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

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 g 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.Tissue transglutaminase (TG) 1 (EC 2.3.2.13) is a widely distributed intra-and extracellular calcium-dependent enzyme, which catalyzes the formation of high molecular mass complexes of its substrate proteins by creating isopeptide crosslinks from glutamine and lysine residues and releasing ammonia (1, 2). TG is suggested to be involved in matrix maturation and stabilize the tissue with cross-links that are resistant to normal proteolysis (1, 2). TG is closely related to wound healing which suggests a role for it in tissue remodeling and repair (3,4). Immunohistochemical data have also demonstrated the 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). The number of proteins serving as glutaminyl substrates for TG is restricted indicating the physiological importance of its functions (1). The roles of TG and the actions of its enzymatic products, meaning high molecular weight proteins, are still unclear.Osteopontin (OPN), a prominent and potentially multifunctional acidic phosphoglycoprotein (7,8), is a substrate of TG (9 -11). OPN is a major product of bone forming cells, osteoblasts, but is not specific to bone. It is also synthesized in other types of tissues and found in, e.g. inner ear, brain, kidney (7), and atherosclerotic plaques (13,14), and it is also secreted into milk (12) and urine (15). Its production is also related to immunity, infection, and cancer (8). Osteoblasts express OPN at an early developmental stage of bone formation (16,17). In bone, OPN is deposited into unmineralized matrix pri...

Osteopontin promotes pathologic mineralization in articular cartilage

“…3). Osteopontin is an important substrate of the transglutaminase enzymes in mineralizing tissues (Beninati et al 1994;Kaartinen et al 1999).…”

“…3). Osteopontin is an important substrate of the transglutaminase enzymes in mineralizing tissues (Beninati et al 1994;Kaartinen et al 1999).Osteopontin is almost ubiquitous at sites of both normal and pathologic mineralization. However, its role in mineral formation remains poorly understood and somewhat controversial.…”

“…Osteopontin contains sites for phosphorylation, glycosylation, transglutamination, and thrombin cleavage. Transglutaminase covalently binds osteopontin to fibronectin, to itself, and to other matrix constituents (Kaartinen et al 1999). Functional alterations due to these post-translational modifications are well-described.…”

OPN‐a induces muscle inflammation by increasing recruitment and activation of pro‐inflammatory macrophages

“…Transglutamination of OPN is thought to modify protein function by altering the conformational state of OPN, thereby increasing its cell‐binding and chemotactic abilities (Kaartinen et al . 1999; Nishimichi et al . 2011).…”

Transcriptional regulation of human osteopontin promoter by C/EBPα and AML-1 in metastatic cancer cells

“…The antiapoptosis effect depends on phosphorylation of OPN (Kaartinen et al, 1999). The host-derived OPN may act as a macrophage chemoattractant (Kaartinen et al, 1999), whereas the tumor-derived OPN can inhibit the function of macrophage and enhance the growth of metastasis (Crawford et al, 1998).…”