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1.
Oncogene ; 25(19): 2807-11, 2006 May 04.
Article in English | MEDLINE | ID: mdl-16369489

ABSTRACT

Natural killer cells are well known to play an important role in immune defense against tumor development and viral infections. To further characterize new functionally relevant structures in these cells, we studied a series of monoclonal antibodies that we have raised against the NK cell line YT. One of these antibodies previously described as AY19, recognizes a 85 kD surface glycoprotein. Here we report the identification of a new secreted isoform of protocadherin 15, PCDH15C, which represents a potential associated protein for p85. Importantly, whereas protocadherins are absent from the surface of normal hematopoietic cells, we describe, for the first time, that PCDH15 is expressed in cytotoxic tumor-derived T- and NK-cell lines as well as in biopsies of nasal NK/T-cell lymphomas.


Subject(s)
Biomarkers, Tumor/metabolism , Cadherins/metabolism , Killer Cells, Natural/metabolism , Lymphoma, T-Cell/metabolism , Nose Neoplasms/metabolism , Protein Precursors/metabolism , Animals , Biopsy , COS Cells , Cadherin Related Proteins , Chlorocebus aethiops , Humans , Protein Isoforms/metabolism , Transfection , Tumor Cells, Cultured
2.
Biochem Biophys Res Commun ; 276(2): 515-23, 2000 Sep 24.
Article in English | MEDLINE | ID: mdl-11027506

ABSTRACT

During oogenesis, maternal mRNAs are synthesised and stored in a translationally dormant form due to the presence of regulatory elements at the 3' untranslated regions (3'UTR). In Xenopus oocytes, several studies have described the presence of RNA-binding proteins capable to repress maternal-mRNA translation. The testis-brain RNA-binding protein (TB-RBP/Translin) is a single-stranded DNA- and RNA-binding protein which can bind the 3' UTR regions (Y and H elements) of stored mRNAs and can suppress in vitro translation of the mRNAs that contain these sequences. Here we report the cloning of the Xenopus homologue of the TB-RBP/Translin protein (X-translin) as well as its expression, its localisation, and its biochemical association with the protein named Translin associated factor X (Trax) in Xenopus oocytes. The fact that this protein is highly present in the cytoplasm from stage VI oocytes until 48 h embryos and that it has been described as capable to inhibit paternal mRNA translation, indicates that it could play an important role in maternal mRNA translation control during Xenopus oogenesis and embryogenesis. Moreover, we investigated X-translin localisation during cell cycle in XTC cells. In interphase, although a weak and diffuse nuclear staining was observed, X-translin was mostly present in the cytoplasm where it exhibited a prominent granular staining. Interestingly, part of X-translin underwent a remarkable redistribution throughout mitosis and associated with centrosomes, which may suggest a new unknown role for this protein in cell cycle.


Subject(s)
Centrosome/metabolism , DNA-Binding Proteins/genetics , Mitosis/genetics , Amino Acid Sequence , Animals , Carrier Proteins/metabolism , Consensus Sequence , DNA, Complementary/analysis , DNA, Single-Stranded/metabolism , DNA-Binding Proteins/chemistry , DNA-Binding Proteins/metabolism , Humans , Molecular Sequence Data , Nuclear Proteins/metabolism , Oocytes/metabolism , Sequence Homology, Amino Acid , Xenopus laevis/embryology
3.
J Exp Med ; 191(10): 1807-12, 2000 May 15.
Article in English | MEDLINE | ID: mdl-10811873

ABSTRACT

Little is known about innate immunity to bacteria after birth in the hitherto sterile fetal intestine. Breast-feeding has long been associated with a lower incidence of gastrointestinal infections and inflammatory and allergic diseases. We found in human breast milk a 48-kD polypeptide, which we confirmed by mass spectrometry and sequencing to be a soluble form of the bacterial pattern recognition receptor CD14 (sCD14). Milk sCD14 (m-sCD14) concentrations were up to 20-fold higher than serum sCD14 from nonpregnant, pregnant, or lactating women. In contrast, lipopolysaccharide (LPS)-binding protein was at very low levels. Mammary epithelial cells produced 48-kD sCD14. m-sCD14 mediated activation by LPS and whole bacteria of CD14 negative cells, including intestinal epithelial cells, resulting in release of innate immune response molecules. m-sCD14 was undetectable in the infant formulas and commercial (cows') milk tested, although it was present in bovine colostrum. These findings indicate a sentinel role for sCD14 in human milk during bacterial colonization of the gut, and suggest that m-sCD14 may be involved in modulating local innate and adaptive immune responses, thus controlling homeostasis in the neonatal intestine.


Subject(s)
Bacteria/immunology , Lipopolysaccharide Receptors/metabolism , Milk, Human/immunology , Milk, Human/microbiology , Amino Acid Sequence , Animals , Cattle , Colostrum/immunology , Female , Humans , Immunity, Innate , Immunity, Mucosal , Infant Food/analysis , Infant, Newborn , Intestines/immunology , Intestines/microbiology , Lipopolysaccharide Receptors/blood , Lipopolysaccharide Receptors/genetics , Molecular Sequence Data , Pregnancy , Solubility
4.
Biochemistry ; 38(10): 3133-9, 1999 Mar 09.
Article in English | MEDLINE | ID: mdl-10074368

ABSTRACT

Polyglycylation is a posttranslational modification specific to tubulin. This modification was originally identified in highly stable microtubules from Paramecium cilia. As many as 34 posttranslationally added glycine residues have been located in the C-terminal domains of Paramecium alpha- and beta-tubulin. In this study, post source decay matrix-assisted laser desorption/ionization mass spectrometry (PSD MALDI MS) and electrospray ionization on a hybrid quadrupole orthogonal time-of-flight tandem mass spectrometer (ESI Q-TOF MS/MS) were both used to demonstrate that a single molecule of beta-tubulin, from either dynamic cytoplasmic microtubules or stable axonemal microtubules, can be glycylated on each of the last four C-terminal glutamate residues Glu437, Glu438, Glu439, and Glu441 in the sequence 427DATAEEEGEFEEEGEQ442. In both dynamic and stable microtubules the most abundant beta-tubulin isoform contains six posttranslationally added glycine residues: two glycine residues on both Glu437 and Glu438 and one glycine residue on both Glu439 and Glu441. The number and relative abundance of glycylated isoforms of beta-tubulin in both cytoplasmic and axonemal microtubules were compared by MALDI MS.1 The abundance of the major glycylated isoforms in axonemal tubulin decreases regularly with glycylation levels from 6 to 19 whereas it drops abruptly in cytoplasmic tubulin with glycylation levels from 6 to 9. However, the polyglycine chains are similarly distributed on the four C-terminal glutamate residues of cytoplasmic and axonemal tubulin. The polyglycylation results in bulky C-terminal domains with negatively charged surfaces, all surrounding the microtubular structure.


Subject(s)
Peptides/metabolism , Protein Processing, Post-Translational , Tubulin/metabolism , Amino Acid Sequence , Animals , Cytoplasm/metabolism , Mass Spectrometry , Microtubules/metabolism , Molecular Sequence Data , Paramecium tetraurelia , Peptide Fragments/chemical synthesis , Peptide Fragments/metabolism , Peptides/chemistry , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Tubulin/chemistry
5.
Anal Chem ; 69(19): 3979-85, 1997 Oct 01.
Article in English | MEDLINE | ID: mdl-9322434

ABSTRACT

Sequencing conditions for postsource decay and collision-induced dissociation/postsource decay matrix-assisted laser desorption/ionization time-of-flight mass spectrometry have been optimized to elucidate the structure of polyglycylation of tubulin. This posttranslational modification involves the linkage of multiple glycine residues through the gamma-carboxyl of glutamic acid residues in the carboxyl termini of the protein. Individual alpha- and beta-tubulin polypeptides contain respectively three and four potential glycylation sites. The sample preparation we used was the thin-layer preparation of the target specimen in the presence of alpha-cyano-4-hydroxycinnamic acid and nitrocellulose. The study of different synthetic polyglycylated peptides fragmentation (modified peptides with the linear sequence DATAEEEGEFEEEGEQ) shows that the peptides fragment regularly to form major fragments of b- and y-type ions with negligible side-chain fragmentation. The rules were applied to the structural elucidation of a Paramecium beta-tubulin hexaglycylated peptide available in the subpicomole range. Polyglycylation was identified on the last four glutamic acid residues.


Subject(s)
Protein Processing, Post-Translational , Sequence Analysis/methods , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization/methods , Tubulin/chemistry , Amino Acid Sequence , Animals , Glycine/chemistry , Microchemistry , Molecular Sequence Data , Paramecium , Peptide Mapping/methods , Protein Conformation , Protozoan Proteins/chemistry
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