Domain IV of mouse laminin β1 and β2âchains: Structure, glycosaminoglycan modification and immunochemical analysis of tissue contents
2002
Sasaki, Takako | Mann, Karlheinz | Miner, Jeffrey H. | Miosge, Nicolai | Timpl, Rupert
Domain IV, consisting of about 230 residues, represents a particular protein module so far found only in laminin β1 and β2âchains. Both domains were obtained by recombinant production in mammalian cells. They showed a globular structure, as expected from electron microscopic examination of laminins. Fragment β1IV was obtained as a monomer and a disulfideâbonded dimer, and both were modified to ≈â50% by a single chondroitin sulfate chain attached to Ser721 of an SGD consensus sequence. Dimerization is caused by an odd number of cysteines, with three of them having a partial thiol character. Whether both modifications also occur in tissue forms of laminin remains to be established. Fragment β2IV was only obtained as a monomer, as it lacked one crucial cysteine and the SGD sequence. It required, however, the presence of two adjacent LE modules for proper folding. Polyclonal antibodies raised against both fragments showed no crossâreaction with each other and allowed establishment of βâchainâspecific radioimmunoassays and light and electron microscopic immunostaining of tissues. This demonstrated a 5–25âfold lower content of β2 compared with β1 chains in various tissue extracts of adult mice. Tissues derived from β2âdeficient mice failed to react with the β2âspecific antibodies but showed a twofold higher content of β1 than heterozygotes. The antibodies to β2 showed broader tissue staining than reported previously, including in particular a distinct reaction with the extrasynaptic endomysium of skeletal muscle. Immunogold staining localized both βâchains primarily to basement membranes of kidney, muscle and various other tissues.
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