tailieunhanh - Báo cáo khoa học: Domain IV of mouse laminin b1 and b2 chains Structure, glycosaminoglycan modi®cation and immunochemical analysis of tissue contents

Domain IV, consisting of about 230 residues, represents a particular protein module so far found only in lamininb1 andb2 chains. Both domains were obtained by recombi-nant production in mammalian cells. They showed a globular structure, as expected from electron microscopic examination of laminins. Fragmentb1IV was obtained as a monomer and a disul®de-bonded dimer, and both were modi®ed to 50% by a single chondroitin sulfate chain attached to Ser721 of an SGD consensus sequence. | Eur. J. Biochem. 269 431-442 2002 FEBS 2002 Domain IV of mouse laminin pi and p2 chains Structure glycosaminoglycan modification and immunochemical analysis of tissue contents Takako Sasaki1 Karlheinz Mann1 Jeffrey H. Miner2 Nicolai Miosge3 and Rupert Timpl1 1 Max-Planck-Institut fur Biochemie Martinsried Germany 2Renal Division and Department of Cell Biology and Physiology Washington University School of Medicine St Louis MO USA 3Center of Anatomy Department of Histology University of GoÈttingen Germany Domain IV consisting of about 230 residues represents a particular protein module so far found only in laminin p1 and p2 chains. Both domains were obtained by recombinant production in mammalian cells. They showed a globular structure as expected from electron microscopic examination of laminins. Fragment piIV was obtained as a monomer and a disulhde-bonded dimer and both were modihed to w 50 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 modihcations also occur in tissue forms of laminin remains to be established. Fragment p2IV 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 cross-reaction with each other and allowed establishment of p chain-specihc radioimmunoassays and light and electron microscopic immunostaining of tissues. This demonstrated a 5-25-fold lower content of p2 compared with p1 chains in various tissue extracts of adult mice. Tissues derived from p2-dehcient mice failed to react with the p2-specihc antibodies but showed a twofold higher content of p1 than heterozygotes. The antibodies to p2 showed broader tissue staining than reported previously including in particular a distinct reaction with the extra-synaptic .