Studies of the permeation properties of glomerular basement membrane: cross-linking renders glomerular basement membrane permeable to protein.

Cross-linking glomerular basement membrane (GBM) has been shown to render it more permeable to protein. Isolated pig GBM was cross-linked with dimethylmalonimidate which reacts selectively with lysine epsilon-NH2 groups or with glutaraldehyde, a less selective cross-linking agent. Studies of the ultrafiltration properties of these materials in vitro using cytochrome c, myoglobin, bovine serum albumin and immunoglobulin showed that cross-linking had markedly increased solvent and protein fluxes as compared with native membranes particularly at higher pressures. Filtration studies with serum demonstrated that the cross-linked membranes were more permeable to serum proteins. Thickness measurements under pressure indicated that cross-linked membrane was less compressed than native membrane as pressure was increased. Pore theory did not provide a suitable model for analysis of the results, but analysis of the results using the fibre-matrix hypothesis indicated that cross-linking had the effect of bundling together the fibres (type IV collagen) in the GBM matrix. The effect of cross-linking on filtration could be explained by a combination of contraction of the membrane, fibre bundling and increased rigidity compared with native membrane. Cross-linking of GBM might lead to long-term damage of the glomerular capillary wall in nephritis, so promoting proteinuria.     

Non-collagen protein and proteoglycan in renal glomerular basement membrane

Extraction of rat glomerular basement membrane, purified by osmotic lysis and sequential detergent treatment, with 8 M urea containing protease inhibitors solubilizes protein that is devoid of hydroxyproline and hydroxylysine. This material represents 8–12% of total membrane protein, elutes mainly as two high molecular weight peaks on agarose gel filtration, and is associated with glycosaminoglycans. Isolated rat renal glomeruli incorporate [³⁵S]sulfate into basement membrane from which this non-collagenous ³⁵S-labeled fraction can be subsequently solubilized. The radioactivity incorporated into urea-soluble glomerular basement membrane eluted primarily with the higher molecular weight peak (M_r greater than 250 000). Cellulose acetate electrophoresis after pronase digestion of the urea-soluble fraction revealed glycosaminoglycan that was resistant to digestion with Streptomyces hyaluronidase and chondroitinase ABC, sensitive to nitrous acid treatment, and contained [³⁵S]-sulfate. The findings indicate that one of the non-collagenous components of glomerular basement membrane is a proteoglycan containing heparan sulfate.     

Ultrastructural organization of the glomerular basement membrane as revealed by a deep-etch replica method

Summary The fine structure of the glomerular basement membrane was re-evaluated by using a deep-etch replica method.The structure of the laminae rarae interna and externa of the rat glomerular basement membrane was basically identical in that 6 to 8 nm fibrils were interconnected to form a three-dimensional, polygonal network. The size of the mesh was quite variable but most often ranged from 20 to 25 nm in width. In addition, a zipper-like substructure of the epithelial slit diaphragm was observed. By contrast, the lamina densa was composed of closely packed particles.After exposure of the bovine glomerular basement membrane to ultrasonic waves or trypsin, the particles of the lamina densa were effectively removed. The underlying structure showed the fibrillar network closely resembled that seen in the laminae rarae of the rat glomerular basement membrane.The glomerular basement membrane thus revealed was as principally composed of a fibrillar network, which might be regularly arranged units of type-IV collagen. Numerous fine particles, most likely proper components of the glomerular basement membrane, were attached onto this basic fibrillar structure, giving rise to a morphologic appearance different from that of the laminae rarae.     

To form and function: on the role of basement membrane mechanics in tissue development,homeostasis and disease

The basement membrane (BM) is a special type of extracellular matrix that lines the basal side of epithelial and endothelial tissues. Functionally, the BM is important for providing physical and biochemical cues to the overlying cells, sculpting the tissue into its correct size and shape. In this review, we focus on recent studies that have unveiled the complex mechanical properties of the BM. We discuss how these properties can change during development, homeostasis and disease via different molecular mechanisms, and the subsequent impact on tissue form and function in a variety of organisms. We also explore how better characterization of BM mechanics can contribute to disease diagnosis and treatment, as well as development of better in silico and in vitro models that not only impact the fields of tissue engineering and regenerative medicine, but can also reduce the use of animals in research.     

Fine structure of the glomerular basement membrane of the rat kidney visualized by high-resolution scanning electron microscopy

Summary The fine structure of the glomerular basement membrane (GBM) of the rat kidney was studied by means of high resolution scanning electron microscopy. Specimens were taken from kidneys perfused with paraformaldehyde, freeze-fractured and then processed with conductive staining. The fractured surface of glomerular tufts exhibited the inner and outer surface of the GBM uncovered by endothelial and epithelial cells. The lamina densa was composed of densely packed granular material together with scattered fibrils. The laminae rarae interna and externa were composed of a meshwork that showed some structural heterogeneities. The meshwork composing the lamina rara interna contained 5-to 9-nm-thick fibrils, had pores 11–30 nm wide, and was associated with granular material except in those places that corresponded with endothelial fenestrae. The meshwork of the lamina rara externa was made up of 6- to 11-nm-thick fibrils, and had smaller pores under the foot processes (10–24 nm wide) than those near the filtration slits (16–32 nm wide). In addition to the meshwork, the lamina rara interna contained microfibrils that were arranged differently depending on the topography of the capillary wall: scattered fibrils had no predominant orientation at the convex side, circumferential bundles lay at the concave side of the peripheral capillary wall, and had a circumferential arrangement in the paramesangial wall.     

Scarface,a secreted serine protease‐like protein,regulates polarized localization of laminin A at the basement membrane of the Drosophila embryo

Cell–matrix interactions brought about by the activity of integrins and laminins maintain the polarized architecture of epithelia and mediate morphogenetic interactions between apposing tissues. Although the polarized localization of laminins at the basement membrane is a crucial step in these processes, little is known about how this polarized distribution is achieved. Here, in Drosophila, we analyse the role of the secreted serine protease‐like protein Scarface in germ‐band retraction and dorsal closure—morphogenetic processes that rely on the activity of integrins and laminins. We present evidence that scarface is regulated by c‐Jun amino‐terminal kinase and that scarface mutant embryos show defects in these morphogenetic processes. Anomalous accumulation of laminin A on the apical surface of epithelial cells was observed in these embryos before a loss of epithelial polarity was induced. We propose that Scarface has a key role in regulating the polarized localization of laminin A in this developmental context.     

Binding of mouse nidogen-2 to basement membrane components and cells and its expression in embryonic and adult tissues suggest complementary functions of the two nidogens

Nidogen-1 binds several basement membrane components by well-defined, domain-specific interactions. Organ culture and gene targeting approaches suggest that a high-affinity nidogen-binding site of the laminin gamma1 chain (gamma1III4) is important for kidney development and for nerve guidance. Other proteins may also bind gamma1III4, although human nidogen-2 binds poorly to the mouse laminin gamma1 chain. We therefore characterized recombinant mouse nidogen-2 and its binding to basement membrane proteins and cells. Mouse nidogen-1 and -2 interacted at comparable levels with collagen IV, perlecan, and fibulin-2 and, most notably, also with laminin-1 fragments P1 and gamma1III3-5, which both contain the gamma1III4 module. In embryos, nidogen-2 mRNA was produced by mesenchyme at sites of epithelial-mesenchymal interactions, but the protein was deposited on epithelial basement membranes, as previously shown for nidogen-1. Hence, binding of both nidogens to the epithelial laminin gamma1 chain is dependent on epithelial-mesenchymal interactions. Epidermal growth factor stimulated expression of both nidogens in embryonic submandibular glands. Both nidogens were found in all studied embryonic and adult basement membranes. Nidogen-2 was more adhesive than nidogen-1 for some cell lines and was mainly mediated by alpha3beta1 and alpha6beta1 integrins as shown by antibody inhibition. These findings revealed extensive coregulation of nidogen-1 and -2 expression and much more complementary functions of the two nidogens than previously recognized.     

Overproduction of the proFhuA outer membrane receptor protein of Escherichia coli K-12: isolation,properties, and immunocytochemical localization at the inner side of the cytoplasmic membrane

The fhu operon of Escherichia coli K-12 comprises four genes, termed fhuA,C,D,B, which are involved in the uptake of iron-hydroxamate compounds. The fhuA gene encodes the outer membrane receptor protein. Cells that contained three copies of the fhuACD fragment on the thermoamplifiable plasmid pHK232 accumulated at 37° C large amounts of the proFhuA protein. Most of the overproduced proFhuA protein was not translocated into the outer membrane but instead precipitated at the cytoplasmic side of the inner membrane, presumably at the sites of synthesis. Despite inhibition of export proFhuA synthesis continued.The precipitate formed was sedimented by centrifugation at 8,000xg. The proFhuA protein could be solubilized in 1% sodium dodecyl sulfate. Replacement of sodium dodecyl sulfate by Triton X-100 resulted in a proFhuA protein which exhibited 10% of the phage T5 binding activity of renatured mature FhuA protein. Binding of phage T5 was inhibited by the FhuA-specific ligands ferrichrome, albomycin and colicin M. Limited proteolysis of the isolated pro- and mature form of the FhuA protein with trypsin yielded similar oligopeptide patterns. Addition of ferrichrome affected trypsin cleavage of both proteins in the same way. The common proteolytic intermediates together with phage inactivation indicate a similar conformation of the pro- and mature form.Dedicated to Prof. G. Braunitzer on the occasion of his 60th birthday     

Studies of albumin binding to rat liver plasma membranes: Implications for the albumin receptor hypothesis

To characterize a previously proposed hepatocyte albumin receptor, we examined the binding of native and defatted ¹²⁵I-labeled rat albumin to rat liver plasma membranes. After incubation for 30 min, binding was determined from the distribution of radioactivity between membrane pellet and supernatant following initial centrifugation (15 000 × g for 15 min), after repeated cycles of washing with buffer and re-centrifugation. ¹²⁵I-labeled albumin recovered in the initial membrane pellet averaged only 4% of that incubated. Moreover, this albumin was only loosely associated with the membrane, as indicated by recovery in the pellet of under 0.5% of the counts after three washes. Binding of ¹²⁵I-labeled albumin to the plasma membranes was no greater than to erythrocyte ghosts, was not inhibited by excess unlabeled albumin, and was not decreased by heat denaturation of the membranes, all suggestive of a lack of specific binding. Failure to observe albumin binding to the membranes was not due to a rapid dissociation rate or ‘off-time’, as incubations in the presence of sufficient ultraviolet light to promote covalent binding of ligands to receptors did not increase ¹²⁵I counts bound to the membrane. Finally, affinity chromatography over albumin/agarose gel of solubilized membrane proteins provided no evidence of a membrane protein with a high affinity for albumin. These studies, therefore, do not support the hypothesis that liver cell plasma membranes contain a specific albumin receptor.     

Reduced expressions of calmodulin genes and protein and reduced ability of calmodulin to activate plasma membrane Ca2+‐ATPase in the brain of protein undernourished rats: modulatory roles of selenium and zinc supplementation

The roles of protein undernutrition as well as selenium (Se) and zinc (Zn) supplementation on the ability of calmodulin (CaM) to activate erythrocyte ghost membrane (EGM) Ca²⁺‐ATPase and the calmodulin genes and protein expressions in rat's cortex and cerebellum were investigated. Rats on adequate protein diet and protein‐undernourished (PU) rats were fed with diet containing 16% and 5% casein, respectively, for a period of 10 weeks. The rats were then supplemented with Se and Zn at a concentration of 0.15 and 227 mg l⁻¹, respectively, in drinking water for 3 weeks. The results obtained from the study showed significant reductions in synaptosomal plasma membrane Ca²⁺‐ATPase (PMCA) activity, Ca²⁺/CaM activated EGM Ca²⁺ATPase activity and calmodulin genes and protein expressions in PU rats. Se or Zn supplementation improved the ability of Ca²⁺/CaM to activate EGM Ca²⁺‐ATPase and protein expressions. Se or Zn supplementation improved gene expression in the cerebellum but not in the cortex. Also, the activity of PMCA was significantly improved by Zn. In conclusion, it is postulated that Se and Zn might be beneficial antioxidants in protecting against neuronal dysfunction resulting from reduced level of calmodulin such as present in protein undernutrition. Copyright © 2016 John Wiley & Sons, Ltd.     

Strained contacts with the cell membrane may influence ligand affinity to G protein coupled receptors: a case of free fatty acid receptor 1 agonists

A set of 1,3,4-thiadiazole-2-carboxamides bearing a substituted biphenyl in the amide portion was synthesised and tested for agonistic activity towards free fatty acid receptor 1 (FFA1). The observed activity trends were impossible to rationalised based solely on the docking energy scores of Glide SP. On the contrary, when the phospholipid cell membrane bilayer was reconstructed around FFA1, it became apparent that inactive compounds displayed significant strained contacts with the membrane while for active compounds the strain was noticeably lower. These findings justify using the improved docking protocol for modelling GPCR-ligand interactions which uses the crystal structure of the receptor and a reconstructed portion of a cell membrane.     

Attachment of Madin-Darby canine kidney cells to extracellular matrix: role of a laminin binding protein related to the 37/67 kDa laminin receptor in the development of plasma membrane polarization.

Madin-Darby canine kidney (MDCK) cells have been extensively used as a model for the study of epithelial polarization. The contacts between the cell and extra-cellular matrix (ECM) provide a signal for the polarization of apical membrane markers. In order to study the molecular basis of these contacts, MDCK cells extracts in Triton X-100 were affinity-purified on laminin, yielding polypeptides of 100-110 and 36 kDa, but only the second one could be enzymatically iodinated from the cell surface. This protein was also recognized by an antibody against the 37/67-kDa laminin/elastin family of proteins. Different polypeptides were purified by the same method on type I collagen. An antibody developed against the polypeptides purified on laminin recognized also a 67-kDa protein, blocked 125I-laminin binding to a population of high affinity (1.5 nM KD) binding sites and caused a significant decrease in cell attachment and spreading to laminin or endogenous ECM. This antibody did not interfere with MDCK cell attachment to fibronectin or collagen matrices, but still impaired cell spreading. An apical MDCK plasma membrane protein (184 kDa), fully polarized in untreated cells, was partially mispolarized after treatment with anti-36 kDa antibody. These results are consistent with a model of various ECM receptors operating together in these cells, and show an important role of a non-integrin 36-kDa laminin binding protein related to the 67-kDa laminin receptor family in cell attachment, spreading and polarization.     

Application of three-dimensional molecular hydrophobicity potential to the analysis of spatial organization of membrane domains in proteins: I. Hydrophobic properties of transmembrane segments of Na+, K+-ATPase

A new computer-aided molecular modeling approach based on the concept of three-dimensional (3D) molecular hydrophobicity potential has been developed to calculate the spatial organization of intramembrane domains in proteins. The method has been tested by calculating the arrangement of membrane-spanning segments in the photoreaction center ofRhodopseudomonas viridis and comparing the results obtained with those derived from the X-ray data. We have applied this computational procedure to the analysis of interhelical packing in membrane moiety of Na⁺, K⁺-ATPase. The work consists of three parts. In Part I, 3D distributions of electrostatic and molecular hydrophobicity potentials on the surfaces of transmembrane helical peptides were computed and visualized. The hydrophobic and electrostatic properties of helices are discussed from the point of view of their possible arrangement within the protein molecule. Interlocation of helical segments connected with short extramembrane loops found by means of optimization of their hydrophobic/hydrophilic contacts is considered in Part II. The most probable 3D model of packing of helical peptides in the membrane domain of Na⁺, K⁺-ATPase is discussed in the final part of the work.     

Ca2+ responses to thyrotropin-releasing hormone and angiotensin II: the role of plasma membrane integrity and effect of G11alpha protein overexpression on homologous and heterologous desensitization

The molecular mechanisms involved in GPCR-initiated signaling cascades where the two receptors share the same signaling cascade, such as thyrotropin-releasing hormone (TRH) and angiotensin II (ANG II), are still far from being understood. Here, we analyzed hormone-induced Ca(2+) responses and the process of desensitization in HEK-293 cells, which express endogenous ANG II receptors. These cells were transfected to express exogenously high levels of TRH receptors (clone E2) or both TRH receptors and G(11)alpha protein (clone E2M11). We observed that the characteristics of the Ca(2+) response, as well as the process of desensitization, were both strongly dependent on receptor number and G(11)alpha protein level. Whereas treatment of E2 cells with TRH or ANG II led to significant desensitization of the Ca(2+) response to subsequent addition of either hormone, the response was not desensitized in E2M11 cells expressing high levels of G(11)alpha. In addition, stimulation of both cell lines with THR elicited a clear heterologous desensitization to subsequent stimulation with ANG II. On the other hand, ANG II did not affect a subsequent response to TRH. ANG II-mediated signal transduction was strongly dependent on plasma membrane integrity modified by cholesterol depletion, but signaling through TRH receptors was altered only slightly under these conditions. It may be concluded that the level of expression of G-protein-coupled receptors and their cognate G-proteins strongly influences not only the magnitude of the Ca(2+) response but also the process of desensitization and resistance to subsequent hormone addition.