Kathrin Weyer

Mutational analysis of the proteolytic domain of pregnancy-associated plasma protein-A (PAPP-A): classification as a metzincin

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.

Endocytic Receptors in the Renal Proximal Tubule

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.

Mouse model of proximal tubule endocytic dysfunction

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.

Megalin-mediated specific uptake of chitosan/siRNA nanoparticles in mouse kidney proximal tubule epithelial cells enables AQP1 gene silencing.

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.

Renal Uptake of 99mTc-Dimercaptosuccinic Acid Is Dependent on Normal Proximal Tubule Receptor–Mediated Endocytosis

99mTc-labeled dimercaptosuccinic acid (99mTc-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 99mTc-DMSA. Methods: Control or megalin/cubilin-deficient mice were injected intravenously with 0.5 MBq of 99mTc-DMSA or 99mTc-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 99mTc-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 99mTc-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 99mTc-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 99mTc-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: 99mTc-DMSA is filtered bound to α1-microglobulin and accumulates in the kidneys by megalin/cubilin-mediated endocytosis of the 99mTc-DMSA protein complex. Renal accumulation of 99mTc-DMSA is thus critically dependent on megalin/cubilin receptor function and therefore is a marker of proximal tubule endocytic activity.

A substrate specificity-determining unit of three Lin12-Notch repeat modules is formed in trans within the pappalysin-1 dimer and requires a sequence stretch C-terminal to the third module

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.

Generation of Urinary Albumin Fragments Does Not Require Proximal Tubular Uptake

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.

Increased lysosomal proteolysis counteracts protein accumulation in the proximal tubule during focal segmental glomerulosclerosis

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.

Papp-a2 modulates development of cranial cartilage and angiogenesis in zebrafish embryos

ABSTRACT 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.

Placental Regulation of Peptide Hormone and Growth Factor Activity by proMBP

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.