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

The extracellular matrix protein BM-40 (osteonectin, SPARC) has recently been shown to be a major target for transglutaminase catalyzed cross-linking in differentiating cartilage. In the present study we demonstrate that recombinant human BM-40 can be modified with [H-3]putrescine 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 deletions in the N-terminal domain I of BM-40, site-directed mutagenesis of the reactive glutamines, and fusion of the seven-amino acid-long N-terminal sequence (APQQEAL) to an unrelated protein, The results showed that the N-terminal target site is sufficient for modification by transglutaminase but at a low level, For high efficiency amine incorporation an intact domain I is required, The conservation of at least one of 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.