Cross-linking of osteopontin by tissue transglutaminase increases its collagen binding properties.
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.
Kaartinen
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MT
Department of Biochemistry and Biotechnology, University of Kuopio, FIN-70210 Kuopio, Finland. Mari.Kaartinen@yjy,fi
Pirhonen
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Linnala-Kankkunen
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Mäenpää
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PH
D000818:Animals; D001345:Autoradiography; D016723:Bone Remodeling; D002364:Caseins / Q000378:metabolism; D002417:Cattle; D002852:Chromatography, Ion Exchange; D002942:Circular Dichroism; D003094:Collagen / Q000378:metabolism*; D004797:Enzyme-Linked Immunosorbent Assay; D008892:Milk / Q000737:chemistry; D053495:Osteopontin; D011485:Protein Binding; D011487:Protein Conformation; D012795:Sialoglycoproteins / Q000378:metabolism*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D011503:Transglutaminases / Q000378:metabolism*
D002364:Caseins; D012795:Sialoglycoproteins; D053495:Osteopontin; D003094:Collagen; D011503:Transglutaminases