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S Sheriff

Publications and source records attributed to S Sheriff.

At least 55 records · Page 3Linked to original sources
PubMed1995

The x-ray structure of an anti-tumour antibody in complex with antigen.

The crystal structures of the murine BR96 Fab and its human chimera have been determined in complex with the nonoate methyl ester derivative of Lewis Y (nLey) at 2.8 A and 2.5 A resolution, respectively. BR96 binds the carbohydrate in a large pocket which is formed by residues of all CDR loops except L2. The binding of the carbohydrate is mediated predominantly by aromatic residues in BR96. Analysis of the structure suggests that BR96 is capable of recognizing a structure larger than the Le(y) tetrasaccharide, providing a possible explanation for its high tumour selectivity. The structure provides a rationale for mutagenesis experiments that have resulted in BR96 CDR loop mutants with increased affinity for nLey and/or tumour cells.

Animals↗
PubMed1994

Crystallization and preliminary X-ray analysis of anti-digoxin antibodies.

The Fab fragments of several monoclonal antibodies that bind digoxin and other cardiac glycosides have been screened for crystallization conditions. We have crystallized two of these in forms suitable for X-ray analysis. The anti-digoxin antibody 40-50 in complex with ouabain crystallizes with symmetry consistent with space group C2, with a = 92.6, b = 85.0, c = 73.0 A and beta = 131.7 degrees. This crystal form shows considerable non-isomorphism between crystals. A second anti-digoxin antibody, 49-10, crystallizes with symmetry consistent with space group P2(1)2(1)2, with a = 95.3, b = 147.1 and c = 76.2 A.

Journal Article↗
PubMed1994

Crystallization and preliminary X-ray analysis of a trimeric form of human mannose binding protein.

A trimeric form of the carbohydrate recognition domain of human mannose binding protein has been crystallized in two different forms. The first form crystallizes with symmetry consistent with space group P2(1)2(1)2(1) and a = 61 A; b = 144 A; c = 107 A with presumably two trimers in the asymmetric unit. The second form crystallizes with symmetry consistent with space group P321 and a = b = 77 A; c = 58 A and one monomer per asymmetric unit. The molecular and crystallographic 3-folds must be coincident in this crystal form.

Carrier Proteins↗
PubMed1994

Crystallization and preliminary X-ray analysis of an anti-staphylococcal nuclease-staphylococcal nuclease complex and of a second anti-staphylococcal nuclease antibody.

The Fab fragments of several monoclonal antibodies that bind Staphylococcal nuclease have been screened for crystallization conditions. Two of these, N10 and N25, have been crystallized in forms suitable for X-ray structural analysis. The anti-Staphylococcal nuclease antibody complex N10 Fab-nuclease crystallizes with symmetry consistent with space group C2 and cell parameters of a = 234.7 A; b = 43.5 A; c = 74.4 A; beta = 106.4 degrees. A second anti-Staphylococcal nuclease antibody, N25, although crystallized starting with the Fab-nuclease complex, apparently crystallizes as uncomplexed N25 Fab with symmetry consistent with space group P3(1)21 (or its enantiomorph P3(2)21) and cell parameters of a = b = 80.9 A; c = 138.4 A.

Antibodies, Bacterial↗
PubMed1994

[D-TRP32]neuropeptide Y: a competitive antagonist of NPY in rat hypothalamus.

Neuropeptide Y (NPY) is a potent orexigenic peptide. Structure-activity studies have revealed that nearly the entire sequence of NPY is required to elicit feeding responses. Therefore, in order to develop antagonistic peptides for NPY-induced feeding, we synthesized full-length analogs of NPY, substituting D-Trp in the C-terminal receptor binding region, and screened their activity in rat hypothalamus. Although [D-Trp36]NPY and [D-Trp34]NPY inhibited isoproterenol-stimulated hypothalamic membrane adenylate cyclase activity, [D-Trp32]NPY exhibited no intrinsic activity. Furthermore, [D-Trp32]NPY inhibited [125I]NPY binding to rat hypothalamic membranes with a potency comparable to that of NPY. The presence of 30 and 300 nM concentrations of [D-Trp32]NPY shifted the inhibitory dose-response curve of NPY on isoproterenol-stimulated hypothalamic membrane adenylate cyclase activity parallel to the right with comparable KB values. Moreover, in vivo experiments in rats revealed that [D-Trp32]NPY (10 micrograms) significantly attenuated the 1-h feeding response induced by NPY (1 microgram). Several other substitutions at position 32 including 2-D-Nal resulted in agonist activity, suggesting that there are strict structural requirements to induce the antagonistic property in NPY. These findings confirm that [D-Trp32]NPY is a competitive antagonist of NPY in both in vitro and in vivo systems. Analogs based on [D-Trp32]NPY may have potential clinical application, since NPY has been implicated in the pathophysiology of a number of feeding disorders including obesity, anorexia, and bulimia.

Adenylyl Cyclase Inhibitors↗
PubMed1994

Crystallization and preliminary X-ray analysis of the monoclonal anti-tumor antibody BR96 and its complex with the Lewis Y determinant.

The monoclonal anti-tumor antibody BR96 binds a tetrasaccharide, Lewis y (Le(y)), in vitro and recognizes a Le(y)-bearing or Le(y)-related tumor-associated antigen in vivo. The Fab of the murine monoclonal antibody, mBR96 (IgG3, kappa), and the Fab' of its human chimera, cBR96 (IgG1, kappa), and their complexes with Le(y) have been screened for crystallization conditions. Crystals suitable for X-ray diffraction have been obtained for uncomplexed cBR96 Fab', cBR96 Fab' in complex with Le(y) and mBR96 Fab in complex with Le(y). The symmetry of the cBR96 Fab' crystals is consistent with space group P2(1)2(1)2, a = 61.1 A; b = 174.3 A; c = 45.6 A; the symmetry of the cBR96 Fab'-Le(y) complex crystals with space group P4(3)2(1)2 (or its enantiomorph), a = b = 82.2 A; c = 167.1 A and the symmetry of the mBR96 Fab-Le(y) complex crystals with space group P2(1)2(1)2(1), a = 69.4 A; b = 84.9 A; c = 86.8 A.

Animals↗
PubMed1994

Possible role of neuropeptide Y in experimental cancer anorexia.

The efficacy of NPY to elicit feeding in TB rats was reduced prior to the onset of overt anorexia, with the feeding response decreasing further as anorexia developed. Hypothalamic concentration of NPY was reduced in TB rats, with the magnitude of the decrease paralleling the degree of anorexia. Binding affinity of NPY to hypothalamic membranes taken from TB rats suggested decreased binding affinity with no change in receptor number. Infusing ammonium salts at a concentration and rate necessary to increase blood ammonia levels to the degree observed in TB rats, produced anorexia and decreased NPY feeding. These results suggest that NPY feeding systems are abnormal in TB rats and that hyperammonemia may be of primary importance in this dysfunction.

Ammonia↗
PubMed1994

Human mannose-binding protein carbohydrate recognition domain trimerizes through a triple alpha-helical coiled-coil.

Human mannose-binding protein is a hexamer of trimers with each subunit consisting of an amino-terminal region rich in cysteine, 19 collagen repeats, a 'neck', and a carbohydrate recognition domain that requires calcium to bind ligand. A 148-residue peptide, consisting of the 'neck' and carbohydrate recognition domains forms trimers in solution and in crystals. The structure of this trimeric peptide has been determined in two different crystal forms. The 'neck' forms a triple alpha-helical coiled-coil. Each alpha-helix interacts with a neighbouring carbohydrate recognition domain. The spatial arrangement of the carbohydrate recognition domains suggest how MBP trimers form the basic recognition unit for branched oligosaccharides on microorganisms.

Amino Acid Sequence↗
PubMed1993

26-10 Fab-digoxin complex: affinity and specificity due to surface complementarity.

We have determined the three-dimensional structures of the antigen-binding fragment of the anti-digoxin monoclonal antibody 26-10 in the uncomplexed state at 2.7 A resolution and as a complex with digoxin at 2.5 A resolution. Neither the antibody nor digoxin undergoes any significant conformational changes upon forming the complex. Digoxin interacts primarily with the antibody heavy chain and is oriented such that the carbohydrate groups are exposed to solvent and the lactone ring is buried in a deep pocket at the bottom of the combining site. Despite extensive interactions between antibody and antigen, no hydrogen bonds or salt links are formed between 26-10 and digoxin. Thus the 26-10-digoxin complex is unique among the known three-dimensional structures of antibody-antigen complexes in that specificity and high affinity arise primarily from shape complementarity.

Amino Acid Sequence↗
PubMed1993

Heavy chain position 50 is a determinant of affinity and specificity for the anti-digoxin antibody 26-10.

Antibody produced by a variant of the murine antidigoxin hybridoma 26-10 has reduced affinity for digoxin but enhanced recognition of the digoxin 12-hydroxyl due to a Tyr to His substitution at heavy chain position 50 (Schildbach, J. F., Panka, D. J., Parks, D. R., Jager, G. C., Novotny, J., Herzenberg, L. A., Mudgett-Hunter, M., Bruccoleri, R. E., Haber, E., and Margolies, M. N. (1991) J. Biol. Chem. 266, 4640-4647). Consistent with these data, the 26-10 Fab-digoxin x-ray crystal structure (Jeffrey, P. D., Strong, R. K., Sieker, L. C., Chang, C. Y., Campbell, R. L., Petsko, G. A., Haber, E., Margolies, M. N., and Sheriff, S. (1993) Proc. Natl. Acad. Sci. U. S. A., in press) reveals that Tyr-50 contacts a region of digoxin that includes the hapten-12 carbon. To determine the effects of other heavy chain position 50 substitutions, mutant antibodies were engineered, and their affinities for digoxin and digoxin analogues were measured. The affinity of the mutant antibodies for digoxin roughly correlates with the size of the position 50 side chain. Substitutions of Trp or Phe have no effect on affinity, whereas substitutions of Asn, His, Leu, Ala, Gly, and Asp confer progressively lower affinities. Although Trp and Phe mutants exhibit wild-type specificity, Asn and Asp mutants have improved affinity for digoxin relative to digitoxin (12-deshydroxydigoxin). Leu, Ala, and Gly mutants have improved affinity for 12-acetyldigoxin relative to digoxin as compared with 26-10. These results indicate that position 50 is a determinant of both antibody affinity and fine specificity for antibody 26-10 and that single-amino acid substitutions can alter antibody fine specificity. Models of the mutants were computationally constructed, and haptens were docked into the modeled binding sites. The results suggest that 12-acetyldigoxigenin occupies different orientations in the 26-10 and in the Ala mutant binding sites, resulting in altered binding.

Amino Acids↗
PubMed1993

Modulation of antibody affinity by a non-contact residue.

Antibody LB4, produced by a spontaneous variant of the murine anti-digoxin monoclonal antibody 26-10, has an affinity for digoxin two orders of magnitude lower than that of the parent antibody due to replacement of serine with phenylalanine at position 52 of the heavy chain variable region (Schildbach, J.F., Panka, D.J., Parks, D.R., et al., 1991, J. Biol. Chem. 266, 4640-4647). To examine the basis for the decreased affinity, a panel of engineered antibodies with substitutions at position 52 was created, and their affinities for digoxin were measured. The antibody affinities decreased concomitantly with increasing size of the substituted side chains, although the shape of the side chains also influenced affinity. The crystal structure of the 26-10 Fab complexed with digoxin (P.D.J., R.K. Strong, L.C. Sieker, C. Chang, R.L. Campbell, G.A. Petsko, E.H., M.N.M., & S.S., submitted for publication) shows that the serine at heavy chain position 52 is not in contact with hapten, but is adjacent to a tyrosine at heavy chain position 33 that is a contact residue. The mutant antibodies were modeled by applying a conformational search procedure to position side chains, using the 26-10 Fab crystal structure as a starting point. The results suggest that each of the substituted side chains may be accommodated within the antibody without substantial structural rearrangement, and that none of these substituted side chains are able to contact hapten. These modeling results are consistent with the substituents at position 52 having only an indirect influence upon antibody affinity.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1993

Characterization of the backbone dynamics of an anti-digoxin antibody VL domain by inverse detected 1H-15N NMR: comparisons with X-ray data for the Fab.

The dynamic behavior of the polypeptide backbone of a recombinant antidigoxin antibody VL domain has been characterized by measurements of 15NT1 and T2 relaxation times, 1H-15N NOE values, and 1H-2H exchange rates. These data were acquired with 2D inverse detected heteronuclear 1H-15N NMR methods. The relaxation data are interpreted in terms of model free spectral density functions and exchange contributions to transverse relaxation rates R2 (= 1/T2). All characterized residues display low-amplitude picosecond time-scale librational motions. Fifteen residues undergo conformational changes on the nanosecond timescale, and 24 residues have significant R2 exchange contributions, which reflect motions on the microsecond to millisecond time-scale. For several residues, microsecond to millisecond motions of nearby aromatic rings are postulated to account for some or all of their observed R2 exchange contributions. The measured 1H-2H exchange rates are correlated with hydrogen bonding patterns and distances from the solvent accessible surface. The degree of local flexibility indicated by the NMR measurements is compared to crystallographic B-factors derived from X-ray analyses of the native Fab and the Fab/digoxin complex. In general, both the NMR and X-ray data indicate enhanced flexibility in the turns, hypervariable loops, and portions of beta-strands A, B, and G. However, on a residue-specific level, correlations among the various NMR data, and between the NMR and X-ray data, are often absent. This is attributed to the different dynamic processes and environments that influence the various observables. The combined data indicate that certain regions of the VL domain, including the three hypervariable loops, undergo dynamic changes upon VL:VH association and/or complexation with digoxin. Overall, the 26-10 VL domain exhibits relatively low flexibility on the ps-ns timescale. The possible functional consequences of this result are considered.

Digoxin↗
PubMed1992

Amylin inhibits insulin-stimulated glucose uptake in C2C12 muscle cell line through a cholera-toxin-sensitive mechanism.

Rat amylin inhibits insulin-stimulated glucose uptake with an IC50 of 12.1 +/- 4.1 pM in C2C12 myotubes. The maximal inhibition is 64 +/- 5.4% observed at a 100-pM dose of the peptide. Consistently, presence of 100 pM amylin shifted the dose-response curve of insulin to the right, increasing the ED50 from 0.71 to 16 nM. No effect of amylin is observed on basal glucose uptake in these cells. Cholera-toxin treatment of the cells did not affect the insulin-stimulated glucose uptake, while the inhibitory effect is completely lost in toxin-treated cells. These findings strongly suggest that rat amylin is active at a physiological concentration and the amylin inhibition of glucose uptake is mediated through a cholera-toxin-sensitive mechanism.

Amyloid↗
PubMed1992

Inhibitory and stimulatory effects of neuropeptide Y(17-36) on rat cardiac adenylate cyclase activity. Structure-function studies.

Neuropeptide Y (NPY) inhibits cardiac adenylate cyclase activity by interacting with specific receptors coupled to a pertussis toxin-sensitive G protein. Structure-activity studies revealed that only C-terminal fragments can exhibit an NPY-like inhibitory effect on 125I-NPY binding and adenylate cyclase activity of rat cardiac ventricular membranes. Although NPY(17-36) inhibited 125I-NPY binding with high potency, it produced a biphasic effect on basal (GTP, 10 and 100 microM or guanosine 5'-gamma-O-(thio)triphosphate (GTP gamma S, 10 microM) adenylate cyclase activity. Low concentrations (less than 1 nM) of NPY(17-36) inhibited the adenylate cyclase activity whereas high concentrations (greater than 1 nM) reversed this action. GTP gamma S (100 microM) reversed the biphasic effect of NPY(17-36). NPY(17-36) exhibited only a stimulatory effect in the membranes from pertussis toxin-treated rats and an inhibitory effect with membranes from cholera toxin-treated rats. Low concentrations (less than 1 nM) of NPY(17-36) inhibited isoproterenol-stimulated adenylate cyclase activity whereas high doses (greater than 1 nM) reversed this activity. The cardiac NPY receptor antagonist, NPY(18-36) (1 microM), completely blocked the biphasic effect of NPY(17-36) on isoproterenol-stimulated activity. The inhibitory dose-response curve of NPY on isoproterenol-stimulated adenylate cyclase activity was shifted parallel to the right by NPY(17-36) (1 microM), suggesting that it is an antagonist of NPY at high concentrations. N-alpha-acetylated and C-terminally deamidated analogs of NPY(17-36) had no effect on the adenylate cyclase activity. [im-DNP-His26] NPY exhibited a more pronounced biphasic effect whereas N-alpha-myristoyl-NPY(17-36) elicited only a stimulatory effect. These investigations suggest that: 1) the inhibitory and stimulatory effects of NPY(17-36) are mediated by high affinity NPY receptors coupled to a pertussis toxin-sensitive G protein and a distinct population of low affinity receptors coupled to a cholera toxin-sensitive G protein, respectively; and 2) the stimulatory effect of NPY(17-36) is dissociable.

Adenylate Cyclase Toxin↗
PubMed1992

Roux-en-Y jejunal bypass abolishes postprandial neuropeptide Y release.

Numerous physiologic aberrations occur after Roux-en-Y bypass procedures. Neuropeptide Y (NPY), a 36 amino acid polypeptide, has been shown to have many effects on gastrointestinal physiology, including alterations in blood flow, motility, and secretion and absorption. Recent work demonstrating a postprandial increase in circulating NPY prompted this investigation into its potential roles after Roux-en-Y bypass. Three groups of rats underwent Roux-en-Y cholangiojejunostomy, jejunojejunostomy, or proximal jejunal transection with reanastomosis. After a 3-month recovery, the animals were tested with both mixed and fat meals. Control animals had rapid increases in circulating NPY after the mixed meal. This response was not seen in either of the Roux-en-Y groups (P less than 0.05). No animals had circulating changes in NPY after the fat meal. Additionally, small intestinal NPY receptor analysis revealed high NPY affinity to the epithelial cells of the proximal small intestine. Our results demonstrate a dependence of postprandial NPY release on proximal small intestinal continuity that is abolished by Roux-en-Y bypass of a jejunal segment. The absence of postprandial elevation in plasma NPY after proximal jejunal bypass and the abundance of NPY receptors in the proximal small intestine merits further investigation into the physiologic roles of NPY in the foregut.

Anastomosis, Roux-en-Y↗
PubMed1990

Neuropeptide Y (18-36) is a competitive antagonist of neuropeptide Y in rat cardiac ventricular membranes.

Neuropeptide Y (NPY), a hexatriacontapeptide amide, is present in high concentrations in the mammalian heart. Specific receptors of NPY in rat cardiac ventricular membranes have been characterized recently in our laboratory. Structure-activity studies with selected partial sequences of NPY revealed that NPY(18-36) inhibited the binding of 125I-NPY to rat cardiac ventricular membranes but had no effect on the cardiac adenylate cyclase activity. NPY, as previously reported, inhibited the cardiac adenylate cyclase activity. These observations suggested that NPY (18-36) may be an antagonist of NPY in cardiac membranes. Consistent with this observation, the presence of NPY (18-36) (1 microM) shifted the inhibitory adenylate cyclase activity dose-response curve of NPY to the right in a parallel fashion. Furthermore, NPY(18-36) (1 microM) completely abolished the effect of NPY (10 nM) that alone caused 80% of the maximum inhibition of adenylate cyclase activity. These findings confirm that NPY(18-36) is a competitive antagonist of NPY in rat cardiac ventricular membranes. NPY cardiac receptor antagonist, NPY(18-36), or analogs based on this sequence may have potential clinical application, since NPY has been implicated in the pathophysiology of congestive heart failure.

Adenylyl Cyclases↗
PubMed1990

Neuropeptide Y and peptide YY stimulate the growth of exocrine pancreatic carcinoma cells.

Neuropeptides exert inhibitory effects on pancreatic secretion, but their role in the regulation of growth is unknown. This study was executed to evaluate the effects of PYY and NPY on cell growth and 3H-thymidine incorporation in human (MiaPaCa-2, Capan-2) and hamster (H2T) exocrine pancreatic carcinoma cells in vitro. A significant increase in the number of cells after 96 h of treatment with NPY was observed at 0.01 microM in H2T, 0.1 microM in MiaPCa-2 and at 1 microM in Capan-2 cells. PYY was less potent and did not increase significantly cell growth in MiaPaCa-2, but did at 0.1 microM in Capan-2 and at 1 microM concentration in H2T. Stimulation for 48h with NPY increased 3H-thymidine incorporation significantly at 0.01 microM in all cell lines. With PYY, stimulation of 3H-thymidine incorporation occurred in H2T cells at 0.01 microM. 3H-thymidine incorporation after PYY treatment was significantly increased at 0.1 microM in MiaPaCa-2 and at 1 microM in Capan-2 cells. Receptor studies showed low but definite specific binding of both NPY and PYY in all cell lines. The results suggest that NPY and PYY may have a role in the regulation of growth of exocrine pancreatic carcinoma cells.

Adenocarcinoma↗