Results: 5

1.
Figure 2

Figure 2. From: Mutations within a furin consensus sequence block proteolytic release of ectodysplasin-A and cause X-linked hypohidrotic ectodermal dysplasia.

Inhibition of EDA-HA proteolytic processing. (A) BSC-40 cells were first infected with an adenoviral construct, α1-PDX, a potent inhibitor of furin activity, and subsequently infected with VV:EDA-HA. (B) LoVo cells were coinfected with VV:EDA-HA and either the furin construct, VV:fur/f (fur +), or, in control cultures, wild-type virus (fur −). Cell and media fractions were analyzed by SDS/PAGE and immunoblot with the anti-HA antibody.

Yanwen Chen, et al. Proc Natl Acad Sci U S A. 2001 June 19;98(13):7218-7223.
2.
Figure 5

Figure 5. From: Mutations within a furin consensus sequence block proteolytic release of ectodysplasin-A and cause X-linked hypohidrotic ectodermal dysplasia.

Expression of recombinant EDA harboring mutations in the furin cleavage site. (A) The biosynthesis of recombinant EDA harboring XLHED mutations in the putative furin cleavage site (see Fig. 1A). (B) VV:EDA-HA(K158N) was expressed in the presence or absence of tunicamycin (10 μM). All constructs were expressed in BSC-40 cells, and the media and cell extracts were analyzed by SDS/PAGE and immunoblotting with HA antibody.

Yanwen Chen, et al. Proc Natl Acad Sci U S A. 2001 June 19;98(13):7218-7223.
3.
Figure 3

Figure 3. From: Mutations within a furin consensus sequence block proteolytic release of ectodysplasin-A and cause X-linked hypohidrotic ectodermal dysplasia.

Determination of the site of EDA processing by furin. (A) Synthetic peptide, pEDA (EDA residues 148–165) (100 μM), was digested with furin, and an aliquot was subjected directly to Edman sequencing. The results for each cycle are shown. The multiple sequence was ordered from the sequence of the starting peptide. Residues in parentheses were present in trace amounts. (B) EDA-HA and EDA-HA with the R159A mutation were expressed in BSC-40 cells and analyzed by SDS/PAGE and immunoblotting.

Yanwen Chen, et al. Proc Natl Acad Sci U S A. 2001 June 19;98(13):7218-7223.
4.
Figure 4

Figure 4. From: Mutations within a furin consensus sequence block proteolytic release of ectodysplasin-A and cause X-linked hypohidrotic ectodermal dysplasia.

Kinetics of furin cleavage of normal and mutant peptide substrates. The internally quenched control peptide pEDA, containing the furin site (Fig. 3) as well as mutated versions of this peptide, pR153C, pR155C, pR156H, pR156C, pK158N, and pR159A (Fig. 1A), were digested with furin (Materials and Methods), and the time course of proteolysis was followed by increased fluorescence at 400 nm and presented as the degree of conversion (Materials and Methods). (Inset) kcat, Km, and kcat/Km for pEDA, R153C, and R155C cleavage by furin.

Yanwen Chen, et al. Proc Natl Acad Sci U S A. 2001 June 19;98(13):7218-7223.
5.
Figure 1

Figure 1. From: Mutations within a furin consensus sequence block proteolytic release of ectodysplasin-A and cause X-linked hypohidrotic ectodermal dysplasia.

Structure of EDA and the EDA-HA construct. The EDA protein is a type II transmembrane protein. Cyt, the N-terminal cytoplasmic domain; TM, the transmembrane domain. EC1, the first extracellular domain, contains a compound consensus furin cleavage site, Arg-X-Lys/Arg-Arg (italics, upstream site; underline, downstream site), near its C terminus. TH, the collagenous triple helical domain; EC2, the second extracellular domain, which contains an embedded TNF core motif. This domain also contains two consensus N-linked glycosylation sites, as indicated in the schematic (5). The recombinant EDA-HA molecule (400 aa) used in this study is identical to the EDA-A1 sequence (391 aa), with the addition of the 9-aa hemagglutinin (HA) epitope tag as indicated. The positions of Arg156 and Arg159 are indicated. Mutations within the putative furin site(s) of EDA in patients with XLHED are identified by downward arrows (5, 6, 23, 54) and are abbreviated R153C, R155C, R156S, R156H, R156C, and K156N. The position of each amino acid is given relative to the downstream site (Arg159), P1-P8, N-terminal and P1′-P2′, C-terminal. (B) BSC-40 cells were infected with VV:EDA-HA or wild-type virus (VV:WT) for 16 h, and the cell (c) and medium (m) compartments were analyzed by SDS/PAGE and immunoblotting with HA antibody. Cell and medium samples were also analyzed after digestion with bacterial collagenase (C'ase). A schematic of collagenase digestion products is on the right. After collagenase digestion, the HA epitope tag remains with the N-terminal EC1 domain. The triple helix appears to be more resistant to bacterial collagenase in the context of the parent molecule compared with the 36/27-kDa fragments. (C) BSC-40 cells were infected with VV:EDA-HA for 4 h in the presence (+) or absence (−) of tunicamycin (Tm) to inhibit N-glycosylation. The same proteolytic processing and N-glycosylation patterns were detected when EDA was expressed in other cell lines, HaCaT (epidermal epithelia), 293 (kidney epithelia), COS-7 (kidney fibroblast), and A204 (rhabdomyosarcoma) (data not shown).

Yanwen Chen, et al. Proc Natl Acad Sci U S A. 2001 June 19;98(13):7218-7223.

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