2 ADJACENT N-TERMINAL GLUTAMINES OF BM-40 (OSTEONECTIN, SPARC) ACT ASAMINE ACCEPTOR SITES IN TRANSGLUTAMINASE(C)-CATALYZED MODIFICATION

The extracellular matrix protein BM-40 (osteonectin, SPARC) has recent ly been shown to be a major target for transglutaminase catalyzed cros s-linking in differentiating cartilage. In the present study we demons trate that recombinant human BM-40 can be modified with [H-3]putrescin e in a 1:1 molar ratio by transglutaminase(C) (tissue transglutaminase ). Residues Gln(3) and Gln(4) were identified as major amine acceptor sites, This was confirmed with several mutant proteins, including dele tions in the N-terminal domain I of BM-40, site-directed mutagenesis o f the reactive glutamines, and fusion of the seven-amino acid-long N-t erminal sequence (APQQEAL) to an unrelated protein, The results showed that the N-terminal target site is sufficient for modification by tra nsglutaminase but at a low level, For high efficiency amine incorporat ion an intact domain I is required, The conservation of at least one o f the transglutaminase target glutamines in the known vertebrate BM-40 sequences and their absence in an invertebrate homologue point to an important, but yet unknown, role of this modification in vertebrates.