The chemokine receptor CCR5 is the major coreceptor for R5 human immunodeficiency virus type-1 strains. We mapped the epitope specificities of 18 CCR5 monoclonal antibodies (mAbs) to identify domains of CCR5 required for chemokine binding, gp120 binding, and for inducing conformational changes in Env that lead to membrane fusion. We identified mAbs that bound to N-terminal epitopes, extracellular loop 2 (ECL2) epitopes, and multidomain (MD) epitopes composed of more than one single extracellular domain. N-terminal mAbs recognized specific residues that span the first 13 amino acids of CCR5, while nearly all ECL2 mAbs recognized residues Tyr-184 to Phe-189. In addition, all MD epitopes involved ECL2, including at least residues Lys-171 and Glu-172. We found that ECL2-specific mAbs were more efficient than NH 2 -or MD-antibodies in blocking RAN-TES or MIP-1␤ binding. By contrast, N-terminal mAbs blocked gp120-CCR5 binding more effectively than ECL2 mAbs. Surprisingly, ECL2 mAbs were more potent inhibitors of viral infection than N-terminal mAbs. Thus, the ability to block virus infection did not correlate with the ability to block gp120 binding. Together, these results imply that chemokines and Env bind to distinct but overlapping sites in CCR5, and suggest that the N-terminal domain of CCR5 is more important for gp120 binding while the extracellular loops are more important for inducing conformational changes in Env that lead to membrane fusion and virus infection. Measurements of individual antibody affinities coupled with kinetic analysis of equilibrium binding states also suggested that there are multiple conformational states of CCR5. A previously described mAb, 2D7, was unique in its ability to effectively block both chemokine and Env binding as well as coreceptor activity. 2D7 bound to a unique antigenic determinant in the first half of ECL2 and recognized a far greater proportion of cell surface CCR5 molecules than the other mAbs examined. Thus, the epitope recognized by 2D7 may represent a particularly attractive target for CCR5 antagonists.Cellular entry by HIV-1 1 requires the presence of both CD4 and certain members of the chemokine receptor family (1-5). While numerous molecules can serve as coreceptors for one or more virus strains, the chemokine receptors CCR5 and CXCR4 are clearly the major coreceptors for R5 and X4 virus strains, respectively (6, 7). R5 virus strains are largely responsible for virus transmission, and individuals who lack CCR5 due to a natural knock-out mutation in the CCR5 gene (ccr5 ⌬32 allele) are highly resistant to HIV-1 infection (8 -10). The importance of CCR5 for viral entry and replication is further underscored by the observation that individuals heterozygous for the ccr5 ⌬32 allele have a 2-4-year delayed progression to AIDS (8,9,11,12), most likely due to reduced expression levels of CCR5 (13,14). On the other hand, X4 viruses tend to emerge years after infection, and the switch from R5 to X4 viruses correlates with progression to .The HIV-1 envelope (Env) glycoprotein...

Two genes, MF alpha 1 and MF alpha 2, coding for the alpha-factor in yeast Saccharomyces cerevisiae were identified by in situ colony hybridization of synthetic probes to a yeast genomic library. The probes were designed on the basis of the known amino acid sequence of the tridecapeptide alpha-pheromone. The nucleotide sequence revealed that the two genes, though similar in their overall structure, differ from each other in several striking ways. MF alpha 1 gene contains 4 copies of the coding sequence for the alpha-factor, which are separated by 24 nucleotides encoding the octapeptide Lys-Arg-Glu-Ala-Glu(or Asp)-Ala-Glu-Ala. The first alpha-factor coding block is preceded by a sequence for the hexapeptide Lys-Arg-Glu-Ala and 83 additional amino acids. MF alpha 2 gene contains coding sequences for two copies of the alpha-factor that differ from each other and from alpha-factor encoded by MF alpha 1 gene by a Gln leads to Asn and a Lys leads to Arg substitution. The first copy of the alpha-factor is preceded by a sequence coding for 87 amino acids which ends with Lys-Arg-Glu-Ala-Val-Ala-Asp-Ala. The coding blocks of the two copies of the pheromone are separated by the sequence for Lys-Arg-Glu-Ala-Asn-Ala-Asp-Ala. Thus, the alpha-factor can be derived from 2 different precursor proteins of 165 and 120 amino acids containing, respectively, 4 and 2 copies of the pheromone.

An inverted membrane vesicle fraction isolated from uninfected Escherichia coli and largely derived from the inner membrane has been shown to contain an endoproteolytic activity that cleaves nascent bacteriophage fI pre-coat protein into two identifiable products. The electrophoretic mobility on sodium dodecyl sulfate/urea/polyacrylamide gels and the partial amino-terminal sequence of the larger fragment were indistinguishable from those of the mature phage coat [15][16][17][18][19][20][21][22][23][24][25][26][27][28][29][30] "extra" amino acids at the amino terminus (see ref. 1 for a recent example). It has been proposed in the signal hypothesis (2) that this extension (the signal peptide) functions to establish a ribosome-membrane junction and thus provides the topological condition for a cotranslational and unidirectional transfer of the nascent chain across the membrane. Translation of mRNAs for eukaryotic secretory proteins in cell-free systems in the absence of microsomal membranes-i.e., in the absence of signal peptidase-therefore made it possible to detect the synthesis of these larger forms still containing the signal peptide and referred to as "pre"-secretory proteins. Translation in the presence of microsomal membranes has been shown to result in the removal of the signal peptide (3, 4). That mechanisms identical to those proposed in the signal hypothesis may also operate in prokaryotic cells was suggested by recent findings that demonstrated in vitro synthesis of several Escherichia colf proteins as larger than mature size molecules. Among these are the secretory protein alkaline phosphatase (5) and several outer membrane proteins (6), among them the lipoprotein (7). In the latter case it was shown that pre-lipoprotein contains an amino-terminal extension of 20 amino acid residues which resembled the signal peptide of eukaryotic presecretory proteins (7).The aim of the present study was to investigate whether other Our results demonstrate that a vesicle preparation largely derived from the inner membrane of uninfected E. coli contains a specific endoproteolytic activity that acts on nascent pre-coat protein to generate its mature form.METHODS AND MATERIALS Protein was synthesized in a coupled transcription-translation system as previously described (11, 12). [35S]Methionine was used as the radiolabeled amino acid unless specified otherwise. The translation products were then analyzed by electrophoresis in 1-mm-thick slab gels, which were generally of the sort described by Laemmli (13), except that they contained urea (8 M) in the separating gel, and the separating gel was prepared so that it contained a continuous exponential gradient of acrylamide ranging from 22% acrylamide/0.4% bisacrylamide at the bottom to 15% acrylamide/0.27% bisacrylamide at the top. Such gradients are needed to give good resolution of precursor and mature fi coat protein. Slab gels were stained, destained, dried, and subjected to autoradiography as previously described (2). In some instances (Figs. 1 and 4) the ne...

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Increasing evidence has shown that death signaling in T cells is regulated in a complicated way. Molecules other than death receptors can also trigger T-cell death. Here, we demonstrate for the first time that P-selectin glycoprotein ligand-1 (PSGL-1) or CD162 molecules crosslinked by an anti-PSGL-1 monoclonal antibody, TAB4, can trigger a death signal in activated T cells. In contrast to classic cell death, PSGL-1-mediated T-cell death is caspase independent. It involves translocation of apoptosis-inducing factor from mitochondria to nucleus and mitochondrial cytochrome c release. Ultrastructurally, both peripheral condensation of chromatin and apoptotic body were observed in PSGL-1-mediated T-cell death. Collectively, this study demonstrates a novel role for PSGL-1 in controlling activated T-cell death and, thus, advances our understanding of immune regulation. IntroductionDeath of T cells is fundamental in proper immune responses. In central and peripheral lymphoid organs, T-cell death occurs as a regulated event to ensure self-tolerance. [1][2][3][4] As the consequence of an immune response in normal conditions, most antigen-activated T cells die subsequently. [5][6][7] Activated T-cell death plays an important role in shaping and maintaining the T-cell repertoire and in avoiding undesired immune responses. Therefore, clarification of molecular mechanisms that determine death of activated T cells becomes imperative.According to current understanding, activated T-cell death occurs in at least 2 conditions: cytokine withdrawal and repeated antigenic stimulation. 8 In the former, when there is no further antigen stimulation, both interleukin 2 (IL-2) production and IL-2 receptor expression decline, and the death because of cytokine withdrawal ensues. 9,10 The latter is antigen driven and mediated through death receptors such as Fas and tumor necrosis factor (TNF) receptor. [11][12][13] Both conditions of activated T-cell death occur by way of activation of caspases and are strictly dependent on caspase-3. 14,15 Such classic caspase-activating death pathways may culminate mitochondrial dysfunction and cytochrome c release. 16 There is now increasing evidence for the existence of alternative, caspase-independent machinery of cell death in T-cell development and activation. [17][18][19][20][21][22][23] Fas-deficient lpr mice are able to eliminate T cells. 24 Additionally, by repeated antigenic stimulation, activated T-cell death in FLIP (FLICE [FADD (Fas receptor-associated death domain)-like IL-1␤-converting enzyme]-inhibitory protein) transgenic mice does occur. These mice do not develop lymphoproliferative disease despite their resistance to Fas-triggered death. 25 In line with these observations, several reports have shown that caspase inhibition fails to prevent cell death on some death stimuli, 17,18,26 suggesting the involvement of other alternative mechanisms. However, these caspase-independent pathways may be triggered in activated T cells when subjected to some death stimuli other than death receptors. ...