Figure 4

The extracellular domain of LPHN3 consists of two globular domains separated by an O-glycosylated stalk. (A) Coomassie blue stained gel of purified ecto-LPHN3 protein that was subjected to SEC in (B). (B) SEC of the purified extracellular domain of LPHN3. The dashed line at volume 0 indicates the beginning of the injection. Fractions were collected between red lines. Fraction C3 was used for EM analysis. Ev and Iv indicate the excluded and included volumes, respectively. (C) Representative raw image of the extracellular domain of LPHN3. Scale bar is 100 nm. (D) Nine representative class averages of the individual particles. Scale bar is 20 nm. (E) Histogram of the distribution of inter-domain distances of individual particles. No particles were found to be separated by less than 33 Å or more than 153 Å. (F) Schematic model of the extracellular domain of LPHN3. Because of the slight difference is size of each domain, we tentatively assigned the larger domain to the Lectin and Olfactomedin domains, and the smaller domain to the HRD, GAIN, and GPS domains. In the proximal globular domain we manually superimposed a ribbon model of the available crystal structure [17]. (G) O-linked sugar analysis of the N-terminal domain of LPHN3 truncated at residue 571, just before the HRD. Several O-linked sugars and one N-linked glycosylation were present, as predicted by bioinformatics tools. Known glycans are indicated above the corresponding spectra. Sugars are represented as follows: yellow circle, galactose; blue square, N-acetylglucosamine; orange square, N-acetylgalactosamine; pink diamond, N-acetylneuraminic acid; red triangle, fucose; cyan diamond, N-glycolylneuraminic acid. (H) Domain organization of LPHN3. The extracellular domain of LPHN3 is composed of two globular regions separated by a flexible linker. The distal globular domain contains Lectin and Olfactomedin homology domains, while the proximal globular domain contains the HRD and GAIN domains.