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. 2022 Jun 27;11:e79990. doi: 10.7554/eLife.79990

Figure 1. Isolation of Birbeck granules.

(A) Domain organization of langerin. Human langerin is a 328-amino acids protein composed of an N-terminal cytoplasmic tail, a transmembrane domain (TM), a coiled-coil neck, and a C-terminal carbohydrate-recognition domain (CRD). We fused a SNAP-tag at the N-terminus of langerin and inserted an HRV3C protease cleavage site between the tag and the langerin sequences. For streptavidin-mediated precipitation of Birbeck granules, the SNAP-tag was biotinylated (star). (B) Model of langerin oligomerization within Birbeck granules. Langerin trimers bind to each other face-to-face via the CRDs, bringing the two layers of the plasma membrane closer together. (C) Birbeck granules formed in 293T cells overexpressing langerin. Addition of yeast mannan induced the formation of Birbeck granules a few micrometers long. Inset shows a magnified view of Birbeck granules. (D) SDS-PAGE of isolated Birbeck granules. Purified langerin (arrowhead) was released from streptavidin-agarose by HRV3C digestion. M: molecular weight marker; and BG: isolated Birbeck granules. (E) Cryo-electron microscopy of isolated Birbeck granules. Black square indicates the position of the close-up view shown in F. (F) Wavy lamellar structure of the Birbeck granule. Black dots were gold nanoparticles used as fiducial markers. (G) Class averages of the projection images of Birbeck granules. The image dimension is 34 nm2. Although 2D classification did not converge well due to the continuity of the structure, some classes showed a porous structure with a honeycomb-like lattice.

Figure 1—source data 1. Original gel image of Figure 1D.
Figure 1—source data 2. Annotated gel image of Figure 1D.

Figure 1.

Figure 1—figure supplement 1. Isolation of Birbeck granules.

Figure 1—figure supplement 1.

(A) Fluorescence microscopy image of 293T cells expressing langerin. IRES-driven GFP expression was detected. The transformation efficiency was approximately 40%. (B) Negative-stain electron microscopy image of an isolated Birbeck granule stained with 2% uranium acetate. (C) Cryo-electron microscopy image of a twisted Birbeck granule. (D) Coiled-coil prediction profile of langerin.