Cyclic RGD peptide inhibits alpha 4 beta 1 interaction with connecting segment 1 and vascular cell adhesion molecule.

The integrin supergene family includes receptors for a variety of extracellular matrix as well as cell surface proteins. Integrin alpha 4 has been shown to play an important role in leukocyte adhesion and extravasation during immune and inflammatory reactions. One recognition sequence known to interact with alpha 4 is the Leu-Asp-Val (LDV) site contained in the connecting segment 1 region of fibronectin. Here we present evidence that shows that a conformationally restricted RGD-containing peptide is a potent inhibitor of cell adhesion mediated by alpha 4 beta 1, a receptor not convincingly documented to interact with RGD peptides. This peptide, 1-adamantaneacetyl-Cys-Gly-Arg-Gly-Asp-Ser-Pro-Cys (disulfide bridge between residues 1-8), blocks Jurkat cell adhesion to connecting segment 1-containing peptides as well as cell adhesion to cytokine-activated endothelial cells. Adhesion of Jurkat cells to either vascular cell adhesion molecule-expressing cells or recombinant vascular cell adhesion molecule-coated plates was likewise inhibited by this peptide. Furthermore, alpha 4 beta 1 can bind directly to a cyclic RGD peptide immobilized to Sepharose. Integrins, alpha 5 beta 1, alpha v beta 3, alpha IIb/beta IIIa, alpha 2 beta 1, alpha v beta 1, alpha v beta 5, alpha v beta 6, and alpha 3 beta 1, have been shown to recognize the Arg-Gly-Asp (RGD) sequence present in a variety of extracellular matrix proteins, and our data support the addition of alpha 4 beta 1 to this group. Further studies using molecular modeling of such cyclic RGD peptides could help in the design of more potent peptides or nonpeptide mimetics that could effectively block alpha 4-mediated activity and have potential application in a number of inflammatory diseases.

A novel cyclic pentapeptide inhibits alpha 4 beta 1 and alpha 5 beta 1 integrin-mediated cell adhesion.

Lymphocytes and monocytes initiate and modulate inflammatory and immune responses for host defense. This process is dependent upon extravasation of leukocytes from the circulation to sites of antigenic challenge and is controlled, in part, by various integrins, including alpha 4 beta 1 and alpha 5 beta 1. A small cyclic pentapeptide that inhibits, in vitro, both alpha 4 beta 1 and alpha 5 beta 1 activity is described. This peptide, Arg-Cys-Asp-Thioproline-Cys (RC*D[ThioP]C*), is cyclized by a disulfide bond through the cysteine residues (the asterisks denote cyclizing residues). RC*D(ThioP)C* inhibits alpha 5 beta 1-mediated leukocyte adhesion to the 120-kDa Arg-Gly-Asp (RGD)-containing binding site of fibronectin. Two different adhesion activities of alpha 4 beta 1 are also inhibited: alpha 4 beta 1-mediated cell adhesion to the alternatively spliced CS-1 site of fibronectin and the alpha 4 beta 1-dependent binding of leukocytes to cytokine-activated endothelial cells. Both alpha 4 beta 1 and alpha 5 beta 1 can be purified by affinity chromatography using the immobilized pentapeptide. The peptide does not inhibit adhesion to other extracellular matrix proteins including laminin and vitronectin. The specificity of the RC*D(ThioP)C* peptide for alpha 4 beta 1 and alpha 5 beta 1 suggests potential therapeutic utility for inhibiting inflammatory disease.

Initiation of human cytomegalovirus infection requires initial interaction with cell surface heparan sulfate.

In this report, we demonstrate that the initial event in human cytomegalovirus (HCMV) infection is attachment to extracellular heparan sulfate. Further, this interaction is important for initiation of infection in fibroblast cells. Using microbinding assays to specifically monitor virus attachment as well as plaque titration assays to measure infectivity, we found that heparin competition as well as enzymatic digestion of cells with heparinase blocked virus attachment, initiation of immediate-early gene expression and infectivity. Other major glycosaminoglycans were found not to be involved in HCMV attachment and infectivity. In addition, HCMV was unable to attach to mutant derivatives of Chinese hamster ovary cells deficient in synthesis of heparan sulfate proteoglycans. Basic fibroblast growth factor, which requires initial interaction with extracellular heparin prior to binding to its high affinity receptor, also inhibited HCMV attachment to cells. Time-course experiments revealed that the initial HCMV binding was sensitive to heparin competition (10 micrograms/ml) or 0.75 M salt washes. The initial heparin-dissociable binding converted rapidly to high affinity (heparin resistant) HCMV attachment. These data suggest that sequential receptor interactions may mediate HCMV adsorption to cells. Heparin affinity chromatography revealed that multiple HCMV envelope glycoproteins, including gB, are capable of binding to heparin.

Human cytomegalovirus penetrates host cells by pH-independent fusion at the cell surface.

Biochemical, genetic, and morphological criteria were used to demonstrate that human cytomegalovirus penetrates permissive fibroblasts and nonpermissive Chinese hamster ovary (CHO) cells by pH-independent fusion between the virus envelope and the host cell plasma membrane and not by low pH-induced fusion within endosomes. Viral immediate early (IE) gene expression and infectivity were unaffected by conditions which block various stages of endocytosis or agents that alter the acidic pH of the endosome. IE gene expression was also evident in a mutant CHO cell line which is defective in endosomal acidification. Morphological analysis of the entry process at the electron microscopic level revealed viral particles in various stages of virion-plasma membrane fusion. In contrast, intact enveloped virions were not observed sequestered within coated pits or vesicular structures. Collectively, the data indicate that the entry pathway by which HCMV gains access to the cytoplasm of fibroblasts and CHO cells in order to initiate infection is via pH independent, virion envelope-plasma membrane fusion.

Expression of a human cytomegalovirus receptor correlates with infectibility of cells.

Previous studies have demonstrated that human cytomegalovirus (HCMV) specifically binds to a fibroblast membrane glycoprotein(s) with a molecular mass from 30 to 34 kDa. In this study, the distribution of the putative receptor proteins was analyzed in a variety of cell types, including cell types representative of those that are infected in vivo. Using a sensitive microbinding assay (to score virus attachment) and an indirect detection method (to score HCMV-binding proteins), we found that the 34- and 32-kDa HCMV binding proteins are ubiquitous molecules, broadly distributed among diverse cell types. In addition, the level of virus attachment was found to correlate with the abundance of the 34- and 32-kDa cellular proteins, while the ability of the virus to penetrate cells and initiate infection did not. The results support the hypothesis that the 34- and 32-kDa cellular proteins represent the HCMV (attachment) receptor. The data also support the notion that additional cellular components are required for virus entry and fusion.

Predominant low-molecular-weight proteins in isolated brain capillaries are histones.

Blood-brain barrier (BBB) function is endowed by the expression of unique proteins within the brain capillary endothelium. In the absence of knowing the function of BBB-specific proteins, one strategy for identification of these proteins is the purification and amino acid sequencing of proteins within the brain capillary that are not found in other cells. Earlier studies have shown that a 16-18K triplet of low-molecular-weight proteins in isolated brain capillaries is not found in either erythrocytes or in capillary-free preparations of synaptosomal proteins. Therefore, the present studies describe the purification of the 16-18K triplet of proteins as well as a 14K protein in isolated brain capillaries using sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and C4 reverse-phase HPLC. Amino acid sequencing of the N-terminus of the 14K, 17K, and 18K proteins and of two tryptic peptides of the 16K protein showed that these proteins are alpha-globin, histone 2B, histone 3, and histone 2A, respectively. SDS-PAGE of subcellular fractions of bovine brain capillaries demonstrated that the 16-18K triplet of histone proteins migrated in the nuclear fraction. In addition, a 34K doublet and a 200K protein were localized in the nuclear pellet. Therefore, the present studies demonstrate that the predominant 14-18K proteins seen on SDS-PAGE of isolated brain capillaries are known proteins and provide a general scheme for purification of brain capillary proteins isolated following SDS-PAGE.

Brain capillary 46,000 dalton protein is cytoplasmic actin and is localized to endothelial plasma membrane.

The most abundant protein of the brain capillary, which makes up the blood-brain barrier (BBB) in vivo, is a protein that migrates at a molecular weight of approximately 46 kDa on sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The bovine brain capillary 46 kDa protein was purified by SDS-PAGE and Sephadex G-25 gel filtration. The purified protein migrated as a single band of molecular weight of approximately 42,000 Da on subsequent SDS-PAGE followed by silver staining. The protein was digested by trypsin and tryptic peptides were analyzed by reverse phase high-performance liquid chromatography (HPLC). Two of these peptides, 11 and 18 amino acids in length, were sequenced and found to be identical to amino acid sequences corresponding to portions of cytoplasmic actin. The SDS-PAGE gel-purified 46 kDa protein was also subjected to limited proteolysis using S. aureus V8 protease, and this resulted in the formation of a prominent 31 kDa doublet as well as smaller proteolytic fragments, and these fragments were of identical molecular weight to those generated from limited proteolysis of bovine actin. Electron microscopic immunoperoxidase studies with primary cultures of bovine brain capillary endothelium showed that immunoreactive actin is intimately associated with the plasma membranes. In conclusion, the brain capillary 46 kDa protein is cytoplasmic actin and is localized to the endothelial plasma membrane. Modulations of brain capillary endothelial actin may play a role in the regulation of BBB permeability.

Adaptational "crosstalk" and the crucial role of methylation in chemotactic migration by Escherichia coli.

We investigated roles of methylation in bacterial chemotaxis by characterizing a methyl-accepting transducer protein incapable of methylation because of amino acid substitutions at the modification sites. Mutant Trg protein recognized ligand and generated excitatory signals that affected flagella but was unable to mediate efficient adaptation or net cellular migration in a relevant chemical gradient. Defects caused by lack of methyl-accepting sites on Trg were suppressed by a sufficient cellular content of other transducer molecules with functional methyl-accepting sites. These observations establish directly that methylation is crucial for transducer-mediated chemotaxis and that neither phosphotransfer reactions among the soluble Che proteins nor other interaction among those chemotactic components can effectively fulfill the functions of methylation. Suppression was correlated with adaptational "crosstalk" in which unoccupied methyl-accepting transducers acquired methyl groups, thus apparently substituting effectively for blocked methyl-accepting sites on the transducer. A plausible model for this phenomenon is that increased methylation of unstimulated transducers results from global inhibition of the demethylating enzyme in a cell with a normally active methyltransferase and no available methyl-accepting sites on the stimulated, mutant transducer. Thus methylation can perform its roles in adaptation and gradient sensing even if modification occurs on molecules different from those that recognize the stimulating compound. This observation emphasizes the central role of methylation and the modular nature of the chemosensory system.

Differential binding of testosterone and estradiol to isoforms of sex hormone-binding globulin: selective alteration of estradiol binding in cirrhosis.

The MCR of testosterone is decreased but the MCR of estradiol is unchanged in men with cirrhosis and elevated serum sex hormone-binding globulin (SHBG) concentrations. Previous studies indicated that SHBG from cirrhotic men selectively delivers estradiol, but not testosterone, to peripheral tissues of rats in vivo. These results suggest that estradiol and testosterone may bind to different SHBG isoforms in serum and that the estradiol-binding isoforms may be selectively altered in cirrhosis. This hypothesis was tested by polyacrylamide gel isoelectric focusing and fast protein liquid chromatography chromatofocusing. After Concanavalin-A affinity purification of serum glycoproteins from pregnant women, normal men, normal women, and cirrhotic men, the glycoprotein fraction was reconstituted, labeled with [3H]testosterone or [3H]estradiol, and applied to isoelectric focusing gels. Testosterone was bound selectively by the most acidic isoforms of SHBG, pI 4.5-5.4, and there was a significant anodal shift of the estradiol-binding isoforms in serum from cirrhotic men compared to that in serum from normal men. The selective binding of testosterone to the most acidic isoforms of SHBG was confirmed by fast protein liquid chromatography chromatofocusing, wherein the binding reactions were measured at neutral pH after separation of the isoforms. These biochemical studies and previous physiological experiments question the conventional view that testosterone and estradiol bind to a single competitive binding site on SHBG. Rather, testosterone is selectively bound by the most acidic SHBG isoforms. The estradiol-binding isoforms undergo a significant anodal shift in cirrhosis; this abnormality may result in the lack of decrease in estradiol MCR in cirrhosis.

Site-directed mutations altering methyl-accepting residues of a sensory transducer protein.

The Trg protein is one of a family of transducer proteins that mediate chemotactic response in Escherichia coli. Transducers are methyl-accepting proteins that gain or lose methyl esters on specific glutamyl residues during sensory adaptation. In this study, the significance of multiple sites of methylation on transducer proteins was addressed by using oligonucleotide-directed, site-specific mutagenesis to substitute an alanyl residue at each of the five methyl-accepting sites in Trg. The resulting collection of five mutations, each inactivating a single site, was analyzed for effects on covalent modification at the remaining sites on Trg and for the ability of the altered proteins to mediate sensory adaptation. Most of the alanyl substitutions had substantial biochemical effects, enhancing or reducing methyl-accepting activity of other sites, including one case of activation of a site not methylated in wild-type protein. Analysis of the altered proteins provided explanations for many features of the complex pattern of electrophoretic forms exhibited by Trg. The mutant proteins were less efficient than normal Trg in mediating adaptation. Correlation of biochemical and behavioral data indicated that reduction in the number of methyl-accepting sites on the transducer lengthened the time required to reach an adapted state.

Sites of covalent modification in Trg, a sensory transducer of Escherichia coli.

The Trg protein mediates chemotactic response of Escherichia coli to the attractants ribose and galactose. Like other transducers, Trg is a transmembrane protein that undergoes post-translational covalent modification. The modifications are hydrolysis (deamidation) of certain glutamine side chains to create glutamate residues and methylation of specific glutamates to form carboxyl methyl esters. Analysis of radiolabeled, tryptic peptides by high performance liquid chromatography and gas-phase sequencing allowed direct identification of the modified residues of Trg. The protein has 5 methyl-accepting residues. Four, at positions 304, 310, 311, and 318, are contained in a 23-residue tryptic peptide ending in lysine. The fifth, at position 500, is within a 25-residue tryptic peptide ending in arginine. At two sites, 311 and 318, glutamines are deamidated to create methyl-accepting glutamates. There is not a required order of modification among the sites. However, there is a substantial preference for methylation on the arginine peptide and, among sites on the lysine peptide, for the middle pair. Comparison of sequences surrounding modified residues identified in this work for Trg and previously for Tsr and Tar suggests a consensus sequence for methyl-accepting sites of Ala/Ser-Xaa-Xaa-Glu-Glu*-Xaa-Ala/OH-Ala-OH/Ala, where OH signifies Ser or Thr and the asterick marks the site of modification.

Chemotactic transducer proteins of Escherichia coli exhibit homology with methyl-accepting proteins from distantly related bacteria.

Transducers are transmembrane, methyl-accepting proteins central to the chemotactic systems of the enteric bacteria Escherichia coli and Salmonella typhimurium. Methyl-accepting proteins have been reported in a number of species in addition to these enteric bacteria. Those species include Bacillus subtilis and Spirochaeta aurantia, representatives of groups that diverged from ancestral enteric bacteria and from each other very early in bacterial evolution. An antiserum that reacts with all transducers of E. coli precipitated specifically methyl-accepting proteins from B. subtilis and S. aurantia, indicating that these proteins share antigenic determinants with transducers of E. coli. In addition, analysis of tryptic peptides by high-pressure liquid chromatography revealed similarities in the regions of methyl-accepting sites for proteins from all three species. These observations imply that structural features have been preserved in the three species from transducers contained in a common ancestor of eubacteria. It is thus reasonable to predict that other flagellated, chemotactic bacteria will be found to contain methyl-accepting proteins homologous to transducers of enteric bacteria.