Enhanced Membrane-tethered Mucin 3 (MUC3) Expression by a Tetrameric Branched Peptide with a Conserved TFLK Motif Inhibits Bacteria Adherence*

Background: A tetrameric branched peptide (BP) containing a TFLK motif was designed to induce MUC3 expression and inhibit bacteria adherence. Results: TFLK-containing 10-mer BP induced MUC3 expression and dramatically inhibited bacteria adherence to the HT-29-Gal cells. Conclusion: This peptide acts through transcriptional mechanisms. Significance: This peptide has potential therapeutic value against gastrointestinal infection. We investigated whether a synthetic tetrameric branched peptide based on the conserved TFLK motif from mammary-associated serum amyloid A3 (M-SAA3) is more efficient than the monomeric peptide at up-regulating MUC3 expression and examined the possible mechanism(s) and biological significance of this process. We used standard solid-phase methods to synthesize a tetrameric branched peptide (sequence GWLTFLKAAG) containing a trilysine core, termed the TFLK-containing 10-mer BP. The aberrant expression of transcription factors was analyzed using a transcription factor protein/DNA array. MUC3 and relevant transcription factors were detected using real-time PCR and/or Western blots. The luciferase assay, EMSA, and ChIP assays were used to analyze the activity of the human MUC3 promoter. The bacterial adherence assay was used to evaluate the in vitro inhibition of enteropathogenic Escherichia coli or enterohemorrhage E. coli serotype O157:H7 (EHEC O157:H7) adherence to HT-29-Gal cells after treatment with the TFLK-containing 10-mer BP. In HT-29-Gal cells, the TFLK-containing 10-mer BP induced higher levels of MUC3 expression than the M-SAA3-derived N-terminal 10-mer monomeric peptide, and MUC3 expression was activated through transcriptional mechanisms, including the induction of multiple transcription factors and further binding with their cis-elements between nucleotides −242 and −62 within MUC3 promoter. Interestingly, the TFLK-containing 10-mer BP dramatically inhibited enteropathogenic E. coli and EHEC O157:H7 adherence to the HT-29-Gal cells compared with the controls. This finding suggests a potential therapeutic use for this peptide to prevent gastrointestinal infection.

[1]  Oliver Lieleg,et al.  Mucin biopolymers as broad-spectrum antiviral agents. , 2012, Biomacromolecules.

[2]  J. Boyer,et al.  Elevated hepatic multidrug resistance‐associated protein 3/ATP‐binding cassette subfamily C 3 expression in human obstructive cholestasis is mediated through tumor necrosis factor alpha and c‐Jun NH2‐terminal kinase/stress‐activated protein kinase–signaling pathway , 2012, Hepatology.

[3]  N. K. Ho,et al.  Enterohemorrhagic Escherichia coli O157:H7 Shiga Toxins Inhibit Gamma Interferon-Mediated Cellular Activation , 2012, Infection and Immunity.

[4]  J. Raynes,et al.  Recombinant expression of goat milk serum amyloid A: preliminary studies of the protein and derived peptides on macrophage phagocytosis. , 2012, Protein and peptide letters.

[5]  H. Sakai,et al.  Antigen challenge influences various transcription factors of rat bronchus: protein/DNA array study. , 2011, International immunopharmacology.

[6]  C. Maletzki,et al.  Prophylactic application of bovine colostrum ameliorates murine colitis via induction of immunoregulatory cells. , 2011, The Journal of nutrition.

[7]  M. Ruvo,et al.  Branched peptides for the modulation of protein-protein interactions: more arms are better than one? , 2011, Current medicinal chemistry.

[8]  L. N. Fink,et al.  Bovine colostrum is superior to enriched formulas in stimulating intestinal function and necrotising enterocolitis resistance in preterm pigs , 2010, British Journal of Nutrition.

[9]  Y. S. Kim,et al.  Intestinal Goblet Cells and Mucins in Health and Disease: Recent Insights and Progress , 2010, Current gastroenterology reports.

[10]  S. Batra,et al.  Activity of recombinant cysteine-rich domain proteins derived from the membrane-bound MUC17/Muc3 family mucins. , 2010, Biochimica et biophysica acta.

[11]  S. Batra,et al.  Human intestinal MUC17 mucin augments intestinal cell restitution and enhances healing of experimental colitis. , 2010, The international journal of biochemistry & cell biology.

[12]  A. Pini,et al.  A novel tetrabranched antimicrobial peptide that neutralizes bacterial lipopolysaccharide and prevents septic shock in vivo , 2010, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[13]  Y. Fujibayashi,et al.  Effect of multimerization of a linear Arg-Gly-Asp peptide on integrin binding affinity and specificity. , 2010, Biological & pharmaceutical bulletin.

[14]  P. Edwards.  Current parallel chemistry principles and practice: application to the discovery of biologically active molecules. , 2009, Current opinion in drug discovery & development.

[15]  R. P. Ross,et al.  Evaluation of colostrum-derived human mammary-associated serum amyloid A3 (M-SAA3) protein and peptide derivatives for the prevention of enteric infection: in vitro and in murine models of intestinal disease. , 2009, FEMS immunology and medical microbiology.

[16]  M. McGuckin,et al.  Mucin Dynamics in Intestinal Bacterial Infection , 2008, PloS one.

[17]  J. Ambati,et al.  Modulation of Angiogenesis by a Tetrameric Tripeptide That Antagonizes Vascular Endothelial Growth Factor Receptor 1* , 2008, Journal of Biological Chemistry.

[18]  B. Thapa,et al.  Role of colostrum in gastrointestinal infections , 2008, Indian journal of pediatrics.

[19]  A. Pini,et al.  Molecular Basis of Branched Peptides Resistance to Enzyme Proteolysis , 2007, Chemical biology & drug design.

[20]  C. De Simone,et al.  The VSL#3 probiotic formula induces mucin gene expression and secretion in colonic epithelial cells. , 2007, American journal of physiology. Gastrointestinal and liver physiology.

[21]  Yoon-Sik Lee,et al.  Combinatorial Solid Phase Peptide Synthesis and Bioassays , 2005 .

[22]  S. Gout,et al.  Early enterocytic differentiation of HT-29 cells: biochemical changes and strength increases of adherens junctions. , 2004, Experimental cell research.

[23]  S. Crawley,et al.  Initiation of Transcription of the MUC3A Human Intestinal Mucin from a TATA-less Promoter and Comparison with the MUC3B Amino Terminus* , 2003, Journal of Biological Chemistry.

[24]  S. Ho,et al.  Characterization of the mouse Muc3 membrane bound intestinal mucin 5' coding and promoter regions: regulation by inflammatory cytokines. , 2003, Biochimica et biophysica acta.

[25]  M. Hollingsworth,et al.  Extracellular MUC3 mucin secretion follows adherence of Lactobacillus strains to intestinal epithelial cells in vitro , 2003, Gut.

[26]  D. Mack,et al.  Human serum amyloid A3 peptide enhances intestinal MUC3 expression and inhibits EPEC adherence. , 2003, Biochemical and biophysical research communications.

[27]  D. Mack,et al.  The Conserved TFLK Motif of Mammary-Associated Serum Amyloid A3 Is Responsible for Up-regulation of Intestinal MUC3 Mucin Expression In Vitro , 2003, Pediatric Research.

[28]  R. Lai,et al.  Identification of upstream stimulatory factor as an activator of the human dipeptidyl peptidase IV gene in Caco-2 cells. , 2000, Gene.

[29]  R. Playford,et al.  Colostrum and milk-derived peptide growth factors for the treatment of gastrointestinal disorders. , 2000, The American journal of clinical nutrition.

[30]  M. Hollingsworth,et al.  Probiotics inhibit enteropathogenic E. coliadherence in vitro by inducing intestinal mucin gene expression. , 1999, American journal of physiology. Gastrointestinal and liver physiology.

[31]  Y. Kim,et al.  Identification and characterization of the MUC2 (human intestinal mucin) gene 5'-flanking region: promoter activity in cultured cells. , 1997, The Biochemical journal.

[32]  P. Traber,et al.  Intestine-specific gene transcription. , 1996, Annual review of physiology.

[33]  J. Gum,et al.  Human dipeptidyl peptidase IV gene promoter: tissue-specific regulation from a TATA-less GC-rich sequence characteristic of a housekeeping gene promoter. , 1995, The Biochemical journal.

[34]  T. McDaniel,et al.  Enteropathogenic Escherichia coli contains a putative type III secretion system necessary for the export of proteins involved in attaching and effacing lesion formation. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[35]  L. Augenlicht,et al.  Patterns of expression of lineage-specific markers during the in vitro-induced differentiation of HT29 colon carcinoma cells. , 1995, Cell growth & differentiation : the molecular biology journal of the American Association for Cancer Research.

[36]  M. Hollingsworth,et al.  Alteration in expression of MUC2 and MUC3 mRNA levels in HT29 colonic carcinoma cells. , 1994, Biochemical and biophysical research communications.

[37]  J. Hughes,et al.  Diarrhea in Developed and Developing Countries: Magnitude, Special Settings, and Etiologies , 1990, Reviews of infectious diseases.