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WO1997039122A9 - PHOSPHOPROTEINE DE 78 kDa ISOLEE ET CLONEE DE MASTOCYTES (AGENT INHIBITEUR DE LA DEGRANULATION DES MASTOCYTES) ET SON UTILISATION - Google Patents

PHOSPHOPROTEINE DE 78 kDa ISOLEE ET CLONEE DE MASTOCYTES (AGENT INHIBITEUR DE LA DEGRANULATION DES MASTOCYTES) ET SON UTILISATION

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Publication number
WO1997039122A9
WO1997039122A9 PCT/US1997/006042 US9706042W WO9739122A9 WO 1997039122 A9 WO1997039122 A9 WO 1997039122A9 US 9706042 W US9706042 W US 9706042W WO 9739122 A9 WO9739122 A9 WO 9739122A9
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WIPO (PCT)
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glu
lys
leu
ala
gin
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PCT/US1997/006042
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English (en)
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WO1997039122A2 (fr
WO1997039122A3 (fr
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Priority to AU24563/97A priority Critical patent/AU2456397A/en
Publication of WO1997039122A2 publication Critical patent/WO1997039122A2/fr
Publication of WO1997039122A3 publication Critical patent/WO1997039122A3/fr
Publication of WO1997039122A9 publication Critical patent/WO1997039122A9/fr

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Definitions

  • Moesin is evidently not phosphorylated in any cell type other than MC (Krieg et al . , J. Biol . Chem . 2662:19258 (1992), Franck et al . , J. Cel l Sci . , 105:219 (1993)); which distinguishes moesin from ezrin which is phosphorylated on tyrosine residues, not serine or threonine, by epidermal growth factor (EGF) in human carcinoma A-431 cells.
  • EGF epidermal growth factor
  • the tyrosine phosphorylation is also stimulated by platelet-derived growth factor (PDGF) . These tyrosine factors do not stimulate the phosphorylation of moesin.
  • PDGF platelet-derived growth factor
  • Moesin itself has diagnostic utility in, for example, the production of anti -moesin antibodies that are useful in diagnosing for the presence of moesin in MC, e.g, in nasal polyps in which the level of this protein may be low to absent.
  • Fig. 1 shows the size and overlapping nature of moesin cDNA fragments obtained from a library screening. The coding region is boxed.
  • Fig. 2 shows the nucleotide sequence of rat moesin cDNA and deduced amino acid sequence (SEQ ID NOS: 1-2).
  • Fig. 3 shows the homology of amino acid sequences of moesin from different species. The underlined amino acids are the putative sites phosphorylated by PKC (consensus: K/R X X T/S) . The amino acid residues in bold face are essential to the recognition and phosphorylation.
  • the consensus sequence of moesin was generated by the PRETTY program. Only the amino acid residues different among the species are shown vertically, in addition to the consensus sequence (SEQ ID NOS: 3-7 are shown in this figure) .
  • the cDNA has been cloned for a 78 kDa rat mast cell protein (also referred to as moesin) whose in vivo phosphorylation on specific serine and threonine residues by protein kinase C ("PKC") produces a phosphorylated form of the 78 kDa protein. It has also been discovered that the phosphorylated form of the 78 kDa protein inhibits mast cell degranulation. This inhibition of degranulation, in turn, inhibits secretion of mast cell granular substances such as preformed substances (histamine, heparin, protease and .
  • PKC protein kinase C
  • cytokines cytokines
  • de novo synthesized mediators prostaglandins, leukotrienes, lipoxins, platelet activating factor, etc.
  • Inhibition of the secretion of these substances has clinical consequences. For example, inhibition of histamine secretion has an anti -allergic effect.
  • Other released substances produce migraine headaches, interstitial cystitis and irritable bowel syndrome.
  • the phosphorylated 78 kDa protein has surprising and profound clinical effects.
  • the RBL moesin cDNA may be expressed in E. coli as follows. A cloned cDNA is amplified with a primer, subcloned into a plasmid, cleaved by a restriction enzyme, and subcloned into a T7 expression vector (e.g., pET 28) . This recombinant plasmid may then be transformed into an E. coli strain, and expression induced by IPTG (Stadier et al . , Enzymol . , 185:60 (1990). The expressed moesin may be purified to homogeneity using a nickel column under denaturing conditions (Invitrogen) .
  • Moesin is then dialyzed against a buffer-detergent mixture, then against just buffer.
  • cDNA expressed in E. col i by the T7 expression systems produces a fusion protein with 6 histidine residues at the N- terminus .
  • E. coli produces a protein which has a mass of 78 kDa by SDS-PAGE and represents about 50% of E. coli ' s protein content.
  • Purified, expressed moesin can be phosphorylated in vi tro or in vivo .
  • the protein may be incubated in a reaction mixture containing, inter alia, PKC, activators (e.g., diolein and phosphatdyeserime) and 32 P [ATP].
  • PKC phospholipase
  • activators e.g., diolein and phosphatdyeserime
  • 32 P [ATP 32 P [ATP].
  • the phosphorylated moesin is incubated with a phosphatase, and the products separated by SDS page.
  • For in vivo phosphorylation MC may be isolated, purified, loaded with 32 Pi, then treated with cromolyn (Theoharides et al . , Science, 207:80 (1980) .
  • the phosphoprotein may be cleaved by cyanogen bromide and the fragments thus obtained separated by SDS-PAGE, electroblotted onto a membrane, and autoradiographed. Radiolabeled fragments may then cut out and sequenced by automatic Edman degradation reactions (e.g., using an Applied Biosystems sequencer) .
  • tryptic/chymotryptic digests of the protein may be completely digested with HCl at 100 °C, and the individual amino acids separated on electro TLC plates and phosphorylated amino acids identified.
  • purified human MC may be treated with a phosphorylation stimulator (e.g., cromolyn), and the phosphorylated 78 kDa phosphoprotein isolated and purified to homogeneity (as described above and below) to a state suitable for administration to humans.
  • a phosphorylation stimulator e.g., cromolyn
  • the human moesin cDNA portion critical for inhibition of degranulation may be expressed in bacteria, and then phosphorylated in vi tro .
  • Phosphomoesin, in an appropriate pharmaceutic vehicle, as in liposomes may be administered routinely to patients by appropriate routes leading to inhibition of MC degranulation stimulated by allergic or other means.
  • the high homology between human and animal moesins suggests that the latter may be used in humans without untoward effects.
  • peritoneal mast cells were obtained by lavage of the peritoneum of male 225 g rats (Taconic, Germantown, NY) injected with Locke's buffer (150 mM NaCl, 5 mM KC1, 2 mM CaCl 2 , 5 mM Hepes , 1 g/liter dextrose and 1 g/liter BSA, pH 7.2) . Cells were then purified to greater than 90% purity by centrifugation at 350xg for 10 min at room temperature through metrizamide (Accurate Chemical and Scientific Corp, Westbury, NY) Shapiro et al . , above.
  • RBL rat basophilic leukemia cells
  • NASH Dr. H. Metzger
  • Protein Assay The Pierce BCA Protein Assay kit was used for the spectrophotometric determination (at 562 nm) of protein concentration. For small samples (5-250 ⁇ g range) , the enhanced protocol using 60°C for 30 min was utilized.
  • Mast cells were prepared and treated as described in Methods except that the SDS gel was 7.5%. A23187 was used at 0.1 ⁇ g/ l for 1 min and C48/80 at 0.5 ⁇ g/ml for 1 min. No calcium indicates that no extracellular calcium was added.
  • Sample preparation with phenol extraction Cells were first lysed in 0.5 ml of Extraction Buffer (0.7M Sucrose, 0.5M Tris, 30mM HCl, 50mM EDTA, 0.1M KC1 , 2% BME and 2 mM PMSF) and were sonicated with multiple short bursts of maximum intensity. The volume was adjusted to 1.5 ml, incubated for 10 min on ice and an equal volume of water-saturated phenol was then added. After 10 min with shaking at room temperature, the phases were separated by centrifugation. Proteins were precipitated from the phenol phase with 5 volumes of 0.1 M ammonium acetate in methanol and kept at - 20°C overnight.
  • Extraction Buffer 0.7M Sucrose, 0.5M Tris, 30mM HCl, 50mM EDTA, 0.1M KC1 , 2% BME and 2 mM PMSF
  • the precipitate was washed 3 times with ammonium acetate and once with acetone .
  • the pellet was then dried under nitrogen gas and was solubilized in O'Farrell 's buffer [0.5% SDS, 9.5 M urea, 2% ampholytes (pH 3.5-10) from Bio-Rad Labs
  • IEF gels were composed of 4% Bis-Acrylamide, 8.0 M Urea, 4% NP-
  • Electroblottin The method of Paul Matsudaira, JBC 262:10035 (1987) was used where proteins were directly electroblotted onto polyvinylidene difluoride (PVDF) .
  • PVDF polyvinylidene difluoride
  • the gel was first soaked in Transfer Buffer (10 mM 3 - (cyclohexylamino) -1- propane sulfonic acid 10% methanol, pH 11, for 5 min to reduce the amount of Tris and glycine.
  • PVDF membrane was then rinsed in 100% methanol and stored in Transfer Buffer. The proteins were electroblotted onto PVDF.
  • kinase inhibitors Radiolabeled mast cells were pretreated with different concentrations and for different times with various cell permeable serine/threonine kinase inhibitors which included inhibitors of PKA, PKG, PKC and calcium/calmodulin kinase II (CAMK IIMBIOMOL Research Labs, PA), followed by cromolyn for 30 sec at 37°C, lysed and run on one dimension SDS- PAGE.
  • the RBL moesin gene was subcloned into pET-28 vector and transformed into E . coli JM109 (DE3); after IPTG induction, a fusion protein with polyhistidine on the N-terminus was expressed.
  • the protein was purified to homogeneity using a nickel column under denaturing conditions. The purified protein was injected into rabbits and polyclonal antisera were generated by I muno Dynamics (San Diego, Ca) and were used without further purification.
  • Rabbit polyclonal anti- calf moesin serum was kindly supplied by Dr. Furthmayr. It crossreacted with both ezrin and moesin and recognized two bands in RBL cell lysate.
  • a monoclonal antibody which recognizes both ezrin and moesin was purchased from Zymed.
  • Immunocvtochemistr Purified mast cells were treated with 100 ⁇ M cromolyn for 30 sec and were immediately fixed in 4% paraformaldehyde. Frozen sections were cut at 7 ⁇ m and treated with 1:200 dilution of rabbit anti-rat moesin polyclonal antibody at room temperature for 1 hr . The sections were then incubated with 1:200 dilution of goat anti-rabbit IgG-biotin (Vector Labs, CA) for 30 min, followed by a further exposure to streptavidine- rhodamine (Pierce, Rockford, IL) for 30 min. The sections were then mounted in aqueous mounting medium and observed under a light microscope (Nikon, Don Santo Corp, Natick, MA) .
  • Rat PKC (a . ⁇ and ⁇ mixture) .
  • the isozyme mixture was purified from rat brain.
  • Recombinant rabbit PKC a and ⁇ expressed in Baculovirus system and purified to >90%, were all purchased from Upstate Biotechnology Inc. (Lake Placid, NY).
  • Phosphotatases were also purchased from Upstate Biotechnology Inc.
  • the purified moesin was dialyzed against phosphate- buffered saline (PBS) containing 1% Triton X-100, then against PBS without the detergent.
  • PBS phosphate- buffered saline
  • the nucleotide sequence and deduced amino acid sequence of the rat moesin cDNA are shown on Fig. 2.
  • the protein contains 577 amino acids, same as human moesin, and has 99% homology with human moesin.
  • the calculated molecular mass of the protein was 67.3 kDa and the pi was 6.37.
  • PKC can recognize specific motifs and phosphorylate serine and threonine residues within them. These are (R/Kl-3, X2-0)- S/T-(X 2 - 8 , R/K 1"3 ), S/T(X 2 -°, R/K 1"3 ) and R/K 1"3 , X 2 ⁇ )-S/T.
  • R/Kl-3, X2-0 S/T-(X 2 - 8 , R/K 1"3
  • S/T(X 2 -°, R/K 1"3 ) and R/K 1"3 , X 2 ⁇ )-S/T When the deduced amino acid sequence was searched for those phosphorylation sites, twelve sites were found, which are shown in Fig. 3. These twelve sequences are apparently conserved between the species, as sequences from different species published showed little variation in those regions.
  • the coding region of the moesin cDNA was cloned in expression vector pET-28.
  • E. coli JM109(DE3) with the recombinant plasmid (pET28-RMl) were treated with IPTG, which induced synthesis of T7 RNA polymerase and in turn led to the expression of cloned gene, the E. coli produced a protein that appeared by SDS-PAGE to have a molecular mass of about 78 kDa. This protein represented almost 50% of total E . col i protein and was purified to homogeneity with a nickel column under denaturing conditions .
  • Moesin purified from E. coli did not have any major hydrophobic regions according to its sequence, suggesting that it probably would not refold properly in solution. It was dissolved in PBS containing 1% Triton X-100 but precipitated in PBS without detergent, suggesting that the solubility of the protein was low. The precipitated moesin was again dissolved in PBS containing 1% Triton, was dialyzed against PBS without detergent, then centrifuged to remove the precipitant. About 75% of precipitated moesin was dissolved by this method.
  • the presence of PKC isozymes was explored using Western blot analysis in purified rat peritoneal mast cells and the a , ⁇ , ⁇ and f isozymes were identified.
  • the ⁇ and e isozymes could not be identified in rat peritoneal mast cells even though the e isozyme was present in RBL cells (results not shown) .
  • the ⁇ and ⁇ isozymes are calcium-dependent, while the others are calcium- independent. Ozawa et al . , J. Biol . Chem . 268:1749 (1993). However, the ⁇ and ⁇ isozymes are linked to stimulation cf secretion, while the ⁇ - * and e to its inhibition.
  • Pretreatment with sphingosine (100 ⁇ M for 2 min), a specific inhibitor of PKC, and staurosporine (0.1 ⁇ M for 15 min) also completely prevented the incorporation of phosphate into the 78 kDa protein.
  • Radiolabeled control or cromolyn- treated MC extracts were first immunoprecipitated with anti-rat moesin serum and the precipitate was analyzed by SDS-PAGE. Autoradiography revealed a single phosphorylated 78 kDa protein. Immunoprecipitation removed all of the 78 kDa phosphoprotein from the supernatant fluid, thus indicating that the phosphoprotein could not be simply "co-precipitated" along with moesin.
  • EXAMPLE 14 Western blot analysis of the distribution of moesin in permeabilized RBL and rat mast cells
  • the specific anti-rat moesin clearly localized the 78 kDa protein at discrete punctate structures at the plasma membrane and no cytoplasmic staining was observed. Treatment with cromolyn did not alter its distribution.
  • ATTORNE /AGENT INFORMATXON (A) NAME: BLECHER, Melvin (B) REGISTRATION NUMBER: 33,649
  • GCC AAG GAG CGT CAA GAA GCT GAA GAA GCC AAG GAG GCC CTG CTG CAG 1313 Ala Lys Glu Arg Gin Glu Ala Glu Glu Ala Lys Glu Ala Leu Leu Gin
  • Lys Glu Lys lie Glu Arg Glu Lys Glu Glu Leu Met Glu Lys Leu Lys 340 345 350
  • Gin Ile Glu Glu Gin Thr Lys Lys Ala Gin Gin Glu Leu Glu Glu Gin 355 360 365 Thr Arg Ser Pro Leu Glu Leu Glu Gin Glu Arg Lys Arg Ala Gin Ser
  • Glu Phe Ala lie Gin Pro Asn Thr Thr Gly Lys Gin Leu Phe Asp Gin 20 25 30
  • Lys lie Ala Gin Asp Leu Glu Met Tyr Gly Val Asn Tyr Phe Ser Ser 195 200 205
  • Ile Lys Pro lie Asp Lys Lys Ala Pro Asp Phe Val Phe Tyr Ala Pro 260 265 270
  • Glu Phe Ala lie Gin Pro Asn Thr Thr Gly Lys Gin Leu Phe Asp Gin 20 25 30 Val Val Lys Thr Ile Gly Leu Arg Glu Val Trp Phe Phe Gly Leu Gin
  • Leu Arg Ile Asn Lys Arg lie Leu Ala Leu Cys Met Gly Asn His Glu 275 280 285

Abstract

On a cloné une protéine de mastocyte de 78 kDa (moesine) depuis le rat et on a déterminé son ADNc et sa séquence d'acides aminés. Cette protéine est phosphorylée sur des résidus spécifiques de serine et de threonine sous l'effet exercé dans les mastocytes par un ou plusieurs isozymes de protéine kinase C, en particulier, l'isozyme κ, ce qui permet d'obtenir une phosphoprotéine qui inhibe la dégranulation des mastocytes (agent inhibiteur de la dégranulation des mastocytes). On peut stimuler la phosphorylation in vivo de cette protéine dans les mastocytes au moyen de médicaments, tels que cromolyne, nedocromil, de flavonoïdes, tels que quercetine et kaempherol, et de lodoxamide, On peut inhiber la dégranulation des mastocytes au moyen de l'administration d'inhibiteurs de phosphomoesine phosphatase. On peut identifier des tissus déficients en moesine de mastocytes au moyen d'un anticorps marqué anti-moesine ou anti-phosphomoesine et les traiter au moyen de la transfection de ces mastocytes par de l'ADNc de moesine dans un vecteur viral, tel que le vecteur du virus de la grippe.
PCT/US1997/006042 1996-04-12 1997-04-11 PHOSPHOPROTEINE DE 78 kDa ISOLEE ET CLONEE DE MASTOCYTES (AGENT INHIBITEUR DE LA DEGRANULATION DES MASTOCYTES) ET SON UTILISATION WO1997039122A2 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU24563/97A AU2456397A (en) 1996-04-12 1997-04-11 Isolated and cloned mast cell 78 kda phosphoprotein (mast cell degranulation inhibitory agent) and use thereof

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US63118496A 1996-04-12 1996-04-12
US08/631,184 1996-04-12

Publications (3)

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WO1997039122A2 WO1997039122A2 (fr) 1997-10-23
WO1997039122A3 WO1997039122A3 (fr) 1997-12-24
WO1997039122A9 true WO1997039122A9 (fr) 1998-03-26

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US6280739B1 (en) * 1996-04-18 2001-08-28 Genetics Institute, Inc. Method of inhibiting angiogenesis using secreted proteins
US5872234A (en) 1997-06-27 1999-02-16 Incyte Pharmaceuticals, Inc. Human extracellular matrix proteins
EP1557426A3 (fr) * 1998-02-09 2005-08-03 Human Genome Sciences, Inc. 45 Protéines humaines secrétées
KR20010043090A (ko) * 1998-04-28 2001-05-25 우에노 도시오 신규인 폴리펩티드, 그 폴리펩티드를 코드화하는 ｃＤＮＡ및 그 용도
US8410248B2 (en) 1999-03-12 2013-04-02 Human Genome Sciences Inc. HWBAO62 polypeptides
CA2374412A1 (fr) * 1999-05-28 2000-12-07 Zymogenetics, Inc. Proteine-31 a helice alpha secretee
US7893218B2 (en) * 2003-06-16 2011-02-22 Stowers Institute For Medical Research Antibodies that specifically bind SOST peptides
RU2380100C1 (ru) * 2008-08-27 2010-01-27 Научно-исследовательский институт гриппа Северо-Западного отделения Российской академии медицинских наук (НИИ гриппа СЗО РАМН) Средство для профилактики и лечения гриппа а и в

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US5707863A (en) * 1993-02-25 1998-01-13 General Hospital Corporation Tumor suppressor gene merlin

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