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A biologically active peptide mimetic of N-acetylgalactosamine/galactose.

Eggink LL, Hoober JK - BMC Res Notes (2009)

Bottom Line: The secretion of IL-21 was stimulated as strongly with the peptide as with interferon-gamma.The data indicate that the quadravalent peptide has biological activity with a degree of specificity.These effects occurred at concentrations in the nanomolar range, in contrast to free sugars that generally bind to proteins in the micro- to millimolar range.

View Article: PubMed Central - HTML - PubMed

Affiliation: Faculty of Biomedicine and Biotechnology, School of Life Sciences, Arizona State University, Tempe, AZ 85287-4501, USA. leggink@asu.edu

ABSTRACT

Background: Glycosylated proteins and lipids are important regulatory factors whose functions can be altered by addition or removal of sugars to the glycan structure. The glycans are recognized by sugar-binding lectins that serve as receptors on the surface of many cells and facilitate initiation of an intracellular signal that changes the properties of the cells. We identified a peptide that mimics the ligand of an N-acetylgalactosamine (GalNAc)-specific lectin and asked whether the peptide would express specific biological activity.

Findings: A 12-mer phage display library was screened with a GalNAc-specific lectin to identify an amino acid sequence that binds to the lectin. Phage particles that were eluted from the lectin with free GalNAc were considered to have been bound to a GalNAc-binding site. Peptides were synthesized with the selected sequence as a quadravalent structure to facilitate receptor crosslinking. Treatment of human peripheral blood mononuclear cells for 24 h with the peptide stimulated secretion of interleukin-8 (IL-8) but not of IL-1beta, IL-6, IL-10, or tumor necrosis factor-alpha (TNF-alpha). The secretion of IL-21 was stimulated as strongly with the peptide as with interferon-gamma.

Conclusion: The data indicate that the quadravalent peptide has biological activity with a degree of specificity. These effects occurred at concentrations in the nanomolar range, in contrast to free sugars that generally bind to proteins in the micro- to millimolar range.

No MeSH data available.


Related in: MedlinePlus

Binding of lectins to L4. (A) For each lectin, 4, 20 and 100 ng were spotted in triplicate. One set of a total of four sets is shown in the figure. Spots in rows A-C, columns 1–3 contained H. pomatia lectin (HPA), rows A-C, columns 4–6 contained T. vulgare lectin (WGA), rows D-F, columns 1–3 contained B. simplicifolia B4 (BS) and rows D-F, columns 4–6 contained C. ensiformis lectin (ConA). The slide was incubated with 2% gelatin to block non-specific sites. Biotin-tagged peptide was incubated with the slide and then the amount of peptide bound was detected by binding of Cy3-conjugated streptavidin and measuring fluorescence of the spots. In columns marked B, buffer A was spotted as blanks. (B) Biotin-tagged L4 or control peptide was bound in wells of a streptavidin-coated microtiter plate. The wells were blocked with 2% gelatin, washed, and then equal amounts (50 ng) of lectins conjugated with peroxidase were added. After washing, the amount of lectin bound was detected by peroxidase activity. Standard curves were determined with each lectin-peroxidase conjugate to quantitate binding. Data are expressed as the amount of lectin bound minus a blank without peptide. Light blue bars, peptide L4; dark blue bars, control peptide.
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Figure 2: Binding of lectins to L4. (A) For each lectin, 4, 20 and 100 ng were spotted in triplicate. One set of a total of four sets is shown in the figure. Spots in rows A-C, columns 1–3 contained H. pomatia lectin (HPA), rows A-C, columns 4–6 contained T. vulgare lectin (WGA), rows D-F, columns 1–3 contained B. simplicifolia B4 (BS) and rows D-F, columns 4–6 contained C. ensiformis lectin (ConA). The slide was incubated with 2% gelatin to block non-specific sites. Biotin-tagged peptide was incubated with the slide and then the amount of peptide bound was detected by binding of Cy3-conjugated streptavidin and measuring fluorescence of the spots. In columns marked B, buffer A was spotted as blanks. (B) Biotin-tagged L4 or control peptide was bound in wells of a streptavidin-coated microtiter plate. The wells were blocked with 2% gelatin, washed, and then equal amounts (50 ng) of lectins conjugated with peroxidase were added. After washing, the amount of lectin bound was detected by peroxidase activity. Standard curves were determined with each lectin-peroxidase conjugate to quantitate binding. Data are expressed as the amount of lectin bound minus a blank without peptide. Light blue bars, peptide L4; dark blue bars, control peptide.

Mentions: Whether L4 expressed characteristics of a sugar was tested by two methods. Lectins whose primary specificities are GalNAc (Helix pomatia, HPA); GlcNAc or NeuNAc (wheat germ agglutinin, WGA); Gal [Griffonia (Bandeiraea) simplicifolia isolectin B4, BS]; and Man or Glc (concanavalin A, ConA), were fixed onto a glass slide. Non-specific sites were blocked with gelatin, and the slide was then incubated with biotin-tagged L4. Binding was detected by adding streptavidin labeled with the fluorescent dye Cy3. Lectins were spotted in triplicate to minimize conclusions from inaccurate delivery, as indicated by the top row of ConA. All four lectins bound L4, with strongest binding found with the Gal-specific lectin, BS (Fig. 2A). Binding was also observed with HPA and with WGA, which binds to clusters of GalNAc [14,15].


A biologically active peptide mimetic of N-acetylgalactosamine/galactose.

Eggink LL, Hoober JK - BMC Res Notes (2009)

Binding of lectins to L4. (A) For each lectin, 4, 20 and 100 ng were spotted in triplicate. One set of a total of four sets is shown in the figure. Spots in rows A-C, columns 1–3 contained H. pomatia lectin (HPA), rows A-C, columns 4–6 contained T. vulgare lectin (WGA), rows D-F, columns 1–3 contained B. simplicifolia B4 (BS) and rows D-F, columns 4–6 contained C. ensiformis lectin (ConA). The slide was incubated with 2% gelatin to block non-specific sites. Biotin-tagged peptide was incubated with the slide and then the amount of peptide bound was detected by binding of Cy3-conjugated streptavidin and measuring fluorescence of the spots. In columns marked B, buffer A was spotted as blanks. (B) Biotin-tagged L4 or control peptide was bound in wells of a streptavidin-coated microtiter plate. The wells were blocked with 2% gelatin, washed, and then equal amounts (50 ng) of lectins conjugated with peroxidase were added. After washing, the amount of lectin bound was detected by peroxidase activity. Standard curves were determined with each lectin-peroxidase conjugate to quantitate binding. Data are expressed as the amount of lectin bound minus a blank without peptide. Light blue bars, peptide L4; dark blue bars, control peptide.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2657794&req=5

Figure 2: Binding of lectins to L4. (A) For each lectin, 4, 20 and 100 ng were spotted in triplicate. One set of a total of four sets is shown in the figure. Spots in rows A-C, columns 1–3 contained H. pomatia lectin (HPA), rows A-C, columns 4–6 contained T. vulgare lectin (WGA), rows D-F, columns 1–3 contained B. simplicifolia B4 (BS) and rows D-F, columns 4–6 contained C. ensiformis lectin (ConA). The slide was incubated with 2% gelatin to block non-specific sites. Biotin-tagged peptide was incubated with the slide and then the amount of peptide bound was detected by binding of Cy3-conjugated streptavidin and measuring fluorescence of the spots. In columns marked B, buffer A was spotted as blanks. (B) Biotin-tagged L4 or control peptide was bound in wells of a streptavidin-coated microtiter plate. The wells were blocked with 2% gelatin, washed, and then equal amounts (50 ng) of lectins conjugated with peroxidase were added. After washing, the amount of lectin bound was detected by peroxidase activity. Standard curves were determined with each lectin-peroxidase conjugate to quantitate binding. Data are expressed as the amount of lectin bound minus a blank without peptide. Light blue bars, peptide L4; dark blue bars, control peptide.
Mentions: Whether L4 expressed characteristics of a sugar was tested by two methods. Lectins whose primary specificities are GalNAc (Helix pomatia, HPA); GlcNAc or NeuNAc (wheat germ agglutinin, WGA); Gal [Griffonia (Bandeiraea) simplicifolia isolectin B4, BS]; and Man or Glc (concanavalin A, ConA), were fixed onto a glass slide. Non-specific sites were blocked with gelatin, and the slide was then incubated with biotin-tagged L4. Binding was detected by adding streptavidin labeled with the fluorescent dye Cy3. Lectins were spotted in triplicate to minimize conclusions from inaccurate delivery, as indicated by the top row of ConA. All four lectins bound L4, with strongest binding found with the Gal-specific lectin, BS (Fig. 2A). Binding was also observed with HPA and with WGA, which binds to clusters of GalNAc [14,15].

Bottom Line: The secretion of IL-21 was stimulated as strongly with the peptide as with interferon-gamma.The data indicate that the quadravalent peptide has biological activity with a degree of specificity.These effects occurred at concentrations in the nanomolar range, in contrast to free sugars that generally bind to proteins in the micro- to millimolar range.

View Article: PubMed Central - HTML - PubMed

Affiliation: Faculty of Biomedicine and Biotechnology, School of Life Sciences, Arizona State University, Tempe, AZ 85287-4501, USA. leggink@asu.edu

ABSTRACT

Background: Glycosylated proteins and lipids are important regulatory factors whose functions can be altered by addition or removal of sugars to the glycan structure. The glycans are recognized by sugar-binding lectins that serve as receptors on the surface of many cells and facilitate initiation of an intracellular signal that changes the properties of the cells. We identified a peptide that mimics the ligand of an N-acetylgalactosamine (GalNAc)-specific lectin and asked whether the peptide would express specific biological activity.

Findings: A 12-mer phage display library was screened with a GalNAc-specific lectin to identify an amino acid sequence that binds to the lectin. Phage particles that were eluted from the lectin with free GalNAc were considered to have been bound to a GalNAc-binding site. Peptides were synthesized with the selected sequence as a quadravalent structure to facilitate receptor crosslinking. Treatment of human peripheral blood mononuclear cells for 24 h with the peptide stimulated secretion of interleukin-8 (IL-8) but not of IL-1beta, IL-6, IL-10, or tumor necrosis factor-alpha (TNF-alpha). The secretion of IL-21 was stimulated as strongly with the peptide as with interferon-gamma.

Conclusion: The data indicate that the quadravalent peptide has biological activity with a degree of specificity. These effects occurred at concentrations in the nanomolar range, in contrast to free sugars that generally bind to proteins in the micro- to millimolar range.

No MeSH data available.


Related in: MedlinePlus