Cryo-electron tomography of Birbeck granules reveals the molecular mechanism of langerin lattice formation

  1. Toshiyuki Oda
  2. Haruaki Yanagisawa
  3. Hideyuki Shinmori
  4. Youichi Ogawa
  5. Tatsuyoshi Kawamura

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Abstract

Langerhans cells are specialized antigen-presenting cells localized within the epidermis and mucosal epithelium. Upon contact with Langerhans cells, pathogens are captured by the C-type lectin langerin and internalized into a structurally unique vesicle known as a Birbeck granule. Although the immunological role of Langerhans cells and Birbeck granules have been extensively studied, the mechanism by which the characteristic zippered membrane structure of Birbeck granules is formed remains elusive. In this study, we observed isolated Birbeck granules using cryo-electron tomography and reconstructed the 3D structure of the repeating unit of the honeycomb lattice of langerin at 6.4 Å resolution. We found that the interaction between the two langerin trimers was mediated by docking the flexible loop at residues 258–263 into the secondary carbohydrate-binding cleft. Mutations within the loop inhibited Birbeck granule formation and the internalization of HIV pseudovirus. These findings suggest a molecular mechanism for membrane zippering during Birbeck granule biogenesis and provide insight into the role of langerin in the defense against viral infection.

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  1. Version published to 10.7554/elife.79990 on eLife
  2. Version published to 10.1101/2022.02.24.481763v3 on bioRxiv
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    Reply to the reviewers

    We thank the referees for their valuable suggestions. We have revised the text accordingly and already conducted most of the requested experiments.

    Reviewer #1

    1. The authors state that addition of mannan increases length of Birbeck granules however, no data are presented. It would make this more convincing when the length is compared between conditions with and without mannan (as shown in Fig 4, where the condition without mannan is lacking).

    Reply: Thank you for pointing out the missing data. We added an EM image of Birbeck granules and quantification of Birbeck granules formation in the absence of mannan (Figure 4A-D).

    Supp, fig 1B perhaps as a panel in main figure as this is an important control to show that Birbeck granules are isolated.

    Reply: We moved the supplemental figure 1B to main figure 1D.

    1. Only the(total) length of Birbeck granules is taken into account, but not the number of Birbeck granules. Is it possible to quantify the number of Birbeck granules.

    Reply: We added Figure 4D to show the number of Birbeck granules. Note that the difference in the number of Birbeck granules was less significant than that of total length because there were numerous short fragments in the mutant specimen.

    Fig 5. Only the condition (ARGK) where there is virtually no Birbeck granules formation is included, however, is virus still internalized in the other conditions (MRGD or MRGK) as Birbeck granule formation was less effective but still present? It would be interesting to include those mutants. A more specific quantification would be by p24 ELISA. Is there a reason why immunoblotting has been chosen? In the supernatant condition, explain why the virus p24 seems less in the control condition whereas one would expect max concentration in that condition.

    Reply: Thank you for suggesting the use of ELISA. We chose immunoblotting because of its higher sensitivity and lower cost. But ELISA is advantageous when it comes to comparing large number of samples. We performed p24 ELISA and quantified the virus internalization in all the mutants available (Figure 5C). As you pointed out, the transfer efficiency of the immunoblot in Figure 5A was not uniform across the membrane; Pr55 bands became denser toward the right, while p24 bands had a gradient in the opposite direction. The immunoblots and ELISA showed that about ~1% of the viruses were attached or internalized and ~99% did not interact with the cells. Thus, the attached/internalized viruses did not affect the amount of viruses in the supernatant. Results of ELISA also showed the amount of viruses in the supernatant were nearly equal among the samples (Figure S3B).

    Abstract First sentence: not mucosal tissue but mucosal epithelium Last sentence: Virual should be viral

    Reply: We corrected the typo. Thank you.

    Discussion The last section comparing DC-SIGN and langerin is not clear and some overstatements are made. "Considering that DC-SIGN serves as an attachment receptor for viruses but not as an entry receptor, the possible structural coupling of lateral ligand binding and internalization implies that langerin functions as a more efficient entry receptor for viruses than DC-SIGN or other C-type lectins." It is not correct that langerin but not DC-SIGN can function as an entry receptor. DC-SIGN has been shown to facilitate infection of different viruses such DENV and ZIKV. In contrast, langerin can restrict viruses such as HIV-1 but also facilitate infection for example Influenza A and DENV. So attachment or entry is more likely a consequence of the internalization and dependence on pH changes for fusion as some viruses such as DENV fuse in acidic vesicles. This needs to be discussed more clearly.

    Reply: Thank you for pointing out our wrong statement. We replaced the statement with weakened one as below:

    Page 13, line 213: “The difference in the ligand-binding manner between langerin and DC-SIGN may contribute to their different carbohydrate recognition preferences (Valverde et al., 2020; Takahara et al., 2004).“

    Reviewer #2

    1. Langerin can exist on the cell surface and in Birbeck granules. They should examine langerin cell surface expression in the 3 states, wildtype, mutated and lectin - . __Do the mutations change cell surface expression? __

    Reply: We performed surface labeling experiments and showed that those mutations did not affect surface expression of langerin (Figure S3A).

    1. Birbeck granules are present in the absence of mannan and pathogens (see Pena-Cruz JCI 2018, PMID: 29723162). Thus, this suggests that Birbeck granules are present even without langerin clathrin coated pit internalization from the cell surface. How does their model account for this observation?

    Reply: We think there are two possibilities:

    1. Birbeck granules were shown to stem from the endoplasmic reticulum (Valladeau et al Immunity 2000; Lenormand et al PlosONE 2013). Since the rER is the site of glycosylation, langerin is likely to capture the oligo-mannose-glycosylated proteins within the rER and form Birbeck granules.
    2. Blood plasma proteins such as immunoglobulin D, immunoglobulin E, and apolipoprotein B-100 are reported to carry high-mannose glycans (Clerc et al Glycoconj J. 2016). Those glycoproteins in the cell culture media can induce Birbeck granule formation.
    1. Different cell types can have varied Langerin levels (see Pena-Cruz JCI 2018, PMID: 29723162). Is Birbeck granule formation depend on certain level of langerin expression? Do Birbeck granules form when Langerin is present at low as compared to high levels?

    Reply: In the course of the experiments, we isolated a cell line stably expressing langerin. However, langerin expressing cells were extremely slow in proliferation and the expression levels were low. To answer this question, we recovered this “failed” stable cell line and found that the low langerin-expressing cells can form Birbeck granules, but with lower efficiency (Figure S3C-E).

    1. Authors use immunoblots to show that HIV is present in intra-cellular Langerin structures. It would be ideal to visualize HIV with presumably internal Birbeck granules using imaging techniques such as cryo-electron micrography or another form of high resolution imaging.

    Reply: We are currently working on ultra-thin section electron microscopy of HIV-infected langerin-expressing cells. Visualization of HIV-containing Birbeck granules using cryo-electron microscopy is highly challenging because the current precision of cryo-FIB-SEM milling technique is too low to target a specific intracellular structure. We believe conventional electron microscopy will provide sufficiently convincing evidence that HIV is present within Birbeck granules.

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    Referee #2

    Evidence, reproducibility and clarity

    In this manuscript, investigators used cryo-electron tomography to reconstruct the structure of langerin and langerin composed organelle, termed Birbeck granule. They find that langerin trimers interact via carbohydrate binding cleft, mediated via 258 - 262 residues. Mutations in the residue prevent Birbeck granule structures. They propose a molecular structure for HIV binding and internalization.

    Significance

    This is highly interesting work with significance for understanding pathogen, such as HIV, recognition and clearance in mucosal antigen presenting cells.

    I am not an expert in structural studies but the cryo-electron tomography is impressive and convincing. I have concerns with some of the HIV - Birbeck granule aspects. Cell transfected with langerin and mutated langerin were exposed to HIV pseudotypes. They show that HIV binding occurs in the absence of mannan with both wildtype and mutated langerin. On the other hand, a langerin that lacks calcium binding does not bind virus (lectin -). They show that the mutated langerin has limited internalization, presumably because of lack of Birbeck granule formation.

    1. Langerin can exist on the cell surface and in Birbeck granules. They should examine langerin cell surface expression in the 3 states, wildtype, mutated and lectin - . Do the mutations change cell surface expression?
    2. Birbeck granules are present in the absence of mannan and pathogens (see Pena-Cruz JCI 2018, PMID: 29723162). Thus, this suggests that Birbeck granules are present even without langerin clathrin coated pit internalization from the cell surface. How does their model account for this observation?
    3. Different cell types can have varied Langerin levels (see Pena-Cruz JCI 2018, PMID: 29723162). Is Birbeck granule formation depend on certain level of langerin expression? Do Birbeck granules form when Langerin is present at low as compared to high levels?
    4. Authors use immunoblots to show that HIV is present in intra-cellular Langerin structures. It would be ideal to visualize HIV with presumably internal Birbeck granules using imaging techniques such as cryo-electron micrography or another form of high resolution imaging.

    All microbiologists, immunologists, and investigators interested in infectious disease will be interested in this work.

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    Referee #1

    Evidence, reproducibility and clarity

    The authors here have used cryo-electron tomography, 3D reconstruction and modelling on isolated Birbeck granules to provide a molecular mechanism for langerin-induced Birbeck granule formation. Their data revealed a structure of the repeating unit of the honeycomb lattice of langerin in Birbeck granules. Their model suggests that the interaction between the two langerin trimers is mediated by docking the flexible loop at residues 258-262 into the secondary carbohydrate-binding cleft. Mutational analysis within the loop suggests that these interactions are important for Birbeck granule formation and virus internalization.

    The results presented in the manuscript are very interesting and propose an novel mechanism how langerin induces Birbeck granule formation and how two langerin trimers are able to interact with virus and induce Birbeck granule formation.

    Comments.

    Fig. 1. The authors state that addition of mannan increases length of Birbeck granules however, no data are presented. It would make this more convincing when the length is compared between conditions with and without mannan (as shown in Fig 4, where the condition without mannan is lacking).

    Supp, fig 1B perhaps as a panel in main figure as this is an important control to show that Birbeck granules are isolated.

    Fig. 4. Only the(total) length of Birbeck granules is taken into account, but not the number of Birbeck granules. Is it possible to quantify the number of Birbeck granules.

    Fig 5. Only the condition (ARGK) where there is virtually no Birbeck granules formation is included, however, is virus still internalized in the other conditions (MRGD or MRGK) as Birbeck granule formation was less effective but still present? It would be interesting to include those mutants. A more specific quantification would be by p24 ELISA. Is there a reason why immunoblotting has been chosen? In the supernatant condition, explain why the virus p24 seems less in the control condition whereas one would expect max concentration in that condition.

    Minor comments

    Abstract First sentence: not mucosal tissue but mucosal epithelium Last sentence: Virual should be viral

    Discussion The last section comparing DC-SIGN and langerin is not clear and some overstatements are made. "Considering that DC-SIGN serves as an attachment receptor for viruses but not as an entry receptor, the possible structural coupling of lateral ligand binding and internalization implies that langerin functions as a more efficient entry receptor for viruses than DC-SIGN or other C-type lectins." It is not correct that langerin but not DC-SIGN can function as an entry receptor. DC-SIGN has been shown to facilitate infection of different viruses such DENV and ZIKV. In contrast, langerin can restrict viruses such as HIV-1 but also facilitate infection for example Influenza A and DENV. So attachment or entry is more likely a consequence of the internalization and dependence on pH changes for fusion as some viruses such as DENV fuse in acidic vesicles. This needs to be discussed more clearly.

    Significance

    There is little known about the molecular mechanism of birbeck granule formation and the role of langerin as well as its ligand (HIV or mannan). here the authors convincingly reveal a mechanism which is corroborated by mutational analyses. This is important in the field. the major drawback which is that a cell-line has been used, 293T, and overexpression of langerin. I understand the reason (manipulation in other cells more difficult, no good LC cell-lines, primary cells probably impossible) but it makes the significance a bit less. overall this is a significant contribution to the field.

  6. Version published to 10.1101/2022.02.24.481763v2 on bioRxiv
  7. Version published to 10.1101/2022.02.24.481763v1 on bioRxiv