Tissue transglutaminase and its substrates in bone.
Tissue transglutaminase (tTG) is an intra- and extracellular, protein-cross-linking enzyme that has been implicated in apoptosis, matrix stabilization, and cell attachment in a variety of tissues. This study provides in vivo evidence in bone of TG activity, its tissue localization, and identification of its substrates. In microplate- and blotting-based activity assays using biotinylated primary amine as a probe, we show TG activity in protein extracts from the mineralized compartment of intramembranous rat bone. Avidin affinity purification of bone extract labeled with biotinylated primary amine in the presence of tTG, in conjunction with Western blotting, permitted identification of three major noncollagenous TG substrates in bone: osteopontin (OPN), bone sialoprotein (BSP), and alpha2 HS-glycoprotein (AHSG), of which the latter two are novel substrates. Cross-linking and labeling of purified proteins confirmed their ability to serve as TG substrates, because they readily incorporated biotinylated primary amine and formed large protein aggregates in the presence of tTG. All three proteins were also identified in the high molecular weight complexes extractable from the mineralized compartment of bone. Two-dimensional (2D) gel electrophoretic analysis combined with Western blotting indicated that the proteins are not cross-linked to each other, but form distinct homotypic polymers. In the extracellular matrix of bone, tTG and isopeptide bonds were localized by immunohistochemistry in the osteoid and in the pericellular matrix surrounding osteocytes. At the cellular level, osteoblasts and osteocytes were immunostained for tTG. Collectively, these data suggest a role for tTG and its covalently cross-linked substrates in cell adhesion and possibly also in bone matrix maturation and calcification.
Kaartinen
Mari T
MT
Faculty of Dentistry, McGill University, Montreal, Quebec, Canada.
El-Maadawy
Sherif
S
Räsänen
Niina H
NH
McKee
Marc D
MD
D000818:Animals; D015153:Blotting, Western; D001842:Bone and Bones / Q000201:enzymology*; D002846:Chromatography, Affinity; D015180:Electrophoresis, Gel, Two-Dimensional; D007150:Immunohistochemistry; D051381:Rats; D013379:Substrate Specificity; D011503:Transglutaminases / Q000378:metabolism*
D011503:Transglutaminases