Specificity determinants in the attachment sites of bacteriophages HK022 and lambda.

The Int proteins of bacteriophages HK022 and lambda promote recombination between phage and bacterial attachment sites. Although the proteins and attachment sites of the two phages are similar, neither protein promotes efficient recombination between the pair of attachment sites used by the other phage. To analyze this difference in specificity, we constructed and characterized chimeric attachment sites in which segments of one site were replaced with corresponding segments of the other. Most such chimeras recombined with appropriate partner sites in vivo and in vitro, and their differential responses to the Int proteins of the two phages allowed us to locate determinants of the specificity difference in the bacterial attachment sites and a central segment of the phage attachment sites. The location of these determinants encompasses three of the four core-type binding sites for lambda Int: C, B, and most importantly, B'. The regions corresponding to the C' core binding site and the arm-type binding sites of lambda Int play no role in the specificity difference and, indeed, are well conserved in the two phages. We found, unexpectedly, that the effect of replacement of an Int-binding region on the recombinational potency of one chimeric site was reversed by a change of partner. This novel context effect suggests that postsynaptic interactions affect the specificity of recognition of attachment sites by Int.     

Attachment of cells to basement membrane collagen type IV

Of ten different cell lines examined, three showed distinct attachment and spreading on collagen IV substrates, and neither attachment nor spreading was enhanced by adding soluble laminin or fibronectin. This reaction was not inhibited by cycloheximide or antibodies to laminin, indicating a direct attachment to collagen IV without the need of mediator proteins. Cell-binding sites were localized to the major triple-helical domain of collagen IV and required an intact triple helical conformation for activity. Fibronectin showed preferential binding to denatured collagen IV necessary to mediate cell binding to the substrate. Fibronectin binding sites of collagen IV were mapped to unfolded structures of the major triple-helical domain and show a similar specificity to fibronectin-binding sites of collagen I. The data extend previous observations on biologically potential binding sites located in the triple helix of basement membrane collagen IV.     

Syndecan binding sites in the laminin alpha1 chain G domain

The laminin alpha1 chain G domain has multiple biological activities. Previously, we identified cell binding sequences in the laminin alpha1 chain G domain by screening 113 synthetic peptide-polystyrene beads for cell attachment activity. Here, we have used a recombinant protein of the laminin alpha1 G domain (rec-alpha1G) and a large set of synthetic peptides to further identify and characterize heparin, cell, and syndecan-4 binding sites in the laminin alpha1 chain G domain. The rec-alpha1G protein promoted both cell attachment and heparin binding (K(D) = 19 nM). Cell attachment to the rec-alpha1G protein was inhibited 60% by heparin and 30% by EDTA. The heparin binding sites were identified by competing heparin binding to the rec-alpha1G protein with 110 synthetic peptides in solution. Only two peptides, AG73 (IC(50) = 147 microM) and AG75 (IC(50) = 206 microM), inhibited heparin binding to rec-alpha1G. When the peptides were compared in a solid-phase heparin binding assay, AG73 showed more heparin binding than AG75. AG73 also inhibited fibroblast attachment to the rec-alpha1G protein, but AG75 did not. Cell attachment to the peptides was studied using peptide-coated plates and peptide-conjugated sepharose beads. AG73 promoted cell attachment in both assays, but AG75 only showed cell attachment activity in the bead assay. Additionally, AG73, but not AG75, inhibited branching morphogenesis of mouse submandibular glands in organ culture. Furthermore, the rec-alpha1G protein bound syndecan-4, and both AG73 and AG75 inhibited this binding. These results suggest that the AG73 and AG75 sites are important for heparin and syndecan-4 binding in the laminin alpha1 chain G domain. These sites may play a critical role in the diverse biological activities involving heparin and syndecan-4 binding.     

Stereoselective binding of ketoprofen enantiomers to human serum albumin studied by high-performance liquid affinity chromatography

A chiral stationary phase based on immobilized human serum albumin (HSA) was used to study the stereoselective binding of ketoprofen enantiomers by means of high-performance liquid affinity chromatography. The technique of zonal elution was applied together with a novel mathematical approach describing attachment to more than one type of binding site. Phenylbutazon (PBZ) and diazepam (DAZ) were used as markers for the major believed binding regions on HSA. Both R- and S-ketoprofen (KTR and KTS) display high affinity to the primary PBZ- and DAZ-binding sites and low-affinity to the secondary DAZ sites. The binding to high-affinity regions is accepted to be a stepwise process initiated by the binding to the primary DAZ sites and followed by the attachment to the primary PBZ sites. The chiral recognition is attributed to the high-affinity PBZ-binding sites and to the low-affinity DAZ-binding sites.     

An integration-proficient int mutant of bacteriophage lambda

Summary We have isolated and characterized a novel int mutant of phage . This mutant promotes efficient recombination between the phage and bacterial attachment sites, but, unlike wild type, does not promote efficient recombination of any other pair of attachment sites tested in most conditions. IN particular, recombination between two phage or two prophage attachment sites is poor relative to the wild type frequency. We attribute this unusual phenotype to differences in the distribution of int protein binding sites among different attachment sites (Ross and Landy 1982, 1983). We suggest that int protein molecules bound to one of two recombining DNAs interact with empty sites or with bound proteins on the other, and that the mutant protein acts efficiently only if the distribution of protein binding sites within the two attachment sites is that of the attP-attB pair. Similar discrimination among attachment site pairs by wild type int protein may also modulate recombination frequencies.     

Role of Escherichia coli IHF protein in lambda site-specific recombination. A mutational analysis of binding sites

The phage lambda attachment site, attP, contains three binding sites for an Escherichia coli protein, IHF, that is needed for efficient integrative recombination. We have used synthetic oligodeoxyribonucleotides to direct multiple base changes at each of these three sites. Alteration by two base-pairs of the consensus sequence for the leftmost binding site specifically interferes with IHF binding to that site and modestly depresses recombination in vitro. For each of the three binding sites, alteration of the consensus sequence by four base-pairs strongly depresses recombination in vitro, indicating that all three sites are important for attP function. The mutated attP sites are also depressed for recombination in vivo but some of the mutants are less affected than they are in vitro. The disparity between effects in vivo and in vitro for some mutants but not others suggests that the three binding sites are not functionally equivalent and that at some sites additional E. coli factors may replace or assist IHF. The non-equivalence of the three IHF sites is also indicated by the behavior of prophage attachment sites carrying mutations in the binding sites.     

Hepadnavirus infection requires interaction between the viral pre-S domain and a specific hepatocellular receptor.

To better define the molecules involved in the initial interaction between hepadnaviruses and hepatocytes, we performed binding and infectivity studies with the duck hepatitis B virus (DHBV) and cultured primary duck hepatocytes. In competition experiments with naturally occurring subviral particles containing DHBV surface proteins, these DNA-free particles were found to interfere with viral infectivity if used at sufficiently high concentrations. In direct binding saturation experiments with radiolabelled subviral particles, a biphasic titration curve containing a saturable component was obtained. Quantitative evaluation of both the binding and the infectivity data indicates that the duck hepatocyte presents about 10(4) high-affinity binding sites for viral and subviral particles. Binding to these productive sites may be preceded by reversible virus attachment to a large number of less specific, nonsaturable primary binding sites. To identify which of the viral envelope proteins is responsible for hepatocyte-specific attachment, subviral particles containing only one of the two DHBV surface proteins were produced in Saccharomyces cerevisiae. In infectivity competition experiments, only particles containing the large pre-S/S protein were found to markedly reduce the efficiency of DHBV infection, while particles containing the small S protein had only a minor effect. Similarly, physical binding of radiolabelled serum-derived subviral particles to primary duck hepatocytes was inhibited well only by the yeast-derived pre-S/S particles. Together, these results strongly support the notion that hepadnaviral infection is initiated by specific attachment of the pre-S domain of the large DHBV envelope protein to a limited number of hepatocellular binding sites.     

Collagen receptors mediate early events in the attachment of epithelial (MDCK) cells

Summary Madin-Darby canine kidney (MDCK) cells kept in suspension culture for 12–15 hr displayed high-affinity binding sites for¹²⁵I-lathyritic (soluble) collagen (120,000/cell,K _D =30nm) and preferred collagens types I and IV over laminin or fibronectin as substrates during the first hour of attachment. On the other hand, after 4 hr, attachment to all four substrates was equally efficient. Upon challenge with a collagen substrate, the high-affinity sites were rapidly recruited on it (T1/2=6 min). Their occupancy by soluble collagen triggered the exocytosis of a second large population of low-affinity collagen binding sites that included laminin and seems to be involved in a second cell-attachment mechanism. These results are compatible with a twostep model of MDCK cell attachment to the substrate: first, via high-affinity collagen binding sites, and second, via laminin of cellular origin.     

Regulation of enzyme activity by enzyme orientation: A hypothesis

In addition to substrate binding sites, many enzymes must possess supersubstrate binding sites that regulate attachment and orientation of the enzyme toward the matrix (micelle, membrane) in which the substrate molecules are embedded, the supersubstrate.     

Ultraviolet photosensitivity of the estrogen binding protein from rat uterus. Wavelength and ligand dependence. Photocovalent attachment of estrogens to proteins.

Ultraviolet irradiation of the estrogen binding protein in rat uterine cytosol results in a progressive photoinactivation which is rapid at 254 nm and slower at greater than 315 nm. Both unfilled and estradiol-filled sites are inactivated at approimately the same rates at 254 nm (t 1/2 equals 8 min and 11 min, respectively), but at 315 nm, empty sites are consumed much more rapidly (t 1/2 equals 3.4 hr) than filled ones (t 1/2 equals 24 hr). The protective effect of the estrogen ligand at this wavelength appears to depend on its binding to the estrogen-specific binding site, as inactivation rate studies at different concentrations of estrone, estradiol, and estriol show a good correlation between the extent of protection and the fractional saturation of the high affinity estrogen binding sites. Scatchard analysis indicates that inactivation is the result of a loss of binding sites and not a decrease in their affinity, and sedimentation analysis shows that increased heterogeneity and aggregation of the estrogen binding species accompanies the photoinactivation process. Photoinactivation appears to be the result of direct irradiative damage of the animo acid residues, as the inactivation rate is the same under air and nitrogen atmospheres, and is unaffected by nucleophiles, reductants, and radical scavengers. When photoinactivation is measureed by irradiation of cytosol containing [3-H]estradiol, a concurrent photocovalent attachment process is noted; the steroid becomes linked to protein in a solvent-extractable manner (boiling ethanol inextractable). This attachment, however, does not appear to be related to the steroid binding at the estrogen binding site. Its rate is affected by reductants and scavengers. A similar photocovalent attachment reaction occurs when bovine serum albumin or ovalbumin is irradiated in the presence of [3-H]estradiol or [3-H]diethylstilbestrol. The detailed reactions involved in this photocovalent attachment process have not been defined at present.     

Gestational alterations in phospholipase C coupled muscarinic response

In the pregnant rat, carbachol-induced phosphoinositol hydrolysis by myometrium at the placental attachment region progressively decreased toward term, whereas hydrolysis was relatively stable in the myometrium of the non-attachment region. Tritium-quinuclidinyl benzilate binding increased in the myometrium of non-attachment regions as pregnancy progressed. At placental attachment sites binding remained relatively stable until parturition when it increased. Apparently the myometrium associated with the placental attachment site is less sensitive to cholinergic influence during pregnancy compared with the non-attachment site when evaluated by muscarinic activation of phospholipase C or ligand binding.     

Synaptic intermediates in bacteriophage lambda site-specific recombination: integrase can align pairs of attachment sites.

Bacteriophage lambda uses site-specific recombination to move its DNA into and out of the Escherichia coli genome. The recombination event is mediated by the recombinase integrase (Int) together with several accessory proteins through short specific DNA sequences known as attachment sites. A gel mobility shift assay has been used to show that, in the absence of accessory proteins, Int can align and hold together two DNA molecules, each with an attachment site, to form stable non-covalent 'bimolecular complexes'. Each attachment site must have both core and arm binding sites for Int to participate in a bimolecular complex. These stable structures can be formed between pairs of attL and attP attachment sites, but cannot include attB or attR sites; they are inhibited by integration host factor (IHF) protein. The bimolecular complexes are shown to represent a synaptic intermediate in the reaction in which Int protein promotes the IHF-independent recombination of two attL sites. These complexes should enable a detailed analysis of synapsis for this pathway.     

Inhibition of reovirus type 3 binding to host cells by sialylated glycoproteins is mediated through the viral attachment protein.

The interaction of mammalian reoviruses with sialylated glycoproteins was studied and found to be highly serotype specific in that attachment of type 3 Dearing reovirus to murine L cell receptors could be strongly inhibited by bovine submaxillary mucin (BSM), fetuin, and alpha 1 acid glycoprotein, albeit at different efficiencies, whereas attachment of type 1 Lang reovirus was inhibited only by fetuin. We subsequently demonstrated, by using reassortants between type 3 and 1 reoviruses, that inhibition of reovirus attachment to cell receptors was specified by the viral attachment protein gene S1. Using a solid-phase binding assay, we further demonstrated that the ability of reovirus type 3 or reassortant 1HA3 and the inability of reovirus type 1 or reassortant 3HA1 to bind avidly to BSM was a property of the viral S1 genome segment and required the presence of sialic acid residues on BSM oligosaccharides. Taken together, these results demonstrated that there is a serotype-specific difference in the ability of the reovirus attachment protein, sigma 1, to interact with sialylated oligosaccharides of glycoproteins. Interaction of reovirus type 3 with sialylated oligosaccharides of BSM is dramatically affected by the degree of O-acetylation of their sialic acid residues, as indicated by the findings that chemical removal of O-acetyl groups stimulated reovirus type 3 attachment to BSM, whereas preferential removal of residues lacking or possessing reduced amounts of O-acetyl groups per sialic acid molecule with Vibrio cholerae sialidase abolished binding. We also demonstrated that BSM was 10 times more potent in inhibiting attachment of infectious reovirus to L cells than was V. cholerae-treated BSM. The results are consistent with the hypothesis that sialylated oligosaccharides on host cells or erythrocytes may act as binding sites or components of binding sites for type 3 reovirus through a specific interaction with the virus attachment protein.     

Effect of glycosaminoglycans on fibronectin-mediated cell attachment

In this study, the role of glycosaminoglycans in fibronectin-mediated cell attachment to collagen has been investigated. While it has been established that fibronectin possesses binding sites for several glycosaminoglycans, it was found that only dextran sulfate and macromolecular heparin could decrease the initial rate of cell attachment to collagen. Low molecular weight heparin was inactive. This study suggests that the glycosaminoglycan binding site of fibronectin plays a role in the mechanism of cell attachment.     

Studies on the binding of lambda Int protein to attachment site DNA: identification of a tight-binding site in the P'' region.

We have used three approaches to studying the interaction of lambda Int protein with bacteriophage attachment site DNA, POP': location of binding sites by retention of DNA fragments in a filter binding assay, reconstruction of a binding site by DNA synthesis and protection of a binding site from an exonuclease. Retention of restriction fragments on nitrocellulose filters in the presence of Int protein was used to locate binding sites. A high affinity binding site lies in P' between base pairs -6 and +173 from the center of the common core sequence, and low affinity sites are found in the 200 base pair region left of position -6. Reconstruction of the high affinity binding site region from the right using primed DNA synthesis and testing for filter binding in the presence of Int protein shows that sequences sufficient for tight binding of Int protein lie to the right of position +66. When attachment site DNA is protected by bound Int protein against digestion by exonuclease III, four Int dependent protection bands are seen in positions +58, +68, +79 and +88. This can be interpreted either as showing that four Int protein monomers bind to the high affinity region in series, or as evidence for wrapping of the DNA around Int protein, leading to structural changes resembling those occurring to DNA in nucleosomes.     

Determinants of site-specific recombination in the lambdoid coliphage HK022. An evolutionary change in specificity

The temperate bacteriophage HK022, like its relative lambda, inserts its chromosome into a specific site in the bacterial chromosome during lysogenization and excises it after induction. However, we find that the recombinational specificities of the two phages differ: they use different bacterial sites, and neither promotes efficient insertion or excision of the other phage chromosome. In order to determine the basis for this difference in specificity, we sequenced the HK022 elements that are involved in insertion and excision, and compared them to the corresponding lambda elements. The location, orientation, size and overall arrangement of the int and xis genes and the phage attachment sites are nearly identical in the two genomes, as is common for other functionally related elements in lambdoid phages. The Xis proteins of the two phages are functionally interchangeable, and their predicted amino acid sequences differ by but one residue. In contrast, the two Int proteins are not functionally interchangeable, and their sequences, although similar, differ at many positions. These sequence differences are not uniformly distributed: the amino-terminal 55 residues are completely conserved, but the remaining 302 show a pattern of differences interspersed with identities and conservative changes. These findings imply that the specificity difference between HK022 and lambda site-specific recombination is a consequence of the inability of the respective Int proteins to recognize pairs of heterologous attachment sites. The two phage attachment sites are remarkably similar, especially the two "arm" segments, which in lambda contain binding sites for Int, Xis and integration host factor. They are less similar in the segment between the two arms, which in lambda contains the points of recombinational strand exchange and a second class of binding site for Int protein (the "core-type" sites). The two bacterial attachment sites are quite different, although both have a short stretch of perfect homology with their respective phage partners at the points of strand exchange. We propose that the two Int proteins recognize similar or identical sites in the arms of their cognate attachment sites, and that differences in binding or action at the core-type sites is responsible for the divergent specificities. Genetic experiments and sequence comparisons suggest that both proteins recognize different but overlapping families of core-type sites, and that divergence in specificity has been achieved by an alternating succession of small, mutually compatible changes in protein and site.     

Mechanism for chromosome and minichromosome segregation in Escherichia coli

A mechanism for the segregation of chromosomes and minichromosomes into daughter cells during division of Escherichia coli is presented. It is based on the idea that the cell envelope contains a large number of sites capable of binding to the chromosomal replication origin, oriC, and that a polymerizing DNA strand becomes attached to one of the sites at initiation of a round of replication. The attachment sites are distributed throughout the actively growing cell envelope, i.e. lateral envelope and septum, but not in the existing cell poles. This asymmetric distribution of oriC attachment sites accounts for the experimentally observed non-random chromosome and minichromosome segregation, and for the variation in the degree of non-random segregation with cell strain and growth rate. The multi-site attachment concept also accounts for the unstable maintenance of minichromosomes.     

Identification of biologically active sequences in the laminin alpha 4 chain G domain

Laminins are a family of trimeric extracellular matrix proteins consisting of alpha, beta, and gamma chains. So far five different laminin alpha chains have been identified. The laminin alpha 4 chain, which is present in laminin-8/9, is expressed in cells of mesenchymal origin, such as endothelial cells and adipocytes. Previously, we identified heparin-binding sites in the C-terminal globular domain (G domain) of the laminin alpha 4 chain. Here we have focused on the biological functions of the laminin alpha 4 chain G domain and screened active sites using a recombinant protein and synthetic peptides. The rec-alpha 4G protein, comprising the entire G domain, promoted cell attachment activity. The cell attachment activity of rec-alpha 4G was completely blocked by heparin and partially inhibited by EDTA. We synthesized 116 overlapping peptides covering the entire G domain and tested their cell attachment activity. Twenty peptides showed cell attachment activity, and 16 bound to heparin. We further tested the effect of the 20 active peptides in competition assays for cell attachment and heparin binding to rec-alpha 4G protein. A4G6 (LAIKNDNLVYVY), A4G20 (DVISLYNFKHIY), A4G82 (TLFLAHGRLVFM), and A4G83 (LVFMFNVGHKKL), which promoted cell attachment and heparin binding, significantly inhibited both cell attachment and heparin binding to rec-alpha 4G. These results suggest that the four active sites are involved in the biological functions of the laminin alpha 4 chain G domain. Furthermore, rec-alpha 4G, A4G6, and A4G20 were found to interact with syndecan-4. These active peptides may be useful for defining of the molecular mechanism laminin-receptor interactions and laminin-mediated cellular signaling pathways.     

Active Peptide Sequences in the Cell Binding Domain of Thrombospondins

Methods for measuring the attachment of viable cells to immobilized peptides to identify sequences possessing cell binding activity are described. The 212-residue C-terminal cell binding domain (CBD) of human thrombospondin-1 (TS1 or TS) was thought to contain a site or sites for the attachment of a number of cell types based on inhibition of cell attachment to TS1 with monoclonal antibodies. The presence of such a site was confirmed by expression of this region of TS1 in Escherichia coli and demonstration of its cell binding activity independent of other known cell attachment sites in TS1. To further define active sequences within the CBD, a set of eight overlapping peptides that spanned the 212-residue domain was synthesized with standard techniques. Several of these long (30-mer) peptides were found to be insoluble in aqueous buffers; thus, they could not be tested as soluble inhibitors of the attachment of cells to TS1, a standard approach to identification of active cell binding sequences. Using chemically derivatized plates prepared by Costar Corp., we devised assays to test the direct attachment of cells to the peptides immobilized on plastic wells. We also found that the peptides were adsorbed to underivatized plastic when solubilized in 6 M guanidine hydrochloride. Even rather short peptides (5 residues) were efficiently immobilized with these methods. Peptides immobilized directly on plastic gave higher levels of cell attachment than the same peptides conjugated to a carrier protein and then immobilized. Using these methods, two of the 30-mers were found to support cell attachment. The active sequences within the 30-mers were then narrowed down to a hexamer in one case and a pentamer, both containing the tripeptide VVM. Shorter, soluble peptides containing these active sequences act synergistically to inhibit the attachment of cells to TS1 and its recombinant CBD.