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A Peptide Mimetic of 5-Acetylneuraminic Acid-Galactose Binds with High Avidity to Siglecs and NKG2D.

Eggink LL, Spyroulias GA, Jones NG, Hanson CV, Hoober JK - PLoS ONE (2015)

Bottom Line: Incubation of neuraminidase-treated human peripheral blood mononuclear cells with svH1C resulted in binding of the peptide to a subset of the CD14+ monocyte population.Tyrosine phosphorylation of siglecs decreased dramatically when peripheral blood mononuclear cells were treated with 100 nM svH1C.These results support the conclusion that svH1C mimics Neu5Ac-containing sequences and interacts with cell-surface receptors with avidities sufficient to induce biological responses at low concentrations.

View Article: PubMed Central - PubMed

Affiliation: Susavion Biosciences, Inc., Tempe, AZ, 85281-3257, United States of America.

ABSTRACT
We previously identified several peptide sequences that mimicked the terminal sugars of complex glycans. Using plant lectins as analogs of lectin-type cell-surface receptors, a tetravalent form of a peptide with the sequence NPSHPLSG, designated svH1C, bound with high avidity to lectins specific for glycans with terminal 5-acetylneuraminic acid (Neu5Ac)-galactose (Gal)/N-acetylgalactosamine (GalNAc) sequences. In this report, we show by circular dichroism and NMR spectra that svH1C lacks an ordered structure and thus interacts with binding sites from a flexible conformation. The peptide binds with high avidity to several recombinant human siglec receptors that bind preferentially to Neu5Ac(α2,3)Gal, Neu5Ac(α2,6)GalNAc or Neu5Ac(α2,8)Neu5Ac ligands. In addition, the peptide bound the receptor NKG2D, which contains a lectin-like domain that binds Neu5Ac(α2,3)Gal. The peptide bound to these receptors with a KD in the range of 0.6 to 1 μM. Binding to these receptors was inhibited by the glycoprotein fetuin, which contains multiple glycans that terminate in Neu5Ac(α2,3)Gal or Neu5Ac(α2,6)Gal, and by sialyllactose. Binding of svH1C was not detected with CLEC9a, CLEC10a or DC-SIGN, which are lectin-type receptors specific for other sugars. Incubation of neuraminidase-treated human peripheral blood mononuclear cells with svH1C resulted in binding of the peptide to a subset of the CD14+ monocyte population. Tyrosine phosphorylation of siglecs decreased dramatically when peripheral blood mononuclear cells were treated with 100 nM svH1C. Subcutaneous, alternate-day injections of svH1C into mice induced several-fold increases in populations of several types of immune cells in the peritoneal cavity. These results support the conclusion that svH1C mimics Neu5Ac-containing sequences and interacts with cell-surface receptors with avidities sufficient to induce biological responses at low concentrations. The attenuation of inhibitory receptors suggests that svH1C has characteristics of a checkpoint inhibitor.

No MeSH data available.


Circular dichroism spectra of svH1C as a function of concentration.(A) 100 μM peptide in 50 mM borate, pH 9.0; (B) peptide solution in (A) diluted 1:3 with water; (C) peptide solution in (A) diluted 1:9 with water; (D) 100 μM peptide in 50 mM borate adjusted to pH 2.
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pone.0130532.g001: Circular dichroism spectra of svH1C as a function of concentration.(A) 100 μM peptide in 50 mM borate, pH 9.0; (B) peptide solution in (A) diluted 1:3 with water; (C) peptide solution in (A) diluted 1:9 with water; (D) 100 μM peptide in 50 mM borate adjusted to pH 2.

Mentions: As an initial approach to structural analysis, CD spectra were performed with svH1C in borate buffer, pH 9. The spectrum for a 100 μM solution of the peptide had a strong negative signal at 234 nm (Fig 1, curve A). The minimum broadened and shifted to 227 nm when diluted to 25 μM (Fig 1, curve B), and disappeared when the peptide was diluted to 10 μM, with a new minimum at 203 (Fig 1, curve C). The unusual spectra at higher concentrations may be a function of the tetravalent structure of the peptide, because similar negative peaks were observed between 225 nm and 235 nm with 100 μM solutions of several other tetravalent peptides with similarly short but different sequences. When the sample that provided spectrum A in Fig 1 was acidified to pH 2, the minimum at 234 nm was replaced with a minimum at 211 nm and a shoulder at about 230 nm (Fig 1, curve D). This spectrum suggests a weak α-helical structure [56], but formation of helical structures is restrained by the two proline residues in each arm. These spectra suggest that the peptide aggregates at the higher concentrations. However, because the receptor binding studies were performed with 2 μM peptide, svH1C was likely in a random, flexible structure under those conditions.


A Peptide Mimetic of 5-Acetylneuraminic Acid-Galactose Binds with High Avidity to Siglecs and NKG2D.

Eggink LL, Spyroulias GA, Jones NG, Hanson CV, Hoober JK - PLoS ONE (2015)

Circular dichroism spectra of svH1C as a function of concentration.(A) 100 μM peptide in 50 mM borate, pH 9.0; (B) peptide solution in (A) diluted 1:3 with water; (C) peptide solution in (A) diluted 1:9 with water; (D) 100 μM peptide in 50 mM borate adjusted to pH 2.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4482410&req=5

pone.0130532.g001: Circular dichroism spectra of svH1C as a function of concentration.(A) 100 μM peptide in 50 mM borate, pH 9.0; (B) peptide solution in (A) diluted 1:3 with water; (C) peptide solution in (A) diluted 1:9 with water; (D) 100 μM peptide in 50 mM borate adjusted to pH 2.
Mentions: As an initial approach to structural analysis, CD spectra were performed with svH1C in borate buffer, pH 9. The spectrum for a 100 μM solution of the peptide had a strong negative signal at 234 nm (Fig 1, curve A). The minimum broadened and shifted to 227 nm when diluted to 25 μM (Fig 1, curve B), and disappeared when the peptide was diluted to 10 μM, with a new minimum at 203 (Fig 1, curve C). The unusual spectra at higher concentrations may be a function of the tetravalent structure of the peptide, because similar negative peaks were observed between 225 nm and 235 nm with 100 μM solutions of several other tetravalent peptides with similarly short but different sequences. When the sample that provided spectrum A in Fig 1 was acidified to pH 2, the minimum at 234 nm was replaced with a minimum at 211 nm and a shoulder at about 230 nm (Fig 1, curve D). This spectrum suggests a weak α-helical structure [56], but formation of helical structures is restrained by the two proline residues in each arm. These spectra suggest that the peptide aggregates at the higher concentrations. However, because the receptor binding studies were performed with 2 μM peptide, svH1C was likely in a random, flexible structure under those conditions.

Bottom Line: Incubation of neuraminidase-treated human peripheral blood mononuclear cells with svH1C resulted in binding of the peptide to a subset of the CD14+ monocyte population.Tyrosine phosphorylation of siglecs decreased dramatically when peripheral blood mononuclear cells were treated with 100 nM svH1C.These results support the conclusion that svH1C mimics Neu5Ac-containing sequences and interacts with cell-surface receptors with avidities sufficient to induce biological responses at low concentrations.

View Article: PubMed Central - PubMed

Affiliation: Susavion Biosciences, Inc., Tempe, AZ, 85281-3257, United States of America.

ABSTRACT
We previously identified several peptide sequences that mimicked the terminal sugars of complex glycans. Using plant lectins as analogs of lectin-type cell-surface receptors, a tetravalent form of a peptide with the sequence NPSHPLSG, designated svH1C, bound with high avidity to lectins specific for glycans with terminal 5-acetylneuraminic acid (Neu5Ac)-galactose (Gal)/N-acetylgalactosamine (GalNAc) sequences. In this report, we show by circular dichroism and NMR spectra that svH1C lacks an ordered structure and thus interacts with binding sites from a flexible conformation. The peptide binds with high avidity to several recombinant human siglec receptors that bind preferentially to Neu5Ac(α2,3)Gal, Neu5Ac(α2,6)GalNAc or Neu5Ac(α2,8)Neu5Ac ligands. In addition, the peptide bound the receptor NKG2D, which contains a lectin-like domain that binds Neu5Ac(α2,3)Gal. The peptide bound to these receptors with a KD in the range of 0.6 to 1 μM. Binding to these receptors was inhibited by the glycoprotein fetuin, which contains multiple glycans that terminate in Neu5Ac(α2,3)Gal or Neu5Ac(α2,6)Gal, and by sialyllactose. Binding of svH1C was not detected with CLEC9a, CLEC10a or DC-SIGN, which are lectin-type receptors specific for other sugars. Incubation of neuraminidase-treated human peripheral blood mononuclear cells with svH1C resulted in binding of the peptide to a subset of the CD14+ monocyte population. Tyrosine phosphorylation of siglecs decreased dramatically when peripheral blood mononuclear cells were treated with 100 nM svH1C. Subcutaneous, alternate-day injections of svH1C into mice induced several-fold increases in populations of several types of immune cells in the peritoneal cavity. These results support the conclusion that svH1C mimics Neu5Ac-containing sequences and interacts with cell-surface receptors with avidities sufficient to induce biological responses at low concentrations. The attenuation of inhibitory receptors suggests that svH1C has characteristics of a checkpoint inhibitor.

No MeSH data available.