Journal: The Journal of Biological Chemistry

Article Title: Homodimerization of the Lymph Vessel Endothelial Receptor LYVE-1 through a Redox-labile Disulfide Is Critical for Hyaluronan Binding in Lymphatic Endothelium *

doi: 10.1074/jbc.M116.736926

Figure Lengend Snippet: LYVE-1 in lymphatic endothelium is organized largely as homodimers linked via the critical disulfide Cys-201. The self-association status of LYVE-1 in native HDLEC and transfected cells and the potential involvement of Cys-201 were assessed using non-reducing SDS-PAGE and Western blotting. A , schematic representations of wild-type hLYVE-1 and hLYVE-1 C201A in which the unpaired cysteine 201 in the membrane proximal domain predicted to form an intermolecular disulfide was mutated to alanine; also shown is the soluble full ectodomain construct hLYVE-1 Δ238 His 10 . Positions of the three disulfides in the extended Link module and two further unpaired cysteines in the transmembrane and cytoplasmic domains are indicated. N -Linked sugars are shown in green , and O -linked sugars are in purple. B , reducing and non-reducing SDS-PAGE/Western blots of LYVE-1 in primary HDLEC showing predominant organization as the homodimer and its disassembly in HDLEC lysed in the absence of NEM as indicated. C , bar chart showing quantitative comparison of monomer and dimer band intensities from equivalent HDLEC Western blots (mean ± S.E. n = 4) as determined by densitometric scanning. Statistical p values were obtained using the two-tailed unpaired t test ( n.s. , non-significant). D and E , non-reducing SDS-PAGE/Western blots of LYVE-1 in different mouse tissues and transfected COS7 and Jurkat T cells lysed in the presence of NEM as indicated. F , reducing and non-reducing SDS-PAGE/Western blots of 293T cells transfected with either full-length wild-type LYVE-1 or C201A mutants and of CHO cell-derived soluble hLYVE-1 Δ238 His 10 protein showing the critical involvement of Cys-201 in homodimer formation. Samples were prepared in the presence of NEM as indicated. All blots were probed with affinity-purified polyclonal goat anti-human or mouse LYVE-1 antibodies followed by anti-goat IRdye® 800 before imaging with a Licor® Odyssey scanner. All data are from representative experiments that were repeated at least three times.

Article Snippet: The following LYVE-1-specific antibodies were used: polyclonal goat anti-human (AF20891, R&D Systems), a biotinylated polyclonal (BAF20891, R&D Systems), and the mAbs 20891, 20982, 20893 (all R&D Systems), 6A, 8C, and 3A (all made in-house).

Techniques: Transfection, SDS Page, Western Blot, Membrane, Construct, Comparison, Two Tailed Test, Derivative Assay, Affinity Purification, Imaging

Journal: The Journal of Biological Chemistry

Article Title: Homodimerization of the Lymph Vessel Endothelial Receptor LYVE-1 through a Redox-labile Disulfide Is Critical for Hyaluronan Binding in Lymphatic Endothelium *

doi: 10.1074/jbc.M116.736926

Figure Lengend Snippet: The Cys-201 disulfide linkage in the LYVE-1 homodimer is highly labile. The sensitivity to reduction of the Cys-201 intermolecular disulfide was investigated after exposure of soluble hLYVE-1 homodimers to varying concentrations of TCEP and assessment of the consequences for both HA binding and dimer disassembly ( A–C ) A and B , concentration-dependent effects of TCEP treatment on HMW bHA binding to immobilized hLYVE-1 (Δ238 His 10 ) homodimers and hLYVE-1 (Δ238 His 10 ) monomer (non-dimerizing C201A mutant), respectively, as detected with a streptavidin HRP conjugate. Values in each case are the mean ± S.E. n = 3. Statistics p values were obtained using the two-tailed unpaired t test ( n.s. , non-significant). C , non-reducing SDS-PAGE analysis showing concentration-dependent effects of TCEP treatment on soluble homodimer disassembly assessed by Western blotting with LYVE-1 polyclonal antibody and green IRdye® 800 conjugate. D , schematic showing the location and numbering of individual cysteines ( yellow circles ) forming Link domain disulfides in the LYVE-1 homodimer; N - and O -linked glycan chains are colored green and red , respectively. Data shown are from representative experiments that were repeated three times.

Article Snippet: The following LYVE-1-specific antibodies were used: polyclonal goat anti-human (AF20891, R&D Systems), a biotinylated polyclonal (BAF20891, R&D Systems), and the mAbs 20891, 20982, 20893 (all R&D Systems), 6A, 8C, and 3A (all made in-house).

Techniques: Binding Assay, Concentration Assay, Mutagenesis, Two Tailed Test, SDS Page, Western Blot, Glycoproteomics