Two adjacent N-terminal glutamines of BM-40 (osteonectin, SPARC) act as amine acceptor sites in transglutaminaseC-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 [3H]putrescine in a 1:1 molar ratio by transglutaminaseC (tissue transglutaminase). Residues Gln3 and Gln4 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.
Hohenadl
C
C
Max-Planck-Institut für Biochemie, Martinsried, Federal Republic of Germany.
Mann
K
K
Mayer
U
U
Timpl
R
R
Paulsson
M
M
Aeschlimann
D
D
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D003432:Cross-Linking Reagents; D017931:DNA Primers / Q000235:genetics; D015195:Drug Design; D005973:Glutamine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006168:Guinea Pigs; D006801:Humans; D008969:Molecular Sequence Data; D015394:Molecular Structure; D016297:Mutagenesis, Site-Directed; D015676:Osteonectin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011700:Putrescine; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity; D011503:Transglutaminases / Q000378:metabolism*
D003432:Cross-Linking Reagents; D017931:DNA Primers; D015676:Osteonectin; D011994:Recombinant Proteins; D005973:Glutamine; D011503:Transglutaminases; D011700:Putrescine