Tissue-nonspecific Alkaline Phosphatase Is Required for the Calcification of Collagen in Serum

Abstract

Previous studies have shown that the type I collagen of tendon and demineralized bone both calcify rapidly in serum. The speed, collagen matrix-type specificity, and extent of the re-calcification of demineralized bone in serum suggest that the serum calcification activity identified in these studies may participate in normal biomineralization. Because of its presence in serum and its long history of association with the normal mineralization of the collagen matrix of bone, tissue-nonspecific alkaline phosphatase (TNAP) is an obvious candidate for a protein that could be a component of serum calcification activity, and experiments were therefore carried out to test this possibility. These experiments show that the inactivation of TNAP in serum prevents collagen calcification, and that the addition of physiological levels of purified TNAP restores the ability of TNAP-deficient serum to calcify collagen. Additional experiments show that the role of TNAP in collagen calcification is to activate a serum nucleator of apatite crystal formation. Based on these and earlier studies, the mechanism of collagen calcification in serum requires at least four elements as follows. Our goal is to understand the biochemical mechanism responsible for the calcification of collagen fibrils in normal bone formation. In the course of our investigations, we have discovered that purified type I collagen and demineralized bone matrix both calcify rapidly when incubated in serum in the absence of cells (1-4). The calcification of collagen is because of the presence of a serum calcification activity, one sufficiently potent that collagen calcifies when incubated in media containing as little as 1.5% serum but not in serum-free media alone (1-4). This serum calcification activity consists of one or more proteins that are 50 -150 kDa in size (3, 4).Although serum-driven collagen calcification is an in vitro, cell-free assay, there are several reasons to believe that it could be relevant to understanding mechanisms by which collagen fibrils are mineralized in nature. 1) The assay conditions are physiologically relevant; collagen added to serum calcifies when incubated at the temperature and pH of mammalian blood, without the need to add anything to serum to promote mineralization, such as ␤-glycerophosphate or phosphate (see Ref. 1 and references therein). 2) Serum is relevant to bone mineralization; osteoblasts form bone in a vascular compartment (5), and proteins in serum have direct access to the site of collagen fibril formation and mineralization, whereas proteins secreted by the osteoblast appear rapidly in serum. 3) Serum-driven calcification is evolutionarily conserved; the serum calcification activity appeared in animals at the time vertebrates acquired the ability to form calcium phosphate mineral structures, with no evidence for a similar activity in the serum of invertebrates (2). 4) Serum-driven calcification is specific; calcification is restricted to those structures that were calcified in bone prior to demineralization, with n...

Bone Alkaline Phosphatase and Mortality in Dialysis Patients

“…Inactivation of AP prevents serum collagen calcification. AP was furthermore reported to activate a serum nucleator of apatite crystal formation (6). In line with this, clinical studies have found serum AP to be associated with coronary artery calcification and all-cause mortality in patients with CKD and on hemodialysis (7)(8)(9).…”

Functional and molecular structural analysis of dentine interfaces promoted by a Zn-doped self-etching adhesive and an in vitro load cycling model

“…It has been shown that this local increase of Ca ++ concentration may inhibit Zn ++ binding, depending on the relative abundance of these two divalent ions (Rosenberg et al, 1998). The association among the in situ release of Zn ++ , mineral precipitation and in vitro load cycling on partially demineralised and infiltrated dentine is supported by the effect of compressive loads on the stimulation of the tissuenonspecific alkaline phosphatase (McAllister and Frangos 1999), a zinc-metalloenzyme that hydrolyzes a broad range of phosphate monoesters (Price et al, 2009;Posner et al, 1986). At high phosphate concentration, calcium pyrophosphate, calcium phosphate and unstable and non-crystalline amorphous complexes are formed (Cheng and Pritzker, 1983) (Figs 4J, 5J) around the collagen fibrils, keeping the alkaline phosphatase and other enzymes "fossilized" (Van Meerbeek et al, 2001), thus hindering the complete remineralisation.…”

Untitled

“…This deficiency results in accumulation of extracellular pyrophosphate and causes the inhibition of skeletal and dental mineralization [80][81][82][83]. There are six different clinical forms of the disease, and they are divided according to the age of the patient and degree of severity.…”