Cross-linking of Osteopontin by Tissue Transglutaminase Increases Its Collagen Binding Properties

Mari T Kaartinen,Arja Pirhonen,Annikka Linnala-Kankkunen,Pekka H Mäenpää

doi:10.1074/jbc.274.3.1729

Abstract

Osteopontin, a major noncollagenous bone protein, is an in vitro and in vivo substrate of tissue transglutaminase, which catalyzes formation of cross-linked protein aggregates. The roles of the enzyme and the polymeric osteopontin are presently not fully understood. In this study we provide evidence that transglutaminase treatment significantly increases the binding of osteopontin to collagen. This was tested with an enzyme-linked immunosorbent assay. The results also show that this increased interaction is clearly calcium-dependent and specific to osteopontin. In dot blot overlay assay 1 microgram of collagen type I was able to bind 420 ng of in vitro prepared and purified polymeric osteopontin and only 83 ng of monomeric osteopontin, indicating that the transglutaminase treatment introduces a 5-fold amount of osteopontin onto collagen. Assays using a reversed situation showed that the collagen binding of the polymeric form of osteopontin appears to be dependent on its conformation in solution. Circular dichroism analysis of monomeric and polymeric osteopontin indicated that transglutaminase treatment induces a conformational change in osteopontin, probably exposing motives relevant to its interactions with other extracellular molecules. This altered collagen binding property of osteopontin may have relevance to its biological functions in tissue repair, bone remodeling, and collagen fibrillogenesis.

Highlights

Presence of TG in mineralizing cartilage and bone [5, 6] and the enzyme is thought to participate in matrix cross-linking before the tissue undergoes calcification [5, 6]
Since recent gene knock-out experiments have demonstrated that osteocalcin is an inhibitor of bone formation [21], our results suggest that TG activity and the OPN aggregates may be involved in enhancement of biomineralization or matrix maturation that precedes it
Casein appeared to bind to collagens both in the presence and absence of calcium ions, but the binding decreased as the amount of TG increased in the reaction mixture, suggesting that TG was competing with casein in collagen binding

Summary

The abbreviations used

TG, transglutaminase; OPN, osteopontin; HPLC, high performance liquid chromatography; PAGE, polyacrylamide gel electrophoresis; MALDI-TOF, matrix-assisted laser desorption ionization-time of flight; ELISA, enzyme-linked immunosorbent assay; BSA, bovine serum albumin; BAG-75, bone acidic glycoprotein; FPLC, fast protein liquid chromatography. OPN is a major product of bone forming cells, osteoblasts, but is not specific to bone. Recent knock-out mice experiments by Liaw et al [19] indicate that OPN, functions in tissue repair, matrix organization, and collagen fibrillogenesis. Since recent gene knock-out experiments have demonstrated that osteocalcin is an inhibitor of bone formation [21], our results suggest that TG activity and the OPN aggregates may be involved in enhancement of biomineralization or matrix maturation that precedes it. We provide evidence that OPN, as a high molecular weight complex form, exhibits significantly increased binding ability to all tested collagens. This may result from an altered conformation of the OPN after polymerization as observed by circular dichroism measurements. A more stabile structure and amplified collagen binding property, after treating OPN with an enzyme that is intimately involved in tissue repair, brings further support to OPNЈs role as a tissue remodeling protein and gives an insight into the functions of the polymeric OPN

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION