Acid Test: Lipid Antigens Get into the Groove

Immunity

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Acid Test: Lipid Antigens Get into the Groove Mitchell Kronenberg1,* and Barbara A. Sullivan1 1La Jolla Institute for Allergy & Immunology, La Jolla, CA 92037, USA *Correspondence: [email protected] DOI 10.1016/j.immuni.2008.05.003

How do CD1 molecules load lipid antigens? In this issue of Immunity, Relloso et al. (2008) uncover how lysosomal pH targets amino acids in CD1b, causing it to open and attain a conformation more receptive to lipid antigens. X-ray crystallographic studies have revealed the structures of several complexes of lipid antigens bound to different CD1 isoforms (Zajonc and Kronenberg, 2007). Although these structures show how the aliphatic chains of diverse antigens fit into pockets in the grooves of CD1 proteins, they reveal a fundamental topologic problem. Unlike MHC class I and class II molecules, the grooves of CD1 molecules are not open along their length. The lipid tails are buried, and a portal over what is usually called the F0 pocket, toward the carboxyl terminal of the a helices, permits the emergence of the hydrophilic head groups of CD1-bound antigens (Zajonc and Kronenberg, 2007). Antigens with long hydrophobic chains therefore somehow must traverse a relatively small portal in the roof of the CD1 molecules to enter the antigen-binding groove. Now, a comprehensive report in this issue (Relloso et al., 2008) has combined modeling with immunochemical methods to provide a better understanding of the mechanism of insertion of lipids into the CD1 groove. CD1 proteins are a family of antigen-presenting molecules found in most vertebrates. Their hydrophobic grooves present lipid antigens, which are mostly glycolipids. The CD1 gene family contains evolutionarily divergent members that are not uniformly represented in different

species (Dascher, 2007). For example, in humans there are four CD1 isotypes— CD1a, CD1b, CD1c, and CD1d—but only a CD1d ortholog in mice. CD1-reactive T cells have a unique biology. Natural killer T (NK T) cells with an invariant TCRa chain recognize CD1d. These NKT cells share features with innate immune cells, and, at least in mice, they are critical for the regulation of many immune responses (Bendelac et al., 2007). The human CD1a, CD1b, and CD1c proteins, by contrast, present lipids from mycobacterial species to T lymphocytes with diverse antigen receptors. The properties of these mycobacterial-reactive T cells suggest that they constitute part of the adaptive immune response for host defense (Barral and Brenner, 2007). Working with lipids presents special challenges, as they are famously sticky, and, moreover, unlike peptides they mostly exist in organized macromolecular structures, such as micelles and bilayers. Despite this, it has been possible to make substantial progress in understanding how CD1 proteins get loaded with lipid-containing antigens, in part by following the route of CD1 molecules in antigen-presenting cells (APCs). As outlined in Figure 1, CD1 polypeptides assemble with autologous phospholipids, such as phosphatidylcholine, and spacer lipids in the endoplasmic reticulum (Barral and Brenner, 2007; De

Libero and Mori, 2008; Garcia-Alles et al., 2006). After transport to the cell surface, either directly from the Golgi network or through an endosomal intermediate, CD1 isoforms subsequently are internalized, and they recycle through endosomal compartments, where self-phospholipids are exchanged for various self- and foreign antigens. Interestingly, different CD1 molecules are adapted to survey different endosomal compartments. For example, CD1a recycles through early endosomes, but human CD1b and mouse CD1d make their way deep into lysosomal compartments to acquire antigens (Barral and Brenner, 2007; De Libero and Mori, 2008). Sphingolipid activator proteins, including the four saposins and the GM2 activator protein, as well as the Niemann-Pick type C2 protein, are important for lipid antigen exchange in lysosomes, thereby augmenting CD1-mediated antigen presentation (Barral and Brenner, 2007; De Libero and Mori, 2008). Some of these proteins perturb membranes, thereby making glycolipids more susceptible to interaction with other proteins, whereas others may directly facilitate lipid exchange with membranes. Not only do they colocalize in late endosomes and lysosomes with CD1 molecules, but several sphingolipid activator proteins also bind to CD1. Despite these recent advances,

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Immunity

Previews the molecular details of the anin short-term presentation astigen exchange process remain says, in which antigen loading largely unknown. of CD1b occurs predominantly Here, Relloso et al. (2008) foon the cell surface, the tether cused on human CD1b and its mutations enhanced the prepresentation of the mycobactesentation of C80 GMM. The opposite effect on C32 GMM rial cell wall antigen glucose presentation was attributed to monomycolate (GMM) to an increased off rate from binda cloned T cell. CD1b is distining the more open mutant guished by its ability to bind CD1b molecules. This concluand present lipids with longer sion was supported by the rehydrophobic chains, and by its sults from a pulse-chase assay, preferential trafficking to lysoin which APC pulsed briefly somes. The GMM antigen has with antigen were chased in ana single glucose, and forms tigen-free media before stimuthat contain either 32 (C32 GMM) or 80 (C80 GMM) carbons lation of T cells. With APC exwere used. Previous work had pressing wild-type CD1b, the shown that acidic pH caused ability to respond to C32 GMM decayed more quickly during conformational changes in the chase than the ability to reCD1b that enhanced its ability spond to C80 GMM, consistent to bind hydrophobic comwith earlier data indicating that pounds (Ernst et al., 1998; Im more hydrophobic antigens et al., 2004), including GMM have stable binding to CD1b. with longer hydrophobic tails Furthermore, when presented (Cheng et al., 2006). This sugFigure 1. Depiction of CD1b Trafficking in APCs and the pH-Dependent Conformational Change by CD1b proteins with tether gested to Relloso et al. (2008) (Top) Intracellular CD1b trafficking route. After exiting the trans-Golgi netmutations, the decay in the that acidic amino acids could work, CD1b molecules arrive at the cell surface and then recycle through ability to present either antigen be important for lipid antigen endosomal compartments of decreasing pH before returning to the cell surface. EE, early endosomes; LE, late endosomes; TGN, trans-Golgi netwas accelerated. loading, as glutamic (E) and aswork; and ER, endoplasmic reticulum. When antigen loading ocpartic (D) acids will lose nega(Bottom) Schematic of pH-dependent conformational change in CD1b. At curred at low pH, changes in tive charge as a result of protonthe acidic pH of late endosomes, protonation of acidic amino acids leads to the loss of negative charge. The result is the neutralization of ionic antigen binding and presentaation as the pH decreases. The tethers, which allows the CD1b groove to open and bind lipid antigens tion by wild-type CD1b were authors propose that the partial with longer alkyl chains. similar to those induced by the loss of negative charge in the tether mutations, including inacidic lysosomal environment might unfurl the CD1b groove, allowing it in the roof near the portal at the top of the creased binding of fluorescent probes to more easily bind lipids, especially those protein were judged to most likely have and increased GMM loading at the cell with longer alkyl chains (Figure 1). A corol- a direct involvement in antigen loading. surface. In fact, the C80 GMM could not lary is that ionic interactions at the neutral This information helped in the choice of be loaded on the cell surface unless the pH of the cell surface might tether CD1b, acidic amino acids to mutate, and five po- pH were 5.5 or less during the loading remaking it less accessible to bulky lipid an- sitions were selected. Of the five, muta- action, although C32 GMM could be tigens. The authors therefore offer the ap- tions that removed negative charges at loaded to some extent at neutral pH. Furpealing hypothesis that ‘‘ionic tethers’’ act D60 or E62 led to increased antigen pre- thermore, binding of either GMM antigen as ‘‘molecular switches’’ that regulate the sentation, a gain of function suggestive was less stable at low pH. The combined conformation of CD1b as it responds of increased lipid accessibility. The exist- effects of acid pH and tether mutations to pH changes during endolysosomal ing structures of CD1b show that amino were less than additive, however, sugtrafficking. acids at these positions stabilize the gesting that low pH and disruption of the Relloso et al. (2008) carried out experi- CD1b structure in a more closed confor- tethers by mutation were affecting prements to address the more specific ques- mation by linking the a1 helix to a2, or dominantly the same pathway. Although the data are convincing, one tions of which acidic amino acids are by stabilizing a flexible loop at the end of might question what fraction of the CD1b titrated to permit increased lipid binding the a1 helix. and how they help to lock the CD1b molSeveral experiments demonstrated in- molecules were protonated at the key ecule in its slow-on and slow-off state at creased lipid accessibility in the absence amino acids. The pKa is the pH at which neutral pH. Molecular dynamics simula- of the anionic tethers. First, soluble ver- half of an acid is protonated, and for free tions were used to identify regions of the sions of the mutant CD1b proteins inter- D and E amino acids, the pKa values are CD1b molecule that have a more flexible acted better with a hydrophobic fluores- 3.9 and 4.1, respectively. The chemical conformation. One in the lateral wall of cent probe, showing greater accessibility formula predicts at pH 5.0, typical of late a pocket in the CD1b groove and another of the antigen-binding groove. Second, endosomes and a value that substantially 728 Immunity 28, June 2008 ª2008 Elsevier Inc.

Immunity

Previews increased antigen loading at the cell surface, less than 10% of the CD1b molecules would be protonated at position 60. With a similar percentage protonated at position 62, very few CD1b molecules would have both anionic tethers neutralized. However, because the surrounding amino acids in a folded protein can substantially influence ionization, the authors asserted that estimates based on the pKa of free amino acids are not accurate. In fact, a predictive model that takes protein context into account suggested that more than a few percent of the CD1b molecules would be protonated at the pH of late endosomes and lysosomes. Although the results provided by Relloso et al. (2008) give great insight into the dynamics of CD1b lipid antigen loading, an unresolved issue is how the functions of the sphingolipid activator proteins in lysosomes, such as the saposins, interplay

with the direct effects of pH on CD1b conformation. For example, do these accessory proteins interact preferentially with the protonated form of CD1b, or do they stabilize the more open conformation? Also, do anionic switches influence the behavior of the other CD1 isoforms? Further studies clearly are needed to determine if the current findings can be generalized to lipid loading of other CD1 isoforms.

Dascher, C.C. (2007). Curr. Top. Microbiol. Immunol. 314, 3–26.

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Bridging Toll-like- and B Cell-Receptor Signaling: Meet Me at the Autophagosome John G. Monroe1,* and Mary E. Keir1,* 1Genentech, Inc., 1 DNA Way, MS 93b, South San Francisco, CA 94080 *Correspondence: [email protected] (J.G.M.), [email protected] (M.E.K.) DOI 10.1016/j.immuni.2008.05.006

In this issue of Immunity, Chaturvedi et al. (2008) describe a mechanism for the bridging of innate and adaptive immune receptor functions. In their model, B cell-receptor signaling induces the fusion of Toll-like receptor 9 (TLR9)-containing endosomes with internalized signaling-competent BCR into autophagosomes. Pattern-recognition receptors (PRRs) act as sentinels of the immune system, alerting both the innate and the adaptive immune system to the presence of microbial pathogens (Medzhitov, 2007). Invariant regions within microbes, such as cellwall components of bacteria and fungi, trigger signaling through PRR and induce immune activation. Toll-like receptors (TLRs) are a well-characterized subset of PRR and play a key role in the detection of microbes, as in the case of TLR4 triggering by lipopolysaccharide (LPS), a Gram-negative bacterial-cell-wall component. Perhaps one of the more intriguing problems inherent to TLR specificity

is their ability to differentiate viral- from self-antigens, because viruses share almost all of their constitutive parts with host cells and exploit host cell machinery in order to replicate. How TLR7 and TLR9, which detect single-stranded RNA and unmethylated DNA, respectively, respond only to foreign nucleic acids remains incompletely understood. The influence of TLR signaling on the adaptive immune response has been the subject of intense study over the past several years. B cells express germ-lineencoded TLRs, including both TLR7 and TLR9, as well as B cell receptors (BCR), which are highly diversified through so-

matic hypermutation of the V(D)J region. BCR ligation results in activation of MAPkinase-signaling cascades, receptor-antigen internalization, and the induction of antigen processing for presentation on MHC class II molecules. Signals induced via TLR9 ligation in B cells independently activate downstream signals through MAP kinases and result in nuclear factorkappa B (NF-kB) activation and the initiation of proliferation and upregulation of activation markers. The induction of signaling via TLR9 litigation in B cells is not dependent on BCR ligation, but coligation of the two receptors induces increased activation of NF-kB and phosphorylation

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