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A novel 54-KDA AU-rich RNA binding protein in human intestine with a role in apolipoprotein B MRNA editing
A186 AGA ABSTRACTS
1197 A NOVEL 54·KDA AU·RICH RNA BINDING PROTEIN IN HU· MAN INTESTINE WITH A ROLE IN APOLIPOPROTEIN B MRNA EDITING. Jeffrey O. Henderson, Shrikant Anant, Jing Min, V. S. Sankaranand, Masahiro aka, Nicholas O. Davidson, Washington Univ, St. Louis, MO. Apolipoprotein B (apo B), an obligate component of intestinal and hepatic lipoproteins, circulates in two forms, apo B 100 and apo B48, both encoded by a single nuclear APOB gene. Apo B48, is produced exclusively in the human small intestine, by an RNA editing event that targets a specific cytidine for enzymatic deamination, creating a UAA stop codon. Apo B RNA editing is mediated by a multicomponent protein complex, containing a catalytic subunit, apobec-l, as well as other, currently unknown protein components which have a wide tissue distribution. We have now cloned a novel 54-kDa protein (p54) from chicken small intestine, using a yeast two-hybrid screen, with apobec-l as bait. The human, rat, mouse and bovine homologs of p54 exhibit >95% homology. p54 is a nove! member of the Hu family of RNA binding proteins, containing three RNA recognition motifs (RRMs), with the third RRM separated from the first two by a unique bridging region rich in alanines (A) and glutamines (Q). Mutagenesis of the RRMs and the NQ region indicates that the RRMs are not required for interaction with apobec-l. However, mutations within the NQ region disrupt binding of p54 to recombinant apobec-l. Using UV cross linking and EMSA, we demonstrate that p54 is an AU-rich RNA binding protein. More specifically, p54 binds to apoB RNA. These findings suggest that p54 may playa role in binding the appropriate region of apo B RNA in preparation for C to U editing of the nascent transcript. p54 mRNA is expressed in multiple human tissues including the small intestine, lung, brain, heart and liver. This contrasts with the distribution of apobec-l, which in humans is confined to the luminal GI tract. Biochemical fractionation of bovine liver extracts demonstrates that p54 coelutes with the peak of apo B editing activity. Only those fractions that were highly enriched for p54 demonstrated high levels of editing. Furthermore, immunodepletion of p54 from the highly enriched fractions resulted in the complete abrogation of editing activity.Immunofluorescence analysis by confocal microscopy demonstrated that apobec-l and p54 are colocalized in the cell, with a predominantly cytoplasmic and perinuclear localization. Taken together, the data strongly suggest that p54 is a novel, integral and required auxiliary component of the mammalian apo B RNA C to U editing enzyme complex. The colocalization of apobec-l and p54, together with their RNA binding activity, suggests that these factors together regulate the specificity of C to U editing. 1198 NOVEL SYSTEM FOR ENHANCED SPECIFIC TRANSEPITHE· LIAL ANTIGEN TRANSPORT IN SENSITIZED RATS IS MEDI· ATED BY IGE/CD23. Mary H. Perdue, Ping-Chang Yang, M. Cecilia Berin, Linda C. Yu, Daniel D. Conrad, McMaster Univ, Hamilton, ON, Canada; Virgina Commonwealth Univ, Richmond, VA. We previously reported that sensitization of rats resulted in the appearance of a unique system for rapid and specific transepithelial transport of antigen. Two min after its addition to the apical surface of enterocytes, the specific antigen to which the rat had been sensitized was delivered across the cell (via endosomes) into the lamina propria. This transport preceded mast cell activation and the rise in the short-circuit current (lsc) indicative of the hypersensitivity response. The specificity of the protein transport suggested the involvement of epithelial immunoglobulin receptors. Aims: The current studies were designed to define the essential components of this antigen transport system. Methods: Horseradish peroxidase (HRP) was used as a model antigen. Rats were actively sensitized (AS) to HRP or passively sensitized (PS) by ip injection of serum from AS rats; studies were conducted at 14 or 3 days, respectively. Sham-sensitized naive rats served as controls (C). Jejunal tissues were mounted in Ussing chambers
GASTROENTEROLOGY Vol. 118, No.4
and challenged on the luminal side with HRP. After 2 min, tissues were removed and fixed for electron microscopic studies. Separate tissues were used to examine the hypersensitivity reaction (A in Isc and conductance). Results: Enhanced HRP uptake into enterocytes (significantly greater area of HRP-containing endosomes) was shown in AS and PS rats compared with C rats. Whole serum, but not IgE-depleted serum, transferred the enhanced specific antigen transport phenomenon. Immunohistochemistry demonstrated that expression of CD23, the low affinity IgE receptor (FceRII), on epithelial cells was induced by sensitization. The number of CD23 immunogold-labelled receptors on the microvillous membrane of enterocytes was significantly increased in AS vs C rats and was reduced in AS rats following HRP challenge. In addition, high magnification EM revealed CD23 and HRP within the same endosomes. Finally, pre-treatment of sensitized tissues with anti-CD23 antibody significantly reduced the epithelial uptake of HRP and the subsequent hypersensitivity reaction. Conclusions: Our results provide convincing evidence that IgE antibodies bound to CD23 receptors on epithelial cells are responsible for the specific nature of this enhanced transepithelial antigen transport system in sensitized rats. Interfering with this pathway inhibits the hypersensitivity reaction, thus suggesting that it may be a novel target for therapeutic intervention in food allergic conditions. 1199 GALANIN·l RECEPTOR (GAUR) UP·REGULATION IS ESSEN· TIAL FOR MEDIATING THE EXCESS FLUID SECRETION IN INFECTIOUS DIARRHEA. Kristina A. Matkowskyj, Alexey V. Danilkovich, Richard V. Benya, Univ of Illinois at Chicago, Chicago, IL. Although GallR are not normally expressed by epithelial cells lining the mouse colon, we recently showed that infection with enterohemorrhagic E. coli (EHEC) markedly increased the expression of this protein by an NF-KB-mediated mechanism (J Clin Invest 1999; 104: 253). When expressed by colonocytes, GallR causes chloride secretion (Am J Phys 1999; 276: G64). Thus the purpose of this study was to determine: 1), if other pathogens increase GallR expression; and 2) elucidate the absolute contribution of this receptor to mediating the excessive fluid secretion observed post-infection. To create mice genetically incapable of expressing GallR, we first cloned the mouse GALIR gene from a murine Pjlibrary using human GallR eDNA as a probe. Homologous recombination (HR) with the native gene was achieved using linearized pNTK vector containing 5 Kb of the GALlR gene, but which had been disrupted by insertion of a Neomycin resistance gene in exon 1. ES cells were transfected by electroporation and cultured in the presence of G-4l8. Resistant clones undergoing successful HR were injected into pseudopregnant mice. Chimeric offspring were then mated with C57BLl6J mice to produce animals that were heterozygous or homozygous negative for the GallR. Colonic fluid secretion was assessed by closed loop experiments in wild type mice (ie, GallR+'+), and in those that had one (GallR+/') or both (GallR-/') alleles deleted. Mice of all three genotypes were infected with log growth EHEC, Salmonella, or Shigella by gavage, and colonic fluid secretion assessed after exposure to intraluminal galanin (I J.tM) for 4 hrs (Table). Conclusion: Elimination of GALlR does not alter basal colonic fluid secretion, but markedly attenuates the increase in secretion due to pathogen infection. These data support a critical central role for the Gall R in mediating the excess fluid secretion observed in infectious diarrhea. Colonic Fluid Secretion (mg/cm) inthe Presence ofLuminal Galanin Pathogen None
EHEC Salmonella Shigella
Peak Infection
GallR'/'
GallW"
GaI1R<'
N/A 3days 6 days 4 days
346±25 71.2±8,7 178,4±11.2 830±5.2
39.3±1,8 43,8±2,4 42,O±1.1 55,4+0.8
36.9±O,2 32,9±7.3 38.8±6.8 40.9±O.5
1197 A NOVEL 54·KDA AU·RICH RNA BINDING PROTEIN IN HU· MAN INTESTINE WITH A ROLE IN APOLIPOPROTEIN B MRNA EDITING. Jeffrey O. Henderson, Shrikant Anant, Jing Min, V. S. Sankaranand, Masahiro aka, Nicholas O. Davidson, Washington Univ, St. Louis, MO. Apolipoprotein B (apo B), an obligate component of intestinal and hepatic lipoproteins, circulates in two forms, apo B 100 and apo B48, both encoded by a single nuclear APOB gene. Apo B48, is produced exclusively in the human small intestine, by an RNA editing event that targets a specific cytidine for enzymatic deamination, creating a UAA stop codon. Apo B RNA editing is mediated by a multicomponent protein complex, containing a catalytic subunit, apobec-l, as well as other, currently unknown protein components which have a wide tissue distribution. We have now cloned a novel 54-kDa protein (p54) from chicken small intestine, using a yeast two-hybrid screen, with apobec-l as bait. The human, rat, mouse and bovine homologs of p54 exhibit >95% homology. p54 is a nove! member of the Hu family of RNA binding proteins, containing three RNA recognition motifs (RRMs), with the third RRM separated from the first two by a unique bridging region rich in alanines (A) and glutamines (Q). Mutagenesis of the RRMs and the NQ region indicates that the RRMs are not required for interaction with apobec-l. However, mutations within the NQ region disrupt binding of p54 to recombinant apobec-l. Using UV cross linking and EMSA, we demonstrate that p54 is an AU-rich RNA binding protein. More specifically, p54 binds to apoB RNA. These findings suggest that p54 may playa role in binding the appropriate region of apo B RNA in preparation for C to U editing of the nascent transcript. p54 mRNA is expressed in multiple human tissues including the small intestine, lung, brain, heart and liver. This contrasts with the distribution of apobec-l, which in humans is confined to the luminal GI tract. Biochemical fractionation of bovine liver extracts demonstrates that p54 coelutes with the peak of apo B editing activity. Only those fractions that were highly enriched for p54 demonstrated high levels of editing. Furthermore, immunodepletion of p54 from the highly enriched fractions resulted in the complete abrogation of editing activity.Immunofluorescence analysis by confocal microscopy demonstrated that apobec-l and p54 are colocalized in the cell, with a predominantly cytoplasmic and perinuclear localization. Taken together, the data strongly suggest that p54 is a novel, integral and required auxiliary component of the mammalian apo B RNA C to U editing enzyme complex. The colocalization of apobec-l and p54, together with their RNA binding activity, suggests that these factors together regulate the specificity of C to U editing. 1198 NOVEL SYSTEM FOR ENHANCED SPECIFIC TRANSEPITHE· LIAL ANTIGEN TRANSPORT IN SENSITIZED RATS IS MEDI· ATED BY IGE/CD23. Mary H. Perdue, Ping-Chang Yang, M. Cecilia Berin, Linda C. Yu, Daniel D. Conrad, McMaster Univ, Hamilton, ON, Canada; Virgina Commonwealth Univ, Richmond, VA. We previously reported that sensitization of rats resulted in the appearance of a unique system for rapid and specific transepithelial transport of antigen. Two min after its addition to the apical surface of enterocytes, the specific antigen to which the rat had been sensitized was delivered across the cell (via endosomes) into the lamina propria. This transport preceded mast cell activation and the rise in the short-circuit current (lsc) indicative of the hypersensitivity response. The specificity of the protein transport suggested the involvement of epithelial immunoglobulin receptors. Aims: The current studies were designed to define the essential components of this antigen transport system. Methods: Horseradish peroxidase (HRP) was used as a model antigen. Rats were actively sensitized (AS) to HRP or passively sensitized (PS) by ip injection of serum from AS rats; studies were conducted at 14 or 3 days, respectively. Sham-sensitized naive rats served as controls (C). Jejunal tissues were mounted in Ussing chambers
GASTROENTEROLOGY Vol. 118, No.4
and challenged on the luminal side with HRP. After 2 min, tissues were removed and fixed for electron microscopic studies. Separate tissues were used to examine the hypersensitivity reaction (A in Isc and conductance). Results: Enhanced HRP uptake into enterocytes (significantly greater area of HRP-containing endosomes) was shown in AS and PS rats compared with C rats. Whole serum, but not IgE-depleted serum, transferred the enhanced specific antigen transport phenomenon. Immunohistochemistry demonstrated that expression of CD23, the low affinity IgE receptor (FceRII), on epithelial cells was induced by sensitization. The number of CD23 immunogold-labelled receptors on the microvillous membrane of enterocytes was significantly increased in AS vs C rats and was reduced in AS rats following HRP challenge. In addition, high magnification EM revealed CD23 and HRP within the same endosomes. Finally, pre-treatment of sensitized tissues with anti-CD23 antibody significantly reduced the epithelial uptake of HRP and the subsequent hypersensitivity reaction. Conclusions: Our results provide convincing evidence that IgE antibodies bound to CD23 receptors on epithelial cells are responsible for the specific nature of this enhanced transepithelial antigen transport system in sensitized rats. Interfering with this pathway inhibits the hypersensitivity reaction, thus suggesting that it may be a novel target for therapeutic intervention in food allergic conditions. 1199 GALANIN·l RECEPTOR (GAUR) UP·REGULATION IS ESSEN· TIAL FOR MEDIATING THE EXCESS FLUID SECRETION IN INFECTIOUS DIARRHEA. Kristina A. Matkowskyj, Alexey V. Danilkovich, Richard V. Benya, Univ of Illinois at Chicago, Chicago, IL. Although GallR are not normally expressed by epithelial cells lining the mouse colon, we recently showed that infection with enterohemorrhagic E. coli (EHEC) markedly increased the expression of this protein by an NF-KB-mediated mechanism (J Clin Invest 1999; 104: 253). When expressed by colonocytes, GallR causes chloride secretion (Am J Phys 1999; 276: G64). Thus the purpose of this study was to determine: 1), if other pathogens increase GallR expression; and 2) elucidate the absolute contribution of this receptor to mediating the excessive fluid secretion observed post-infection. To create mice genetically incapable of expressing GallR, we first cloned the mouse GALIR gene from a murine Pjlibrary using human GallR eDNA as a probe. Homologous recombination (HR) with the native gene was achieved using linearized pNTK vector containing 5 Kb of the GALlR gene, but which had been disrupted by insertion of a Neomycin resistance gene in exon 1. ES cells were transfected by electroporation and cultured in the presence of G-4l8. Resistant clones undergoing successful HR were injected into pseudopregnant mice. Chimeric offspring were then mated with C57BLl6J mice to produce animals that were heterozygous or homozygous negative for the GallR. Colonic fluid secretion was assessed by closed loop experiments in wild type mice (ie, GallR+'+), and in those that had one (GallR+/') or both (GallR-/') alleles deleted. Mice of all three genotypes were infected with log growth EHEC, Salmonella, or Shigella by gavage, and colonic fluid secretion assessed after exposure to intraluminal galanin (I J.tM) for 4 hrs (Table). Conclusion: Elimination of GALlR does not alter basal colonic fluid secretion, but markedly attenuates the increase in secretion due to pathogen infection. These data support a critical central role for the Gall R in mediating the excess fluid secretion observed in infectious diarrhea. Colonic Fluid Secretion (mg/cm) inthe Presence ofLuminal Galanin Pathogen None
EHEC Salmonella Shigella
Peak Infection
GallR'/'
GallW"
GaI1R<'
N/A 3days 6 days 4 days
346±25 71.2±8,7 178,4±11.2 830±5.2
39.3±1,8 43,8±2,4 42,O±1.1 55,4+0.8
36.9±O,2 32,9±7.3 38.8±6.8 40.9±O.5