scholarly journals P-selectin glycoprotein ligand 1 is a ligand for L-selectin on neutrophils, monocytes, and CD34+ hematopoietic progenitor cells.

1996 ◽  
Vol 135 (2) ◽  
pp. 523-531 ◽  
Author(s):  
O Spertini ◽  
A S Cordey ◽  
N Monai ◽  
L Giuffrè ◽  
M Schapira
Keyword(s):  
Critical Role ◽  
Myeloid Cells ◽  
Chimeric Protein ◽  
Amino Acid Residues ◽  
Hematopoietic Progenitors ◽  
Calcium Dependent ◽  
Amino Terminal ◽  
Selectin Ligand ◽  
Selectin Binding

Selectins play a critical role in initiating leukocyte binding to vascular endothelium. In addition, in vitro experiments have shown that neutrophils use L-selectin to roll on adherent neutrophils, suggesting that they express a nonvascular L-selectin ligand. Using a L-selectin/IgM heavy chain (mu) chimeric protein as an immunocytological probe, we show here that L-selectin can bind to neutrophils, monocytes, CD34+ hematopoietic progenitors, and HL-60 and KG-1 myeloid cells. The interaction between L-selectin and leukocytes was protease sensitive and calcium dependent, and abolished by cell treatment with neuraminidase, chlorate, or O-sialoglycoprotein endopeptidase. These results revealed common features between leukocyte L-selectin ligand and the mucin-like P-selectin glycoprotein ligand 1 (PSGL-1), which mediates neutrophil rolling on P- and E-selectin. The possibility that PSGL-1 could be a ligand for L-selectin was further supported by the ability of P-selectin/mu chimera to inhibit L-selectin/mu binding to leukocytes and by the complete inhibition of both selectin interactions with myeloid cells treated with mocarhagin, a cobra venom metalloproteinase that cleaves the amino terminus of PSGL-1 at Tyr-51. Finally, the abrogation of L- and P-selectin binding to myeloid cells treated with a polyclonal antibody, raised against a peptide corresponding to the amino acid residues 42-56 of PSGL-1, indicated that L- and P-selectin interact with a domain located at the amino-terminal end of PSGL-1. The ability of the anti-PSGL-1 mAb PL-1 to inhibit L- and P-selectin binding to KG-1 cells further supported that possibility. Thus, apart from being involved in neutrophil rolling on P- and E-selectin, PSGL-1 also plays a critical role in mediating neutrophil attachment to adherent neutrophils. Interaction between L-selectin and PSGL-1 may be of major importance for increasing leukocyte recruitment at inflammatory sites.

scholarly journals Conservation of Histone Binding and Transcriptional Repressor Functions in a Schizosaccharomyces pombeTup1p Homolog

1999 ◽  
Vol 19 (12) ◽  
pp. 8461-8468 ◽  
Author(s):  
Yukio Mukai ◽  
Eri Matsuo ◽  
Sharon Y. Roth ◽  
Satoshi Harashima
Keyword(s):  
Glucose Repression ◽  
Kluyveromyces Lactis ◽  
Chimeric Protein ◽  
Evolutionary Conservation ◽  
Histone Binding ◽  
Amino Terminal ◽  
Corepressor Complex ◽  
Related Proteins ◽  
Repressor Activity

ABSTRACT The Ssn6p-Tup1p corepressor complex is important to the regulation of several diverse genes in Saccharomyces cerevisiae and serves as a model for corepressor functions. To investigate the evolutionary conservation of these functions, sequences homologous to the S. cerevisiae TUP1 gene were cloned fromKluyveromyces lactis (TUP1) andSchizosaccharomyces pombe (tup11 +). Interestingly, while the K. lactis TUP1 gene complemented an S. cerevisiae tup1 null mutation, the S. pombe tup11 + gene did not, even when expressed under the control of the S. cerevisiae TUP1 promoter. However, anS. pombe Tup11p-LexA fusion protein repressed transcription of a corresponding reporter gene, indicating that this Tup1p homolog has intrinsic repressor activity. Moreover, a chimeric protein containing the amino-terminal Ssn6p-binding domain of S. cerevisiae Tup1p and 544 amino acids from the C-terminal region of S. pombe Tup11p complemented the S. cerevisiae tup1 mutation. The failure of native S. pombe Tup11p to complement loss of Tup1p functions in S. cerevisiaecorresponds to an inability to bind to S. cerevisiae Ssn6p in vitro. Disruption of tup11 + in combination with a disruption of tup12 +, anotherTUP1 homolog gene in S. pombe, causes a defect in glucose repression of fbp1 +, suggesting thatS. pombe Tup1p homologs function as repressors in S. pombe. Furthermore, Tup11p binds specifically to histones H3 and H4 in vitro, indicating that both the repression and histone binding functions of Tup1p-related proteins are conserved across species.

Control of AMP deaminase 1 binding to myosin heavy chain

1998 ◽  
Vol 275 (3) ◽  
pp. C870-C881 ◽  
Author(s):  
Ichiro Hisatome ◽  
Takayuki Morisaki ◽  
Hiroshi Kamma ◽  
Takako Sugama ◽  
Hiroko Morisaki ◽  
...  
Keyword(s):  
Myosin Heavy Chain ◽  
Heavy Chain ◽  
Sequence Similarity ◽  
Critical Role ◽  
Energy Charge ◽  
Adenylate Energy Charge ◽  
Amp Deaminase ◽  
Amino Terminal

AMP deaminase (AMPD) plays a central role in preserving the adenylate energy charge in myocytes following exercise and in producing intermediates for the citric acid cycle in muscle. Prior studies have demonstrated that AMPD1 binds to myosin heavy chain (MHC) in vitro; binding to the myofibril varies with the state of muscle contraction in vivo, and binding of AMPD1 to MHC is required for activation of this enzyme in myocytes. The present study has identified three domains in AMPD1 that influence binding of this enzyme to MHC using a cotransfection model that permits assessment of mutations introduced into the AMPD1 peptide. One domain that encompasses residues 178–333 of this 727-amino acid peptide is essential for binding of AMPD1 to MHC. This region of AMPD1 shares sequence similarity with several regions of titin, another MHC binding protein. Two additional domains regulate binding of this peptide to MHC in response to intracellular and extracellular signals. A nucleotide binding site, which is located at residues 660–674, controls binding of AMPD1 to MHC in response to changes in intracellular ATP concentration. Deletion analyses demonstrate that the amino-terminal 65 residues of AMPD1 play a critical role in modulating the sensitivity to ATP-induced inhibition of MHC binding. Alternative splicing of the AMPD1 gene product, which alters the sequence of residues 8–12, produces two AMPD1 isoforms that exhibit different MHC binding properties in the presence of ATP. These findings are discussed in the context of the various roles proposed for AMPD in energy production in the myocyte.

Decreased expression of myocardial eNOS and caveolin in dogs with hypertrophic cardiomyopathy

2002 ◽  
Vol 282 (1) ◽  
pp. H219-H231 ◽  
Author(s):  
A. Piech ◽  
P. E. Massart ◽  
C. Dessy ◽  
O. Feron ◽  
X. Havaux ◽  
...  
Keyword(s):  
Systolic Function ◽  
Critical Role ◽  
Aortic Pressure ◽  
Heart Tissue ◽  
Caveolin 1 ◽  
Calcium Dependent ◽  
Diastolic Relaxation ◽  
Nos Inhibitor

Because nitric oxide (NO) regulates cardiac and vessel contraction, we compared the expression and activity of the endothelial NO synthase (eNOS) and caveolin, which tonically inhibits eNOS in normal and hypertrophic cardiomyopathic hearts. NOS activity (l-[3H]citrulline formation), eNOS immunostaining, and caveolin abundance were measured in heart tissue of 23 mongrel dogs before and at 3 and 7 wk of perinephritic hypertension (PHT). Hemodynamic parameters in vivo and endothelial NO-dependent relaxation of macro- and coronary microvessels in vitro were assessed in the same animals. eNOS immunostaining and total calcium-dependent NOS activity decreased at 7 wk in all four heart cavities (in left ventricle, from 17.0 ± 1.3 to 0.2 ± 0.2 fmol · min−1 · mg protein−1, P < 0.001). Caveolin-1 and -3 also decreased in PHT dog hearts. Accordingly, basal vascular tone was preserved, but maximal endothelial NO-dependent relaxation was impaired in all vessels from 7-wk PHT dogs. The latter had preserved systolic function but impaired diastolic relaxation [relaxation time constant ( T 1), 25.1 ± 0.9 vs. 22.0 ± 1 ms in controls; P < 0.05]. Peripheral infusion of the NOS inhibitor N G -nitro-l-arginine methyl ester increased mean aortic pressure in both groups and reduced diastolic ( T 1, 31.9 ± 1.4 ms) and systolic function in PHT dogs (DP40, 47.5 ± 2.5 vs. 59.4 ± 3.8 s−1 in control animals). In conclusion, both eNOS and caveolin proteins are decreased in the hypertrophic hearts of PHT dogs. This is associated with altered maximal (but not basal) vascular relaxation and impaired diastolic function. Further degradation of cardiac function after NOS inhibition suggests a critical role of residual NOS activity, probably supported by the concurrent downregulation of caveolin.

Glycosylation-dependent inhibition of cutaneous lymphocyte–associated antigen expression: implications in modulating lymphocyte migration to skin

Blood ◽  
2003 ◽  
Vol 101 (2) ◽  
pp. 602-610 ◽  
Author(s):  
Charles J. Dimitroff ◽  
Ralph J. Bernacki ◽  
Robert Sackstein
Keyword(s):  
T Cells ◽  
Human Skin ◽  
Critical Role ◽  
Metabolic Inhibitors ◽  
Lymphocyte Migration ◽  
Skin Homing ◽  
Selectin Ligand ◽  
Adhesive Interactions ◽  
Selectin Binding ◽  
Ligand Activity

Constitutive E-selectin expression on dermal microvascular endothelial cells plays a critical role in mediating rolling adhesive interactions of human skin–homing T cells and in pathologic accumulation of lymphocytes in skin. The major E-selectin ligand on human skin–homing T cells is cutaneous lymphocyte–associated antigen (CLA), a specialized glycoform of P-selectin glycoprotein ligand-1 (PSGL-1) defined by monoclonal antibody HECA-452. Since HECA-452 reactivity, and not PSGL-1 polypeptide itself, confers the specificity of human T cells to enter dermal tissue, inhibition of HECA-452 expression is a potential strategy for modulating lymphocyte migration to skin. In this study, we examined the efficacy of several well-characterized metabolic inhibitors of glycosylation and of a novel fluorinated analog of N-acetylglucosamine (2-acetamido-1,3,6-tri-O-acetyl-4-deoxy-4-fluoro-D-glucopyranose [4-F-GlcNAc]) to alter HECA-452 expression on human CLA+ T cells and prevent cell tethering and rolling on selectins under shear stress. At concentrations that did not affect PSGL-1 expression, we found that swainsonine (inhibitor of complex-typeN-glycan synthesis) had no effect on HECA-452 expression or selectin ligand activity, whereas benzyl-O-N-acetylgalactosamide (BAG; inhibitor of O-glycan biosynthesis) ablated HECA-452 expression on PSGL-1 and significantly lowered selectin ligand activity. We found that 4-F-GlcNAc (putative inhibitor of poly-N-acetyllactosamine biosynthesis) was more potent than BAG at lowering HECA-452 expression and selectin binding. In addition, we show that 4-F-GlcNAc was directly incorporated into native CLA expressed on T cells, indicating direct inhibition on poly-N-acetyllactosamine elongation and selectin-binding determinants on PSGL-1 O-glycans. These observations establish a potential treatment approach for targeting pathologic lymphocyte trafficking to skin and indicate that 4-F-GlcNAc may be a promising agent for treatment of dermal tropism associated with malignancies and inflammatory disorders.

scholarly journals Annexin VI: an intracellular target for ATP.

1999 ◽  
Vol 46 (3) ◽  
pp. 801-812 ◽  
Author(s):  
J Bandorowicz-Pikuła ◽  
M Danieluk ◽  
A Wrzosek ◽  
R Buś ◽  
R Buchet ◽  
...  
Keyword(s):  
Nucleotide Binding ◽  
Dependent Manner ◽  
Amino Acid Residues ◽  
Binding Motifs ◽  
Calcium Dependent ◽  
Annexin Vi ◽  
Self Association ◽  
Nucleotide Binding Proteins

Annexin VI (AnxVI), an Ca2+- and phospholipid-binding protein, interacts in vitro with ATP in a calcium-dependent manner. Experimental evidence indicates that its nucleotide-binding domain which is localized in the C-terminal half of the protein differs structurally from ATP/GTP-binding motifs found in other nucleotide-binding proteins. The amino-acid residues of AnxVI directly involved in ATP binding have not been yet defined. Binding of ATP to AnxVI induces changes in the secondary and tertiary structures of protein, affecting the affinity of AnxVI for Ca2+ and, in consequence, influencing the Ca2+-dependent activities of AnxVI: binding to F-actin and to membranous phospholipids, and self-association of the annexin molecules. These observations suggest that ATP is a functional ligand for AnxVI in vivo, and ATP-sensitive AnxVI may play the role of a factor coupling vesicular transport and calcium homeostasis to cellular metabolism.

The Nature of the P-Selectin Ligands on Sickle Cells.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 363-363
Author(s):  
Stephen H. Embury ◽  
Christine E. Baran ◽  
Colleen A. Hefner ◽  
Christi K. Seto ◽  
Neil M. Matsui
Keyword(s):  
Flow Cytometry ◽  
Sialic Acid ◽  
Transferrin Receptor ◽  
Cell Disease ◽  
Selectin Ligands ◽  
Selectin Ligand ◽  
Selectin Binding ◽  
Binding Determinants

Abstract Elucidation of the adhesive interactions that effect microvascular occlusion in sickle cell disease both increases our understanding of the pathophysiology of vasoocclusion and identifies molecular targets for the development of therapeutic interventions. Work from our laboratory has established that sickle RBC adhere to P-selectin on thrombin-activated endothelial cells and to immobilized, recombinant P-selectin in vitro (Matsui et al. Blood 98:1955, 2001) and that this adhesion can be inhibited by agents that block P-selectin (Matsui et al. Blood 100:3790, 2002). Based on these findings, we established that sickle RBC adherence to endothelial P-selectin has a substantial influence on microvascular blood flow in vivo and that blocking P-selectin enhances microvascular flow (Embury et al. Blood In Press). We reasoned that characterization of the cognate ligands for P-selectin ligand on sickle RBC could identify additional targets for therapeutic intervention. We had determined that that sickle RBC did not express the P-selectin ligand, P-selectin glycoprotein-1, but that membrane sialic acid is required for sickle RBC binding to P-selectin. Here we describe further characterization of the P-selectin binding determinants on sickle RBC membranes. We assessed the expression of sialyl Lewis X (sLeX) on sickle RBC using flow cytometry and the importance of sLeX expression to the rolling adhesion of sickle RBC to P-selectin in vitro. Using the monoclonal antibodies (mAb) HECA-452 and CSLEX-1 in flow cytometry we detected significant expression of sLeX on sickle RBC (p < 0.003 and p < 0.02, respectively) but not on non-sickle RBC (p < 0.07 and p < 0.3, respectively). Treatment of sickle RBC with sialidase caused a partial, dose dependant reduction of the level of detectable sLeX and of rolling adhesion to immobilized P-selectin (approximately 40% and 85%, respectively), which correlated positively. To assess the possible selective contribution of reticulocytes as a subset of higher sLeX expressing sickle RBC we employed dual label flow cytometry to determine whether sLeX and the transferrin receptor (CD71) are co-expressed. Using mAb YDJ1.2.2 for the transferrin receptor as a reticulocyte marker and CSLEX-1 showed that sLeX was expressed both on sickle reticulocytes and on older sickle RBC. Treatment of sickle RBC with O-sialoglycoprotein endopeptidase, which cleaves sialylated O-glycans, also reduced both their sLeX expression and rolling adhesion on P-selectin (approximately 30% and 65%, respectively). Treatment of sickle RBC with N-glycosidase F did not reduce sLeX or adhesion levels, trypsin treatment produced inconsistent effects, and phosphatidylinositol-specific phospholipase C caused a significant decrease in adhesion but not a significant reduction in sLex expression. These findings suggest that sickle RBC possess more than one type of glycoprotein as a ligand for P-selectin. We also used a solid-phase binding assay to detect a significant level of P-selectin binding to membrane lipids extracted from sickle RBC. Thus, the P-selectin binding determinants on sickle RBC include sialic acid, sLeX, O-linked glycans, PI-linked glycoproteins, and glycolipids. Each of these P-selectin ligands represents a potential target of new adhesion blocking drugs for the treatment of sickle cell disease.

scholarly journals Chemical Modifications of PhTX-I Myotoxin fromPorthidium hyoproraSnake Venom: Effects on Structural, Enzymatic, and Pharmacological Properties

2013 ◽  
Vol 2013 ◽  
pp. 1-12 ◽  
Author(s):  
Salomón Huancahuire-Vega ◽  
Daniel H. A. Corrêa ◽  
Luciana M. Hollanda ◽  
Marcelo Lancellotti ◽  
Carlos H. I. Ramos ◽  
...  
Keyword(s):  
Catalytic Activity ◽  
Critical Role ◽  
High Catalytic Activity ◽  
Pharmacological Properties ◽  
Amino Acid Residues ◽  
Chemical Modifications ◽  
Specific Amino Acid ◽  
Catalytic Sites

We recently described the isolation of a basic PLA2(PhTX-I) fromPorthidium hyoprorasnake venom. This toxin exhibits high catalytic activity, inducesin vivomyotoxicity, moderates footpad edema, and causesin vitroneuromuscular blockade. Here, we describe the chemical modifications of specific amino acid residues (His, Tyr, Lys, and Trp), performed in PhTX-I, to study their effects on the structural, enzymatic, and pharmacological properties of this myotoxin. After chemical treatment, a single His, 4 Tyr, 7 Lys, and one Trp residues were modified. The secondary structure of the protein remained unchanged as measured by circular dichroism; however other results indicated the critical role played by Lys and Tyr residues in myotoxic, neurotoxic activities and mainly in the cytotoxicity displayed by PhTX-I. His residue and therefore catalytic activity of PhTX-I are relevant for edematogenic, neurotoxic, and myotoxic effects, but not for its cytotoxic activity. This dissociation observed between enzymatic activity and some pharmacological effects suggests that other molecular regions distinct from the catalytic site may also play a role in the toxic activities exerted by this myotoxin. Our observations supported the hypothesis that both the catalytic sites as the hypothetical pharmacological sites are relevant to the pharmacological profile of PhTX-I.

A novel 49-kilodalton protein from Artemia cross-links microtubules in vitro

1992 ◽  
Vol 70 (10-11) ◽  
pp. 1055-1063 ◽  
Author(s):  
Jianshe Zhang ◽  
Thomas H. MacRae
Keyword(s):  
Protein A ◽  
Cross Linking ◽  
Amino Acid Residues ◽  
Microtubule Organization ◽  
Western Blots ◽  
Microtubule Associated Proteins ◽  
Amino Terminal ◽  
Apparent Homogeneity ◽  
Associated Proteins

A 49 kilodalton (kDa) protein, previously proposed to cross-link microtubules, was purified to apparent homogeneity from cell-free extracts of the brine shrimp Artemia. When incubated with tubulin under assembly conditions, the purified 49-kDa protein cross-linked the resulting microtubules. Preformed microtubules were also cross-linked when incubated with the 49-kDa protein. Upon centrifugation through sucrose cushions the 49-kDa protein cosedimented with microtubules, suggesting a stable association between the cross-linking protein and tubulin. Such microtubules were interconnected by particles which were circular, bilobed, or elongated in shape. Disruption of microtubule cross-linking and dissociation of the 49-kDa protein from microtubules occurred in the presence of ATP and 5′-adenylylimidodiphosphate (AMP–PNP), a nonhydrolyzable analogue of ATP. The 49-kDa protein was moderately resistant to heat, it did not stimulate tubulin assembly, and it did not react with antibodies to neural microtubule-associated proteins (MAPs) and kinesin. These observations indicate that the 49-kDa protein is different from many known MAPs, a conclusion strengthened by the inability of antibodies raised to the 49-kDa protein to recognize these proteins. The amino terminal 15 amino acid residues of the 49-kDa protein were determined by Edman digestion and an antibody raised to this peptide reacted with the 49-kDa protein on Western blots. Microtubule cross-linking was unaffected by the synthetic amino-terminal peptide, even when it was present at a fivefold molar excess over the 49-kDa protein. A search of three protein databanks revealed that the amino terminus of the 49-kDa protein is unique among published sequences. The findings verify our earlier proposal that Artemia contains a 49-kDa microtubule cross-linking protein and demonstrate that it has a novel set of characteristics. The 49-kDa protein has the potential to play an important role in microtubule organization and function.Key words: microtubule cross-linking, microtubule-associated proteins, Artemia.

scholarly journals Transformation by v-myb correlates with trans-activation of gene expression.

1990 ◽  
Vol 10 (6) ◽  
pp. 2591-2598 ◽  
Author(s):  
T Lane ◽  
C Ibanez ◽  
A Garcia ◽  
T Graf ◽  
J Lipsick
Keyword(s):  
Gene Expression ◽  
Dna Binding ◽  
Cell Nucleus ◽  
Myeloid Cells ◽  
Protein Product ◽  
Avian Myeloblastosis Virus ◽  
Amino Terminal ◽  
Acute Myelomonocytic Leukemia ◽  
The Relationship

The v-myb oncogene of avian myeloblastosis virus causes acute myelomonocytic leukemia in chickens and transforms avian myeloid cells in vitro. Its protein product p48v-myb is a nuclear, sequence-specific, DNA-binding protein which activates gene expression in transient DNA transfection studies. To investigate the relationship between transformation and trans-activation by v-myb, we constructed 15 in-frame linker insertion mutants. The 12 mutants which transformed myeloid cells also trans-activated gene expression, whereas the 3 mutants which did not transform also did not trans-activate. This implies that trans-activation is required for transformation by v-myb. One of the transformation-defective mutants localized to the cell nucleus but failed to bind DNA. The other two transformation-defective mutants localized to the cell nucleus and bound DNA but nevertheless failed to trans-activate. These latter mutants define two distinct domains of p48v-myb which control trans-activation by DNA-bound protein, one within the amino-terminal DNA-binding domain itself and one in a carboxyl-terminal domain which is not required for DNA binding.

Transformation by v-myb correlates with trans-activation of gene expression

1990 ◽  
Vol 10 (6) ◽  
pp. 2591-2598 ◽  
Author(s):  
T Lane ◽  
C Ibanez ◽  
A Garcia ◽  
T Graf ◽  
J Lipsick
Keyword(s):  
Gene Expression ◽  
Dna Binding ◽  
Cell Nucleus ◽  
Myeloid Cells ◽  
Protein Product ◽  
Avian Myeloblastosis Virus ◽  
Amino Terminal ◽  
Acute Myelomonocytic Leukemia ◽  
The Relationship

The v-myb oncogene of avian myeloblastosis virus causes acute myelomonocytic leukemia in chickens and transforms avian myeloid cells in vitro. Its protein product p48v-myb is a nuclear, sequence-specific, DNA-binding protein which activates gene expression in transient DNA transfection studies. To investigate the relationship between transformation and trans-activation by v-myb, we constructed 15 in-frame linker insertion mutants. The 12 mutants which transformed myeloid cells also trans-activated gene expression, whereas the 3 mutants which did not transform also did not trans-activate. This implies that trans-activation is required for transformation by v-myb. One of the transformation-defective mutants localized to the cell nucleus but failed to bind DNA. The other two transformation-defective mutants localized to the cell nucleus and bound DNA but nevertheless failed to trans-activate. These latter mutants define two distinct domains of p48v-myb which control trans-activation by DNA-bound protein, one within the amino-terminal DNA-binding domain itself and one in a carboxyl-terminal domain which is not required for DNA binding.

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