice, correlating with a reduction in renal megalin/cubilin expression in knockout mice to about 10% of normal. The reduced renal uptake in megalin/cubilin-deficient mice was accompanied by an increase in the urinary excretion of (99m)Tc-DMSA. Size separation of the urine by ultracentrifugation and sodium dodecyl sulfate polyacrylamide gel electrophoresis demonstrated that in megalin/cubilin-deficient mice an increased amount of (99m)Tc-DMSA was excreted in an approximately 27-kDa form, which by mass spectrometry was identified as the plasma protein α1-microglobulin, an established megalin/cubilin ligand. CONCLUSION: (99m)Tc-DMSA is filtered bound to α1-microglobulin and accumulates in the kidneys by megalin/cubilin-mediated endocytosis of the (99m)Tc-DMSA protein complex. Renal accumulation of (99m)Tc-DMSA is thus critically dependent on megalin/cubilin receptor function and therefore is a marker of proximal tubule endocytic activity.

Protein reabsorption is a predominant feature of the renal proximal tubule. Animal studies show that the ability to rescue plasma proteins relies on the endocytic receptors megalin and cubilin. Recently, studies of patients with syndromes caused by dysfunctional receptors have supported the importance of these for protein clearance of human ultrafiltrate. This review focuses on the molecular biology and physiology of the receptors and their involvement in renal pathological conditions.

BACKGROUND: Several studies have indicated the central role of the megalin/cubilin multiligand endocytic receptor complex in protein reabsorption in the kidney proximal tubule. However, the poor viability of the existing megalin-deficient mice precludes further studies and comparison of homogeneous groups of mice.METHODS: Megalin- and/or cubilin-deficient mice were generated using a conditional Cre-loxP system, where the Cre gene is driven by the Wnt4 promoter. Kidney tissues from the mice were analysed for megalin and cubilin expression by quantitative reverse transcription-polymerase chain reaction, western blotting and immunohistochemistry. Renal albumin uptake was visualized by immunohistochemistry. Twenty-four-hour urine samples were collected in metabolic cages and analysed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and western blotting. Urinary albumin/creatinine ratios were measured by ELISA and the alkaline picrate method.RESULTS: The Meg(lox/lox);Cre(+), Cubn(lox/lox);Cre(+) and Meg(lox/lox),Cubn(lox/lox);Cre(+) mice were all viable, fertile and developed normal kidneys. Megalin and/or cubilin expression, assessed by immunohistology and western blotting, was reduced by >89%. Consistent with this observation, the mice excreted megalin and cubilin ligands such as transferrin and albumin in addition to low-molecular weight proteins. We further show that megalin/cubilin double-deficient mice excrete albumin with an average of 1.45 ± 0.54 mg/day, suggesting a very low albumin concentration in the glomerular ultrafiltrate.CONCLUSIONS: We report here the efficient genetic ablation of megalin, cubilin or both, using a Cre transgene driven by the Wnt4 promoter. The viable megalin/cubilin double-deficient mice now allow for detailed large-scale group analysis, and we anticipate that the mice will be of great value as an animal model for proximal tubulopathies with disrupted endocytosis.

The gene PRG2, encoding the proform of eosinophil major basic protein (proMBP), is one of the most highly expressed genes during human pregnancy, and low proMBP levels predict Down syndrome and poor pregnancy outcome. Reminiscent of a magnet, the primary structure of proMBP is extremely charge polarized, consisting of an N-terminal acidic propiece followed by a highly basic MBP domain in the C-terminal. Many tissues synthesize and secrete full-length proMBP, but only distinct cell types of the immune system process and store mature MBP in intracellular granules. MBP is released upon degranulation of eosinophil leukocytes and is toxic to bacteria, parasites, and mammalian cells. In contrast, proMBP is apparently nontoxic and functions in the inhibition of proteolysis and prohormone conversion. Recent research has revealed the complexity of proMBP biology and shed light on the process of MBP generation. ProMBP specifically forms disulfide-mediated, covalent complexes with the metzincin metalloproteinase pregnancy-associated plasma protein A (PAPPA) and the prohormone angiotensinogen (AGT). In both processes, PAPPA and AGT have reduced biological activity in the resulting complexes. In addition, proMBP is a component of high-molecular-weight AGT and, therefore, is potentially involved in the development of preeclampsia and in pregnancy-induced hypertension.

The Lin12-Notch repeat (LNR) module of about 35 residues is a hallmark of the Notch receptor family. Three copies, arranged in tandem, are invariably present in the extracellular portion of the Notch receptors. Although their function is unknown, genetic and biochemical data indicate that the LNR modules participate in the regulation of ligand-induced proteolytic cleavage of the Notch receptor, a prerequisite to intramembrane cleavage and Notch signaling. Outside the Notch receptor family, the LNR module is present only in the metalloproteinase pregnancy-associated plasma protein-A (PAPP-A) and its homologue PAPP-A2, which also contain three copies. Curiously, LNR modules 1 and 2 are present within the proteolytic domain of PAPP-A/A2, but LNR3 is separated from LNR2 by more than 1000 amino acids. The growth factor antagonists insulin-like growth factor-binding protein (IGFBP)-4 and -5 are both substrates of PAPP-A. We provide here evidence that the PAPP-A LNR modules function together to determine the proteolytic specificity of PAPP-A. Analysis of C-terminally truncated PAPP-A mutants followed by the analysis of LNR deletion mutants demonstrated that each of the three PAPP-A LNR modules is strictly required for proteolytic activity against IGFBP-4 but not for proteolytic activity against IGFBP-5. Individual substitution of conserved LNR residues predicted to participate in calcium coordination caused elimination (D341A, D356A, D389A, D1484A, D1499A, and D1502A) or a significant reduction (D359A and E392A) of IGFBP-4 proteolysis, whereas IGFBP-5 proteolysis was unaffected. The activity of the latter mutants against IGFBP-4 could be partially rescued by calcium, and the addition of the calcium-binding protein calbindin D9k to wild-type PAPP-A eliminated activity against IGFBP-4 but not against IGFBP-5, demonstrating that the PAPP-A LNR modules bind calcium ions. We propose a model in which LNR3 is spatially localized in proximity to LNR1 and -2, forming a single functional unit.

The metalloproteinase pregnancy-associated plasma protein-A (PAPP-A) cleaves a subset of insulin-like growth factor binding proteins (IGFBP), which inhibit the activities of insulin-like growth factor (IGF). Through this proteolytic activity, PAPP-A is believed to regulate IGF bioavailability in several biological systems, including the human reproductive system and the cardiovascular system. PAPP-A adheres to mammalian cells by interactions with glycosaminoglycan (GAG), thus targeting the proteolytic activity of PAPP-A to the cell surface. Based on site-directed mutagenesis, we here delineate the PAPP-A GAG-binding site in the C-terminal modules CCP3 and CCP4. Using heparin affinity chromatography, commonly employed in such studies, we define three clusters of arginines and lysines of CCP3, which are important for the interaction of PAPP-A with heparin. In a model of PAPP-A CCP3-CCP4, basic residues of these sequence clusters form a contiguous patch located on one side of the structure. Binding to the unknown, natural cell surface receptor of PAPP-A, assessed by flow cytometry, also depends on residues of these three basic clusters. However, single or double residue substitutions generally have a modest effect on PAPP-A heparin binding assessed by chromatography, but cell surface adhesion was critically reduced by several of these substitutions, emphasizing the relevance of analysis by flow cytometry. The contributions of positively charged residues located in CCP4 were all minor when analyzed by heparin affinity chromatography. However, the mutation of CCP4 residues Arg1459 and Lys1460 to Ala almost abrogated cell surface adhesion. Furthermore, when acidic residues of the homologous proteinase PAPP-A2 (Asp1547, Glu1555 and Glu1567) were introduced into the corresponding positions in the sequence of PAPP-A, located in each of the three basic clusters of CCP3, binding to heparin was strongly impaired and cell surface binding was abrogated. This explains, at least in part, why PAPP-A2 lacks the ability of cell surface adhesion, and further emphasizes the role of the basic clusters defined in PAPP-A.

The activities of insulin-like growth factor (IGF)-I and -II are regulated by IGF-binding proteins (IGFBPs). Cleavage of IGFBP-4 by the metalloproteinase pregnancy-associated plasma protein-A (PAPP-A) causes release of bound IGF and has been established in several biological systems including the human reproductive system. Using flow cytometry, we first demonstrate that PAPP-A reversibly binds to the cell surface of several cell types analyzed. Heparin and heparan sulfate, but not dermatan or chondroitin sulfate, effectively compete for PAPP-A surface binding, and because incubation of cells with heparinase abrogated PAPP-A adhesion, binding is probably mediated by a cell surface heparan sulfate proteoglycan. Furthermore, the proteolytic activity of PAPP-A is preserved while bound to cells, suggesting that adhesion functions to target its activity to the vicinity of the IGF receptor, decreasing the probability that released IGF is captured by another IGFBP molecule before receptor binding. This mechanism potentially functions in both autocrine and paracrine regulation, as PAPP-A need not be synthesized in a cell to which it adheres. A truncated PAPP-A variant without the five short consensus repeats in the C-terminal third of the 1547-residue PAPP-A subunit, lacked surface binding. We also show that PAPP-A2, a recently discovered IGFBP-5 proteinase with homology to PAPP-A, does not bind cells. This finding allowed further mapping of the PAPP-A adhesion site to short consensus repeat modules 3 and 4 by the expression and analysis of nine PAPP-A/PAPP-A2 chimeras. Interestingly, the proteolytically inactive, disulfide-bound complex of PAPP-A and the proform of eosinophil major basic protein (proMBP), PAPP-A.proMBP, shows only weak surface binding, probably because the adhesion site of PAPP-A is occupied by heparan sulfate, known to be covalently bound to proMBP. This hypothesis was further substantiated by demonstrating that heparinase treatment of PAPP-A.proMBP restores surface binding. We finally propose a model in which IGF bioactivity is regulated by reversible cell surface binding of PAPP-A, which in turn is regulated by proMBP.

Pregnancy-associated plasma protein A2 (PAPP-A2, also known as pappalysin-2) is a large metalloproteinase that is known to be required for normal postnatal growth and bone development in mice. We here report the detection of zebrafish papp-a2 mRNA in the chordamesoderm, notochord and lower jaw of zebrafish ( Danio rerio ) embryos, and that papp-a2 -knockdown embryos display broadened axial mesoderm, notochord bends and severely reduced cranial cartilages. Genetic data link these phenotypes to insulin-like growth factor (Igf)-binding protein-3 (Igfbp-3) and bone morphogenetic protein (Bmp) signaling, and biochemical analysis show specific Igfbp-3 proteolysis by Papp-a2, implicating Papp-a2 in the modulation of Bmp signaling by Igfbp-3 proteolysis. Knockdown of papp-a2 additionally resulted in angiogenesis defects, strikingly similar to previous observations in embryos with mutations in components of the Notch system. Accordingly, we find that Notch signaling is modulated by Papp-a2 in vivo , and, furthermore, that human PAPP-A2 is capable of modulating Notch signaling independently of its proteolytic activity in cell culture. Based on these results, we conclude that Papp-a2 modulates Bmp and Notch signaling by independent mechanisms in zebrafish embryos. In conclusion, these data link pappalysin function in zebrafish to two different signaling pathways outside the IGF system.

The bioavailability of insulin-like growth factor (IGF)-I and -II is controlled by six IGF-binding proteins (IGFBPs 1-6). Bound IGF is not active, but proteolytic cleavage of the binding protein causes release of IGF. Pregnancy-associated plasma protein-A (PAPP-A) has recently been found to cleave IGFBP-4 in an IGF-dependent manner. To experimentally support the hypothesis that PAPP-A belongs to the metzincin superfamily of metalloproteinases, all containing the elongated zinc-binding motif HEXXHXXGXXH (His-482-His-492 in PAPP-A), we expressed mutants of PAPP-A in mammalian cells. Substitution of Glu-483 with Ala causes a complete loss of activity, defining this motif as part of the active site of PAPP-A. Interestingly, a mutant with Glu-483 replaced by Gln shows residual activity. Known metzincin structures contain a so-called Met-turn, whose strictly conserved Met residue is thought to interact directly with residues of the active site. By further mutagenesis we provide experimental evidence that Met-556 of PAPP-A, 63 residues from the zinc-binding motif, is located in a Met-turn of PAPP-A. Our hypothesis is also supported by secondary-structure prediction, and the ability of a 55-residue deletion mutant (d[S498-Y552]) to express and retain antigenecity. However, because PAPP-A differs in the features defining the individual established metzincin families, we suggest that PAPP-A belongs to a separate family. We also found that PAPP-A can undergo autocleavage, and that autocleaved PAPP-A is inactive. A lack of unifying elements in the sequences around the found cleavage sites of PAPP-A and a variant suggests steric regulation of substrate specificity.