Henrik Vorum

An Endocytic Pathway Essential for Renal Uptake and Activation of the Steroid 25-(OH) Vitamin D3

Steroid hormones may enter cells by diffusion through the plasma membrane. However, we demonstrate here that some steroid hormones are taken up by receptor-mediated endocytosis of steroid-carrier complexes. We show that 25-(OH) vitamin D3 in complex with its plasma carrier, the vitamin D-binding protein, is filtered through the glomerulus and reabsorbed in the proximal tubules by the endocytic receptor megalin. Endocytosis is required to preserve 25-(OH) vitamin D3 and to deliver to the cells the precursor for generation of 1,25-(OH)2 vitamin D3, a regulator of the calcium metabolism. Megalin-/- mice are unable to retrieve the steroid from the glomerular filtrate and develop vitamin D deficiency and bone disease.

Improved silver staining protocols for high sensitivity protein identification using matrix-assisted laser desorption/ionization-time of flight analysis.

In proteomics it is essential to be able to detect proteins separated by gel electrophoresis at high sensitivity. Silver staining is currently the most popular method. Here we present silver staining protocols that are optimized for staining sensitivity, peptide recovery and compatibility with digestion and mass spectrometry.

Megalin Knockout Mice as an Animal Model of Low Molecular Weight Proteinuria

Megalin is an endocytic receptor expressed on the luminal surface of the renal proximal tubules. The receptor is believed to play an important role in the tubular uptake of macromolecules filtered through the glomerulus. To elucidate the role of megalin in vivo and to identify its endogenous ligands, we analyzed the proximal tubular function in mice genetically deficient for the receptor. We demonstrate that megalin-deficient mice exhibit a tubular resorption deficiency and excrete low molecular weight plasma proteins in the urine (low molecular weight proteinuria). Proteins excreted include small plasma proteins that carry lipophilic compounds including vitamin D-binding protein, retinol-binding protein, alpha(1)-microglobulin and odorant-binding protein. Megalin binds these proteins and mediates their cellular uptake. Urinary loss of carrier proteins in megalin-deficient mice results in concomitant loss of lipophilic vitamins bound to the carriers. Similar to megalin knockout mice, patients with low molecular weight proteinuria as in Fanconi syndrome are also shown to excrete vitamin/carrier complexes. Thus, these results identify a crucial role of the proximal tubule in retrieval of filtered vitamin/carrier complexes and the central role played by megalin in this process.

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.

Solubility of long-chain fatty acids in phosphate buffer at pH 7.4

The solubility of the saturated fatty acids lauric, myristic, palmitic, and stearic acid and the unsaturated oleic acid at 37 degrees C in phosphate buffer (pH 7.4) was estimated by using two independent methods. The one was a conventional solubility technique measuring the concentration of dissolved fatty acid in buffer by using radioactive compounds. The other was a dialysis exchange technique monitoring possible aggregation of solvated fatty acid anions by measuring the rate of diffusion of labelled compound across a dialysis membrane under conditions of chemical equilibrium. It was found that the results were strongly dependent on the radiochemical purity of the fatty acids. Using highly purified samples of radioactively labelled fatty acids, the solubility of monomeric laurate was shown to be greater than 500 microM, whereas the solubility of monomeric myristate was found to be 20-30 microM. Palmitate, stearate, and oleate solutions, on the other hand, showed a tendency to aggregation even at concentrations below 1 microM. Special attention was given to palmitate, as a reference compound for long-chain fatty acids, and the solubility of monomeric palmitate was estimated to be lower than 10(-10) M.

The CREC family, a novel family of multiple EF-hand, low-affinity Ca2+-binding proteins localised to the secretory pathway of mammalian cells

The CREC family consists of a number of recently discovered multiple (up to seven) EF‐hand proteins that localise to the secretory pathway of mammalian cells. At present, the family includes reticulocalbin, ERC‐55/TCBP‐49/E6BP, Cab45, calumenin and crocalbin/CBP‐50. Similar proteins are found in quite diverse invertebrate organisms such as DCB‐45 and SCF in Drosophila melanogaster, SCF in Bombyx mori, CCB‐39 in Caenorhabditis elegans and Pfs40/PfERC in Plasmodium falciparum. The Ca2+ affinity is rather low with dissociation constants around 10−4–10−3 M. The proteins may participate in Ca2+‐regulated activities. Recent evidence has been obtained that some CREC family members are involved in pathological activities such as malignant cell transformation, mediation of the toxic effects of snake venom toxins and putative participation in amyloid formation.

Heterogeneous nuclear ribonucleoproteins H, H', and F are members of a ubiquitously expressed subfamily of related but distinct proteins encoded by genes mapping to different chromosomes.

Molecular cDNA cloning, two-dimensional gel immunoblotting, and amino acid microsequencing identified three sequence-unique and distinct proteins that constitute a subfamily of ubiquitously expressed heterogeneous nuclear ribonucleoproteins corresponding to hnRNPs H, H′, and F. These proteins share epitopes and sequence identity with two other proteins, isoelectric focusing sample spot numbers 2222 (37.6 kDa; pI 6.5) and 2326 (39.5 kDa; pI 6.6), indicating that the subfamily may contain additional members. The identity between hnRNPs H and H′ is 96%, between H and F 78%, and between H′ and F 75%, respectively. The three proteins contain three repeats, which we denote quasi-RRMs (qRRMs) since they have a remote similarity to the RNA recognition motif (RRM). The three qRRMs of hnRNP H, with a few additional NH-terminal amino acids, were constructed by polymerase chain reaction amplification and used for ribohomopolymer binding studies. Each qRRM repeat bound poly(rG), while only the NH-terminal qRRM bound poly(rC) and poly(rU). None of the repeats bound detectable amounts of poly(rA). The expression levels of hnRNPs H and F were differentially regulated in pairs of normal and transformed fibroblasts and keratinocytes. In normal human keratinocytes, the expression level of H was unaffected by treatment with several substances tested including two second messengers and seven cytokines. Likewise the expression level of F was independent of these substances, although it was strikingly down-regulated by long term treatment with 4β-phorbol 12-myristate 13-acetate, indicating that the protein kinase C signaling pathway regulates its expression. No effect of 4β-phorbol 12-myristate 13-acetate was observed on the expression of hnRNP H. The genes coding for hnRNPs H, H′, and F were chromosome-mapped to 5q35.3 (HNRPH1), 6q25.3-q26, and/or Xq22 (HNRPH2) and 10q11.21-q11.22 (HNRPF), respectively.

Identification of a Phospholemman-like Protein from Shark Rectal Glands EVIDENCE FOR INDIRECT REGULATION OF Na,K-ATPase BY PROTEIN KINASE C VIA A NOVEL MEMBER OF THE FXYDY FAMILY

The Na,K-ATPase provides the driving force for many ion transport processes through control of Na+and K+ concentration gradients across the plasma membranes of animal cells. It is composed of two subunits, α and β. In many tissues, predominantly in kidney, it is associated with a small ancillary component, the γ-subunit that plays a modulatory role. A novel 15-kDa protein, sharing considerable homology to the γ-subunit and to phospholemman (PLM) was identified in purified Na,K-ATPase preparations from rectal glands of the shark Squalus acanthias, but was absent in pig kidney preparations. This PLM-like protein from shark (PLMS) was found to be a substrate for both PKA and PKC. Antibodies to the Na,K-ATPase α-subunit coimmunoprecipitated PLMS. Purified PLMS also coimmunoprecipitated with the α-subunit of pig kidney Na,K-ATPase, indicating specific association with different α-isoforms. Finally, PLMS and the α-subunit were expressed in stoichiometric amounts in rectal gland membrane preparations. Incubation of membrane bound Na,K-ATPase with non-solubilizing concentrations of C12E8resulted in functional dissociation of PLMS from Na,K-ATPase and increased the hydrolytic activity. The same effects were observed after PKC phosphorylation of Na,K-ATPase membrane preparations. Thus, PLMS may function as a modulator of shark Na,K-ATPase in a way resembling the phospholamban regulation of the Ca-ATPase.

Influence of fatty acids on the binding of warfarin and phenprocoumon to human serum albumin with relation to anticoagulant therapy.

Warfarin and phenprocoumon binding to human serum albumin was studied by equilibrium dialysis.

Human 2-D PAGE databases for proteome analysis in health and disease: http ://biobase.dk/cgi-bin/celis

Human 2‐D PAGE Databases established at the Danish Centre for Human Genome Research are now available on the World Wide Web (http://biobase.dk/cgi‐bin/celis). The databanks, which offer a comprehensive approach to the analysis of the human proteome both in health and disease, contain data on known and unknown proteins recorded in various IEF and NEPHGE 2‐D PAGE reference maps (non‐cultured keratinocytes, non‐cultured transitional cell carcinomas, MRC‐5 fibroblasts and urine). One can display names and information on specific protein spots by clicking on the image of the gel representing the 2‐D gel map in which one is interested. In addition, the database can be searched by protein name, keywords or organelle or cellular component. The entry files contain links to other databases such as Medline, Swiss‐Prot, PIR, PDB, CySPID, OMIM, Methabolic pathways, etc. The on‐line information is updated regularly.