Volume 119, Issue 2, Pages 395-405 (August 2000) Identification and characterization of a novel gastric peptide hormone: The motilin- related peptide  Catherine Tomasetto, Sherif M. Karam, Stephane Ribieras, Régis Masson, Olivier Lefèbvre, Adrien Staub, Glory Alexander, Marie-Pierre Chenard, Marie-Christine Rio  Gastroenterology  Volume 119, Issue 2, Pages 395-405 (August 2000) DOI: 10.1053/gast.2000.9371 Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 1 Expression analysis of m40, m46, m49, and m53 genes. The Northern blot contained approximately 10 μg of total RNAs isolated from mouse cerebral cortex (lane 1), stomach (lane 2), colon (lane 3), and small intestine (lane 4). The blot was successively hybridized using m40, m46, m49, and m53 32P-labeled probes. Note that m53 is evenly expressed in all tissues tested, m49 throughout the GI tract, m40 in the stomach and small intestine, and m46 only in the stomach. The approximate size of each transcript is indicated on the left (kilobase values). Gastroenterology 2000 119, 395-405DOI: (10.1053/gast.2000.9371) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 2 cDNA and protein sequences of m46 and homology with the promotilin. (A) Nucleotides and amino acids are numbered on the left and right, respectively. Amino acid residues involved in the signal peptide sequence are printed in italics. The dibasic cleavage sites are printed in bold. The EMBL/Genbank accession number for this sequence is AJ243503. The sequences of the synthetic peptides used to generate anti-m46Nt and anti-m46Ct antibodies are boxed. (B) Alignment of the m46 and pig promotilin (accession no.P01307) protein sequences. (C) Alignment of rat, mouse, and human m46 protein sequences. Matching residues are indicated as homologous (*), conserved (:), and semiconserved (.). Gastroenterology 2000 119, 395-405DOI: (10.1053/gast.2000.9371) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 3 Expression analysis of m46 transcript by Northern blot and in situ hybridization of mouse tissues. (A) The Northern blot contained approximatively 10 μg of total RNAs isolated from the olfactory bulb (lane 1), hippocampus (lane 2), cerebellum (lane 3), cerebral cortex (lane 4), striatum (lane 5), mammary gland (lane 6), uterus (lane 7), ovary (lane 8), testis (lane 9), kidney (lane 10), thymus (lane 11), liver (lane 12), spleen (lane 13), colon (lane 14), ileum (lane 15), jejunum (lane 16), duodenum (lane 17), and stomach (lane 18). The blot was successively hybridized using m46 and the internal loading control 36B4 32P-labeled probes. RNA sizes are indicated on the left (kilobase values). (B) Stomach corpus section hybridized with an m46-specific antisense probe. Scattered cells present in the lower portion of the gastric units are labeled (dark staining) (original magnification 20×). Gastroenterology 2000 119, 395-405DOI: (10.1053/gast.2000.9371) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 4 Immunohistochemical localization of m46 protein in adult mouse GI tissues. (A) Section of gastric mucosa probed with rabbit polyclonal anti-m46Ct and visualized with FITC-labeled goat anti-rabbit IgG (green). m46 is present in several round scattered cells. (B) Section of gastric mucosa incubated with FITC-labeled GSII lectin (green) and anti-m46Ct vizualized with TRITC-labeled anti-rabbit IgG (red). Note that mucus-secreting lectin-labeled neck cells do not express m46. (C) Section of gastric mucosa probed with anti-m46, visualized with FITC-labeled goat anti-rabbit IgG (green), and probed with the monoclonal anti–β subunit of H+,K+-ATPase, visualized as dark brown with peroxidase-labeled anti-mouse IgG and diaminobenzidine tetrahydrochloride (DAB). Note that H+,K+-ATPase is not colocalized with m46. (D) Section of gastric mucosa incubated with TRITC-labeled UEA-1 lectin (red) and anti-m46Ct and visualized with FITC-labeled goat anti-rabbit IgG (green). Lectin-labeled pit cells are seen in the luminal compartment (top) and m46-producing cells in the adluminal compartment (bottom). (E) Section of duodenal mucosa incubated with TRITC-labeled DAB lectin (red) and anti-m46Ct plus FITC-labeled goat anti-rabbit IgG (green). Note the presence of 2 m46-producing cells in the villi. Lectin-labeled enterocytes, goblets cells, and Paneth cells do not produce m46. (F) Section of Brunner's gland incubated as in E with DAB and anti-m46Ct (all pictures at same magnification; bar in A = 40 μm). Gastroenterology 2000 119, 395-405DOI: (10.1053/gast.2000.9371) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 5 Colocalization of m46 and chromogranin A, serotonin, and somatostatin in gastric cells. (A) Section probed with an anti–chromogranin A rabbit polyclonal antibody and visualized with TRITC-labeled anti-rabbit IgG (red). (B, E, and H) Sections were probed with an anti-m46Ct biotinylated rabbit polyclonal antibody and visualized with FITC-labeled avidin (green). (D) Section probed with antiserotonin rabbit polyclonal antibody and visualized with TRITC-labeled anti-rabbit IgG (red). (G) Section probed with antisomatostatin rabbit polyclonal antibody and visualized with TRITC-labeled anti-rabbit IgG (red). (C) Overlay A and B. (F) Overlay D and E. (I) Overlay G and H. Some rare serotonin-expressing cells do not express m46 (F, arrowhead), and some m46-expressing cells do not express chromogranin A or somatostatin (C and F, arrows) (all pictures at same magnification; bar in I = 40 μm). Gastroenterology 2000 119, 395-405DOI: (10.1053/gast.2000.9371) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 6 Immunogold localization of m46 in Lowicryl-embedded ultrathin sections of the oxyntic mucosa of adult mice. (A) Gold particles are specifically located on small dark granules. (B) Portion of an enteroendocrine cell is seen at the bottom, and part of a parietal cell at the top. The endocrine cell contains 2 types of granules; some are round and dark (arrows), and others appear larger and pale (arrowheads). The dark ones contain m46, as indicated by the presence of gold particles. Parietal cell canaliculus (c) and mitochondria (m) are not labeled with gold particles. (C) Portions of an enteroendocrine cell (bottom right) and a zymogenic cell (upper left) showing m46 localization only in the enteroendocrine cell granules (arrows). The type of this enteroendocrine cell is apparently different from that shown in B. The zymogenic granules (z) are not labeled (bars = 500 nm). Gastroenterology 2000 119, 395-405DOI: (10.1053/gast.2000.9371) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 7 Western blot analysis of m46 expression and processing. (A) COS-1 cell supernatants (10 μL) were analyzed by Western blot after transfection of pSG5-m46 antisense (lane 1) and pSG5-m46 sense (lane 2) expression vector. The blot was revealed using the anti-m46Ct antibody. Protein molecular size markers are indicated on the left (kilodalton values). A single protein of approximately 11 kilodaltons could be detected when the sense construct was transfected. (B) Total protein (20 μg) from stomach cytosol (lane 1) and gastric juice (lane 2) was analyzed by Western blotting. The blots were probed with either an anti-m46Ct or an anti-m46Nt antibody. The secreted protein as it is produced in COS-1 cell supernatant (lane 3) is detected with both antibodies (arrow a) in stomach cytosol. Lower-molecular-weight proteins at approximately 8 kilodaltons (arrow b) and 3 kilodaltons (arrow c) are detected with the anti-m46Ct and anti-m46Nt antibodies, respectively. Gastric juice (lane 2) is negative with both anti-m46 antibodies. (C) The same protein extracts were probed with the control antibody anti-mSP. The mSP peptide is detected in both stomach cytosol and gastric juice. Protein molecular size markers are indicated on the left (kilodalton values). (D) Schematic representation of m46 and porcine prepromotilin protein structures, including signal sequence (gray boxes), MTLRP and motilin (hatched boxes), and MTLRPAP and MAP (open boxes) peptides. The sequence homologies between the corresponding parts of both proteins are indicated; identities and similarities are printed in plain and bold characters, respectively. Gastroenterology 2000 119, 395-405DOI: (10.1053/gast.2000.9371) Copyright © 2000 American Gastroenterological Association Terms and Conditions