Mimotope/anti-mimotope probing of structural relationships in platelet glycoprotein Ib alpha.

A bacteriophage library displaying random decapeptides was used to characterize the binding preference of C-34, a monoclonal antibody originally raised against platelet-type von Willebrand disease platelets heterozygous for the mutation 23OWKQ (G --> V)233V234 in the alpha chain of glycoprotein Ib (GPIb alpha). Three rounds of biopanning C-34 against the library resulted in striking convergence upon the sequence WNWRYREYV. Since no portion of this sequence corresponds to a recognizable peptide sequence within human platelet GPIb alpha, it may be considered a "mimotope" of the naturally occurring C-34 epitope, presumably bearing similarity to it in three-dimensional structure. Synthetic AWNWRYREYV peptide preincubated with C-34 fully neutralized the ability of C-34 to inhibit platelet aggregation, with an IC50 of approximately 6 microg/ml. When biotinylated AWNWRYREYV was subsequently bioparmed against the original decapeptide library, the sole clone demonstrating inhibitory activity above background level in a functional platelet assay displayed the sequence RHVAWWRQGV, and chemically synthesized peptide fully inhibited ristocetin-induced aggregation, with an IC50 of 200-400 microg/ml. Synthesized RHVAWWKQGV peptide exerted only slight inhibition, whereas RHVAWWKQVV peptide showed potent inhibitory activity. Moreover, whereas synthesized wild-type 228YVWKQGVDVK237 GPIb alpha peptide was virtually without inhibitory activity, the 228YVWKQ(G -->V) 233VDVK237 peptide fully inhibited ristocetin-induced aggregation, with an IC50 of approximately 400 microg/ml. These studies raise the possibility of an intramolecular association of peptide regions within GPIb alpha that may play a role in the regulation of von Willebrand factor-dependent platelet aggregation.

Mutant cystic fibrosis transmembrane conductance regulator inhibits acidification and apoptosis in C127 cells: possible relevance to cystic fibrosis.

We have shown elsewhere that acidification is an early event in apoptosis, preceding DNA cleavage. Cells expressing the most common mutation (delF508) of the cystic fibrosis transmembrane regulator (CFTR) exhibit a higher resting intracellular pH and are unable to secrete chloride and bicarbonate in response to cAMP. We hypothesized that defective acidification in cells expressing delF508 CFTR would interfere with the acidification that accompanies apoptosis, which in turn, would prevent endonuclease activation and cleavage of DNA. We therefore determined whether the function of the CFTR would affect the process of apoptosis in mouse mammary epithelial C127 cells stably transfected with the wild-type CFTR (C127/wt) or the delF508 mutation of the CFTR (C127/508). C127 cells possessed an acid endonuclease capable of DNA degradation at low pH. Sixteen hours after treatment with cycloheximide, C127/wt cells underwent cytoplasmic acidification. In contrast, C127/508 cells failed to demonstrate acidification. Furthermore, the C127/508 cells did not show nuclear condensation or DNA fragmentation detected by in situ nick-end labeling after treatment with cycloheximide or etoposide, in contrast to the characteristic features of apoptosis demonstrated by the C127/wt cells. Measurement of cell viability indicated a preservation of cell viability in C127/508 cells but not in C127/wt cells. That this resistance to the induction of apoptosis depended upon the loss of CFTR activity is shown by the finding that inhibition of the CFTR with diphenylamine carboxylate in C127/wt cells conferred similar protection. These findings suggest a role for the CFTR in acidification during the initiation of apoptosis in epithelial cells and imply that a failure to undergo programmed cell death could contribute to the pathogenesis of cystic fibrosis.

Identification of a second transmembrane protein tyrosine phosphatase, IA-2beta, as an autoantigen in insulin-dependent diabetes mellitus: precursor of the 37-kDa tryptic fragment.

A novel cDNA, IA-2beta, was isolated from a mouse neonatal brain library. The predicted protein sequence revealed an extracellular domain, a transmembrane region, and an intracellular domain. The intracellular domain is 376 amino acids long and 74% identical to the intracellular domain of IA-2, a major autoantigen in insulin-dependent diabetes mellitus (IDDM). A partial sequence of the extracellular domain of IA-2beta indicates that it differs substantially (only 26% identical) from that of IA-2. Both molecules are expressed in islets and brain tissue. Forty-six percent (23 of 50) of the IDDM sera but none of the sera from normal controls (0 of 50) immunoprecipitated the intracellular domain of IA-2beta. Competitive inhibition experiments showed that IDDM sera have autoantibodies that recognize both common and distinct determinants on IA-2 and IA-2beta. Many IDDM sera are known to immunoprecipitate 37-kDa and 40-kDa tryptic fragments from islet cells, but the identity of the precursor protein(s) has remained elusive. The current study shows that treatment of recombinant IA-2beta and IA-2 with trypsin yields a 37-kDa fragment and a 40-kDa fragment, respectively, and that these fragments can be immunoprecipitated with diabetic sera. Absorption of diabetic sera with unlabeled recombinant IA-2 or IA-2beta, prior to incubation with radiolabeled 37-kDa and 40-kDa tryptic fragments derived from insulinoma or glucagonoma cells, blocks the immunoprecipitation of both of these radiolabeled tryptic fragments. We conclude that IA-2beta and IA-2 are the precursors of the 37-kDa and 40-kDa islet cell autoantigens, respectively, and that both IA-2 and IA-2beta are major autoantigens in IDDM.

Molecular mechanism of transmembrane signaling by the aspartate receptor: a model.

The aspartate receptor of bacterial chemotaxis is representative of a large class of membrane-spanning receptors found in prokaryotic and eukaryotic organisms. These receptors, which regulate histidine kinase pathways and possess two putative transmembrane helices per subunit, appear to control a wide variety of cellular processes. The best characterized subgroup of the two-helix receptor class is the homologous family of chemosensory receptors from Escherichia coli and Salmonella typhimurium, including the aspartate receptor. This receptor binds aspartate, an attractant, in the periplasmic compartment and undergoes an intramolecular, transmembrane conformational change, thereby modulating the autophosphorylation rate of a bound histidine kinase in the cytoplasm. Here, we analyze recent results from x-ray crystallographic, solution 19F NMR, and engineered disulfide studies probing the aspartate-induced structural change within the periplasmic and transmembrane regions of the receptor. Together, these approaches provide evidence that aspartate binding triggers a "swinging-piston" displacement of the second membrane-spanning helix, which is proposed to communicate the signal across the bilayer.

A quantitative test to estimate neutralizing antibodies to the hepatitis C virus: cytofluorimetric assessment of envelope glycoprotein 2 binding to target cells.

Hepatitis C virus (HCV) is a major cause of chronic hepatitis. The virus does not replicate efficiently in cell cultures, and it is therefore difficult to assess infection-neutralizing antibodies and to evaluate protective immunity in vitro. To study the binding of the HCV envelope to cell-surface receptors, we developed an assay to assess specific binding of recombinant envelope proteins to human cells and neutralization thereof. HCV recombinant envelope proteins expressed in various systems were incubated with human cells, and binding was assessed by flow cytometry using anti-envelope antibodies. Envelope glycoprotein 2 (E2) expressed in mammalian cells, but not in yeast or insect cells, binds human cells with high affinity (Kd approximately 10(-8) M). We then assessed antibodies able to neutralize E2 binding in the sera of both vaccinated and carrier chimpanzees, as well as in the sera of humans infected with various HCV genotypes. Vaccination with recombinant envelope proteins expressed in mammalian cells elicited high titers of neutralizing antibodies that correlated with protection from HCV challenge. HCV infection does not elicit neutralizing antibodies in most chimpanzees and humans, although low titers of neutralizing antibodies were detectable in a minority of infections. The ability to neutralize binding of E2 derived from the HCV-1 genotype was equally distributed among sera from patients infected with HCV genotypes 1, 2, and 3, demonstrating that binding of E2 is partly independent of E2 hypervariable regions. However, a mouse monoclonal antibody raised against the E2 hypervariable region 1 can partially neutralize binding of E2, indicating that at least two neutralizing epitopes, one of which is hypervariable, should exist on the E2 protein. The neutralization-of-binding assay described will be useful to study protective immunity to HCV infection and for vaccine development.

Anti-idiotypic antibodies mimicking glycoprotein D of herpes simplex virus identify a cellular protein required for virus spread from cell to cell and virus-induced polykaryocytosis.

Glycoprotein D (gD) of herpes simplex virus 1 (HSV-1) is required for stable attachment and penetration of the virus into susceptible cells after initial binding. We derived anti-idiotypic antibodies to the neutralizing monoclonal antibody HD1 to gD of HSV-1. These antibodies have the properties expected of antibodies against a gD receptor. Specifically, they bind to the surface of HEp-2, Vero, and HeLa cells susceptible to HSV infection and specifically react with a Mr 62,000 protein in these and other (143TK- and BHK) cell lines. They neutralize virion infectivity, drastically decrease plaque formation by impairing cell-to-cell spread of virions, and reduce polykaryocytosis induced by strain HFEM, which carries a syncytial (syn-) mutation. They do not affect HSV growth in a single-step cycle and plaque formation by an unrelated virus, indicating that they specifically affect the interaction of HSV gD) with a cell surface receptor. We conclude that the Mr 62,000 cell surface protein interacts with gD to enable spread of HSV-1 from cell to cell and virus-induced polykaryocytosis.

Uptake of fluorescent dyes associated with the functional expression of the cystic fibrosis transmembrane conductance regulator in epithelial cells.

Specific mutations in the cystic fibrosis transmembrane conductance regulator (CFTR), the most common autosomal recessive fatal genetic disease of Caucasians, result in the loss of epithelial cell adenosine 3',5'-cyclic-monophosphate (cAMP)-stimulated Cl- conductance. We show that the influx of a fluorescent dye, dihydrorhodamine 6G (dR6G), is increased in cells expressing human CFTR after retrovirus- and adenovirus-mediated gene transfer. dR6G influx is stimulated by cAMP and is inhibited by antagonists of cAMP action. Dye uptake is ATP-dependent and inhibited by Cl- removal or the addition of 10 mM SCN-. Increased staining is associated with functional activation of CFTR Cl- permeability. dR6G staining enables both the fluorescent assessment of CFTR function and the identification of successfully corrected cells after gene therapy.

Myristate exchange on the Trypanosoma brucei variant surface glycoprotein.

The glycosyl-phosphatidylinositol (GPI) anchor of the Trypanosoma brucei variant surface glycoprotein (VSG) is unique in having exclusively myristate as its fatty acid component. We previously demonstrated that the myristate specificity is the result of two independent pathways. First, the newly synthesized free GPI, which is not myristoylated, undergoes fatty acid remodeling to replace both its fatty acids with myristate. Second, the myristoylated precursor, glycolipid A, undergoes a myristate exchange reaction, detected by the replacement of unlabeled myristate by [3H]myristate. Remodeling and exchange have different enzymatic properties and apparently occur in different subcellular compartments. We now demonstrate that the GPI anchor linked to VSG is the major substrate for myristate exchange. VSG can be efficiently labeled with [3H]myristate by exchange in the presence of cycloheximide, an inhibitor that prevents new VSG synthesis and thus anchor addition to protein. Not only is newly synthesized VSG subject to exchange, but mature VSG, possibly recycling from the cell surface, also undergoes myristate exchange.

A family of transmembrane proteins with homology to the MET-hepatocyte growth factor receptor.

In hunting for unknown genes on the human X chromosome, we identified a cDNA in Xq28 encoding a transmembrane protein (SEX) of 1871 amino acids. SEX shares significant homology with the extracellular domain of the receptors encoded by the oncogenes MET, RON, and SEA [hepatocyte growth factor (HGF) receptor family]. Further screenings of cDNA libraries identified three additional sequences closely related to SEX: these were named SEP, OCT, and NOV and were located on human chromosomes 3p, 1, and 3q, respectively. The proteins encoded by these genes contain large cytoplasmic domains characterized by a distinctive highly conserved sequence (SEX domain). Northern blot analysis revealed different expression of the SEX family of genes in fetal tissues, with SEX, OCT, and NOV predominantly expressed in brain, and SEP expressed at highest levels in kidney. In situ hybridization analysis revealed that SEX has a distinctive pattern of expression in the developing nervous system of the mouse, where it is found in postmitotic neurons from the first stages of neuronal differentiation (9.5 day postcoitus). The SEX protein (220 kDa) is glycosylated and exposed at the cell surface. Unlike the receptors of the HGF family, p220SEX, a MET-SEX chimera or a constitutively dimerized TPR-SEX does not show tyrosine kinase activity. These data define a gene family (SEX family) involved in the development of neural and epithelial tissues, which encodes putative receptors with unexpected enzymatic or binding properties.

Gangliosides are neuronal ligands for myelin-associated glycoprotein.

Nerve cells depend on specific interactions with glial cells for proper function. Myelinating glial cells are thought to associate with neuronal axons, in part, via the cell-surface adhesion protein, myelin-associated glycoprotein (MAG). MAG is also thought to be a major inhibitor of neurite outgrowth (axon regeneration) in the adult central nervous system. Primary structure and in vitro function place MAG in an immunoglobulin-related family of sialic acid-binding lactins. We report that a limited set of structurally related gangliosides, known to be expressed on myelinated neurons in vivo, are ligands for MAG. When major brain gangliosides were adsorbed as artificial membranes on plastic microwells, only GT1b and GD1a supported cell adhesion of MAG-transfected COS-1 cells. Furthermore, a quantitatively minor ganglioside expressed on cholinergic neurons, GQ1b alpha (also known as Chol-1 alpha-b), was much more potent than GT1b or GD1a in supporting MAG-mediated cell adhesion. Adhesion to either GT1b or GQ1b alpha was abolished by pretreatment of the adsorbed gangliosides with neuraminidase. On the basis of structure-function studies of 19 test glycosphingolipids, an alpha 2,3-N-acetylneuraminic acid residue on the terminal galactose of a gangliotetraose core is necessary for MAG binding, and additional sialic acid residues linked to the other neutral core saccharides [Gal(II) and GalNAc(III)] contribute significantly to binding affinity. MAG-mediated adhesion to gangliosides was blocked by pretreatment of the MAG-transfected COS-1 cells with anti-MAG monoclonal antibody 513, which is known to inhibit oligodendrocyte-neuron binding. These data are consistent with the conclusion that MAG-mediated cell-cell interactions involve MAG-ganglioside recognition and binding.

Three distinct structural environments of a transmembrane domain in the inwardly rectifying potassium channel ROMK1 defined by perturbation.

To probe the protein environment of an ion channel, we have perturbed the structure of a transmembrane domain by substituting side chains with those of two different sizes by using site-specific mutagenesis. We have used Trp and Ala as a high- and a low-impact perturbation probe, respectively, to replace each of 18 consecutive residues within the putative second transmembrane segment, M2, of an inwardly rectifying potassium channel, ROMK1. Our rationale is that a change in the channel function as a consequence of these mutations at a particular position will reflect the structural environment of the altered side chain. Each position can then be assigned to one of three classes of environments, as grated by different levels of perturbation: very tolerant (channel functions with both Trp and Ala substitutions), tolerant (function preserved with Ala but not with Trp substitution), and intolerant (either Ala or Trp substitution destroys function). We identify the very tolerant environment as being lipid-facing, tolerant as protein-interior-facing, and intolerant as pore-facing. We observe a strikingly ordered pattern of perturbation of all three environmental classes. This result indicates that M2 is a straight alpha-helix.

A pregnancy-specific glycoprotein is expressed in the brain and serves as a receptor for mouse hepatitis virus.

Mouse hepatitis virus (MHV), a murine coronavirus known to cause encephalitis and demyelination, uses murine homologues of carcinoembryonic antigens as receptors. However, the expression of these receptors is extremely low in the brain. By low-stringency screening of a mouse brain cDNA library, we have identified a member of the pregnancy-specific glycoprotein (PSG) subgroup of the carcinoembryonic antigen gene family. Unlike other PSG that are expressed in the placenta, it is expressed predominantly in the brain. Transfection of the cDNA into COS-7 cells, which lack a functional MHV receptor, conferred susceptibility to infection by some MHV strains, including A59, MHV-2, and MHV-3, but not JHM. Thus, this is a virus strain-specific receptor. The detection of multiple receptors for MHV suggests the flexibility of this virus in receptor utilization. The identification of this virus in receptor utilization. The identification of a PSG predominantly expressed in the brain also expands the potential functions of these molecules.

The LAR/PTP delta/PTP sigma subfamily of transmembrane protein-tyrosine-phosphatases: multiple human LAR, PTP delta, and PTP sigma isoforms are expressed in a tissue-specific manner and associate with the LAR-interacting protein LIP.1.

The transmembrane protein-tyrosine-phosphatases (PTPases) LAR, PTP delta, and PTP sigma each contain two intracellular PTPase domains and an extracellular region consisting of Ig-like and fibronectin type III-like domains. We describe the cloning and characterization of human PTP sigma (HPTP sigma) and compare the structure, alternative splicing, tissue distribution, and PTPase activity of LAR, HPTP delta, and HPTP sigma, as well their ability to associate with the intracellular coiled-coil LAR-interacting protein LIP.1. Overall, these three PTPases are structurally very similar, sharing 64% amino acid identity. Multiple isoforms of LAR, HPTP delta, and HPTP sigma appear to be generated by tissue-specific alternative splicing of up to four mini-exon segments that encode peptides of 4-16 aa located in both the extracellular and intracellular regions. Alternative usage of these peptides varies depending on the tissue mRNA analyzed. Short isoforms of both HPTP sigma and HPTP delta were also detected that contain only four of the eight fibronectin type III-like domains. Northern blot analysis indicates that LAR and HPTP sigma are broadly distributed whereas HPTP delta expression is largely restricted to brain, as is the short HPTP sigma isoform containing only four fibronectin type III-like domains. LAR, HPTP delta, and HPTP sigma exhibit similar in vitro PTPase activities and all three interact with LIP.1, which has been postulated to recruit LAR to focal adhesions. Thus, these closely related PTPases may perform similar functions in various tissues.

Mutation of conserved negatively charged residues in the S2 and S3 transmembrane segments of a mammalian K+ channel selectively modulates channel gating.

Voltage-gated channel proteins sense a change in the transmembrane electric field and respond with a conformational change that allows ions to diffuse across the pore-forming structure. Site-specific mutagenesis combined with electrophysiological analysis of expressed mutants in amphibian oocytes has previously established the S4 transmembrane segment as an element of the voltage sensor. Here, we show that mutations of conserved negatively charged residues in S2 and S3 of a brain K+ channel, thought of as countercharges for the positively charged residues in S4, selectively modulate channel gating without modifying the permeation properties. Mutations of Glu235 in S2 that neutralize or reverse charge increase the probability of channel opening and the apparent gating valence. In contrast, replacements of Glu272 by Arg or Thr268 by Asp in S3 decrease the open probability and the apparent gating valence. Residue Glu225 in S2 tolerated replacement only by acidic residues, whereas Asp258 in S3 was intolerant to any attempted change. These results imply that S2 and S3 are unlikely to be involved in channel lining, yet, together with S4, may be additional components of the voltage-sensing structure.

Fusogenic selectivity of the envelope glycoprotein is a major determinant of human immunodeficiency virus type 1 tropism for CD4+ T-cell lines vs. primary macrophages.

We investigated the relationship between the fusion selectivity of the envelope glycoprotein (env) and the tropism of different human immunodeficiency virus type 1 (HIV-1) isolates for CD4+ human T-cell lines vs. primary macrophages. Recombinant vaccinia viruses were prepared encoding the envs from several well-characterized HIV-1 isolates with distinct cytotropisms. Cells expressing the recombinant envs were mixed with various CD4+ partner cell types; cell fusion was monitored by a quantitative reporter gene assay and by syncytia formation. With CD4+ continuous cell lines as partners (T-cell lines, HeLa cells expressing recombinant CD4), efficient fusion occurred with the envs from T-cell line-tropic isolates (IIIB, LAV, SF2, and RF) but not with the envs from macrophage-tropic isolates (JR-FL, SF162, ADA, and Ba-L). The opposite selectivity pattern was observed with primary macrophages as cell partners; stronger fusion occurred with the envs from the macrophage-tropic than from the T-cell line-tropic isolates. All the envs showed fusion activity with peripheral blood mononuclear cells as partners, consistent with the ability of this cell population to support replication of all the corresponding HIV-1 isolates. These fusion selectivities were maintained irrespective of the cell type used to express env, thereby excluding a role for differential host cell modification. We conclude that the intrinsic fusion selectivity of env plays a major role in the tropism of a HIV-1 isolate for infection of CD4+ T-cell lines vs. primary macrophages, presumably by determining the selectivity of virus entry and cell fusion.