Ras Pathway Activates Epithelial Na+ Channel and Decreases Its Surface Expression in Xenopus Oocytes

The small G protein K-Ras2A is rapidly induced by aldosterone in A6 epithelia. In these Xenopus sodium reabsorbing cells, aldosterone rapidly activates preexisting epithelial Na+ channels (XENaC) via a transcriptionally mediated mechanism. In the Xenopus oocytes expression system, we tested whether the K-Ras2A pathway impacts on XENaC activity by expressing XENaC alone or together with XK-Ras2A rendered constitutively active (XK-Ras2AG12V). As a second control, XENaC-expressing oocytes were treated with progesterone, a sex steroid that induces maturation of the oocytes similarly to activated Ras. Progesterone or XK-Ras2AG12V led to oocyte maturation characterized by a decrease in surface area and endogenous Na+ pump function. In both conditions, the surface expression of exogenous XENaC′s was also decreased; however, in comparison with progesterone-treated oocytes, XK-ras2AG12V-coinjected oocytes expressed a fivefold higher XENaC-mediated macroscopic Na+ current that was as high as that of control oocytes. Thus, the Na+ current per surface-expressed XENaC was increased by XK-Ras2AG12V. The chemical driving force for Na+ influx was not changed, suggesting that XK-Ras2AG12V increased the mean activity of XENaCs at the oocyte surface. These observations raise the possibility that XK-Ras2A, which is the first regulatory protein known to be transcriptionally induced by aldosterone, could play a role in the control of XENaC function in aldosterone target cells.

A highly digestible sorghum mutant cultivar exhibits a unique folded structure of endosperm protein bodies

The endosperm of a sorghum mutant cultivar, with high in vitro uncooked and cooked protein digestibilities, was examined by transmission electron microscopy and α-, β-, and γ-kafirins (storage proteins) were localized within its protein bodies. Transmission electron microscopy micrographs revealed that these protein bodies had a unique microstructure related to high protein digestibility. They were irregular in shape and had numerous invaginations, often reaching to the central area of the protein body. Protein bodies from normal cultivars, such as P721N studied here, with much lower uncooked and cooked digestibilities are spherical and contain no invaginations. Immunocytochemistry results showed that the relative location of α- and β-kafirins within the protein bodies of the highly digestible genotype were similar to the normal cultivar, P721N. γ-Kafirin, however, was concentrated in dark-staining regions at the base of the folds instead of at the protein body periphery, as is typical of normal cultivars. The resulting easy accessibility of digestive enzymes to α-kafirin, the major storage protein, in addition to the increased surface area of the protein bodies of the highly digestible cultivar appear to account for its high in vitro protein digestibility.

Crystal structure of a Staphylococcus aureus protein A domain complexed with the Fab fragment of a human IgM antibody: Structural basis for recognition of B-cell receptors and superantigen activity

Staphylococcus aureus produces a virulence factor, protein A (SpA), that contains five homologous Ig-binding domains. The interactions of SpA with the Fab region of membrane-anchored Igs can stimulate a large fraction of B cells, contributing to lymphocyte clonal selection. To understand the molecular basis for this activity, we have solved the crystal structure of the complex between domain D of SpA and the Fab fragment of a human IgM antibody to 2.7-Å resolution. In the complex, helices II and III of domain D interact with the variable region of the Fab heavy chain (VH) through framework residues, without the involvement of the hypervariable regions implicated in antigen recognition. The contact residues are highly conserved in human VH3 antibodies but not in other families. The contact residues from domain D also are conserved among all SpA Ig-binding domains, suggesting that each could bind in a similar manner. Features of this interaction parallel those reported for staphylococcal enterotoxins that are superantigens for many T cells. The structural homology between Ig VH regions and the T-cell receptor Vβ regions facilitates their comparison, and both types of interactions involve lymphocyte receptor surface remote from the antigen binding site. However, T-cell superantigens reportedly interact through hydrogen bonds with T-cell receptor Vβ backbone atoms in a primary sequence-independent manner, whereas SpA relies on a sequence-restricted conformational binding with residue side chains, suggesting that this common bacterial pathogen has adopted distinct molecular recognition strategies for affecting large sets of B and T lymphocytes.

Proton long-range migration along protein monolayers and its consequences on membrane coupling

It has been shown with lipid layers and more recently with purple membranes that protons have slow surface-to-bulk transfer. This results in long-range proton lateral conduction along membranes. We report here that such lateral transfer can take place along a pure protein film. It is strongly controlled by the packing. Subtle reorganizations of the protein–protein contact can be biological switches between interfacial and delocalized proton pathways between sources and sinks.

Cell surface expression of mouse macrosialin and human CD68 and their role as macrophage receptors for oxidized low density lipoprotein

We have previously identified a 94- to 97-kDa oxidized low density lipoprotein (LDL)-binding protein in mouse macrophages as macrosialin (MS), a member of the lamp family. Earlier immunostaining studies have shown that MS and its human homolog, CD68, are predominantly intracellular proteins. However, using sensitive techniques such as flow cytometry (FACS) and cell-surface-specific biotinylation, we now show that there is significant surface expression of these proteins. FACS analysis of intact cells using mAb FA/11 showed small but definite surface expression of MS in resident mouse peritoneal macrophages but this was greatly enhanced with thioglycollate elicitation. Biotinylation of intact cells and detergent-solubilized cell preparations followed by immunoprecipitation revealed 10–15% of the total MS content of elicited macrophages on the plasma membrane. Similar results were obtained with untreated RAW 264.7 cells. FACS analysis of intact THP-1 monocytic cells showed minimal surface expression of CD68 on unactivated cells (4% of total cell content). Stimulation with phorbol 12-myristate 13-acetate increased both surface and total CD68 expression considerably. Furthermore, the specific binding at 4°C and uptake at 37°C of 125I-labeled oxidized LDL by activated THP-1 cells was inhibited by 30–50% by CD68 mAbs KP-1 and EBM-11. Thus, although the surface expression of MS/CD68 at steady-state represents only a small percentage of their total cellular content, these proteins can play a significant role in oxidized LDL uptake by activated macrophages in vitro and could contribute to foam cell formation in atherosclerotic lesions.

Folding and activity of circularly permuted forms of a polytopic membrane protein

The transmembrane subunit of the Glc transporter (IICBGlc), which mediates uptake and concomitant phosphorylation of glucose, spans the membrane eight times. Variants of IICBGlc with the native N and C termini joined and new N and C termini in the periplasmic and cytoplasmic surface loops were expressed in Escherichia coli. In vivo transport/in vitro phosphotransferase activities of the circularly permuted variants with the termini in the periplasmic loops 1 to 4 were 35/58, 32/37, 0/3, and 0/0% of wild type, respectively. The activities of the variants with the termini in the cytoplasmic loops 1 to 3 were 0/25, 0/4 and 24/70, respectively. Fusion of alkaline phosphatase to the periplasmic C termini stabilized membrane integration and increased uptake and/or phosphorylation activities. These results suggest that internal signal anchor and stop transfer sequences can function as N-terminal signal sequences in a circularly permuted α-helical bundle protein and that the orientation of transmembrane segments is determined by the amino acid sequence and not by the sequential appearance during translation. Of the four IICBGlc variants with new termini in periplasmic loops, only the one with the discontinuity in loop 4 is inactive. The sequences of loop 4 and of the adjacent TM7 and TM8 are conserved in all phosphoenolpyruvate-dependent carbohydrate:phosphotransferase system transporters of the glucose family.

Manipulating the genetic identity and biochemical surface properties of individual cells with electric-field-induced fusion

A method for cell–cell and cell–liposome fusion at the single-cell level is described. Individual cells or liposomes were first selected and manipulated either by optical trapping or by adhesion to a micromanipulator-controlled ultramicroelectrode. Spatially selective fusion of the cell–cell or cell–liposome pair was achieved by the application of a highly focused electric field through a pair of 5-μm o.d. carbon-fiber ultramicroelectrodes. The ability to fuse together single cells opens new possibilities in the manipulation of the genetic and cellular makeup of individual cells in a controlled manner. In the study of cellular networks, for example, the alteration of the biochemical identity of a selected cell can have a profound effect on the behavior of the entire network. Fusion of a single liposome with a target cell allows the introduction of the liposomal content into the cell interior as well as the addition of lipids and membrane proteins onto the cell surface. This cell–liposome fusion represents an approach to the manipulation of the cytoplasmic contents and surface properties of single cells. As an example, we have introduced a membrane protein (γ-glutamyltransferase) reconstituted in liposomes into the cell plasma membrane.

NMR solution structure of the human prion protein

The NMR structures of the recombinant human prion protein, hPrP(23–230), and two C-terminal fragments, hPrP(90–230) and hPrP(121–230), include a globular domain extending from residues 125–228, for which a detailed structure was obtained, and an N-terminal flexibly disordered “tail.” The globular domain contains three α-helices comprising the residues 144–154, 173–194, and 200–228 and a short anti-parallel β-sheet comprising the residues 128–131 and 161–164. Within the globular domain, three polypeptide segments show increased structural disorder: i.e., a loop of residues 167–171, the residues 187–194 at the end of helix 2, and the residues 219–228 in the C-terminal part of helix 3. The local conformational state of the polypeptide segments 187–193 in helix 2 and 219–226 in helix 3 is measurably influenced by the length of the N-terminal tail, with the helical states being most highly populated in hPrP(23–230). When compared with the previously reported structures of the murine and Syrian hamster prion proteins, the length of helix 3 coincides more closely with that in the Syrian hamster protein whereas the disordered loop 167–171 is shared with murine PrP. These species variations of local structure are in a surface area of the cellular form of PrP that has previously been implicated in intermolecular interactions related both to the species barrier for infectious transmission of prion disease and to immune reactions.

VIP17/MAL, a lipid raft-associated protein, is involved in apical transport in MDCK cells

Apical proteins are sorted and delivered from the trans-Golgi network to the plasma membrane by a mechanism involving sphingolipid–cholesterol rafts. In this paper, we report the effects of changing the levels of VIP17/MAL, a tetraspan membrane protein localized to post-Golgi transport containers and the apical cell surface in MDCK cells. Overexpression of VIP17/MAL disturbed the morphology of the MDCK cell layers by increasing apical delivery and seemingly expanding the apical cell surface domains. On the other hand, expression of antisense RNA directed against VIP17/MAL caused accumulation in the Golgi and/or impaired apical transport of different apical protein markers, i.e., influenza virus hemagglutinin, the secretory protein clusterin (gp80), the transmembrane protein gp114, and a glycosylphosphatidylinositol-anchored protein. However, antisense RNA expression did not affect the distribution of E-cadherin to the basolateral surface. Because VIP17/MAL associates with sphingolipid–cholesterol rafts, these data provide functional evidence that this protein is involved in apical transport and might be a component of the machinery clustering lipid rafts with apical cargo to form apical transport carriers.

E-cadherin induces mesenchymal-to-epithelial transition in human ovarian surface epithelium

Ovarian carcinomas are thought to arise in the ovarian surface epithelium (OSE). Although this tissue forms a simple epithelial covering on the ovarian surface, OSE cells exhibit some mesenchymal characteristics and contain little or no E-cadherin. However, E-cadherin is present in metaplastic OSE cells that resemble the more complex epithelia of the oviduct, endometrium and endocervix, and in primary epithelial ovarian carcinomas. To determine whether E-cadherin was a cause or consequence of OSE metaplasia, we expressed this cell-adhesion molecule in simian virus 40-immortalized OSE cells. In these cells the exogenous E-cadherin, all three catenins, and F-actin localized at sites of cell–cell contact, indicating the formation of functional adherens junctions. Unlike the parent OSE cell line, which had undergone a typical mesenchymal transformation in culture, E-cadherin-expressing cells contained cytokeratins and the tight-junction protein occludin. They also formed cobblestone monolayers in two-dimensional culture and simple epithelia in three-dimensional culture that produced CA125 and shed it into the culture medium. CA125 is a normal epithelial-differentiation product of the oviduct, endometrium, and endocervix, but not of normal OSE. It is also a tumor antigen that is produced by ovarian neoplasms and by metaplastic OSE. Thus, E-cadherin restored some normal characteristics of OSE, such as keratin, and it also induced epithelial-differentiation markers associated with weakly preneoplastic, metaplastic OSE and OSE-derived primary carcinomas. The results suggest an unexpected role for E-cadherin in ovarian neoplastic progression.

Induction of the Tat-binding protein 1 gene accompanies the disabling of oncogenic erbB receptor tyrosine kinases

Conversion of a malignant phenotype into a more normal one can be accomplished either by down-regulation of erbB family surface receptors or by creating inactive erbB heterodimers on the cell surface. In this report, we report the identification and cloning of differentially expressed genes from antibody-treated vs. untreated fibroblasts transformed by oncogenic p185neu. We repeatedly isolated a 325-bp cDNA fragment that, as determined by Northern analysis, was expressed at higher levels in anti-p185neu-treated tumor cells but not in cells expressing internalization defective p185neu receptors. This cDNA fragment was identical in amino acid sequence to the recently cloned mouse Tat binding protein-1 (mTBP1), which has 98.4% homology to the HIV tat-binding protein-1 (TBP1). TBP1 mRNA levels were found to be elevated on inhibition of the oncogenic phenotype of transformed cells expressing erbB family receptors. TBP1 overexpression diminished cell proliferation, reduced the ability of the parental cells to form colonies in vitro, and almost completely inhibited transforming efficiency in athymic mice when stably expressed in human tumor cells containing erbB family receptors. Collectively, these results suggest that the attenuation of erbB receptor signaling seems to be associated with activation/induction or recovery of a functional tumor suppressor-like gene, TBP1. Disabling erbB tyrosine kinases by antibodies or by trans-inhibition represents an initial step in triggering a TBP1 pathway.

A new pathway for the secretion of virulence factors by bacteria: The flagellar export apparatus functions as a protein-secretion system

Biogenesis of the flagellum, a motive organelle of many bacterial species, is best understood for members of the Enterobacteriaceae. The flagellum is a heterooligomeric structure that protrudes from the surface of the cell. Its assembly initially involves the synthesis of a dedicated protein export apparatus that subsequently transports other flagellar proteins by a type III mechanism from the cytoplasm to the outer surface of the cell, where oligomerization occurs. In this study, the flagellum export apparatus was shown to function also as a secretion system for the transport of several extracellular proteins in the pathogenic bacterium Yersinia enterocolitica. One of the proteins exported by the flagellar secretion system was the virulence-associated phospholipase, YplA. These results suggest type III protein secretion by the flagellar system may be a general mechanism for the transport of proteins that influence bacterial–host interactions.

NMR structure of the bovine prion protein

The NMR structures of the recombinant 217-residue polypeptide chain of the mature bovine prion protein, bPrP(23–230), and a C-terminal fragment, bPrP(121–230), include a globular domain extending from residue 125 to residue 227, a short flexible chain end of residues 228–230, and an N-terminal flexibly disordered “tail” comprising 108 residues for the intact protein and 4 residues for bPrP(121–230), respectively. The globular domain contains three α-helices comprising the residues 144–154, 173–194, and 200–226, and a short antiparallel β-sheet comprising the residues 128–131 and 161–164. The best-defined parts of the globular domain are the central portions of the helices 2 and 3, which are linked by the only disulfide bond in bPrP. Significantly increased disorder and mobility is observed for helix 1, the loop 166–172 leading from the β-strand 2 to helix 2, the end of helix 2 and the following loop, and the last turn of helix 3. Although there are characteristic local differences relative to the conformations of the murine and Syrian hamster prion proteins, the bPrP structure is essentially identical to that of the human prion protein. On the other hand, there are differences between bovine and human PrP in the surface distribution of electrostatic charges, which then appears to be the principal structural feature of the “healthy” PrP form that might affect the stringency of the species barrier for transmission of prion diseases between humans and cattle.

NMR structures of three single-residue variants of the human prion protein

The NMR structures of three single-amino acid variants of the C-terminal domain of the human prion protein, hPrP(121–230), are presented. In hPrP(M166V) and hPrP(R220K) the substitution is with the corresponding residue in murine PrP, and in hPrP(S170N) it is with the corresponding Syrian hamster residue. All three substitutions are in the surface region of the structure of the cellular form of PrP (PrPC) that is formed by the C-terminal part of helix 3, with residues 218–230, and a loop of residues 166–172. This molecular region shows high species variability and has been implicated in specific interactions with a so far not further characterized “protein X,” and it is related to the species barrier for transmission of prion diseases. As expected, the three variant hPrP(121–230) structures have the same global architecture as the previously determined wild-type bovine, human, murine, and Syrian hamster prion proteins, but with the present study two localized “conformational markers” could be related with single amino acid exchanges. These are the length and quality of definition of helix 3, and the NMR-observability of the residues in the loop 166–172. Poor definition of the C-terminal part of helix 3 is characteristic for murine PrP and has now been observed also for hPrP(R220K), and NMR observation of the complete loop 166–172 has so far been unique for Syrian hamster PrP and is now also documented for hPrP(S170N).

Role of cysteines in Plasmodium falciparum circumsporozoite protein: Interactions with heparin can rejuvenate inactive protein mutants

Various pathogenic bacteria, viruses, and protozoan bind to glycosaminoglycan-based receptors on host cells and initiate an infection. Sporozoites of Plasmodium predominantly express circumsporozoite (CS) protein on their surface, which binds to heparan sulfate proteoglycans on liver cell surface that subsequently leads to malaria. Here we show that the interaction of free heparin with this parasite ligand has the potential to be a critical component of invasion. CS protein of P. falciparum contains four cysteines at positions 361, 365, 396, and 401. In this study, all four cysteine residues were mutagenized to alanine both individually and in different combinations. Conversion of cysteine 396 to alanine (protein CS3) led to a 10-fold increase in the binding activity of the protein to HepG2 cells. Replacement of cysteines at positions 361, 365, and 401 either alone or in different combinations led to a near total loss of binding. Surprisingly, activity in these inactive mutants could be effectively restored in the presence of submolar concentrations of heparin. Heparin also up-regulated binding of CS3 at submolar concentrations with respect to the protein but down-regulated binding when present in excess. Given the significantly different concentrations of heparin in different organs of the host and the in vitro results described here one can consider in vivo ramifications of this phenomenon for pathogen targeting of specific organs and for the functional effects of antigenic variation on receptor ligand interaction.