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1.
Cell Death Differ ; 22(12): 1893-905, 2015 Dec.
Article in English | MEDLINE | ID: mdl-26450453

ABSTRACT

A significant effort is made by the cell to maintain certain phospholipids at specific sites. It is well described that proteins involved in intracellular signaling can be targeted to the plasma membrane and organelles through phospholipid-binding domains. Thus, the accumulation of a specific combination of phospholipids, denoted here as the 'phospholipid code', is key in initiating cellular processes. Interestingly, a variety of extracellular proteins and pathogen-derived proteins can also recognize or modify phospholipids to facilitate the recognition of dying cells, tumorigenesis and host-microbe interactions. In this article, we discuss the importance of the phospholipid code in a range of physiological and pathological processes.


Subject(s)
Phospholipids/metabolism , Cell Membrane/metabolism , Disease Progression , Host-Pathogen Interactions , Humans , Neoplasms/metabolism , Neoplasms/pathology , Reactive Oxygen Species/metabolism , Signal Transduction , Tumor Microenvironment
2.
Cell Death Differ ; 17(3): 381-97, 2010 Mar.
Article in English | MEDLINE | ID: mdl-20019744

ABSTRACT

Phagocytosis serves as one of the key processes involved in development, maintenance of tissue homeostasis, as well as in eliminating pathogens from an organism. Under normal physiological conditions, dying cells (e.g., apoptotic and necrotic cells) and pathogens (e.g., bacteria and fungi) are rapidly detected and removed by professional phagocytes such as macrophages and dendritic cells (DCs). In most cases, specific receptors and opsonins are used by phagocytes to recognize and bind their target cells, which can trigger the intracellular signalling events required for phagocytosis. Depending on the type of target cell, phagocytes may also release both immunomodulatory molecules and growth factors to orchestrate a subsequent immune response and wound healing process. In recent years, evidence is growing that opsonins and receptors involved in the removal of pathogens can also aid the disposal of dying cells at all stages of cell death, in particular plasma membrane-damaged cells such as late apoptotic and necrotic cells. This review provides an overview of the molecular mechanisms and the immunological outcomes of late apoptotic/necrotic cell removal and highlights the striking similarities between late apoptotic/necrotic cell and pathogen clearance.


Subject(s)
Apoptosis , Necrosis , Phagocytosis , Animals , Apoptosis/immunology , Apoptosis/physiology , Complement Activation/immunology , Humans , Immunity, Innate/immunology , Necrosis/immunology , Phagocytosis/immunology , Phagocytosis/physiology
3.
Immunol Cell Biol ; 79(4): 345-9, 2001 Aug.
Article in English | MEDLINE | ID: mdl-11488981

ABSTRACT

Haematopoietic cell-specific transmembrane-4 (HTm4) is a four-transmembrane protein most closely related to CD20 and the beta subunit of the high affinity receptor for IgE (Fc(epsilon)RIbeta). To date, it has only been described in humans, where it is expressed in haematopoietic cells of both myeloid and lymphoid lineages. The function of HTm4 is unknown; however, as for CD20 and Fc(epsilon)RI-beta, it is likely to play a role in signal transduction as part of a multi-subunit cell surface receptor complex. In this study, we report the cDNA cloning and expression distribution of mouse HTm4. The deduced mouse HTm4 protein is of 213 amino acids, and contains four putative transmembrane domains. Mouse HTm4 shows 62% overall amino acid identity with human HTm4; the transmembrane regions are highly conserved between both species (75% identity), whereas the N- and C-terminal and inter-transmembrane loop regions are more divergent (52%). Interestingly, the N-terminal domain of mouse HTm4 is predicted to be 23 amino acids shorter, and the C-terminal domain 23 amino acids longer, than that of human HTm4. Northern blot and reverse transcriptase (RT)-PCR analysis suggest that mouse HTm4 mRNA is expressed at low levels only in spleen, bone marrow and peripheral blood leucocytes. This is the first report of the cloning of HTm4 from a species other than human, and provides important sequence information towards the understanding of the function of this poorly characterized four-transmembrane molecule.


Subject(s)
Cell Cycle Proteins , Cloning, Molecular , Membrane Proteins/chemistry , Membrane Proteins/genetics , Amino Acid Sequence , Animals , Base Sequence , Bone Marrow/metabolism , DNA, Complementary , Humans , Leukocytes/metabolism , Membrane Proteins/metabolism , Mice , Molecular Sequence Data , Protein Structure, Tertiary , Reverse Transcriptase Polymerase Chain Reaction , Spleen/metabolism , Tissue Distribution
5.
Biochem Biophys Res Commun ; 280(1): 374-9, 2001 Jan 12.
Article in English | MEDLINE | ID: mdl-11162526

ABSTRACT

CD20 and the beta subunit of the high affinity receptor for IgE (FcepsilonRIbeta) are related four-transmembrane molecules that are expressed on the surface of hematopoietic cells and play crucial roles in signal transduction. Herein, we report the identification and characterization of a human gene, TETM4, that encodes a novel four-transmembrane protein related to CD20 and FcepsilonRIbeta. The predicted TETM4 protein is 200 amino acids and contains four putative transmembrane regions, N- and C-terminal cytoplasmic domains, and three inter-transmembrane loop regions. TETM4 shows 31.0 and 23.2% overall identity with CD20 and FcepsilonRIbeta respectively, with the highest identity in the transmembrane regions, whereas the N- and C-termini and inter-transmembrane loops are more divergent. Northern blot and RT-PCR analysis suggest that TETM4 mRNA has a highly restricted tissue distribution, being expressed selectively in the testis. Using fluorescence in situ hybridization and radiation hybrid analysis, the TETM4 gene has been localized to chromosome 11q12. The genes for CD20 and FcepsilonRIbeta have also been mapped to the same region of chromosome 11 (11q12-13.1), suggesting that these genes have evolved by duplication to form a family of four-transmembrane genes. TETM4 is the first nonhematopoietic member of the CD20/FcepsilonRIbeta family, and like its hematopoietic-specific relatives, it may be involved in signal transduction as a component of a multimeric receptor complex.


Subject(s)
Antigens, CD20/chemistry , Chromosomes, Human, Pair 11 , Membrane Proteins/genetics , Receptors, IgE/chemistry , Testis/metabolism , Alternative Splicing , Amino Acid Sequence , Base Sequence , Chromosome Mapping , Female , Genetic Variation , Humans , In Situ Hybridization, Fluorescence , Karyotyping , Male , Membrane Proteins/chemistry , Molecular Sequence Data , RNA, Messenger/genetics , Reverse Transcriptase Polymerase Chain Reaction , Sequence Alignment , Sequence Homology, Amino Acid , Transcription, Genetic
6.
Biochemistry ; 39(51): 15659-67, 2000 Dec 26.
Article in English | MEDLINE | ID: mdl-11123890

ABSTRACT

Heparanase is a beta-D-endoglucuronidase that cleaves heparan sulfate (HS) and has been implicated in many important physiological and pathological processes, including tumor cell metastasis, angiogenesis, and leukocyte migration. We report herein the identification of active-site residues of human heparanase. Using PSI-BLAST and PHI-BLAST searches of sequence databases, similarities were identified between heparanase and members of several of the glycosyl hydrolase families (10, 39, and 51) from glycosyl hydrolase clan A (GH-A), including strong local identities to regions containing the critical active-site catalytic proton donor and nucleophile residues that are conserved in this clan of enzymes. Furthermore, secondary structure predictions suggested that heparanase is likely to contain an (alpha/beta)(8) TIM-barrel fold, which is common to the GH-A families. On the basis of sequence alignments with a number of glycosyl hydrolases from GH-A, Glu(225) and Glu(343) of human heparanase were identified as the likely proton donor and nucleophile residues, respectively. The substitution of these residues with alanine and the subsequent expression of the mutant heparanases in COS-7 cells demonstrated that the HS-degrading capacity of both was abolished. In contrast, the alanine substitution of two other glutamic acid residues (Glu(378) and Glu(396)), both predicted to be outside the active site, did not affect heparanase activity. These data suggest that heparanase is a member of the clan A glycosyl hydrolases and has a common catalytic mechanism that involves two conserved acidic residues, a putative proton donor at Glu(225) and a nucleophile at Glu(343).


Subject(s)
Carcinogens/metabolism , Glucuronidase/metabolism , Neoplasm Metastasis , Amino Acid Sequence , Animals , Binding Sites/genetics , COS Cells , Carcinogens/chemistry , Catalysis , Glucuronidase/chemistry , Glucuronidase/genetics , Heparitin Sulfate/metabolism , Humans , Hydrolysis , Mice , Molecular Sequence Data , Mutagenesis, Site-Directed , Protein Structure, Secondary/genetics , Rats , Sequence Homology, Amino Acid
7.
Allergy ; 55(7): 609-19, 2000 Jul.
Article in English | MEDLINE | ID: mdl-10921459

ABSTRACT

Defining the structure of the human high-affinity receptor for IgE, Fc,RI, is crucial to understand the receptor:ligand interaction, and to develop drugs to prevent IgE-dependent allergic diseases. To this end, a series of four anti-FcepsilonRI monoclonal antibodies (mAbs), including three new mAbs, 47, 54, and 3B4, were used in conjunction with synthetic FcepsilonRI peptides to define functional regions of the Fc IgE-binding site and identify an antagonist of IgE binding. The spatial orientation of the epitopes detected by these antibodies and their relationship to the IgE-binding region of FcepsilonRI was defined by a homology model based on the closely related FcepsilonRIIa. Using recombinant soluble FcRI-alpha as well as FcepsilonRI-alpha expressed on the cell surface, a series of direct and competitive binding experiments indicated that the mAbs detected nonoverlapping epitopes. One antibody (15-1), previously thought to be located close to the IgE-binding site, was precisely mapped to a single loop within the IgE-binding site by both mutagenesis and overlapping synthetic peptides encompassing the entire extracellular domain. A synthetic peptide epsilonRI-11, containing the amino acids 101-120 and the mAb 15-1 epitope, inhibited IgE binding and may form the basis for the development of a useful receptor-based therapy.


Subject(s)
Antibodies, Monoclonal/metabolism , Receptors, IgE/antagonists & inhibitors , Receptors, IgE/metabolism , Amino Acid Sequence , Antigens, CD/genetics , Antigens, CD/metabolism , Binding Sites , Binding, Competitive , Epitope Mapping , Epitopes/metabolism , Humans , Immunoglobulin E/metabolism , Models, Molecular , Molecular Sequence Data , Peptides/metabolism , Receptors, IgE/genetics , Receptors, IgG/genetics , Receptors, IgG/metabolism , Recombinant Fusion Proteins/metabolism , Sequence Alignment
8.
Immunol Cell Biol ; 78(3): 280-7, 2000 Jun.
Article in English | MEDLINE | ID: mdl-10849117

ABSTRACT

Histidine-rich glycoprotein (HRG) is a plasma protein of vertebrates that has been implicated in the regulation of several important biological functions, including the immune response and blood clotting. In the present study, we have isolated and determined the sequence of the cDNAs for both mouse and rat HRG. The deduced amino acid sequences of mouse and rat HRG are 525 and 510 amino acids, respectively, and they show the same three-domain structure that has been predicted for human HRG, with which they share high amino acid identity. Northern blot analysis indicates that the mouse HRG mRNA is 1.7 kb and is localized specifically to the liver. It has been suggested, somewhat controversially, that some immune cells, such as monocytes and megakaryocytes, also synthesize HRG. Reverse transcriptase-polymerase chain reaction analysis has failed to show any HRG mRNA in immune tissues of the mouse, including the spleen, thymus, lymph node, bone marrow and peripheral blood leucocytes. These data suggest that HRG expression by immune cells is due to the acquisition of plasma HRG derived from the liver. Finally, genomic Southern blot analysis of the mouse HRG gene suggests that it is a single copy gene.


Subject(s)
Genes , Proteins/genetics , Animals , Base Sequence , Blotting, Northern , Blotting, Southern , Cloning, Molecular , DNA/analysis , DNA, Complementary/analysis , DNA, Complementary/genetics , Male , Mice , Molecular Sequence Data , RNA, Messenger/analysis , Rats , Reverse Transcriptase Polymerase Chain Reaction , Sequence Alignment , Species Specificity
9.
J Biol Chem ; 275(13): 9664-72, 2000 Mar 31.
Article in English | MEDLINE | ID: mdl-10734118

ABSTRACT

The high affinity receptor for IgE, FcepsilonRI, binds IgE through the second Ig-like domain of the alpha subunit. The role of the first Ig-like domain is not well understood, but it is required for optimal binding of IgE to FcepsilonRI, either through a minor contact interaction or in a supporting structural capacity. The results reported here demonstrate that domain one of FcepsilonRI plays a major structural role supporting the presentation of the ligand-binding site, by interactions generated within the interdomain interface. Analysis of a series of chimeric receptors and point mutants indicated that specific residues within the A' strand of domain one are crucial to the maintenance of the interdomain interface, and IgE binding. Mutation of the Arg(15) and Phe(17) residues caused loss in ligand binding, and utilizing a homology model of FcepsilonRI-alpha based on the solved structure of FcgammaRIIa, it appears likely that this decrease is brought about by collapse of the interface and consequently the IgE-binding site. In addition discrepancies in results of previous studies using chimeric IgE receptors comprising FcepsilonRIalpha with either FcgammaRIIa or FcgammaRIIIA can be explained by the presence or absence of Arg(15) and its influence on the IgE-binding site. The data presented here suggest that the second domain of FcepsilonRI-alpha is the only domain involved in direct contact with the IgE ligand and that domain one has a structural function of great importance in maintaining the integrity of the interdomain interface and, through it, the ligand-binding site.


Subject(s)
Immunoglobulin E/metabolism , Receptors, IgE/metabolism , Amino Acid Sequence , Animals , Binding Sites , COS Cells , Crystallography, X-Ray , Models, Molecular , Molecular Sequence Data , Protein Binding , Receptors, IgE/chemistry , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/metabolism , Sequence Homology, Amino Acid
11.
Nat Med ; 5(7): 803-9, 1999 Jul.
Article in English | MEDLINE | ID: mdl-10395326

ABSTRACT

The endoglycosidase heparanase is an important in the degradation of the extracellular matrix by invading cells, notably metastatic tumor cells and migrating leukocytes. Here we report the cDNA sequence of the human platelet enzyme, which encodes a unique protein of 543 amino acids, and the identification of highly homologous sequences in activated mouse T cells and in a highly metastatic rat adenocarcinoma. Furthermore, the expression of heparanase mRNA in rat tumor cells correlates with their metastatic potential. Exhaustive studies have shown only one heparanase sequence, consistent with the idea that this enzyme is the dominant endoglucuronidase in mammalian tissues.


Subject(s)
Adenocarcinoma/enzymology , Blood Platelets/enzymology , Glucuronidase , Glycoside Hydrolases/genetics , Glycoside Hydrolases/metabolism , Mammary Neoplasms, Experimental/enzymology , Neoplasm Invasiveness/physiopathology , Neoplasm Metastasis/physiopathology , Adenocarcinoma/genetics , Adenocarcinoma/pathology , Amino Acid Sequence , Animals , Base Sequence , Cloning, Molecular , DNA, Complementary , Extracellular Matrix/physiology , Female , Humans , Mammals , Mammary Neoplasms, Experimental/genetics , Mammary Neoplasms, Experimental/pathology , Mice , Molecular Sequence Data , RNA, Messenger/analysis , Rats , Sequence Alignment , Sequence Homology, Amino Acid , Transcription, Genetic
12.
Nat Struct Biol ; 6(5): 437-42, 1999 May.
Article in English | MEDLINE | ID: mdl-10331870

ABSTRACT

Fc gamma receptors bind IgG to initiate cellular responses against pathogens and soluble antigens. We have determined the three-dimensional structure of the extracellular portion of human Fc gammaRIIa to 2.0 A resolution providing a structural basis for the unique functions of the leukocyte FcR family. The receptor is composed of two immunoglobulin domains and arranged to expose the ligand-binding site at one end of domain 2. Using alanine mutants we find that the binding sites for IgG1 and 2 are similar but the relative importance of specific regions on the receptor varies. In crystals, Fc gammaRIIa molecules associate to resemble V(L)V(H) dimers, suggesting that two Fc gammaRIIa molecules could cooperate to bind IgG in an asymmetric manner.


Subject(s)
Antigens, CD/chemistry , Leukocytes/chemistry , Receptors, IgG/chemistry , Amino Acid Sequence , Amino Acid Substitution , Animals , Antigens, CD/genetics , Antigens, CD/metabolism , Binding Sites , COS Cells , Conserved Sequence , Crystallization , Crystallography, X-Ray , Dimerization , Humans , Immunoglobulin G/metabolism , Immunoglobulins/chemistry , Ligands , Models, Molecular , Molecular Sequence Data , Protein Binding , Protein Conformation , Receptors, IgG/genetics , Receptors, IgG/metabolism , Transfection
13.
J Biol Chem ; 274(19): 13345-52, 1999 May 07.
Article in English | MEDLINE | ID: mdl-10224096

ABSTRACT

The high affinity receptor for IgE (FcepsilonRI) plays an integral role in triggering IgE-mediated hypersensitivity reactions. The IgE-interactive site of human FcepsilonRI has previously been broadly mapped to several large regions in the second extracellular domain (D2) of the alpha-subunit (FcepsilonRIalpha). In this study, the IgE binding site of human FcepsilonRIalpha has been further localized to subregions of D2, and key residues putatively involved in the interaction with IgE have been identified. Chimeric receptors generated between FcepsilonRIalpha and the functionally distinct but structurally homologous low affinity receptor for IgG (FcgammaRIIa) have been used to localize two IgE binding regions of FcepsilonRIalpha to amino acid segments Tyr129-His134 and Lys154-Glu161. Both regions were capable of independently binding IgE upon placement into FcgammaRIIa. Molecular modeling of the three-dimensional structure of FcepsilonRIalpha-D2 has suggested that these binding regions correspond to the "exposed" C'-E and F-G loop regions at the membrane distal portion of the domain. A systematic site-directed mutagenesis strategy, whereby each residue in the Tyr129-His134 and Lys154-Glu161 regions of FcepsilonRIalpha was replaced with alanine, has identified key residues putatively involved in the interaction with IgE. Substitution of Tyr131, Glu132, Val155, and Asp159 decreased the binding of IgE, whereas substitution of Trp130, Trp156, Tyr160, and Glu161 increased binding. In addition, mutagenesis of residues Trp113, Val115, and Tyr116 in the B-C loop region, which lies adjacent to the C'-E and F-G loops, has suggested Trp113 also contributes to IgE binding, since the substitution of this residue with alanine dramatically reduces binding. This information should prove valuable in the design of strategies to intervene in the FcepsilonRIalpha-IgE interaction for the possible treatment of IgE-mediated allergic disease.


Subject(s)
Immunoglobulin E/metabolism , Receptors, IgE/metabolism , Animals , Binding Sites , COS Cells , Humans , Models, Molecular , Protein Binding , Receptors, IgE/chemistry , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism
14.
J Immunol ; 162(4): 2146-53, 1999 Feb 15.
Article in English | MEDLINE | ID: mdl-9973489

ABSTRACT

The FcR family contains multiple receptors for Igs, of which the most distantly related ( approximately 20%) is the IgA receptor (human Fc alpha R), being more homologous ( approximately 35%) to another family of killer-inhibitory receptor-related immunoreceptors with a 19q13.4 chromosomal location in humans. This study of the Fc alpha R demonstrated that, like several IgG receptors, Fc alpha R is a low affinity receptor for Ab (Ka approximately 106 M-1). Rapid dissociation of the rsFc alpha R:IgA complex (t1/2 approximately 25 s) suggests that monomer IgA would bind transiently to cellular Fc alpha Rs, while IgA immune complexes could bind avidly. Mutagenesis of histidyl 85 and arginyl 82, in the FG loop of domain 1, demonstrated that these residues were essential for the IgA-binding activity of Fc alpha R, while arginyl 87 makes a minor contribution to the binding activity of the receptor. This site is unusual among the Fc receptors (Fc gamma RII, Fc gamma RIII, and Fc epsilon RI), in which the ligand binding site is in domain 2 rather than domain 1, but like Fc alpha R, the FG loop comprises part of the ligand binding site. The putative F and G strands flanking the Fc alpha R ligand binding site are highly homologous in the other killer-inhibitory receptor-related immunoreceptors, suggesting they comprise a conserved structural element on which divergent FG loops are presented and participate in the specific ligand interactions of each of these receptors.


Subject(s)
Antigens, CD/chemistry , Antigens, CD/metabolism , Immunoglobulin A/metabolism , Receptors, Fc/chemistry , Receptors, Fc/metabolism , Amino Acid Sequence , Antigens, CD/genetics , Arginine/genetics , Arginine/metabolism , Biosensing Techniques , Histidine/genetics , Histidine/metabolism , Humans , Models, Molecular , Molecular Sequence Data , Mutagenesis, Site-Directed , Protein Binding/genetics , Protein Binding/immunology , Protein Structure, Tertiary , Receptors, Fc/genetics , Receptors, Immunologic/chemistry , Receptors, KIR , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism
15.
Mol Immunol ; 35(14-15): 989-96, 1998 Oct.
Article in English | MEDLINE | ID: mdl-9881694

ABSTRACT

FcgammaRI (CD64) is functionally unique as it is the only FcgammaR able to bind monomeric IgG with high affinity. FcgammaRI is also structurally distinct, containing an extracellular Ig-interactive region of three Ig-like domains in contrast to the two domains of the low affinity receptors FcgammaRII and FcgammaRIII. Previous studies have demonstrated that the third domain of FcgammaRI plays a crucial role in high affinity IgG binding of the receptor, with the first and second domains together forming a low affinity IgG binding motif. In this study the individual functional contributions of the first and second domains of FcgammaRI to IgG binding have been investigated. Chimeric FcgammaR were generated by exchanging extracellular domains between mouse FcgammaRI and the structurally related yet distinct low affinity receptor for IgG, mouse FcgammaRII. The replacement of both domains 1 and 2 of FcgammaRI with domains 1 and 2 of FcgammaRII results in a dramatic change in IgG binding characteristics, as this receptor loses the capacity to bind monomeric IgG with high affinity and also demonstrates a broader specificity (binding not only IgG2a but also IgG1 and 2b. IgG3 was not tested). However, the substitution of FcgammaRII domain 2 of this chimeric receptor with domain 2 of FcgammaRI (generating a chimeric receptor with domain 1 of FcgammaRII linked to domains 2 and 3 of FcgammaRI) was found to reconstitute the specific high affinity monomeric IgG2a binding of wild-type FcgammaRI, albeit with a slightly reduced affinity (1.8-fold lower than wild-type FcgammaRI). These findings suggest that it is the specific interaction between domains 2 and 3 of FcgammaRI, with domain 1 playing a supporting role in maintaining the conformational stability of the receptor, that is the major structural requirement to confer the unique Ig binding characteristics of FcgammaRI.


Subject(s)
Immunoglobulin G/immunology , Receptors, IgG/chemistry , Receptors, IgG/immunology , Animals , Antibody Affinity/physiology , Antibody Specificity , COS Cells/cytology , Membrane Proteins/immunology , Mice , Protein Structure, Tertiary , Receptors, IgG/biosynthesis , Recombinant Fusion Proteins/biosynthesis , Recombinant Fusion Proteins/immunology
16.
J Biol Chem ; 271(35): 21214-20, 1996 Aug 30.
Article in English | MEDLINE | ID: mdl-8702893

ABSTRACT

The aim of this study was to construct a single chain chimeric FcepsilonRIalpha receptor capable of effector function in leukocytes, including cytotoxic lymphocytes. To determine the most effective single chain FcepsilonRIalpha receptor with respect to IgE binding and signaling function, a variety of chimeric gene constructs were transiently transfected into COS-7 cells. The most effective chimera consisted of four parts including: wild-type or mutated extracellular domains (Trp130 --> Ala130, W130A) of FcepsilonRIalpha, membrane proximal and transmembrane regions of FcgammaRIIa, and intracellular CD3zeta (epsilonIIaIIazeta). Scatchard analysis indicated that these FcepsilonRIalpha chimeric receptor bound ligand with an affinity of 0.9 to 2.2 x 10(9) -1. Ligand binding capacity was dramatically reduced with the deletion of 11 membrane proximal amino acids of FcepsilonRIalpha; however, function was restored by substitution with the equivalent region of FcgammaRIIa, suggesting a crucial requirement for a "spacer" segment between the transmembrane and extracellular ligand binding domain. Chimeras that bound IgE effectively also mediated phagocytosis. Chimeric receptors that contained transmembrane zeta were expressed as multimers and consequently did not bind IgE effectively; however, cotransfection of these chimeras with gamma-chain largely reconstituted IgE-mediated phagocytosis. The mouse cytotoxic T lymphocyte cell line, CTLLR8 was stably transfected with epsilonIIaIIazeta, and cloned transfectants were demonstrated to lyse target cells in an anti-FcepsilonRIalpha or IgE antibody-dependent manner. Therefore, functional single chain chimeric FcepsilonRIalpha receptors were expressed in the absence or presence of associated zeta or gamma molecules and were used to redirect killer lymphocytes to target cells.


Subject(s)
Receptors, IgE/genetics , Recombinant Fusion Proteins/genetics , T-Lymphocytes, Cytotoxic/immunology , Animals , Antibody-Dependent Cell Cytotoxicity , Base Sequence , Cell Line , Cell Membrane/metabolism , DNA Primers , Haplorhini , Mice , Molecular Sequence Data , Phagocytosis , Receptors, IgE/metabolism , Recombinant Fusion Proteins/metabolism , Rosette Formation
17.
Immunogenetics ; 44(5): 340-50, 1996.
Article in English | MEDLINE | ID: mdl-8781119

ABSTRACT

Met-ase-1 is a 30 000 Mr serine protease (granzyme) that was first isolated in the cytolytic granules of rat CD3(-) large granular lymphocytes. We screened a mouse genomic library with rat Met-ase-1 cDNA, and obtained bacteriophage clones that contained the mouse Met-ase-1 gene. The mouse Met-ase-1 gene comprises five exons spanning approximately 5.2 kilobases (kb) and exhibits a similar structural organization to its rat homologue and a family of neutrophil elastase-like serine proteases. Mouse Met-ase-1 mRNA was only detected in total cellular and poly A mRNA of mouse CD3(-) GM1(+) large granular lymphocytes derived from splenocytes stimulated with IL-2 and the mouse NK1.1(+) cell line 4 - 16. Spleen T-cell populations generated by Concanavalin A stimulation and a number of mouse pre-NK and T cell lines did not express mouse Met-ase-1 mRNA. The 5' flanking region of the mouse Met-ase-1 gene also shares considerable regions of identity with the 5' flanking region of the rat Met-ase-1 gene. A 3.3 kb segment of 5' sequence flanking the mouse Met-ase-1 gene was inserted upstream of the chloramphenicol acetyltransferase reporter gene and this construct transiently transfected into a variety of mouse and rat large granular lymphocyte leukemia and T-cell lines. The transcriptional activity of the mouse Met-ase-1 5' flanking region was significant in the RNK-16 large granular lymphocyte leukemia, strongest in the 4 - 16 mouse NK1.1(+) cell line, and weak in several mouse pre-NK cell lines. Reverse transcriptase polymerase chain reaction of mouse large granular lymphocyte mRNA was used to derive the full-length coding sequence for mouse Met-ase-1. The predicted hexapropeptide of mouse Met-ase-1 (Asn-6 to Gln-1), was deleted by polymerase chain reaction mutagenesis to enable expression of active mouse Met-ase-1 in mammalian COS-7 cells. Northern blot analysis and protease assays of transfected COS cell lysates against a panel of thiobenzyl ester substrates formally demonstrated that the mouse Met-ase-1 gene encodes a serine proteinase that hydrolyzes substrates containing a long narrow hydrophobic amino acids like methionine, norleucine, and leucine in the P1.


Subject(s)
Genes , Killer Cells, Natural/enzymology , Mice/genetics , Recombinant Fusion Proteins/genetics , Serine Endopeptidases/genetics , Amino Acid Sequence , Animals , Base Sequence , Cell Line, Transformed , Chlorocebus aethiops , Cloning, Molecular , Lymphocyte Activation , Mice, Inbred BALB C , Molecular Sequence Data , Polymerase Chain Reaction , Rats , Sequence Alignment , Sequence Deletion , Sequence Homology, Amino Acid , Spleen/cytology , Substrate Specificity , Transfection
18.
Immunology ; 86(3): 392-8, 1995 Nov.
Article in English | MEDLINE | ID: mdl-8550076

ABSTRACT

Recombinant soluble IgE Fc receptors (rsFc epsilon RI) are potent inhibitors of type I hypersensitivity reactions tested in a local inflammatory setting. However, the fate of these receptors in vivo is dependent on the cellular source of the rsFc epsilon RI. We have produced these by transiently transfecting Cos-7 cells with a cDNA encoding the extracellular domains of human Fc epsilon RI alpha-chain. Following affinity purification, the rsFc epsilon RI was characterized as 58,000 MW, which was reduced to 23,000 MW following endoglycosidase F treatment. The purified rsFc epsilon RI could inhibit mouse IgE binding to Fc epsilon RI+ transfected CHO-K1 cells in vitro, bind sIgE+ B lymphoma cells in vitro, and inhibit the passive cutaneous anaphylaxis model in vivo in Sprague-Dawley rats. Pharmacokinetic studies in vivo involving intravenous injection of radiolabelled rsFc epsilon RI in mice revealed the receptor to have a rapid initial blood clearance (t1/2 early phase of 15 min) and to accumulate in the liver before being detected in urine. The localization of rsFc epsilon RI in the liver could be blocked by administration of mannose glycosylated ovalbumin and mannan, demonstrating that liver uptake involved the mannose receptor that is expressed on liver sinusoid cells and Kupffer cells. The production of rsFc epsilon RI using a stable expression system in CHO-K1 cells produced functional receptor of the same molecular weight as the Cos-7 system by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). However, biodistribution studies demonstrated differences; the CHO-K1 cell-produced material did not localize to the liver in comparison to the Cos-7-produced rsFc epsilon RI.


Subject(s)
Immunoglobulin E/metabolism , Receptors, IgE/metabolism , Animals , B-Lymphocytes/metabolism , CHO Cells , Cricetinae , Genetic Techniques , Humans , Hypersensitivity, Immediate/immunology , Liver/metabolism , Metabolic Clearance Rate , Mice , Mice, Inbred BALB C , Mice, Inbred CBA , Molecular Weight , Rats , Rats, Sprague-Dawley , Recombinant Proteins/pharmacokinetics , Tissue Distribution
19.
J Biol Chem ; 270(36): 21188-94, 1995 Sep 08.
Article in English | MEDLINE | ID: mdl-7673151

ABSTRACT

The low affinity receptor for IgG, Fc gamma RII (CD32), has a wide distribution on hematopoietic cells where it is responsible for a diverse range of cellular responses crucial for immune regulation and resistance to infection. Fc gamma RII is a member of the immunoglobulin superfamily, containing an extracellular region of two Ig-like domains. The IgG binding site of human Fc gamma RII has been localized to an 8-amino acid segment of the second extracellular domain, Asn154-Ser161. In this study, evidence is presented to suggest that domain 1 and two additional regions of domain 2 also contribute to the binding of IgG by Fc gamma RII. Chimeric receptors generated by exchanging the extracellular domains and segments of domain 2 between Fc gamma RII and the structurally related Fc epsilon RI alpha chain were used to demonstrate that substitution of domain 1 in its entirety or the domain 2 regions encompassing residues Ser109-Val116 and Ser130-Thr135 resulted in a loss of the ability of these receptors to bind hIgG1 in dimeric form. Site-directed mutagenesis performed on individual residues within and flanking the Ser109-Val116 and Ser130-Thr135 domain 2 segments indicated that substitution of Lys113, Pro114, Leu115, Val116, Phe129, and His131 profoundly decreased the binding of hIgG1, whereas substitution of Asp133 and Pro134 increased binding. These findings suggest that not only is domain 1 contributing to the affinity of IgG binding by Fc gamma RII but, importantly, that the domain 2 regions Ser109-Val116 and Phe129-Thr135 also play key roles in the binding of hIgG1. The location of these binding regions on a molecular model of the entire extracellular region of Fc gamma RII indicates that they comprise loops that are juxtaposed in domain 2 at the interface with domain 1, with the putative crucial binding residues forming a hydrophobic pocket surrounded by a wall of predominantly aromatic and basic residues.


Subject(s)
Immunoglobulin G/metabolism , Receptors, IgG/metabolism , Animals , Base Sequence , Binding Sites , Cell Line , DNA, Complementary , Humans , Models, Molecular , Molecular Sequence Data , Mutagenesis, Site-Directed , Receptors, IgG/chemistry , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/metabolism
20.
Immunogenetics ; 42(2): 101-11, 1995.
Article in English | MEDLINE | ID: mdl-7607701

ABSTRACT

Rat natural killer cell Met-ase-1 (RNK-Met-1) is a 30,000 M(r) serine protease (granzyme) found in the cytolytic granules of CD3- large granular lymphocytes (LGL) with natural killer (NK) activity. To characterize the genomic sequences responsible for the CD3- LGL-restricted expression of this gene, we screened a rat genomic library with RNK-Met-1 cDNA, and obtained bacteriophage clones that contained the RNK-Met-1 gene. The RNK-Met-1 gene comprises 5 exons and spans approximately 5.2 kilobases (kb), exhibiting a similar structural organization to a class of CTL-serine proteases with protease catalytic residues encoded near the borders of exons 2, 3, and 5. The 5'-flanking region of the RNK-Met-1 gene contains a number of putative promoter and enhancer regulatory elements and shares several regions of homology with the 5'-flanking region of the mouse perforin gene. We have prepared nested deletions from approximately 3.3 kb of the 5'-flanking region of the RNK-Met-1 gene, and inserted these upstream of the chloramphenicol acetyltransferase (CAT) reporter gene. These 5'-flanking RNK-Met-1-CAT constructs were transiently transfected into rat LGL leukemia, T-lymphoma, and basophilic leukemia cell lines. The transcriptional activity of the RNK-Met-1 5'-flanking region was strong, restricted to the RNK-16 LGL leukemia and controlled by several positive cis-acting regions spread over at least 3.3 kb. The longest and most active 5'-flanking region (-3341 to -33) was also used to drive specific expression of beta-galactosidase in RNK-16. These data are consistent with the NK cell-specific expression of RNK-Met-1 and suggest the potential utility of this gene promoter in the development of transgene models of NK cell biology in vivo.


Subject(s)
Killer Cells, Natural/enzymology , Serine Endopeptidases/genetics , Amino Acid Sequence , Animals , Base Sequence , Blotting, Southern , Cloning, Molecular , DNA , Molecular Sequence Data , Rats , Serine Endopeptidases/chemistry
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