The cutaneous lymphocyte antigen is an essential component of the L-selectin ligand induced on human vascular endothelial cells.

L-selectin mediates leukocyte rolling on vascular endothelium during inflammation. Although vascular endothelium can be activated with inflammatory cytokines to express functional L-selectin ligands, these ligands have not been well characterized. In this study, fucosyltransferase VII cDNA (Fuc-TVII) transfection of the EA.hy926 human vascular endothelial cell line (926-FtVII) induced functional L-selectin ligand expression and expression of sialyl Lewisx (sLex), as defined by HECA-452 (cutaneous lymphocyte antigen; CLA) and CSLEX-1 mAbs. Cytokine activation of human umbilical vein endothelial cells (HUVEC) also induced functional L-selectin ligand expression, with increased CLA expression and Fuc-TVII transcription. The majority of L-selectin-dependent lymphocyte attachment to activated HUVEC and 926-FtVII cells was blocked specifically by treating the endothelial cells with the HECA-452 mAb, but not the CSLEX-1 mAb. CLA-bearing ligands on vascular endothelium also required sulfation and appropriate molecular scaffolds for functional activity, but were distinct from the L-selectin ligands previously identified by the MECA-79 mAb. These findings demonstrate that the HECA-452- defined antigen, CLA, is an essential carbohydrate component of vascular L-selectin ligands.

L-selectin binds to P-selectin glycoprotein ligand-1 on leukocytes: interactions between the lectin, epidermal growth factor, and consensus repeat domains of the selectins determine ligand binding specificity.

The selectins mediate cellular interactions by binding carbohydrate determinants present on a limited number of glycoprotein ligands. L-selectin binds multiple ligands expressed on endothelial cells, while P-selectin interacts exclusively with P-selectin glycoprotein ligand-1 (PSGL-1) on leukocytes. In this study, L-selectin was shown to bind leukocytes through the P-selectin ligand, PSGL-1, although at lower levels than P-selectin. L-selectin binding to PSGL-1 is specific since it was blocked by Abs to L-selectin or PSGL-1, required appropriate glycosylation of PSGL-1, and was Ca2+ dependent. The contributions of the extracellular domains of the selectins to ligand binding was assessed using a panel of chimeric selectins created by exchange of domains between L-selectin and P- or E-selectin. The lectin and epidermal growth factor domains of L- and P-selectin contributed significantly to binding through similar, if not identical, regions of PSGL-1. The different chimeric selectins revealed that the lectin domain was the dominant determinant for ligand binding, while cooperative interactions between the lectin, epidermal growth factor, and short consensus repeat domains of the selectins also modified ligand binding specificity. L-selectin binding to PSGL-1 expressed by leukocytes may mediate neutrophil rolling on stationary leukocytes bound to cytokine-induced endothelial cells, which was previously reported to be a L-selectin-dependent process.

Mutational analysis of the fusion peptide of the human immunodeficiency virus type 1: identification of critical glycine residues.

The ability of human immunodeficiency virus type 1 (HIV-1) to fuse its membrane with the membrane of the target cell is a function of a approximately 23-amino-acid amino-terminal segment of the gp41 subunit of the envelope glycoprotein complex, known as the fusion peptide. The sequence of the fusion peptide is highly conserved among different variants of HIV-1 and is also very similar to that of HIV-2 and SIV. The fusion peptide is very hydrophobic and has a high content of glycine and alanine residues. Representation of the fusion peptide of HIV-1 as an alpha-helix predicts that most glycine residues would be found on one face of the alpha-helix. To assess the importance of the glycine residues for the fusogenic activity of the envelope glycoprotein complex, we mutagenized each glycine residue in the fusion peptide individually to a valine residue. The mutant envelope constructs were tested for their ability to induce syncytia (cell/cell fusion) and to mediate infection (virus/cell fusion) of CD4-positive cells. The results of our analyses show that two glycine residues (G10 and G13) located within the sequence FLGFLG in the middle of the fusion peptide are critical for syncytium formation and for the establishment of a productive infection, whereas other glycine residues (G3, G5, and G20) are more permissive to substitutions. Mutation of each of the two phenylalanines (F8 and F11) of the FLGFLG sequence to valine also decreased fusion, although to a lesser extent than mutation of G10 and G13. These observations demonstrate that G10 and G13 are critical elements of the fusion peptide and suggest that, in addition to hydrophobicity, the exact amino acid composition and structure of the fusion peptide are critical for function.

Studies on primer binding of HIV-1 reverse transcriptase using a fluorescent probe.

The fluorescent nucleotide analog, 2',3'-trinitrophenyladenosine-5'-triphosphate (TNP-ATP), was utilized to quantify the affinities of human immunodeficiency virus-1 reverse transcriptase (HIV-1 RT) for its substrates. Interaction of this probe with the enzyme brings about a twofold increase in the magnitude of fluorescence emission from the probe, and a blue-shift in wavelength maximum, from 561 to 553 nm. TNP-ATP binds HIV-1 RT with a dissociation constant of 21 microM. The presence of millimolar levels of deoxynucleoside triphosphates or micromolar levels of an oligonucleotide primer analogue, p(dT)12-18, suppressed this enhancement of fluorescence. The fact that inhibition was achieved with much lower levels of primer than of dNTPs suggests that TNP-ATP is a probe for the binding site of primer on the enzyme, rather than that of deoxynucleoside triphosphate. In support of this, the effect of TNP-ATP on the kinetics of DNA synthesis catalyzed by the enzyme indicated that the probe is a competitive inhibitor with respect to template-primer. The ability of primers and primer analogs to reverse the fluorescence enhancement was determined, and the corresponding affinities of these compounds for reverse transcriptase were calculated. The affinity increased with primer length, increasing more than 50-fold from a span of 5 to 15 nucleotide residues. The interaction of polydeoxynucleotides was consistent with a model in which the enzyme bound at adjacent internal sites of about 15 residues in length. Several mammalian and bacterial transfer RNA primers were tested, including the natural primer, tRNA(3Lys). The affinities were found to be between 0.55 and 1.2 microM, with no obvious selectivity for the natural primer, which had a Kd of 0.79 microM. These results are discussed within the context of data for HIV-1 RT obtained by other methodologies.

Uncleaved signals for glycosylphosphatidylinositol anchoring cause retention of precursor proteins in the endoplasmic reticulum.

Glycosylphosphatidylinositol (GPI)-anchored proteins are generally absent from the surface of cells that are defective in GPI biosynthesis. The current study was undertaken to: (a) examine in detail the intracellular localization and fate of precursors of GPI-anchored proteins in cells that fail to add GPI groups and (b) define structural characteristics of the precursor proteins that determine their intracellular localization. By examining GPI-deficient cells, we show that the uncleaved precursor of the GPI-anchored protein, Q7b, is retained in the cisternae of the endoplasmic reticulum (ER) and is largely lost intracellularly with a half-time of 2-4 h. Only a small amount (1-10%) of a proteolytically cleaved form of the protein is secreted into the medium. In cells competent for GPI anchor addition, mutation of the putative cleavage/attachment site for GPI addition in Q7b results in a similar phenotype of ER retention of the uncleaved precursor. An aspartic acid residue (Asp316) within the Q7b GPI anchoring signal, previously found to be essential for GPI anchor addition (Waneck, G. L., Stein, M. E., and Flavell, R. A. (1988) Science 241, 697-699), is also shown to be critical for ER retention. Information leading to ER retention is transferable to another protein leading to ER retention is transferable to another protein by fusion of the GPI anchoring signals from either Q7b or the GPI-anchored form of the IgG Fc receptor type III. Analysis by sedimentation on sucrose gradients shows that Q7b species retained in the ER are multimeric, whereas species that exit the ER are monomeric. This correlation suggests that the presence of an uncleaved signal for GPI anchoring induces changes in the aggregation state of the precursor proteins, which may lead to their retention in the ER.

A general method of curve fitting and error analysis using a spreadsheet: determination of the binding constants of tight binding ligands in variable volume assays.

A generalized iterative method of curve fitting and error estimation is described, using a spreadsheet with Monte Carlo simulations. The method is demonstrated on an equilibrium binding experiment with reverse transcriptase of human immunodeficiency virus-1, in which the concentration of all reactants, including enzyme, can vary arbitrarily and any number of ligands can bind tightly.

Nucleic acid interactive properties of a peptide corresponding to the N-terminal zinc finger domain of HIV-1 nucleocapsid protein.

An 18-residue peptide (NC-F1) with an amino acid sequence corresponding to the N-terminal zinc finger of human immunodeficiency virus-1 nucleocapsid protein has been shown to bind to nucleic acids by fluorescence and NMR methods. Previously, this peptide has been shown to fold into a defined structure when bound to zinc (Summers et al., 1990). We have used a fluorescent polynucleotide, poly(ethenoadenylic acid), to monitor binding of this peptide to nucleic acids. In the presence of zinc, the peptide had a smaller site size (1.75 nucleotide residues/peptide) than in the absence of the metal ion (2.75). The salt sensitivity of the interaction indicated that two ion pairs are involved in the association of Zn2+ (NC-F1) with polynucleotide, whereas one ion pair is found in the metal-free peptide-nucleic acid complex. Competition experiments with single-stranded DNA (ss DNA) in either the presence or absence of Zn2+ showed that the peptide bound to ss DNA. Using NMR methods, we monitored the binding of a synthetic oligonucleotide, d(TTTGGTTT), to Zn(NC-F1). The hydrophobic residues F2 and I10, which are on the surface of the peptide and have been implicated in viral RNA recognition, were shown to interact with the oligomer. In accord with this observation, analysis of the salt dependence of the polynucleotide-peptide interaction indicates a nonelectrostatic component of about -6 kcal/mol, a value consistent with theoretical estimates of stacking energies of phenylalanine with nucleic acid bases.

Involvement of basic amino acids in the activity of a nucleic acid helix-destabilizing protein.

Under conditions of low ionic strength, ribonuclease A, which binds more tightly to single- than to double-stranded DNA, lowers the melting temperature of DNA helices (Jensen and von Hippel (1976) J. Biol. Chem. 251, 7198-7214). The effects of chemical modification of lysine and arginine residues on the helix-destabilizing properties of this protein have been examined. Removal of the positive charge on the lysine epsilon-amino group, either by maleylation or acetylation, destroys the ability of RNAase A to lower the Tm of poly[d(A-T)]. However, reductive alkylation of these residues, which has not effect on charge, yields derivatives which lower the Tm by only about one-half that seen with unmodified controls. Phenylglyoxalation of arginines can largely remove the Tm-depressing activity of RNAase A. RNAase S, which is produced by cleavage of RNAase A between amino acids 20 and 21, possesses DNA helix-destabilizing activity comparable to that of the parent protein, whereas S-protein (residues 21-124) increases poly[d(A-T)] Tm and S-peptide (1-20) has no effect on Tm. These results suggest that specific location of several basic amino acids situated on the surface of RNAase A is largely responsible for this protein's DNA melting activity.