Domain IV of mouse laminin β1 and β2 chains: Structure, glycosaminoglycan modification and immunochemical analysis of tissue contents

Takako Sasaki, Karlheinz Mann, Jeffrey H. Miner, Nicolai Miosge, Rupert Timpl

Research output: Contribution to journalArticlepeer-review

62 Scopus citations

Abstract

Domain IV, consisting of about 230 residues, represents a particular protein module so far found only in laminin β1 and β2 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 β1IV was obtained as a monomer and a disulfide-bonded dimer, and both were modified to ≈ 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 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 cross-reaction with each other and allowed establishment of βchain-specific radioimmunoassays and light and electron microscopic immunostaining of tissues. This demonstrated a 5-25-fold lower content of β2 compared with β1 chains in various tissue extracts of adult mice. Tissues derived from β2-deficient mice failed to react with the β2-specific 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 extra-synaptic endomysium of skeletal muscle. Immunogold staining localized both β chains primarily to basement membranes of kidney, muscle and various other tissues.

Original languageEnglish
Pages (from-to)431-442
Number of pages12
JournalEuropean Journal of Biochemistry
Volume269
Issue number2
DOIs
StatePublished - 2002

Keywords

  • Basement membranes
  • Chondroitin sulfate
  • Laminin domain
  • Radioimmunoassay
  • Recombinant production

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