Human IFN-gamma production is inhibited by a synthetic peptide homologous to retroviral envelope protein

A synthetic 17 amino acid peptide (CKS-17) homologous to a highly conserved region of human and animal retroviral transmembrane proteins was investigated for its influence on the in vitro production of IFN-gamma from human peripheral mononuclear cells. The results showed that CKS-17 coupled to a carrier protein, BSA, inhibited production of IFN-gamma in a dose-dependent manner. Controls, consisting of BSA, which had undergone the coupling procedure or neurotensin coupled to BSA in an identical manner as CKS-17, showed no such inhibition. Reduction in IFN-gamma production could not be attributed to decreased viability of cells, delay of IFN-gamma production or to involvement of suppressor cells. Moreover, inhibition of IFN-gamma production was not related to the inhibition of DNA synthesis. The inhibition appeared to be a direct effect of CKS-17 on IFN-gamma-producing cells. Kinetic studies revealed that this suppression occurred when CKS-17 was introduced to the culture concurrent with or within 48 h after introduction of IFN inducers. Preincubation experiments showed that the presence of CKS-17 in the culture medium was not necessary to exert its inhibitory effect. These results suggest that a portion of retroviral envelope proteins possess important immunomodulatory actions.     

A synthetic peptide homologous to the envelope proteins of retroviruses inhibits monocyte-mediated killing by inactivating interleukin 1

The synthetic peptide CKS-17 has homology to a highly conserved region of the immunosuppressive retroviral envelope protein P15E, to envelope proteins of HTLV I, II, III, and to that encoded by an endogeneous C-type human retroviral DNA. CKS-17 inhibits the immune response of lymphocytes and the respiratory burst of human monocytes. Because P15E-related antigens are present in human malignant cell lines and cancerous effusions, we sought to determine the effect of CKS-17 on monocyte-mediated tumor cell lysis. Lysis of A375 tumor cells by lymphokine or lipopolysaccharide-activated human monocytes was inhibited by 10 microM CKS-17 (control, 79%; CKS-17-treated, 19%). Another synthesized peptide, CS-2, which has partial homology to CKS-17, failed to block monocyte-mediated killing. Thus, the inhibition by CKS-17 appeared to be specific. Because interleukin 1 (IL-1) is a cytocidal factor produced by activated monocytes, we also investigated the effect of CKS-17 on IL-1 production by monocytes and on direct IL-1-mediated cytotoxicity. CKS-17 did not block production or secretion of IL-1 by lipopolysaccharide- or interferon-gamma-activated monocytes. However, the direct cytocidal activity of both recombinant IL-1 alpha and IL-1 beta against A375 tumor cells was blocked by CKS-17. The cytotoxic activity of IL-1 was inhibited by CKS-17 if (a) IL-1 was preincubated with CKS-17 for 1 hr at 37 degrees C or (b) the A375 cells were incubated with CKS-17 for 1 hr prior to the addition of IL-1. CKS-17 also blocked IL-1-induced proliferation of murine thymocytes, the D10 T cell line, and an IL-1-responsive astrocytoma cell line. These data suggest that CKS-17 may be a potent inhibitor of IL-1.     

A synthetic peptide homologous to retroviral transmembrane envelope proteins depresses protein kinase C mediated lymphocyte proliferation and directly inactivated protein kinase C: a potential mechanism for immunosuppression

CKS-17, an immunosuppressive peptide homologous to certain retroviral transmembrane envelope protein, has been shown to inhibit lymphocyte proliferation in response to mitogens or alloantigens when covalently attached to bovine serum albumin (CKS-17-BSA). To define its site of action, we determined if CKS-17 conjugated to human serum albumin (CKS-17-HSA) could block the direct activation of lymphocytes by phorbol-12-myristate-13-acetate (PMA) or by a synthetic diacylglycerol, dioctanoylglycerol (DiC8). CKS-17-HSA inhibited lymphocyte proliferation in response to PMA and ionomycin in a dose-dependent manner with up to 88% inhibition occurring with 15 microM CKS-17-HSA. The conjugated peptide also inhibited the proliferation of lymphocytes in response to DiC8 and ionomycin by up to 57% at 15 microM CKS-17-HSA. Based on these findings we investigated the effect of CKS-17-HSA on the activity of protein kinase C (PKC), an enzyme directly activated by PMA and DiC8. PKC was isolated chromatographically from the cytosol of human neutrophils or the human lymphoblastoid cell line Jurkat. CKS-17-HSA caused a dose-dependent enzyme inhibition with a concentration giving half-maximal inhibition (IC50) of ca.3 microM and greater than 95% inhibition at 15 microM CKS-17-HSA. Inhibition of PKC by the conjugated peptide was not reversed by increasing concentrations of Ca2+, Mg2+, phosphatidylserine, diolein, or adenosine triphosphate (ATP), indicating that the conjugated peptide did not function as a chelator or competitive inhibitor. In contrast to its effects on PKC, CKS-17-HSA did not inhibit the activity of adenosine 3':5'-cyclic monophosphate (cyclic AMP)-dependent protein kinase (PK-A) nor the calcium and phospholipid-independent form of PKC (PK-M). Moreover the peptide inhibited in vivo PKC activity in cytosol of intact cells and in membrane of PMA-stimulated cells. These results suggest that the inhibition of lymphocyte proliferation by CKS-17-HSA may be due to the direct inactivation of PKC.     

Lactoferrin: no evidence for its role in regulation of CSA production by human lymphocytes and monocytes

Lactoferrin (LF) has been recently proposed as a physiologic regulator of the granulocyte monocyte progenitor (CFU-GM). This glycoprotein, when saturated with iron, has been said to limit CFU-GM growth by decreasing production and release of colony stimulating activity (CSA) by monocytes and macrophages. Human milk LF saturated with iron, at concentrations ranging from 10(-18) to 10(-8) M was added either to endogenously stimulated bone marrow cells or to mononucleated cells used as feeder layers for adherent cell-depleted marrow. Irrespective of the concentration of LF within the culture system used, no significant inhibition of CFU-GM growth was observed. Moreover, the CFU-GM stimulating activity of medium conditioned by a 4-day incubation of 1 X 10(6) mononucleated blood cells in the presence or in the absence of LF was the same. Various possible explanations for not confirming the reported inhibiting activity of iron saturated LF were explored: 1) masking inhibition of the system by prostaglandin E2 (PGE2), 2) masking inhibition of the system by bovine LF still detectable in the fetal calf serum after heating, 3) preinhibition of the system by leukemic-associated inhibitory activity (LIA) possibly present in the culture system, 4) the iron and calcium content of the culture medium used, 5) the fixation of LF to plastic compounds, 6) the source of the human LF used, 7) the marrow cell separation methods used. None of these factors was shown to play a role in vitro in the activity of LF and thus no evidence was found for a significant role of LF in the regulation of CSA production by monocytes. Peripheral blood human monocytes isolated by elutriation and incubated in albumin free medium in the presence of either 125I-LF or colloidal gold-labeled LF showed no LF binding.     

Identification of a nonconserved amino acid residue in multidrug resistance protein 1 important for determining substrate specificity: evidence for functional interaction between transmembrane helices 14 and 17.

Murine multidrug resistance protein 1 (mrp1), differs from its human ortholog (MRP1) in that it fails to confer anthracycline resistance and transports the MRP1 substrate, 17beta-estradiol 17-(beta-d-glucuronide) (E(2)17betaG), very poorly. By mutating variant residues in mrp1 to those present in MRP1, we identified Glu(1089) of MRP1 as being critical for anthracycline resistance. However, Glu(1089) mutations had no effect on E(2)17betaG transport. We have now identified a nonconserved amino acid within the highly conserved COOH-proximal transmembrane helix of MRP1/mrp1 that is important for transport of the conjugated estrogen. Converting Ala(1239) in mrp1 to Thr, as in the corresponding position (1242) in MRP1, increased E(2)17betaG transport 3-fold. Any mutation of mrp1 Ala(1239), including substitution with Thr, decreased resistance to vincristine and VP-16 without altering anthracycline resistance. However, introduction of a second murine to human mutation, Q1086E, which alone selectively increases anthracycline resistance, into mrp1A1239T restored resistance to both vincristine and VP-16. To confirm the importance of MRP1 Thr(1242) for E(2)17betaG transport and drug resistance, we mutated this residue to Ala, Cys, Ser, Leu, and Lys. These mutations decreased E(2)17betaG transport 2-fold. Conversion to Asp eliminated transport of the estrogen conjugate and also decreased leukotriene C(4) transport approximately 2-fold. The mutations also reduced the ability of MRP1 to confer resistance to all drugs tested. As with mrp1, introduction of a second mutation based on the murine sequence to create MRP1E1089Q/T1242A restored resistance to vincristine and VP-16, but not anthracyclines, without affecting transport of leukotriene C(4) and E(2)17betaG. These results demonstrate the important role of Thr(1242) for E(2)17betaG transport. They also reveal a highly specific functional relationship between nonconserved amino acids in TM helices 14 and 17 of both mrp1 and MRP1 that enables both proteins to confer similar levels of resistance to vincristine and VP-16.     

Activation of the erythropoietin receptor by the gp55-P viral envelope protein is determined by a single amino acid in its transmembrane domain.

The spleen focus forming virus (SFFV) gp55-P envelope glycoprotein specifically binds to and activates murine erythropoietin receptors (EpoRs) coexpressed in the same cell, triggering proliferation of erythroid progenitors and inducing erythroleukemia. Here we demonstrate specific interactions between the single transmembrane domains of the two proteins that are essential for receptor activation. The human EpoR is not activated by gp55-P but by mutation of a single amino acid, L238, in its transmembrane sequence to its murine counterpart serine, resulting in its ability to be activated. The converse mutation in the murine EpoR (S238L) abolishes activation by gp55-P. Computational searches of interactions between the membrane-spanning segments of murine EpoR and gp55-P provide a possible explanation: the face of the EpoR transmembrane domain containing S238 is predicted to interact specifically with gp55-P but not gp55-A, a variant which is much less effective in activating the murine EpoR. Mutational studies on gp55-P M390, which is predicted to interact with S238, provide additional support for this model. Mutation of M390 to isoleucine, the corresponding residue in gp55-A, abolishes activation, but the gp55-P M390L mutation is fully functional. gp55-P is thought to activate signaling by the EpoR by inducing receptor oligomerization through interactions involving specific transmembrane residues.     

Lymphokine-mediated activation of human monocytes: neutralization by monoclonal antibody to interferon-gamma

Purified natural and recombinant human immune interferon (IFN-gamma) were found to activate human monocytes from peripheral blood to exert enhanced cytotoxicity against human colon adenocarcinoma HT-29 cells. A marked monocyte activation was observed at low concentrations (1 and 10 U/ml) of IFN-gamma. Marked monocyte activation was also obtained with two lymphokine preparations, produced in peripheral blood mononuclear cell (PBM) cultures induced with phytohemagglutinin (PHA) or by combined stimulation with PHA and 12-O-tetradecanoylphorbol 13-acetate (TPA). The component responsible for macrophage activation in such lymphokine preparations in the past was considered to be "macrophage-activating factor" (MAF). When monoclonal antibody specifically neutralizing IFN-gamma was added to these lymphokine preparations, all MAF activity disappeared, indicating that IFN-gamma is the sole protein showing MAF activity in these preparations.     

Coreceptor tropism can be influenced by amino acid substitutions in the gp41 transmembrane subunit of human immunodeficiency virus type 1 envelope protein

Many studies have demonstrated that the third variable region (V3) of the human immunodeficiency virus type 1 (HIV-1) envelope protein (Env) is a major determinant of coreceptor tropism. Other regions in the surface gp120 subunit of Env can modulate coreceptor tropism in a manner that is not fully understood. In this study, we evaluated the effect of env determinants outside of V3 on coreceptor usage through the analysis of (i) patient-derived env clones that differ in coreceptor tropism, (ii) chimeric env sequences, and (iii) site-directed mutants. The introduction of distinct V3 sequences from CXCR4-using clones into an R5-tropic env backbone conferred the inefficient use of CXCR4 in some but not all cases. Conversely, in many cases, X4- and dual-tropic env backbones containing the V3 sequences of R5-tropic clones retained the ability to use CXCR4, suggesting that sequences outside of the V3 regions of these CXCR4-using clones were responsible for CXCR4 use. The determinants of CXCR4 use in a set of dual-tropic env sequences with V3 sequences identical to those of R5-tropic clones mapped to the gp41 transmembrane (TM) subunit. In one case, a single-amino-acid substitution in the fusion peptide of TM was able to confer CXCR4 use; however, TM substitutions associated with CXCR4 use varied among different env sequences. These results demonstrate that sequences in TM can modulate coreceptor specificity and that env sequences other than that of V3 may facilitate efficient CXCR4-mediated entry. We hypothesize that the latter plays an important role in the transition from CCR5 to CXCR4 coreceptor use.     

Alphaherpesviruses possess a gene homologous to the protein kinase gene family of eukaryotes and retroviruses.

The US3 genes of herpes simplex virus serotypes 1 and 2, and the corresponding gene of varicella-zoster virus, encode proteins whose sequences are clearly homologous to members of the protein kinase family of eukaryotes and retroviruses. Similarity is most characteristic, and strongest, in an 80 residue region comprising part of the catalytic structure of the kinases. In this region the herpesvirus proteins are most like a yeast cell division control protein, and least like the retrovirus protein-tyrosine kinases. We consider that the herpesvirus proteins are probably involved in modulation of cellular processes during lytic infection, although other roles are also possible, for example in latent infection.     

Determinants of the substrate specificity of multidrug resistance protein 1: role of amino acid residues with hydrogen bonding potential in predicted transmembrane helix 17

Human multidrug resistance protein 1 (MRP1) confers resistance to many natural product chemotherapeutic agents and actively transports structurally diverse organic anion conjugates. We previously demonstrated that two hydrogen-bonding amino acid residues in the predicted transmembrane 17 (TM17) of MRP1, Thr(1242) and Trp(1246), were important for drug resistance and 17beta-estradiol 17-(beta-d-glucuronide) (E(2)17betaG) transport. To determine whether other residues with hydrogen bonding potential within TM17 influence substrate specificity, we replaced Ser(1233), Ser(1235), Ser(1237), Gln(1239), Thr(1241), and Asn(1245) with Ala and Tyr(1236) and Tyr(1243) with Phe. Mutations S1233A, S1235A, S1237A, and Q1239A had no effect on any substrate tested. In contrast, mutations Y1236F and T1241A decreased resistance to vincristine but not to VP-16, doxorubicin, and epirubicin. Mutation Y1243F reduced resistance to all drugs tested by 2-3-fold. Replacement of Asn(1245) with Ala also decreased resistance to VP-16, doxorubicin, and epirubicin but increased resistance to vincristine. This mutation also decreased E(2)17betaG transport approximately 5-fold. Only mutation Y1243F altered the ability of MRP1 to transport both leukotriene 4 and E(2)17betaG. Together with our previous results, these findings suggest that residues with side chain hydrogen bonding potential, clustered in the cytoplasmic half of TM17, participate in the formation of a substrate binding site.     

The Lxl gene maps to mouse Chromosome 17 and codes for a protein that is homologous to glucose and polyspecific transmembrane transporters

A novel mouse gene, provisionally named Lx1, has been cloned and sequenced. Lx1 most likely represents the mouse homolog of the rat gene OCT1, which encodes a polyspecific transmembrane transporter that is possibly involved in drug elimination. The LX1 predicted protein is highly hydrophobic, possesses twelve putative transmembrane domains, and also shares significant homology with members of the sugar transporter family, particularly the novel liver-specific transporter NLT. Lx1 mRNA is expressed at high levels in mouse liver, kidney, and intestine, and at low levels in the adrenals and in lactating mammary glands. The Lx1 gene maps very close to the imprinted Igf2r/Mpr300 gene on mouse Chromosome (Chr) 17, in a region that is syntenic to human Chr 6q. Chr 6q has been previously associated with transient neonatal diabetes mellitus and breast cancer. Received: 11 March 1996 / Accepted: 5 June 1996     

Conversion of human interferon-β from a secreted to a phosphatidylinositol anchored protein by fusion of a 17 amino acid sequence to its carboxyl terminus

A number of cell-surface proteins are anchored in plasma membranes by a glycosylated phosphatidylinositol (PI) moiety that is covalently attached to the carboxyl-terminal amino acid of the mature protein. We have previously reported the construction of a cDNA clone of a truncated Platelet-derived growth factor (PDGF) receptor that consists of the extracellular domain without the transmembrane and cytoplasmic domains. In the construction of the vector, a sequence of 51 base pairs (bp) from the 3′-untranslated region of the receptor cDNA was linked in frame with the external domain coding sequence. The truncated receptor protein with the peptide VTSGHCHEERVDRHDGE fused to its carboxyl terminus was covalently attached to the membrane by a PI linkage and it was released by phosphatidylinositol specific-phospholipase C (PI-PLC). When the 51 bp sequence was deleted, the external domain receptor protein was secreted into the media. To determine whether the PI linkage of the protein was due to the 17 amino acids added, the peptide was fused to the carboxyl terminus of the secreted protein human Interferon-β (hu-IFN-β). Chinese hamster ovary (CHO) cells transfected with the hu-IFN-β cDNA secreted the protein to theconditioned media, whereas CHO cells transfected with the carboxyl terminus modified-hu-IFN-β cDNA did not secrete detectable levels of protein. CHO cells expressing the carboxyl terminus modified-hu-IFN-β were treated with PI-PLC, the media and cell lysates were analyzed by SDS-PAGE after immunoprecipitation with antibodies against hu-IFN-β. The modified protein is anchored to the plasma membrane by a PI linkage and it is specifically released by PI-PLC, whereas a control preparation of CHO cells expressing wild type hu-IFN-β does not show the same pattern. The 17 amino acid peptide fused to the carboxyl terminus of IFN-β directs attachment of a PI anchor and targets the fusion protein to the plasma membrane.     

Effect of amino acid substitutions on calmodulin binding and cytolytic properties of the LLP-1 peptide segment of human immunodeficiency virus type 1 transmembrane protein.

Previous studies have identified two highly basic amphipathic helical regions in the human immunodeficiency virus type 1 transmembrane protein that, in vitro, display both cytolytic and calmodulin-binding and -inhibitory properties that could contribute to cellular dysfunctions and cytopathogenesis during a persistent viral infection. In the current study, the structural specificity of the cytolytic and calmodulin-binding activities of the human immunodeficiency virus type 1 lentivirus lytic peptide (LLP-1) are examined with synthetic peptide homologs and analogs. The results of these studies demonstrate that even minor changes in LLP-1 amino acid content can markedly affect these properties, suggesting that sequence variation in these highly conserved LLP sequences may correlate with alterations in viral cytopathic properties.     

Characterization of the role of polar amino acid residues within predicted transmembrane helix 17 in determining the substrate specificity of multidrug resistance protein 3

Human multidrug resistance protein (MRP) 3 is the most closely related homologue of MRP1. Like MRP1, MRP3 confers resistance to etoposide (VP-16) and actively transports 17 beta-estradiol 17-(beta-D-glucuronide) (E(2)17 beta G), cysteinyl leukotriene 4 (LTC(4)), and methotrexate, although with generally lower affinity. Unlike MRP1, MRP3 also transports monovalent bile salts. We have previously demonstrated that hydrogen-bonding residues predicted to be in the inner-leaflet spanning segment of transmembrane (TM) 17 of MRP1 are important for drug resistance and E(2)17 beta G transport. We have now examined the importance of the hydrogen-bonding potential of residues in TM17 of MRP3 on both substrate specificity and overall activity. Mutation S1229A reduced only methotrexate transport. Mutations S1231A and N1241A decreased resistance to VP-16 and transport of E(2)17 beta G and methotrexate but not taurocholate. Mutation Q1235A also reduced resistance to VP-16 and transport of E(2)17beta G but increased taurocholate transport without affecting transport of methotrexate. Mutations Y1232F and S1233A reduced resistance to VP-16 and the transport of all three substrates tested. In contrast, mutation T1237A markedly increased VP-16 resistance and transport of all substrates. On the basis of the substrates analyzed, residues Ser(1229), Ser(1231), Gln(1235), and Asn(1241) play an important role in determining the specificity of MRP3, while mutation of Tyr(1232), Ser(1233), and Thr(1237) affects overall activity. Unlike MRP1, the involvement of polar residues in determining substrate specificity extends throughout the TM helix. Furthermore, elimination of the hydrogen-bonding potential of a single amino acid, Thr(1237), markedly enhanced the ability of the protein to confer drug resistance and to transport all substrates examined.     

Association of a pH-sensitive peptide with membrane vesicles: role of amino acid sequence

The solution properties and bilayer association of two synthetic 30 amino acid peptides, GALA and LAGA, have been investigated at pH 5 and 7.5. These peptides have the same amino acid composition and differ only in the positioning of glutamic acid and leucine residues which together compose 47% of each peptide. Both peptides undergo a similar coil to helix transition as the pH is lowered from 7.5 to 5.0. However, GALA forms an amphipathic alpha-helix whereas LAGA does not. As a result, GALA partitions into membranes to a greater extent than LAGA and can initiate leakage of vesicle contents and membrane fusion which LAGA cannot (Subbarao et al., 1987; Parente et al., 1988). Membrane association of the peptides has been studied in detail with large phosphatidylcholine vesicles. Direct binding measurements show a strong association of the peptide GALA to vesicles at pH 5 with an apparent Ka around 10(6). The single tryptophan residue in each peptide can be exploited to probe peptide motion and positioning within lipid bilayers. Anisotropy changes and the quenching of tryptophan fluorescence by brominated lipids in the presence of vesicles also indicate that GALA can interact with uncharged vesicles in a pH-dependent manner. By comparison to the peptide LAGA, the membrane association of GALA is shown to be due to the amphipathic nature of its alpha-helical conformation at pH 5.     

Partial amino acid sequence of human thyroxine-binding globulin. Further evidence for a single polypeptide chain.

The NH₂-terminal amino acid of highly purified thyroxine-binding globulin has been identified by dansyl chloride, cyanate and Edman degradation methods. All three gave alanine as the only amino terminal residue. Carbamylation and Edman degradation of the denatured protein yielded 0.86 and 0.98 – 1.05 mole of alanine per mole of protein, respectively. These data further indicate that thyroxine-binding globulin is composed of a single polypeptide chain. Automated Edman degradation gave the partial sequence as: Ala-Ser-Pro-Glu-Gly-Lys-Val-Thr-Ala-Asp-Ser-Ser-Ser-Gln-(Pro)-X-Ala-(Ser)-Leu-Tyr- A computer search revealed no homology of the NH₂-terminal segment of thyroxine-binding globulin with human prealbumin. The NH₂-terminal portion of prealbumin contains part of the thyroxine binding site.