Dietary supplementation of cystinotic mice by lysine inhibits the megalin pathway and decreases kidney cystine content.

Megalin/LRP2 is a major receptor supporting apical endocytosis in kidney proximal tubular cells. We have previously reported that kidney-specific perinatal ablation of the megalin gene in cystinotic mice, a model of nephropathic cystinosis, essentially blocks renal cystine accumulation and partially preserves kidney tissue integrity. Here, we examined whether inhibition of the megalin pathway in adult cystinotic mice by dietary supplementation (5x-fold vs control regular diet) with the dibasic amino-acids (dAAs), lysine or arginine, both of which are used to treat patients with other rare metabolic disorders, could also decrease renal cystine accumulation and protect cystinotic kidneys. Using surface plasmon resonance, we first showed that both dAAs compete for protein ligand binding to immobilized megalin in a concentration-dependent manner, with identical inhibition curves by L- and D-stereoisomers. In cystinotic mice, 2-month diets with 5x-L-lysine and 5x-L-arginine were overall well tolerated, while 5x-D-lysine induced strong polyuria but no weight loss. All diets induced a marked increase of dAA urinary excretion, most prominent under 5x-D-lysine, without sign of kidney insufficiency. Renal cystine accumulation was slowed down approx. twofold by L-dAAs, and totally suppressed by D-lysine. We conclude that prolonged dietary manipulation of the megalin pathway in kidneys is feasible, tolerable and can be effective in vivo.

Facile generation of monoclonal antibodies suitable for conjugation.

Monoclonal antibodies (mAb) are unique tools in therapeutics and immunodiagnostics applications but many of these applications rely on conjugated mAbs. Whether conjugating drugs or tracers, the conjugation process, frequently taking advantage of primary amines on lysine residues, may affect the binding activity of the antibodies. Furthermore, due to the sticky nature of many mAbs, unfavorable interactions may become eminent, with the result of high background signals. The workload associated with producing mAbs, able to withstand conjugation, preserving stability and affinity and avoiding off-target interactions, is comprehensive and related with only incidental success. We designed a method, where uncloned hybridomas were pre-selected for secretion of mAbs with the above characteristics. Using human collectin K1 (CL-K1, alias CL-11, Colec11) as a model antigen, mAbs present in culture supernatant from uncloned hybridomas were immobilized on Protein A beads, followed by solid phase biotinylation and subsequent elution. ELISA was employed to compare the binding activity of conjugated vs. unconjugated mAbs, and furthermore for their application in combination with other antibodies. From a group of 96 uncloned hybridomas we accomplished in obtaining five suitable mAbs, among which, two mAbs were superior. The successful conjugation of the selected mAbs with fluorophores and subsequent applications in microscopy and flow cytometry were further demonstrated. In conclusion, pre-selection of uncloned hybridomas, by testing of their mAbs' ability to withstand conjugation with tracers or drugs, is a successful strategy to avoid a huge workload of cloning numerous hybridomas, in order to obtain conjugatable mAbs.

Identification of the haemoglobin scavenger receptor.

Intravascular haemolysis is a physiological phenomenon as well as a severe pathological complication when accelerated in various autoimmune, infectious (such as malaria) and inherited (such as sickle cell disease) disorders. Haemoglobin released into plasma is captured by the acute phase protein haptoglobin, which is depleted from plasma during elevated haemolysis. Here we report the identification of the acute phase-regulated and signal-inducing macrophage protein, CD163, as a receptor that scavenges haemoglobin by mediating endocytosis of haptoglobin-haemoglobin complexes. CD163 binds only haptoglobin and haemoglobin in complex, which indicates the exposure of a receptor-binding neoepitope. The receptor-ligand interaction is Ca2+-dependent and of high affinity. Complexes of haemoglobin and multimeric haptoglobin (the 2-2 phenotype) exhibit higher functional affinity for CD 163 than do complexes of haemoglobin and dimeric haptoglobin (the 1-1 phenotype). Specific CD163-mediated endocytosis of haptoglobin-haemoglobin complexes is measurable in cells transfected with CD163 complementary DNA and in CD163-expressing myelo-monocytic lymphoma cells.

Haptoglobin and CD163: captor and receptor gating hemoglobin to macrophage lysosomes.

The plasma protein haptoglobin and the endocytic hemoglobin receptor HbSR/CD163 are key molecules in the process of removing hemoglobin released from ruptured erythrocytes. Hemoglobin in plasma is instantly bound with high affinity to haptoglobin--an interaction leading to the recognition of the complex by HbSR/CD163 and endocytosis in macrophages. The haptoglobin-dependent HbSR/CD163 scavenging system for hemoglobin clearance prevents toxic effects of hemoglobin in plasma and kidney and explains the decrease in the haptoglobin plasma concentration in patients with accelerated hemolysis. The HbSR/CD163 activity may be of quantitative importance for iron uptake in macrophages in general and for some iron-associated pathological processes, e.g. the atherogenesis-promoting oxidation of LDL leading to foam cell formation and apoptosis in the vessel wall.

Mutational analysis of affinity and selectivity of kringle-tetranectin interaction. Grafting novel kringle affinity ontp the trtranectin lectin scaffold.

C-type lectin-like domains are found in many proteins, where they mediate binding to a wide diversity of compounds, including carbohydrates, lipids, and proteins. The binding of a C-type lectin-like domain to a ligand is often influenced by calcium. Recently, we have identified a site in the C-type lectin-like domain of tetranectin, involving Lys-148, Glu-150, and Asp-165, which mediates calcium-sensitive binding to plasminogen kringle 4. Here, we investigate the effect of conservative substitutions of these and a neighboring amino acid residue. Substitution of Thr-149 in tetranectin with a tyrosine residue considerably increases the affinity for plasminogen kringle 4, and, in addition, confers affinity for plasminogen kringle 2. As shown by isothermal titration calorimetry analysis, this new interaction is stronger than the binding of wild-type tetranectin to plasminogen kringle 4. This study provides further insight into molecular determinants of importance for binding selectivity and affinity of C-type lectin kringle interactions.

Tetranectin-binding site on plasminogen kringle 4 involves the lysine-binding pocket and at least one additional amino acid residue.

Kringle domains are found in a number of proteins where they govern protein-protein interactions. These interactions are often sensitive to lysine and lysine analogues, and the kringle-lysine interaction has been used as a model system for investigating kringle-protein interactions. In this study, we analyze the interaction of wild-type and six single-residue mutants of recombinant plasminogen kringle 4 expressed in Escherichia coli with the recombinant C-type lectin domain of tetranectin and trans-aminomethyl-cyclohexanoic acid (t-AMCHA) using isothermal titration calorimetry. We find that all amino acid residues of plasminogen kringle 4 found to be involved in t-AMCHA binding are also involved in binding tetranectin. Notably, one amino acid residue of plasminogen kringle 4, Arg 32, not involved in binding t-AMCHA, is critical for binding tetranectin. We also find that Asp 57 and Asp 55 of plasminogen kringle 4, which both were found to interact with the low molecular weight ligand with an almost identical geometry in the crystal of the complex, are not of equal functional importance in t-AMCHA binding. Mutating Asp 57 to an Asn totally eliminates binding, whereas the Asp 55 to Asn, like the Arg 71 to Gln mutation, was found only to decrease affinity.

Crystallization and molecular-replacement solution of a truncated form of human recombinant tetranectin.

The two C-terminal domains, TN23 (residues 17-181), of human recombinant tetranectin, a plasminogen kringle 4 binding C-type lectin, have been crystallized in two different space groups. Using PEG 8000 as precipitant and at a pH of 8.5, crystals belonging to the monoclinic space group C2 are obtained, with unit-cell parameters a = 160.4, b = 44.7, c = 107.5 A, beta = 127.6 degrees. Using sodium formate as precipitant and at a pH of 5.0, TN23 crystallizes in a rhombohedral space group, with unit-cell parameters a = b = c = 107.4 A, alpha = beta = gamma = 78.3 degrees. A full data set to 4.5 A has been collected from the monoclinic crystals. Using the structure of full-length tetranectin, a molecular-replacement solution has been obtained. The crystal packing shows that TN23 crystallizes as a trimer, with one trimer in the asymmetric unit.

The heparin-binding site in tetranectin is located in the N-terminal region and binding does not involve the carbohydrate recognition domain.

Tetranectin is a homotrimeric plasma and extracellular-matrix protein that binds plasminogen and complex sulphated polysaccharides including heparin. In terms of primary and tertiary structure, tetranectin is related to the collectin family of Ca(2+)-binding C-type lectins. Tetranectin is encoded in three exons. Exon 3 encodes the carbohydrate recognition domain, which binds to kringle 4 in plasminogen at low levels of Ca(2+). Exon 2 encodes an alpha-helix, which is necessary and sufficient to govern the trimerization of tetranectin by assembling into a triple-helical coiled-coil structural element. Here we show that the heparin-binding site in tetranectin resides not in the carbohydrate recognition domain but within the N-terminal region, comprising the 16 amino acid residues encoded by exon 1. In particular, the lysine residues in the decapeptide segment KPKKIVNAKK (tetranectin residues 6-15) are shown to be of primary importance in heparin binding.

The plasminogen binding site of the C-type lectin tetranectin is located in the carbohydrate recognition domain, and binding is sensitive to both calcium and lysine.

Tetranectin, a homotrimeric protein belonging to the family of C-type lectins and structurally highly related to corresponding regions of the mannose-binding proteins, is known specifically to bind the plasminogen kringle 4 protein domain, an interaction sensitive to lysine. Surface plasmon resonance and isothermal calorimetry binding analyses using single-residue and deletion mutant tetranectin derivatives produced in Escherichia coli showed that the kringle 4 binding site resides in the carbohydrate recognition domain and includes residues of the putative carbohydrate binding site. Furthermore, the binding analysis revealed that the interaction is sensitive to calcium in addition to lysine.

Structure of the C-type lectin carbohydrate recognition domain of human tetranectin.

Tetranectin (TN) is a C-type lectin involved in fibrinolysis, being the only endogenous ligand known to bind specifically to the kringle 4 domain of plasminogen. TN was originally isolated from plasma, but shows a wide tissue distribution. Furthermore, TN has been found in the extracellular matrix of certain human carcinomas, whereas none or little is present in the corresponding normal tissue. The crystal structure of full-length trimeric TN (2.8 A resolution) has recently been published [Nielsen et al. (1997). FEBS Lett. 412, 388-396]. The crystal structure of the carbohydrate recognition domain (CRD) of human TN (TN3) has been determined separately at 2.0 A resolution in order to obtain detailed information on the two calcium binding sites. This information is essential for the elucidation of the specificity of TN towards oligosaccharides. TN3 crystallizes as a dimer, whereas it appears as a monomer in solution. The overall fold of TN3 is similar to other known CRDs. Each monomer is built of two distinct regions, one region consisting of six beta-strands and two alpha-helices, and the other region is composed of four loops harboring two calcium ions. The calcium ion at site 1 forms an eightfold coordinated complex and has Asp116, Glu120, Gly147, Glu150, Asn151, and one water molecule as ligands. The calcium ion at site 2, which is believed to be involved in recognition and binding of oligosaccharides, is sevenfold coordinated with ligands Gln143, Asp145, Glu150, Asp165, and two water molecules. One sulfate ion has been located at the surface of TN3, forming contacts to Glu120, Lys148, Asn106 of a symmetry-related molecule, and to an ethanol molecule.

Tetranectin, a trimeric plasminogen-binding C-type lectin.

Tetranectin, a plasminogen-binding protein belonging to the family of C-type lectins, was expressed in E. coli and converted to its native form by in vitro refolding and proteolytic processing. Recombinant tetranectin-as well as natural tetranectin from human plasma-was shown by chemical cross-linking analysis and SDS-PAGE to be a homo-trimer in solution as are other known members of the collectin family of C-type lectins. Biochemical evidence is presented showing that an N-terminal domain encoded within exons 1 and 2 of the tetranectin gene is necessary and sufficient to govern subunit trimerization.

Crystal structure of tetranectin, a trimeric plasminogen-binding protein with an alpha-helical coiled coil.

Tetranectin is a plasminogen kringle 4-binding protein. The crystal structure has been determined at 2.8 A resolution using molecular replacement. Human tetranectin is a homotrimer forming a triple alpha-helical coiled coil. Each monomer consists of a carbohydrate recognition domain (CRD) connected to a long alpha-helix. Tetranectin has been classified in a distinct group of the C-type lectin superfamily but has structural similarity to the proteins in the group of collectins. Tetranectin has three intramolecular disulfide bridges. Two of these are conserved in the C-type lectin superfamily, whereas the third is present only in long-form CRDs. Tetranectin represents the first structure of a long-form CRD with intact calcium-binding sites. In tetranectin, the third disulfide bridge tethers the CRD to the long helix in the coiled coil. The trimerization of tetranectin as well as the fixation of the CRDs relative to the helices in the coiled coil indicate a demand for high specificity in the recognition and binding of ligands.

Human plasminogen binding protein tetranectin: crystallization and preliminary X-ray analysis of the C-type lectin CRD and the full-length protein.

The recombinant human plasminogen binding protein tetranectin (TN) and the C-type lectin CRD of this protein (TN3) have been crystallized. TN3 crystallizes in the tetragonal space group P4(2)2(1)2 with cell dimensions a = b = 64.0, c = 75.7 A and with one molecule per asymmetric unit. The crystals diffract X-rays to at least 2.0 A resolution. A complete diffraction data set has been collected to 2.7 A resolution. The crystals of TN, obtained by the vapour-diffusion reverse salting-in method at 280 K, are rhombohedral, space group R3, with the hexagonal axes a = b = 89.1, c = 75.8 A, and diffract to at least 2.5 A. A full data set has been collected to 3.0 A. The asymmetric unit contains one monomer of TN. Molecular replacement solutions for TN3 and TN have been obtained using the structure of the C-type lectin CRD of rat mannose-binding protein as search model. The rhombohedral space group indicates that trimers of TN are formed in accordance with the observation of trimerization in solution.