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The opioid peptide dynorphin A induces leukocyte responses via integrin Mac-1 (αMβ2, CD11b/CD18).

Podolnikova NP, Brothwell JA, Ugarova TP - Mol Pain (2015)

Bottom Line: Our recent elucidation of the ligand recognition specificity of Mac-1 suggested that dynorphin A and dynorphin B contain Mac-1 recognition motifs and can potentially interact with this receptor.In this study, we have synthesized the peptide library spanning the sequence of dynorphin AB, containing dynorphin A and B, and showed that the peptides bound recombinant αMI-domain, the ligand binding region of Mac-1.Further focusing on dynorphin A, we showed that its interaction with the αMI-domain was activation independent as both the α7 helix-truncated (active conformation) and helix-extended (nonactive conformation) αMI-domains efficiently bound dynorphin A.

View Article: PubMed Central - PubMed

Affiliation: From the Center for Metabolic and Vascular Biology, School of Life Sciences, Arizona State University, Tempe, AZ, 85287, USA. Nataly.Podolnikova@asu.edu.

ABSTRACT

Background: Opioid peptides, including dynorphin A, besides their analgesic action in the nervous system, exert a broad spectrum of effects on cells of the immune system, including leukocyte migration, degranulation and cytokine production. The mechanisms whereby opioid peptides induce leukocyte responses are poorly understood. The integrin Mac-1 (αMβ2, CD11b/CD18) is a multiligand receptor which mediates numerous reactions of neutrophils and monocyte/macrophages during the immune-inflammatory response. Our recent elucidation of the ligand recognition specificity of Mac-1 suggested that dynorphin A and dynorphin B contain Mac-1 recognition motifs and can potentially interact with this receptor.

Results: In this study, we have synthesized the peptide library spanning the sequence of dynorphin AB, containing dynorphin A and B, and showed that the peptides bound recombinant αMI-domain, the ligand binding region of Mac-1. In addition, immobilized dynorphins A and B supported adhesion of the Mac-1-expressing cells. In binding to dynorphins A and B, Mac-1 cooperated with cell surface proteoglycans since both anti-Mac-1 function-blocking reagents and heparin were required to block adhesion. Further focusing on dynorphin A, we showed that its interaction with the αMI-domain was activation independent as both the α7 helix-truncated (active conformation) and helix-extended (nonactive conformation) αMI-domains efficiently bound dynorphin A. Dynorphin A induced a potent migratory response of Mac-1-expressing, but not Mac-1-deficient leukocytes, and enhanced Mac-1-mediated phagocytosis of latex beads by murine IC-21 macrophages.

Conclusions: Together, the results identify dynorphins A and B as novel ligands for Mac-1 and suggest a role for the Dynorphin A-Mac-1 interactions in the induction of nonopiod receptor-dependent effects in leukocytes.

No MeSH data available.


Related in: MedlinePlus

Screening of the peptide library spanning the sequence of dynorphin AB for αMI-domain binding. a, Amino acid sequence of dynorphin AB (residues 1–32). b, Cellulose membrane with the assembled peptide library consisting of 9-mer peptides with three-residue offset was incubated with 125I-labeled αMI-domain and subjected to autoradiography followed by densitometry. The αMI-domain binding was observed as dark spots. Spot #10 contains only the β-Ala spacer. The αMI-domain binding was expressed as a percentage of binding to peptide #1, which was assigned a value 100 % and is shown as the numbers on the right of each sequence. The peptide energies that serve as a measure of probability each peptide can interact with the αMI-domain were calculated as described [17]
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Fig1: Screening of the peptide library spanning the sequence of dynorphin AB for αMI-domain binding. a, Amino acid sequence of dynorphin AB (residues 1–32). b, Cellulose membrane with the assembled peptide library consisting of 9-mer peptides with three-residue offset was incubated with 125I-labeled αMI-domain and subjected to autoradiography followed by densitometry. The αMI-domain binding was observed as dark spots. Spot #10 contains only the β-Ala spacer. The αMI-domain binding was expressed as a percentage of binding to peptide #1, which was assigned a value 100 % and is shown as the numbers on the right of each sequence. The peptide energies that serve as a measure of probability each peptide can interact with the αMI-domain were calculated as described [17]

Mentions: To test the hypothesis that leukocyte integrin Mac-1 binds opioid peptides, we screened the peptide library spanning the sequence of dynorphin AB (Fig. 1a) for αMI-domain binding. The library, consisting of 9-mer peptides with a 3-residue offset (Fig. 1b) covalently attached to a cellulose membrane, was incubated with the 125I-labeled αMI-domain after which the αMI-domain binding was visualized by autoradiography and analyzed by densitometry. The densitometry results demonstrated that all peptides interacted with the αMI-domain, albeit to somewhat different extents (Fig. 1b). A control spot containing only the β-alanine spacer (spot 10) was negative. The dynorphin-derived peptides were also analyzed by the computer program IRMA, which determines the capacity of each peptide in the peptide libraries to interact with the αMI-domain [17]. The program assigns each peptide the energy value which serves as a measure of probability that the αMI-domain binds this sequence: the lower the energy, the higher the likelihood that the sequence binds the αMI-domain. As previously determined, strong αMI-domain binding peptides derived from various Mac-1 ligands have energy values in the range of −20 to 6 kJ/mole. The analyses showed a good relationship between the energy scores and the αMI-binding activity of dynorphin AB-derived peptides revealed in peptide scans (Fig. 1b). In line with previous findings [17, 18], peptides enriched in positively charged and hydrophobic residues had the highest affinities for the αMI-domain (spots 1, 2 and 6 through 10). In particular, the YGGFLRRIR and FLRRIRPKL peptides (spots 1 and 2), contain the strong overlapping αMI-domain binding clusters FLRRI and IRP, which conform to the αMI-domain binding motif HyHyBHy and HyBHy. The presence of negatively charged residues in peptides present in spots 3, 4 and 5 weakened their αMI-domain binding activity.Fig. 1


The opioid peptide dynorphin A induces leukocyte responses via integrin Mac-1 (αMβ2, CD11b/CD18).

Podolnikova NP, Brothwell JA, Ugarova TP - Mol Pain (2015)

Screening of the peptide library spanning the sequence of dynorphin AB for αMI-domain binding. a, Amino acid sequence of dynorphin AB (residues 1–32). b, Cellulose membrane with the assembled peptide library consisting of 9-mer peptides with three-residue offset was incubated with 125I-labeled αMI-domain and subjected to autoradiography followed by densitometry. The αMI-domain binding was observed as dark spots. Spot #10 contains only the β-Ala spacer. The αMI-domain binding was expressed as a percentage of binding to peptide #1, which was assigned a value 100 % and is shown as the numbers on the right of each sequence. The peptide energies that serve as a measure of probability each peptide can interact with the αMI-domain were calculated as described [17]
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
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getmorefigures.php?uid=PMC4481117&req=5

Fig1: Screening of the peptide library spanning the sequence of dynorphin AB for αMI-domain binding. a, Amino acid sequence of dynorphin AB (residues 1–32). b, Cellulose membrane with the assembled peptide library consisting of 9-mer peptides with three-residue offset was incubated with 125I-labeled αMI-domain and subjected to autoradiography followed by densitometry. The αMI-domain binding was observed as dark spots. Spot #10 contains only the β-Ala spacer. The αMI-domain binding was expressed as a percentage of binding to peptide #1, which was assigned a value 100 % and is shown as the numbers on the right of each sequence. The peptide energies that serve as a measure of probability each peptide can interact with the αMI-domain were calculated as described [17]
Mentions: To test the hypothesis that leukocyte integrin Mac-1 binds opioid peptides, we screened the peptide library spanning the sequence of dynorphin AB (Fig. 1a) for αMI-domain binding. The library, consisting of 9-mer peptides with a 3-residue offset (Fig. 1b) covalently attached to a cellulose membrane, was incubated with the 125I-labeled αMI-domain after which the αMI-domain binding was visualized by autoradiography and analyzed by densitometry. The densitometry results demonstrated that all peptides interacted with the αMI-domain, albeit to somewhat different extents (Fig. 1b). A control spot containing only the β-alanine spacer (spot 10) was negative. The dynorphin-derived peptides were also analyzed by the computer program IRMA, which determines the capacity of each peptide in the peptide libraries to interact with the αMI-domain [17]. The program assigns each peptide the energy value which serves as a measure of probability that the αMI-domain binds this sequence: the lower the energy, the higher the likelihood that the sequence binds the αMI-domain. As previously determined, strong αMI-domain binding peptides derived from various Mac-1 ligands have energy values in the range of −20 to 6 kJ/mole. The analyses showed a good relationship between the energy scores and the αMI-binding activity of dynorphin AB-derived peptides revealed in peptide scans (Fig. 1b). In line with previous findings [17, 18], peptides enriched in positively charged and hydrophobic residues had the highest affinities for the αMI-domain (spots 1, 2 and 6 through 10). In particular, the YGGFLRRIR and FLRRIRPKL peptides (spots 1 and 2), contain the strong overlapping αMI-domain binding clusters FLRRI and IRP, which conform to the αMI-domain binding motif HyHyBHy and HyBHy. The presence of negatively charged residues in peptides present in spots 3, 4 and 5 weakened their αMI-domain binding activity.Fig. 1

Bottom Line: Our recent elucidation of the ligand recognition specificity of Mac-1 suggested that dynorphin A and dynorphin B contain Mac-1 recognition motifs and can potentially interact with this receptor.In this study, we have synthesized the peptide library spanning the sequence of dynorphin AB, containing dynorphin A and B, and showed that the peptides bound recombinant αMI-domain, the ligand binding region of Mac-1.Further focusing on dynorphin A, we showed that its interaction with the αMI-domain was activation independent as both the α7 helix-truncated (active conformation) and helix-extended (nonactive conformation) αMI-domains efficiently bound dynorphin A.

View Article: PubMed Central - PubMed

Affiliation: From the Center for Metabolic and Vascular Biology, School of Life Sciences, Arizona State University, Tempe, AZ, 85287, USA. Nataly.Podolnikova@asu.edu.

ABSTRACT

Background: Opioid peptides, including dynorphin A, besides their analgesic action in the nervous system, exert a broad spectrum of effects on cells of the immune system, including leukocyte migration, degranulation and cytokine production. The mechanisms whereby opioid peptides induce leukocyte responses are poorly understood. The integrin Mac-1 (αMβ2, CD11b/CD18) is a multiligand receptor which mediates numerous reactions of neutrophils and monocyte/macrophages during the immune-inflammatory response. Our recent elucidation of the ligand recognition specificity of Mac-1 suggested that dynorphin A and dynorphin B contain Mac-1 recognition motifs and can potentially interact with this receptor.

Results: In this study, we have synthesized the peptide library spanning the sequence of dynorphin AB, containing dynorphin A and B, and showed that the peptides bound recombinant αMI-domain, the ligand binding region of Mac-1. In addition, immobilized dynorphins A and B supported adhesion of the Mac-1-expressing cells. In binding to dynorphins A and B, Mac-1 cooperated with cell surface proteoglycans since both anti-Mac-1 function-blocking reagents and heparin were required to block adhesion. Further focusing on dynorphin A, we showed that its interaction with the αMI-domain was activation independent as both the α7 helix-truncated (active conformation) and helix-extended (nonactive conformation) αMI-domains efficiently bound dynorphin A. Dynorphin A induced a potent migratory response of Mac-1-expressing, but not Mac-1-deficient leukocytes, and enhanced Mac-1-mediated phagocytosis of latex beads by murine IC-21 macrophages.

Conclusions: Together, the results identify dynorphins A and B as novel ligands for Mac-1 and suggest a role for the Dynorphin A-Mac-1 interactions in the induction of nonopiod receptor-dependent effects in leukocytes.

No MeSH data available.


Related in: MedlinePlus