B. M. van den Berg

The Light Chain of Factor VIII Comprises a Binding Site for Low Density Lipoprotein Receptor-related Protein

In the present study, the interaction between the endocytic receptor low density lipoprotein receptor-related protein (LRP) and coagulation factor VIII (FVIII) was investigated. Using purified components, FVIII was found to bind to LRP in a reversible and dose-dependent manner (K d ≈ 60 nm). The interaction appeared to be specific because the LRP antagonist receptor-associated protein readily inhibited binding of FVIII to LRP (IC50 ≈ 1 nm). In addition, a 12-fold molar excess of the physiological carrier of FVIII,i.e. von Willebrand factor (vWF), reduced the binding of FVIII to LRP by over 90%. Cellular degradation of125I-labeled FVIII by LRP-expressing cells (≈ 8 fmol/105 cells after a 4.5-h incubation) was reduced by approximately 70% in the presence of receptor-associated protein. LRP-directed antibodies inhibited degradation to a similar extent, indicating that LRP indeed contributes to binding and transport of FVIII to the intracellular degradation pathway. Degradation of FVIII was completely inhibited by vWF. Because vWF binding by FVIII involves its light chain, LRP binding to this subunit was studied. In ligand blotting experiments, binding of FVIII light chain to LRP could be visualized. More detailed analysis revealed that FVIII light chain interacts with LRP with moderate affinity (k on≈ 5 × 104 m −1s−1; k off ≈ 2.5 × 10−3 s−1; K d ≈ 50 nm). Furthermore, experiments using recombinant FVIII C2 domain showed that this domain contributes to the interaction with LRP. In contrast, no association of FVIII heavy chain to LRP could be detected under the same experimental conditions. Collectively, our data demonstrate that in vitro LRP is able to bind FVIII at the cell surface and to mediate its transport to the intracellular degradation pathway. FVIII-LRP interaction involves the FVIII light chain, and FVIII-vWF complex formation plays a regulatory role in LRP binding. Our findings may explain the beneficial effect of vWF on thein vivo survival of FVIII.

THE SECOND AND FOURTH CLUSTER OF CLASS A CYSTEINE-RICH REPEATS OF THE LOW DENSITY LIPOPROTEIN RECEPTOR-RELATED PROTEIN SHARE LIGAND-BINDING PROPERTIES

The low density lipoprotein receptor-related protein (LRP) is a multifunctional endocytic cell-surface receptor that binds and internalizes a diverse array of ligands. The receptor contains four putative ligand-binding domains, generally referred to as clusters I, II, III, and IV. In this study, soluble recombinant receptor fragments, representing each of the four individual clusters, were used to map the binding sites of a set of structurally and functionally distinct ligands. Using surface plasmon resonance, we studied the binding of these fragments to methylamine-activated α2-macroglobulin, pro-urokinase-type plasminogen activator, tissue-type plasminogen activator (t-PA), plasminogen activator inhibitor-1, t-PA·plasminogen activator inhibitor-1 complexes, lipoprotein lipase, apolipoprotein E, tissue factor pathway inhibitor, lactoferrin, the light chain of blood coagulation factor VIII, and the intracellular chaperone receptor-associated protein (RAP). No binding of the cluster I fragment to any of the tested ligands was observed. The cluster III fragment only bound to the anti-LRP monoclonal antibody α2MRα3 and weakly to RAP. Except for t-PA, we found that each of the ligands tested binds both to cluster II and to cluster IV. The affinity rate constants of ligand binding to clusters II and IV and to LRP were measured, showing that clusters II and IV display only minor differences in ligand-binding kinetics. Furthermore, we demonstrate that the subdomains C3–C7 of cluster II are essential for binding of ligands and that this segment partially overlaps with a RAP-binding site on cluster II. Finally, we show that one RAP molecule can bind to different clusters simultaneously, supporting a model in which RAP binding to LRP induces a conformational change in the receptor that is incompatible with ligand binding.

Ligand-receptor interactions of the low density lipoprotein receptor-related protein, a multi-ligand endocytic receptor

Summary The low density lipoprotein receptor-related protein (LRP) is a large membrane glycoprotein that is a member of the low density lipoprotein (LDL) receptor family of endocytic receptors. In contrast to the restricted ligand specificity of the LDL receptor, LRP can bind and internalize a remarkable spectrum of structurally-unrelated classes of ligands suggesting a role for the receptor in diverse physiological and patho-physiological processes ranging from lipoprotein metabolism, cell growth and cell migration to atherosclerosis and Alzheimers disease. In this review we will summarize the current insights in the biology of LRP and particularly focus on the recent progress in our understanding of the molecular mechanisms that enable LRP to interact specifically with such a multitude of different ligands.

Arteether administration in humans: preliminary studies of pharmacokinetics, safety and tolerance

The recently developed artemisinin derivative arteether was administered by intramuscular injection to healthy male subjects in a single dose (n = 23) and a multiple dose study (n = 27). The drug was well tolerated. Clinical, neurological, electrocardiographic and biochemical monitoring did not reveal significant toxicity. Apart from some increase in eosinophil numbers, no haematological abnormality was seen. Preliminary pharmacokinetic data showed a long elimination half life of 25-72 h and marked accumulation in the multiple dose study.

A comparative study of a cationic peroxidase from peanut and an anionic peroxidase from petunia.

SummaryA comparative study on a pure cationic and a pure anionic protein from peanut cells and petunia stem tissue respectively, both with peroxidative activity, was made. The cationic protein weighs 44 Kd and the anionic 36 Kd. No immunological cross reactivity could be detected between the two proteins. In assays for peroxidative activity using the substrates 4-aminoantipyrine, guaiacol and eugenol it was noted that the anionic protein had 1.9, 12.7, and 27.7 fold greater enzymatic activity, respectively. For overall peroxidative measurements it is suggested that aminoantipyrine is probably the superior substrate. With regard to IAA oxidase activity of the two protein fractions it was noted that the cationic enzyme possessed optimal activity at pH 3.6 and the anionic protein at pH 7.0. The latter value could only be obtained by the addition of H2O2 and dichlorophenol (DCP). Since no additives were needed for the assay of IAA oxidation by the cationic protein it is suggested that this is a true IAA oxidase while the anionic fraction is a peroxidase involved in other reactions such as lignin biosynthesis.

Microvascular Damage in Type 1 Diabetic Patients Is Reversed in the First Year After Simultaneous Pancreas–Kidney Transplantation

Simultaneous pancreas–kidney transplantation (SPK) is an advanced treatment option for type 1 diabetes mellitus (DM) patients with microvascular disease including nephropathy. Sidestreamdarkfield (SDF) imaging has emerged as a noninvasive tool to visualize the human microcirculation. This study assessed the effect of SPK in diabetic nephropathy (DN) patients on microvascular alterations using SDF and correlated this with markers for endothelial dysfunction. Microvascular morphology was visualized using SDF of the oral mucosa in DN (n = 26) and SPK patients (n = 38), healthy controls (n = 20), DM1 patients (n = 15, DM ≥ 40 mL/min) and DN patients with a kidney transplant (KTx, n = 15). Furthermore, 21 DN patients were studied longitudinally up to 12 months after SPK. Circulating levels of angiopoietin‐1 (Ang‐1), angiopoietin‐2 (Ang‐2) and soluble thrombomodulin (sTM) were measured using ELISA. Capillary tortuosity in the DN (1.83 ± 0.42) and DM ≥ 40 mL/min (1.55 ± 0.1) group was increased and showed reversal after SPK (1.31 ± 0.3, p < 0.001), but not after KTx (1.64 ± 0.1). sTM levels were increased in DN patients and reduced in SPK and KTx recipients (p < 0.05), while the Ang‐2/Ang‐1 ratio was normalized after SPK and not after KTx alone (from 0.16 ± 0.04 to 0.08 ± 0.02, p < 0.05). Interestingly, in the longitudinal study, reversal of capillary tortuosity and decrease in Ang‐2/Ang‐1 ratio and sTM was observed within 12 months after SPK. SPK is effective in reversing the systemic microvascular structural abnormalities in DN patients in the first year after transplantation.

Genetics of the peroxidase isoenzymes in Petunia : Part 4. Location and developmental expression of the structural gene prxcC.

SummaryThree electrophoretic variants of the peroxidase b isoenzymes in Petunia have been found. The encoding gene prxB is shown to be located on chromosome I by its linkage with the gene Hfl. Analysis of prxB heterozygotes showed a gradual increase of the electrophoretic mobility of all three PRXb allozymes during development and differential expression in enzyme activity of three prxB alleles. The location of prxB on chromosome I was confirmed by an allelic dosage effect in trisomies I, trisomie segregation and the construction of trisomies I with triple-banded PRXb phenotype. From telotrisomic analysis it was concluded that prxB and Hfl are located on the same arm of chromosome I. The unexpected linkage of prxB and Hfl with the gene Fl in one of the crosses was suggested to be caused by a translocation in line SI, involving the gene Fl.

Genetics of the peroxidase isoenzymes in Petunia : Part 5. Differential temporal expression of prxA alleles.

SummaryIn P. hybrida and the putative progenitor species P. axillaris s. l. and P. integrifolia s. l. five mobility alleles of the structural gene prxA were found. The five alleles show differential expression during development of tissue and plant, caused by internal site mutations. Analysis of young but not yet flowering plants heterozygous for prxA showed that the allele prxA3 is expressed first, followed by the alleles prxA2, prxA5, prxA4 and prxA1, in that order. In mature leaves of young flowering plants the prxA2 allele has the highest expression level, followed by the alleles prxA3, prxA5, prxA4 and prxA1. In mature and old leaves of old plants the expression level of the alleles prxA2, prxA3, prxA4 and prxA5 is about equal, whereas that of the prxA1 allele is about twice as high. Crossing experiments suggested that between the plants used no external site differences exist that cause clearly detectable changes in developmental allozyme balance. Fast moving anodic peroxidases were detected that have a variable mobility over a considerable distance. The probability that these enzymes are precursors of the PRXa enzymes is discussed.

Location of structural genes for glucose phosphate isomerase and for leucyl aminopeptidase on chromosome VII of Petunia.

SummaryA gene termed gpiB, coding for one of the two isoenzyme zones of glucose phosphate isomerase in Petunia, has been mapped to a locus on chromosome VII by means of linkage to the marker An4, and by an allelic dosage effect on enzyme activity in trisomics. The high degree of linkage of electrophoretic alleles of gpiB to the pollen colour allele pair An4/an4, as demonstrated in the ancestral species, P. axillaris s.l. and P. integrifolia s.l., has been conserved in all cultivars of P. hybrida investigated. Another gene, coding for the enzyme leucyl-aminopeptidase could also be mapped to chromosome VII and the gene order An4 — lapB — gpiB determined. Apparently, distribution of lapB alleles is not related to the hybrid descent of P. hybrida.

Genetics of the peroxidase isoenzymes in Petunia : 8. Flower and root peroxidases.

SummaryIn root and flower corolla tissue of Petunia several anodic moving peroxidase isoenzymes are present, which cannot be detected in other organs. Alleles of the gene prxF control the presence or absence of several peroxidases that are only present in flower corolla tissue. Alleles of the gene prxG code for two peroxidases that can only be detected in root tissue. In addition to mutations of prxG that cause a change in the electrophoretic mobility of the PRXg enzymes, a mutation was also found that causes the absence of expression in enzyme activity. Crossing experiments indicated that this mutation is located in the gene prxG. Peroxidases encoded by the gene prxH were only found in root tissue. Two alleles of prxH were identified by electrophoretic variation; one allele is responsible for a single band, whereas the other allele could be recognized by a double-banded phenotype. The double-banded PRXh phenotype is suggested to be caused by tandem duplication, followed by mutation in one of the genes. A third prxH allele could be identified by the absence of PRXh activity. The genes prxF, prxG, and prxH were shown to be located on chromosome VII, with the following gene order: prxG-An4-lapB-gpiB-prxH-prxF.