Structural characterization of two variants of fibulin-1 that differ in nidogen affinity.
Two C-terminal variants C and D of mouse fibulin-1 were purified from the culture medium of stably transfected human kidney cell clones. They showed, after rotary shadowing, a dumbbell-like structure of about 33 nm in length. Pepsin digestion demonstrated stability of the disulfide-bonded domains 1 (anaphylatoxin-like) and II (multiple EGF-like motifs) but not for domain III which is different in the variants. A close similarity of the variants was observed in immunochemical assays indicating that domain III epitopes are not very antigenic. Binding analysis in solid phase assays demonstrated for variant C a 100-fold stronger binding to the basement membrane protein nidogen than for variant D. Both interactions were sensitive to EDTA. Surface plasmon resonance assays confirmed this difference and showed KD = 60 nM for variant C and KD > 1 microM for variant D. Lower binding activities and smaller differences between both variants were observed for the calcium-dependent binding to fibronectin, laminin-1 and collagen IV. Self aggregation into nest-like oligomers was observed at high concentrations of fibulin-1 which was not sensitive to EDTA.
Sasaki
T
T
Max-Planck-Institut für Biochemie, Martinsried, Federal Republic of Germany.
Kostka
G
G
Göhring
W
W
Wiedemann
H
H
Mann
K
K
Chu
M L
ML
Timpl
R
R
D000595:Amino Acid Sequence; D000818:Animals; D002118:Calcium / Q000378:metabolism; D002135:Calcium-Binding Proteins / Q000235:genetics / Q000378:metabolism* / Q000648:ultrastructure; D002999:Clone Cells; D003094:Collagen / Q000378:metabolism; D005353:Fibronectins / Q000378:metabolism; D006801:Humans; D007797:Laminin / Q000378:metabolism; D008562:Membrane Glycoproteins / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D011485:Protein Binding; D011994:Recombinant Proteins / Q000378:metabolism / Q000648:ultrastructure; D013329:Structure-Activity Relationship
D002135:Calcium-Binding Proteins; D005353:Fibronectins; D007797:Laminin; D008562:Membrane Glycoproteins; D011994:Recombinant Proteins; C060820:fibulin; C029967:nidogen; D003094:Collagen; D002118:Calcium