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Binding and antigen presentation of ceramide-containing glycolipids by soluble mouse and human CD1d molecules.

Naidenko OV, Maher JK, Ernst WA, Sakai T, Modlin RL, Kronenberg M - J. Exp. Med. (1999)

Bottom Line: Using surface plasmon resonance, we show that at neutral pH, mouse CD1 and human CD1d bind to immobilized alpha-GalCer, unlike human CD1b, which requires acidic pH for lipid antigen binding.The CD1d molecules can also bind both to the nonantigenic beta-GalCer and to phosphatidylethanolamine, indicating that diverse lipids can bind to CD1d.These studies provide the first quantitative analysis of monomeric lipid antigen-CD1 interactions, and they demonstrate that the orientation of the galactose, or even the nature of the polar head group, are likely to be more important for T cell receptor contact than CD1d binding.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Immunology and the Molecular Biology Institute, University of California at Los Angeles, Los Angeles, California 90095, USA.

ABSTRACT
We have purified soluble mouse and human CD1d molecules to assess the structural requirements for lipid antigen presentation by CD1. Plate-bound CD1d molecules from either species can present the glycolipid alpha-galactosyl ceramide (alpha-GalCer) to mouse natural killer T cells, formally demonstrating both the in vitro formation of antigenic complexes, and the presentation of alpha-GalCer by these two CD1d molecules. Using surface plasmon resonance, we show that at neutral pH, mouse CD1 and human CD1d bind to immobilized alpha-GalCer, unlike human CD1b, which requires acidic pH for lipid antigen binding. The CD1d molecules can also bind both to the nonantigenic beta-GalCer and to phosphatidylethanolamine, indicating that diverse lipids can bind to CD1d. These studies provide the first quantitative analysis of monomeric lipid antigen-CD1 interactions, and they demonstrate that the orientation of the galactose, or even the nature of the polar head group, are likely to be more important for T cell receptor contact than CD1d binding.

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Biotin-modified α-GalCer is antigenic for NK T cells. (A) Antigen structures. α-GalCer is shown on the top, with the α-linkage of the sugar indicated by an arrow and the acyl chain of the ceramide moiety enclosed within a box. The position 1–4 carbons of the sphingosine are indicated. The biotin-modified acyl chain of biotin–α-GalCer and biotin–β-GalCer is shown below. (B) Biotin–α-GalCer can be recognized by NK T cells. mCD1-transfected or control, mock-transfected A20 cells were pulsed with 100 ng/ml of lipid antigen, or with 0.1% DMSO vehicle control, washed, and added to cultures of the 3C3 mouse NK T cell hybridoma for 16 h. IL-2 release was measured by ELISA. Data shown are from one experiment that was repeated five times. (C) Dose-dependent stimulation of the 3C3 hybridoma in response to increasing amounts of either α-GalCer or biotin–α-GalCer presented by 1 μg/well mCD1 immobilized on a microtiter plate. This experiment is representative of three independent experiments.
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Figure 3: Biotin-modified α-GalCer is antigenic for NK T cells. (A) Antigen structures. α-GalCer is shown on the top, with the α-linkage of the sugar indicated by an arrow and the acyl chain of the ceramide moiety enclosed within a box. The position 1–4 carbons of the sphingosine are indicated. The biotin-modified acyl chain of biotin–α-GalCer and biotin–β-GalCer is shown below. (B) Biotin–α-GalCer can be recognized by NK T cells. mCD1-transfected or control, mock-transfected A20 cells were pulsed with 100 ng/ml of lipid antigen, or with 0.1% DMSO vehicle control, washed, and added to cultures of the 3C3 mouse NK T cell hybridoma for 16 h. IL-2 release was measured by ELISA. Data shown are from one experiment that was repeated five times. (C) Dose-dependent stimulation of the 3C3 hybridoma in response to increasing amounts of either α-GalCer or biotin–α-GalCer presented by 1 μg/well mCD1 immobilized on a microtiter plate. This experiment is representative of three independent experiments.

Mentions: The syntheses of biotinylated α- and β-GalCer (biotin–α-GalCer and biotin–β-GalCer) (see Fig. 3 A) have been described elsewhere 29. After synthesis, the biotinylated compounds were purified by silica gel column, which completely removed the biotinylating reagent 29. The purity of biotinylated α- and β-GalCer compounds was verified by mass spectrometry. No unbiotinylated galactosyl ceramide was detectable in the final preparation of the compound 29. N-(biotinoyl)dipalmitoyl-l-α-phosphatidylethanolamine (EZ-Link™ biotin-DPPE; see Fig. 5 A) was purchased from Pierce Chemical Co. Gangliosides GM1 and GD1a were purchased from Calbiochem. Hexasaccharide disialyllacto-N-tetraose (α-Neu5Ac-[2→3]-β-Gal-[1→3]-(α-Neu5Ac-[2→6])-β-GlcNac-[1→3]-β-Gal-[1→4]-Glc) was purchased from Sigma Chemical Co. Polyethylene glycol (PEG)2000 ceramide was purchased from Northern Lipids, Inc. P99 peptide (YEHDFHHIREWGNHWKNCLAVM) and NH2 terminus biotinylated P99 peptide were synthesized at Research Genetics, Inc.


Binding and antigen presentation of ceramide-containing glycolipids by soluble mouse and human CD1d molecules.

Naidenko OV, Maher JK, Ernst WA, Sakai T, Modlin RL, Kronenberg M - J. Exp. Med. (1999)

Biotin-modified α-GalCer is antigenic for NK T cells. (A) Antigen structures. α-GalCer is shown on the top, with the α-linkage of the sugar indicated by an arrow and the acyl chain of the ceramide moiety enclosed within a box. The position 1–4 carbons of the sphingosine are indicated. The biotin-modified acyl chain of biotin–α-GalCer and biotin–β-GalCer is shown below. (B) Biotin–α-GalCer can be recognized by NK T cells. mCD1-transfected or control, mock-transfected A20 cells were pulsed with 100 ng/ml of lipid antigen, or with 0.1% DMSO vehicle control, washed, and added to cultures of the 3C3 mouse NK T cell hybridoma for 16 h. IL-2 release was measured by ELISA. Data shown are from one experiment that was repeated five times. (C) Dose-dependent stimulation of the 3C3 hybridoma in response to increasing amounts of either α-GalCer or biotin–α-GalCer presented by 1 μg/well mCD1 immobilized on a microtiter plate. This experiment is representative of three independent experiments.
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Related In: Results  -  Collection

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Figure 3: Biotin-modified α-GalCer is antigenic for NK T cells. (A) Antigen structures. α-GalCer is shown on the top, with the α-linkage of the sugar indicated by an arrow and the acyl chain of the ceramide moiety enclosed within a box. The position 1–4 carbons of the sphingosine are indicated. The biotin-modified acyl chain of biotin–α-GalCer and biotin–β-GalCer is shown below. (B) Biotin–α-GalCer can be recognized by NK T cells. mCD1-transfected or control, mock-transfected A20 cells were pulsed with 100 ng/ml of lipid antigen, or with 0.1% DMSO vehicle control, washed, and added to cultures of the 3C3 mouse NK T cell hybridoma for 16 h. IL-2 release was measured by ELISA. Data shown are from one experiment that was repeated five times. (C) Dose-dependent stimulation of the 3C3 hybridoma in response to increasing amounts of either α-GalCer or biotin–α-GalCer presented by 1 μg/well mCD1 immobilized on a microtiter plate. This experiment is representative of three independent experiments.
Mentions: The syntheses of biotinylated α- and β-GalCer (biotin–α-GalCer and biotin–β-GalCer) (see Fig. 3 A) have been described elsewhere 29. After synthesis, the biotinylated compounds were purified by silica gel column, which completely removed the biotinylating reagent 29. The purity of biotinylated α- and β-GalCer compounds was verified by mass spectrometry. No unbiotinylated galactosyl ceramide was detectable in the final preparation of the compound 29. N-(biotinoyl)dipalmitoyl-l-α-phosphatidylethanolamine (EZ-Link™ biotin-DPPE; see Fig. 5 A) was purchased from Pierce Chemical Co. Gangliosides GM1 and GD1a were purchased from Calbiochem. Hexasaccharide disialyllacto-N-tetraose (α-Neu5Ac-[2→3]-β-Gal-[1→3]-(α-Neu5Ac-[2→6])-β-GlcNac-[1→3]-β-Gal-[1→4]-Glc) was purchased from Sigma Chemical Co. Polyethylene glycol (PEG)2000 ceramide was purchased from Northern Lipids, Inc. P99 peptide (YEHDFHHIREWGNHWKNCLAVM) and NH2 terminus biotinylated P99 peptide were synthesized at Research Genetics, Inc.

Bottom Line: Using surface plasmon resonance, we show that at neutral pH, mouse CD1 and human CD1d bind to immobilized alpha-GalCer, unlike human CD1b, which requires acidic pH for lipid antigen binding.The CD1d molecules can also bind both to the nonantigenic beta-GalCer and to phosphatidylethanolamine, indicating that diverse lipids can bind to CD1d.These studies provide the first quantitative analysis of monomeric lipid antigen-CD1 interactions, and they demonstrate that the orientation of the galactose, or even the nature of the polar head group, are likely to be more important for T cell receptor contact than CD1d binding.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Immunology and the Molecular Biology Institute, University of California at Los Angeles, Los Angeles, California 90095, USA.

ABSTRACT
We have purified soluble mouse and human CD1d molecules to assess the structural requirements for lipid antigen presentation by CD1. Plate-bound CD1d molecules from either species can present the glycolipid alpha-galactosyl ceramide (alpha-GalCer) to mouse natural killer T cells, formally demonstrating both the in vitro formation of antigenic complexes, and the presentation of alpha-GalCer by these two CD1d molecules. Using surface plasmon resonance, we show that at neutral pH, mouse CD1 and human CD1d bind to immobilized alpha-GalCer, unlike human CD1b, which requires acidic pH for lipid antigen binding. The CD1d molecules can also bind both to the nonantigenic beta-GalCer and to phosphatidylethanolamine, indicating that diverse lipids can bind to CD1d. These studies provide the first quantitative analysis of monomeric lipid antigen-CD1 interactions, and they demonstrate that the orientation of the galactose, or even the nature of the polar head group, are likely to be more important for T cell receptor contact than CD1d binding.

Show MeSH