Structural and cell-adhesive properties of three recombinant fragments derived from perlecan domain III.
Domain III of the basement membrane proteoglycan perlecan was produced as three overlapping fragments in stably transfected mammalian cell clones. These recombinant fragments (43-48 kDa) were obtained in purified form and showed complete immunological cross-reactivity with perlecan, indicating their native structure. Rotary shadowing electron microscopy of each fragment demonstrated a small globular structure connected to a short rod. These data were interpreted to indicate that domain III has an elongated shape of 30 nm in length and consists of alternating globular domains (L4 modules) and short connecting segments attributed to tandem arrays of LE (laminin-type of EGF-like) modules which form rod-like segments in laminins. Sequence analyses of pepsin fragments were consistent with the disulfide-bonding patterns known for these modules from studies with laminin fragments, but two additional disulfide loops were also identified. Several cell lines which attached to mouse perlecan and/or human fibronectin failed to adhere to the domain III fragments, despite the fact that one of them contained an RGD (Arg-Gly-Asp) site in the L4 module. Furthermore, no significant binding was observed in solid phase binding assays with alpha 5 beta 1 and alpha v beta 3 integrins underscoring the low activity or accessibility of the RGD site.
Schulze
B
B
Max-Planck-Institut for Biochemie, Martinsried, Federal Republic of Germany.
Sasaki
T
T
Costell
M
M
Mann
K
K
Timpl
R
R
D000595:Amino Acid Sequence; D000818:Animals; D002448:Cell Adhesion / Q000502:physiology*; D002460:Cell Line; D005353:Fibronectins / Q000378:metabolism; D019812:Heparan Sulfate Proteoglycans; D006497:Heparan Sulfate / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D006868:Hydrolysis; D016023:Integrins / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D011509:Proteoglycans / Q000235:genetics* / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism
D005353:Fibronectins; D019812:Heparan Sulfate Proteoglycans; D006497:Heparan Sulfate; D016023:Integrins; D011509:Proteoglycans; D011994:Recombinant Proteins; C071927:perlecan