The A′ and F′ Pockets of Human CD1b Are Both Required for Optimal Presentation of Lipid Antigens to T Cells

Abstract

CD1 proteins are unique in their ability to present lipid Ags to T cells. Human CD1b shares significant amino acid homology with mouse CD1d1, which contains an unusual putative Ag-binding groove formed by two large hydrophobic pockets, A′ and F′. We investigated the function of the amino acid residues that line the A′ and F′ pockets of CD1b by engineering 36 alanine-substitution mutants and analyzing their ability to present mycobacterial glycolipid Ags. Two lipid Ags presented by CD1b were studied, a naturally occurring glucose monomycolate (GMM) isolated from mycobacteria, which contains two long alkyl chains (C54-C62 and C22-C24) and synthetic GMM (sGMM), which includes two short alkyl chains (C18 and C14). We identified eight residues in both the A′ and F′ pockets that were involved in the presentation of both GMM and sGMM to T cells. Interestingly, four additional residues located in the distal portion of the A′ pocket were required for the optimal presentation of GMM, but not sGMM. Conversely, nine residues located between the center of the groove and the F′ pocket were necessary for the optimal presentation of sGMM, but not GMM. These data indicate that both the A′ and F′ pockets of human CD1b are required for the presentation of lipid Ags to T cells.

Direct Measurement of Antigen Binding Properties of CD1 Proteins Using Fluorescent Lipid Probes

“…Single chain CD1 mutant constructs were generated by replacing the wild type CD1b sequence between the BamHI and NotI sites of the scCD1b/pcDNA3 construct with analogous fragments containing the desired alanine substitution mutations. The mutant CD1b fragments were amplified by PCR from the previously reported CD1b mutant constructs (17,32) by using the following primers: 5Ј-ggatccggttctggaggtggaggttcagaacatgcctcccag and 5Ј-cccgcggccgcccactaatggtgatggtgatggtggcccccaattgagccaatggaggt. The resulting scCD1b mutant constructs were fully sequenced to confirm the specific mutations and were used to produce stable CHO transfectants as described above.…”

“…Proteins-A substantial number of site-directed mutations in the ␣1 and ␣2 domains of human CD1b have been reported to affect specific lipid antigen presentation to CD1b-restricted T cells (17,32). Thus far, it has not been determined directly whether the effects of these mutations are due to a reduction in binding of lipid antigens by CD1b or to interference with the stable docking of the TCR on the surface of CD1b.…”

The Crystal Structure of Human CD1b with a Bound Bacterial Glycolipid

“…In both the CD1b-PI and GM2 structures, the relatively short lipid acyl chains occupy the CЈ and AЈ channels, whereas the FЈ and TЈ channels are occupied by detergent molecules (6). An analysis of the CD1b-PI and GM2 structures when combined with the mutagenesis analysis by Niazi et al (31) on residues affecting glycolipid Ag presentation led to the hypothesis that the meromycolate chain of a GMM ligand would occupy the network of AЈ, TЈ, and FЈ channels (6). The current structural analysis confirms that the meromycolate chain of GMM is bound in the CD1b groove traversing from the AЈ (via TЈ) to the FЈ channel, whereas the ␣-chain occupies the CЈ channel.…”

Immunological Properties of Trehalose Dimycolate (Cord Factor) and Other Mycotic Acid‐Containing Glycolipids–‐A Review

“…This process occurs at an acidic pH, in agreement with the requirement of endosomal acidification for antigen presentation by CD I, and results in high affinity binding (19). Later studies investigated the molecular details of lipid presentation to TCR by CD I (45) and of the interaction of mycolic acid with the binding groove of CD 1b (57). T cell specificity for glycosylated or free mycolic acid has been explained by differential TCR recognition of the hydrophilic caps (50; reviewed in [51]).…”

“…Both genes probably resulted from the duplication of an ancestral gene most closely related to human CDld (11) and consequently would be considered distantly related to human CD Ib based on overall sequence similarity. Interestingly, the homology between amino acids forming the antigen-binding groove is particularly high and close structural similarity between the two proteins has been predicted by computer modelling (57). Upregulation of murine CDldl on macrophages after in vivo stimulation with TDM has been reported, but this might represent the consequence of general activation (76).…”