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...

The amino acid sequence of a region on chromosomal protein HMG-I from human cells that is phosphorylated by casein kinase II has been determined. The sequence is: Leu-Glu-Lys-Glu-Glu-Glu-Glu-Gly-Ile-Ser-Gln-Glu-Ser(P)-Ser(P)-Glu-Glu-Glu-Gln.It corresponds to the C-terminal residues 90-107 of HMG-I [(1989) Mol. Cell. Biol. 9,21142123]. Sequence analysis of the native peptide (g&107) after treatment, which specifically converts phosphoserine residues to S-ethylcysteine, revealed that 7&80% of serine residues 102 and 103 were phosphorylated in vivo. Both residues were fully phosphorylated in vitro by incubation with casein kinase II. These results suggest that casein kinase II is involved in the regulation of HMG-I function in the cells.

Osteocalcin, the most abundant noncollagenous protein of bone matrix, has been demonstrated to inhibit bone growth by gene knockout experiments (Ducy, P., Desbois, C., Boyce, B., Pinero, G., Story, B., Dunstan, C., Smith, E., Bonadio, J., Goldstein, S., Gundberg, C., Bradley, A., and Karsenty, G. (1996) Nature 382, 448 -452). Its specific functional mechanism in bone metabolism is, however, largely unknown. In this study, we provide evidence that osteocalcin has an inhibitory effect on tissue transglutaminase activity, as measured by cross-linking of osteopontin, another bone matrix protein. Using a set of synthetic peptides, we found that the inhibitory activity resided within the first 13 N-terminal amino acid residues of osteocalcin. An N-terminal peptide also inhibited cross-linking of another tissue transglutaminase substrate, ␤-casein. The inhibitory peptide was shown to have affinity for the substrates of transglutaminase rather than for the enzyme. Since the N terminus of osteocalcin exhibits homology to the substrate recognition site sequences of two transglutaminases, we conclude that the inhibitory effect is most likely due to competition with the enzyme for the transglutaminase-binding region of the substrates, osteopontin and ␤-casein, which prevents access of the enzyme to them to perform its function. The interference of osteocalcin with osteopontin cross-linking gives osteocalcin a new potential function as the first protein inhibitor of tissue transglutaminase. This suggests a specific role and a plausible mechanism for it as a modulator of maturation, stabilization, and calcification of bone matrix.The organic matrix of mineralized tissues is composed of several noncollagenous proteins, whose functions in bone, dentine, and cartilage development and remodeling are not presently fully understood. In addition to their possible contribution to the structural integrity of hard tissues, the biochemical and medical evidence suggests their involvement in the regulation of bone turnover. The control of bone remodeling involves numerous extracellular matrix events, including various protein-protein interactions, which eventually lead to the arrangement of proteins into larger complexes, which finally form the strong supramolecular architecture of bone matrix.One of the most abundant noncollagenous proteins of adult bone is osteocalcin (OCN), 1 which is a small osteoblast-specific calcium-binding protein of 46 -50 amino acid residues, containing three vitamin K-dependent Gla residues (1, 2). The abundance of OCN in the mineralized matrix and its well conserved amino acid sequence emphasize the importance of OCN in bone, but its functions have still remained unclear. Data accumulated thus far indicate that OCN acts as a negative regulator of bone turnover and a suppressor of mineralization (3-6). OCN deficiency, resulting from interference with the vitamin K-dependent Gla synthesis, causes poor accumulation of OCN in bone and results in excessive calcification and resistance to bone resorption (4, 5). The...

Protamines isolated from ejaculated human, stallion, bull, boar, and ram spermatozoa were subjected to phosphoserine conversion reaction and protein sequencing. Phosphoserines were detected as S-ethylcysteines. Endogenously phosphorylated protamines have previously been found only in ejaculated human sperm. In this study, we demonstrate that ejaculated sperm from other species also contain protamines phosphorylated at serine residues. In P1-protamines, the endogenously phosphorylated serines were located at the N-terminal region in all species studied, whereas in major forms of human and stallion P2-protamines, the serine residues located in the middle region of the molecule were predominantly phosphorylated. These results support the current DNA binding model in the case of the P1-protamines. The internal location of the phosphorylated serines in the P2-protamines indicates, however, that the binding of these proteins to DNA or their interaction with other protamine molecules may differ from that of P1-protamines. This also suggests that, during sperm maturation, P2-protamines may have a role different from that of P1-protamines.

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