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S J Davis

Publications and source records attributed to S J Davis.

At least 55 records · Page 3Linked to original sources
PubMed1997

Mutational analysis of the active site and antibody epitopes of the complement-inhibitory glycoprotein, CD59.

The Ly-6 superfamily of cell surface molecules includes CD59, a potent regulator of the complement system that protects host cells from the cytolytic action of the membrane attack complex (MAC). Although its mechanism of action is not well understood, CD59 is thought to prevent assembly of the MAC by binding to the C8 and/or C9 proteins of the nascent complex. Here a systematic, structure-based mutational approach has been used to determine the region(s) of CD59 required for its protective activity. Analysis of 16 CD59 mutants with single, highly nonconservative substitutions suggests that CD59 has a single active site that includes Trp-40, Arg-53, and Glu-56 of the glycosylated, membrane-distal face of the disk-like extra-cellular domain and, possibly, Asp-24 positioned at the edge of the domain. The putative active site includes residues conserved across species, consistent with the lack of strict homologous restriction previously observed in studies of CD59 function. Competition and mutational analyses of the epitopes of eight CD59-blocking and non-blocking monoclonal antibodies confirmed the location of the active site. Additional experiments showed that the expression and function of CD59 are both glycosylation independent.

Amino Acid Sequence↗
PubMed1997

Characterisation of the low affinity interaction between rat cell adhesion molecules CD2 and CD48 by analytical ultracentrifugation.

CD2 is a cell adhesion molecule found on the plasma membrane of T-lymphocytes. Its counter-receptor in rat is the structurally related CD48. This interaction is believed to contribute to the adhesion of T-cells to other cells such as cytotoxic targets and antigen presenting cells. Cell-cell adhesion involves the formation of multiple cell adhesion molecule complexes at the cell surface and if cell-cell de-adhesion is to occur, these complexes need to be disrupted. The affinities of cell adhesion molecule interactions are suggested to be relatively weak to allow this de-adhesion of cell-cell interactions. The CD2/CD48 interaction has been studied using recombinant extracellular proteins and the affinity of the interaction of soluble recombinant rat CD2-CD48 has been determined (at 37 degrees C) using surface plasmon resonance (and shown to be weak), with the dissociation constant Kd = 60-90 microM. The values determined by surface plasmon resonance results could be affected by the immobilisation of the ligand on the chip and any self-association on the chip. We used three different analytical ultracentrifuge procedures which each allowed the interaction to be studied in free solution without the need for an immobilisation medium. Both sedimentation equilibrium (using direct analysis of the concentration distribution and also modelling of molecular weight versus concentration data) and sedimentation velocity at 5 degrees C yielded dissociation constants in the range of 20-110 microM, supporting the surface plasmon resonance findings showing that binding between these cell adhesion molecules is relatively weak. These studies also ruled out the presence of any significant self-association of the reactants which could lead to systematic error in the surface plasmon resonance results.

Animals↗
PubMed1997

The impact of consolidation on Medicare reimbursement. Mergers, acquisitions, and other types of consolidations involve a change in ownership that implies termination of Medicare participation for the consolidating providers.

In order to fully understand the ramifications of consolidations, mergers, and acquisitions on Medicare reimbursement issues, healthcare financial managers must be well versed in the nuances of governance and managerial control, legal and regulatory compliance, and reimbursement. For example, when consolidations occur, there may be a desire for more shared control of a consolidated organization than would result if assets were sold outright. In addition, three important legal and regulatory issues must be dealt with when consolidations occur: change of ownership rules, cost-reporting consistency rules, and capital PPS rules. Finally, the consolidation of two or more organizations under a single Medicare provider number has several ongoing effects on Medicare reimbursement. DRG standardized amount and area wage index, rural referral centers, sole community providers, disproportionate share hospitals, and medical education are five areas affected.

Capital Expenditures↗
PubMed1996

NMR analysis of interacting soluble forms of the cell-cell recognition molecules CD2 and CD48.

The T cell glycoprotein, CD2, is one of the best characterized molecules mediating recognition at the cell surface. The ligands of murine and human CD2 are CD48 and CD58, respectively, and interactions between these molecules have been shown to influence antigen recognition and T cell activation. The CD58 binding site of human CD2 has been characterized in mutational studies, and here we use heteronuclear NMR spectroscopy to identify the rat CD48 binding site of the N-terminal domain of rat CD2 (CD2d1). The NMR spectrum of bacterially expressed CD2d1, assigned initially at pH 4.3 in the course of determining the three-dimensional solution structure of this domain [Driscoll, P.C., et al. (1991) Nature 353, 762-765], has been reassigned as a two-dimensional 15N-1H heteronuclear single-quantum coherence (HSQC) spectrum at neutral pH. The CD48 binding surface was identified by monitoring perturbations in the line widths and chemical shifts of cross peaks in the HSQC spectrum of CD2d1 during titrations with a soluble form of CD48 expressed in Chinese hamster ovary cells. This first solution NMR analysis of interacting cell surface molecules shows that the ligand binding site extends across an area of ca. 700-800 A2 of the GFCC'C" face corresponding almost exactly to lattice contacts in crystals of soluble CD2 first proposed as a model of the interaction of CD2 with its ligands. The analysis finds no evidence for any large-scale structural changes in domain 1 of CD2 to accompany CD48 binding. Comparisons of the human and rat CD2 ligand binding sites suggest that species- and ligand-specific binding may be determined by as few as three amino acid residues, corresponding to Thr37, Leu38, and Glu41 in rat CD2 (Lys42, Lys43, and Gln46 in human CD2).

Animals↗
PubMed1995

Ligand binding by the immunoglobulin superfamily recognition molecule CD2 is glycosylation-independent.

The evolutionary success of the immunoglobulin superfamily (IgSF) is thought to reflect the ability of IgSF protein domains to form stable structural units. The role of glycosylation in stabilizing these domains is controversial, however. In this study a systematic analysis of the effect of glycosylation on the ligand-binding properties of the cell-cell recognition molecule CD2, which consists of two IgSF domains, was undertaken. A form of human soluble CD2 (hsCD2) with single N-acetylglucosamine residues at each glycosylation site was produced by inhibiting glucosidase I with N-butyldeoxynojirimycin during expression in Chinese hamster ovary cells and digesting the expressed hsCD2 with endoglycosidase H. The ligand and antibody binding properties of this form of hsCD2 were indistinguishable from those of fully glycosylated hsCD2 as determined by surface plasmon resonance analyses. The protein also formed diffraction quality crystals and analysis of the 2.5-A resolution crystal structure indicated that the single N-acetylglucosamine residue present on domain 1 is unlikely to stabilize the ligand binding face of hsCD2. A second, fully deglycosylated form of hsCD2 also bound the ligand and antibodies although this form of the protein tended to aggregate. In contrast to the results of previous studies, the current data indicate that the structural integrity and ligand binding function of human CD2 are glycosylation-independent.

1-Deoxynojirimycin↗
PubMed1995

Topology of the CD2-CD48 cell-adhesion molecule complex: implications for antigen recognition by T cells.

BACKGROUND: The T-lymphocyte cell-surface molecule, CD2, was the first heterophilic cell-adhesion molecule to be discovered and has become an important paradigm for understanding the structural basis of cell adhesion. Interaction of CD2 with its ligands. CD58 (in humans) and CD48 (in mice and rats), contributes to antigen recognition by T cells. CD2, CD48 and CD58 are closely related members of the immunoglobulin superfamily and their extracellular regions are predicted to have very similar structures. The three-dimensional crystal structure of this region of CD2 has been determined, revealing two immunoglobulin domains with the ligand-binding site situated on an exposed beta sheet in the membrane-distal domain. This GFCC'C" beta sheet is also involved in a homophilic 'head-to-head' interaction in the CD2 crystal lattice, which has been proposed to be a model for the interactions of CD2 with its ligands. RESULTS: We show that the CD2-binding site on rat CD48 lies on the equivalent beta-sheet of its membrane-distal immunoglobulin domain. By making complementary mutations, we have shown that two charged residues in the CD48 ligand-binding site interact directly with two oppositely charged residues in CD2's ligand-binding site. These results indicate that the amino-terminal immunoglobulin domains of CD2 and CD48 bind each other in the same orientation as the CD2-CD2 crystal lattice interaction, strongly supporting the suggestion that CD2 interacts head-to-head with its ligand. Modelling CD48 onto the CD2 structure reveals that the CD2-CD48 complex spans approximately the same distance (134 A) as predicted for the complex between the T-cell receptor and the peptide-bound major histocompatibility complex (MHC) molecule. CONCLUSIONS: Our results, together with recent structural studies of CD2, provide the first indication of the specific topology of a cell-adhesion molecule complex. The similar dimensions predicted for the CD2-CD48 complex and the complex between the T-cell receptor and the peptide-bound MHC molecule suggest that one of the functions of CD2 may be to position the plasma membranes of the T cell and the antigen-presenting (or target) cell at the optimal distance for the low-affinity interaction between the T-cell receptor and the peptide-bound MHC molecule.

Amino Acid Sequence↗
PubMed1995

The alpha-glucosidase inhibitor N-butyldeoxynojirimycin inhibits human immunodeficiency virus entry at the level of post-CD4 binding.

The alpha-glucosidase inhibitor N-butyldeoxynojirimycin (NB-DNJ) is a potent inhibitor of human immunodeficiency virus (HIV) replication and syncytium formation in vitro. However, the exact mechanism of action of NB-DNJ remains to be determined. In this study we have examined the impairment of HIV infectivity mediated by NB-DNJ. By two independent HIV entry assays [PCR-based HIV entry assay and entry of Cocal(HIV) pseudotypes], the reduction in infectivity was found to be due to an impairment of viral entry. No effect of NB-DNJ treatment was seen on the kinetics of the interaction between gp120 and CD4 (surface plasmon resonance; BIAcore) or on the binding of virus particles to H9 cells (using radiolabeled virions). We therefore conclude that a major mechanism of action of NB-DNJ as an inhibitor of HIV replication is the impairment of viral entry at the level of post-CD4 binding, due to an effect on viral envelope components.

1-Deoxynojirimycin↗
PubMed1994

Human cell-adhesion molecule CD2 binds CD58 (LFA-3) with a very low affinity and an extremely fast dissociation rate but does not bind CD48 or CD59.

CD2 is a T lymphocyte cell-adhesion molecule (CAM) belonging to the immunoglobulin superfamily (IgSF) which mediates transient adhesion of T cells to antigen-presenting cells and target cells. Reported ligands for human CD2 include the structurally-related IgSF CAMs CD58 (LFA-3) and CD48 as well as, more controversially, the unrelated cell-surface glycoprotein CD59. Using surface plasmon resonance technology, which avoids several pitfalls of conventional binding assays, we recently reported that rat CD2 binds rat CD48 with a very low affinity (Kd 60-90 microM) and dissociates rapidly (koff > or = 6 s-1) [van der Merwe, P. A., Brown, M. H., Davis, S. J., & Barclay, A. N. (1993) EMBO J. 12, 4945-4954]. In contrast, a study using conventional equilibrium binding methods reported a much higher affinity (Kd 0.4 microM) for human CD2 binding CD58 which suggested that the weak binding of rat CD2 to CD48 may not represent a typical CAM interaction. In the present study we have used surface plasmon resonance to obtain definitive affinity and kinetic data on the interactions of a soluble, recombinant form of human CD2 with soluble forms of CD58, CD48, and CD59. Binding of CD2 to CD58 was readily detected but we were unable to detect any direct interaction between CD2 and either CD59 or CD48 under conditions in which very low affinity interactions (Kd approximately 0.5 mM) would have been detected. In contrast to previous reports we found that human CD2 bound CD58 with a very low affinity (Kd 9-22 microM) and dissociated with an extremely fast dissociation rate constant (koff > or = 4 s-1). The association rate constant (kon) could not be measured directly but was calculated to be > or = 400,000 M-1s-1. Taken together, these results provide conclusive evidence that CAM interactions can have very low affinities and extremely fast dissociation rate constants.

Antigens, CD↗
PubMed1994

Crystal structure of the extracellular region of the human cell adhesion molecule CD2 at 2.5 A resolution.

BACKGROUND: The T-lymphocyte antigen CD2 is an adhesion molecule implicated in immune responses in vivo. The extracellular regions of the human and rat homologues of CD2 share only 45% sequence identity and bind different protein ligands. Comparison of the human and rat soluble CD2 (sCD2) structures should provide insights into the structural basis of cell surface recognition. RESULTS: We therefore determined the crystal structure of a form of human sCD2 with single N-acetylglucosamine residues at each glycosylation site to 2.5 A resolution with an R-factor of 19.3%. It is composed of two immunoglobulin superfamily domains similar to those of rat sCD2, but the relative orientation of the domains in the two homologues differs by up to 20 degrees. An interaction involving the flat, highly charged, ligand binding GFCC'C" faces of crystallographically related human sCD2 molecules duplicates, in a different lattice, that observed in the rat sCD2 crystals. CONCLUSIONS: Intramolecular flexibility appears to be a conserved feature of CD2. The head-to-head interaction between molecules represents a general model for interactions between adhesion molecules of this structural class. Ligand specificity may be influenced by the distribution of charged residues on the binding face.

Amino Acid Sequence↗
PubMed1994

Crosstalk between domains in the regulatory subunit of cAMP-dependent protein kinase: influence of amino terminus on cAMP binding and holoenzyme formation.

The regulatory (R) subunit of cAMP-dependent protein kinase os an asymmetric multidomain protein with a dimerization domain at the N-terminus, an autoinhibitors site, and two cAMP binding domains at the C-terminus. Activation of the tetrameric holoenzyme is mediated by the cooperative binding of cAMP to the two cAMP binding sites. To better understand how the various domains influence each other, the N-terminus (delta 1-91) up to the autoinhibitor site was deleted. Not only did this monomeric deletion mutant, purified from Escherichia coli, still bind cAMP and the catalytic (C) subunit with high affinity, holoenzyme formation was actually accelerated by at least 50-fold. MgATP also was not required for rapid reassociation of (delta 1-91)R(cAMP)2 and C. The Kd(cAMP) and the Ka(cAMP) were similar to those for holoenzyme formed with full-length R; however, cooperatively was lost. Thus the N-terminus, either by inter- or intraprotomer contacts, not only impedes holoenzyme formation but also influences the cooperative binding of cAMP. The 1-91 deletion also renders the remaining fragment resistant to proteolytic degradation. Finally, unlike full-length R, the mutant protein can migrate freely into the nucleus. Surface plasmon resonance studies for the first time enabled direct measurements of the association and dissociation rate constants both for the intact R and for (delta 1-91)R. Both displayed very fast on-rates (1 x 10(-5) M-1 s-1 and 1.1 x 10(-5) M-1 s-1, respectively) and extremely slow off-rates (2.3 x 10(5) M-1 and 4.3 x 10(5) M-1, respectively). Thus, unlike the heat-stable protein kinase inhibitor, the region preceding the autoinhibitor site in R does not contribute in a quantitatively significant way to the high-affinity binding of C.

Adenosine Triphosphate↗
PubMed1994

Enhancement of antineoplastic activity of 5-fluorouracil in mice bearing colon 38 tumor by (6R)5,10-dideazatetrahydrofolic acid.

(6R)5,10-Dideazatetrahydrofolic acid (DDATHF, Lometrexol), a potent antitumor drug in vivo and in vitro, is an inhibitor of the two folate-dependent enzymes in the de novo purine biosynthesis pathway: glycinamide ribonucleotide (GAR) and amino imidazole carboxamide (AICAR) transformylases. A single dose of DDATHF (50 mg/kg, i.p.) in C57/BL6 mice caused a prolonged depletion of purine nucleotides (ATP and GTP) in colon 38 tumor and only a temporary effect in liver. GAR transformylase activity was higher in colon 38 tumor than in liver, but a kinetic analysis on the purified enzyme showed no differences in Ki values for DDATHF or Km values for the folate substrate. As a consequence of de novo purine synthesis inhibition, there was a 2- to 3-fold elevation of 5-phosphoribosyl-1-pyrophosphate pools in colon 38 tumor between 4 and 12 hr after DDATHF administration. When DDATHF (50 mg/kg) was administered 4 or 8 hr prior to 5-fluorouracil (5-FU; 85 mg/kg, i.p., weekly), these biochemical effects significantly increased the antitumor activity of 5-FU, with a modest increase in toxicity. Lower doses of DDATHF (25 and 37.5 mg/kg) when combined with 5-FU also resulted in an improved antitumor activity without additional toxicity. The two different schedules of administration for DDATHF, 4 and 8 hr prior to 5-FU, showed no differences in antitumor activity or toxicity.

Acyltransferases↗
PubMed1994

Three-dimensional solution structure of the extracellular region of the complement regulatory protein CD59, a new cell-surface protein domain related to snake venom neurotoxins.

The cell surface antigen CD59 is an inhibitor of complement-mediated lysis and a member of the Ly6 superfamily (Ly6SF) of cysteine-rich cell-surface molecules whose sequences are related to those of snake venom neurotoxins. The three-dimensional solution structure of a recombinant form of the extracellular region of the molecule (residues 1-70 of the mature protein; sCD59) has been solved by 2D NMR methods. sCD59 is a relatively flat, disk-shaped molecule consisting of a two-standed beta-sheet finger loosely packed against a protein core formed by a three-stranded beta-sheet and a short helix. Structure calculations allowed an unambiguous assignment of the disulfide-bonded cysteine pairs as 3-26, 6-13, 19-39, 45-63, and 64-69. The topology of sCD59 is similar to that of the snake venom neurotoxins and consistent with an evolutionary relationship existing between the Ly6SF and the neurotoxins.

Amino Acid Sequence↗