Annikka Linnala-Kankkunen

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 μg 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.

Transglutaminase-catalyzed Cross-linking of Osteopontin Is Inhibited by Osteocalcin

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, β-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 β-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.

Identification of sites on chromosomal protein HMG-I phosphorylated by casein kinase II

High‐mobility‐group protein; Casein kinase II; Protein phosphorylation; Phosphopeptide; Chromatin

Selective decrease in low-Mr HMG proteins HMG I and HMG Y during differentiation of mouse teratocarcinoma cells

We have studied the presence of high‐mobility‐group (HMG) chromatin proteins in undifferentiated F9 mouse teratocarcinoma cells and F9 cells, which were induced to differentiate by treatment with retinoic acid and dibutyryl‐cAMP for 5 days. Acetic acid/urea‐polyacrylamide gel electrophoresis and reversed‐phase HPLC revealed that the induced F9 cells contained 77 and 62% less HMG I and HMG Y, respectively, than their untreated counterparts. The relative amounts of two other low‐M r HMG proteins HMG 14 and HMG 17 remained essentially unchanged and only a minor decrease was observed in the content of one of the high‐M r HMG proteins, HMG 2. The identity of the low‐M r HMG proteins was verified by amino acid analysis or partial sequencing. These results suggest that HMG I and HMG Y are HMG proteins specific for undifferentiated cells.

Primary structures of two protamine 2 variants (St2a and St2b) from stallion spermatozoa

Protamines were extracted from stallion sperm cell nuclei, alkylated with iodoacetamide and separated by reversed-phase high-performance liquid chromatography. Two main components, protamine 1 and protamine 2, were obtained. The latter contains two subspecies, separable by acetic acid-urea-polyacrylamide gel electrophoresis. The primary structure of protamine 2a (St2a) was determined by analysis of fragments obtained from purified protamine 2 peak by thermolysin digestion. The digested peptides were separated by acetic acid-urea gel electrophoresis and, after electroblotting onto a polyvinylidene difluoride filter, their amino acid sequences were determined by pulse liquid peptide sequencing. The amino acid sequence of protamine 2b was predicted from the double sequence data of protamine 2 peak by eliminating the amino acid of St2a in each cycle. St2a and St2b were found to contain 62 and 58 amino acid residues, respectively, and they seem to be homologous with type 2 protamines from human and mouse spermatozoa.

Differential phosphorylation of high mobility group protein HMG 14 from calf thymus and avian erythrocytes by a cyclic GMP-dependent protein kinase

Phosphorylation of HMG 14 proteins from calf thymus and avian erythrocytes was studied using a cyclic GMP-dependent protein kinase from bovine lung. HMG 14 from calf thymus was a good substrate for the enzyme, but HMG 14 from avian erythrocytes was not phosphorylated. Of the potential phosphorylation sites, the one in the amino terminal sequence Pro-Lys-Arg-Lys-Val-Ser-Ser-Ala-Glu (residues 1-9) is present in HMG 14 from calf thymus but not in HMG 14 from avian erythrocytes suggesting that the phosphorylated amino acid residue in HMG 14 from calf thymus is Ser-6 (and possibly Ser-7).

Identification of a low-Mr acidic nuclear protein as prothymosin α

We have purified to homogeneity a 15‐kDa perchloric acid (PCA)‐soluble protein from rat thymus nuclei. This highly acidic protein showed a M r, of ca. 30 kDa in acetic acid/urea gels, probably due to oligomer formation. Sequence analysis of internal tryptic and thennolytic peptides revealed that the purified protein is, in fact, prothymosin α, a very hydrophilic polypeptide, which has been previously classified as a thymic or immunomodulating hormone. We found that prothymosin α is a rather abundant nuclear protein in rat thymus; its concentration is comparable to that of a well‐characterized nonhistone protein HMG‐14. The subcellular localization and physicochemical properties of prothymosin α suggest that its function is related to those of other long polyacidic regions containing nuclear proteins.

Thiophosphorylation and phosphorylation of chromatin proteins from calf thymus in vitro

Thiophosphorylation and phosphorylation of 5% perchloric acid extractable proteins from calf thymus chromatin were studied using a cyclic GMP-dependent protein kinase from bovine lung and a nuclear protein kinase II from rat liver. The phosphorylation reaction catalyzed by nuclear protein kinase II utilized [gamma -35S]ATP as a phosphate donor almost as efficiently as [gamma -32P]ATP, but the cGMP-dependent protein kinase mediated phosphorylation by [35S]ATP was about 20 times less effective than that by [32P]ATP. In addition, using [35S]ATP instead of [32P]ATP changed markedly the cGMP-dependent phosphorylation pattern of the PCA-extractable proteins as examined by gel electrophoresis. Thus, depending on the type of protein kinase, the results from thiophosphorylation and phosphorylation reactions may vary considerably.

Polyamines and heparin do not appreciably influence phosphorylation of chromatin proteins HMG 14 and HMG 17 by nuclear protein kinase II

Phosphorylation of acidic substrates such as casein and phosvitin by nuclear protein kinase II is stimulated by polyamines and inhibited by heparin, which mimics an endogenous proteoglycan inhibitor. The phosphorylation in vitro of the chromatin proteins HMG 14 and HMG 17 by nuclear protein kinase II were examined in this study focusing on the modifying effects of polyamines and heparin. Both HMG proteins were phosphorylated by the enzyme, but polyamines did not appreciably influence the extent of their phosphorylation. In addition, heparin did not inhibit the kinase reaction with the HMG proteins as substrates. These results indicate that the nuclear protein kinase II does actively phosphorylate HMG 14 and HMG 17 in vitro but that in contrast to some model substrates, polyamines and heparin do not appreciably affect their phosphorylation.

Phosphorylation alters the affinity of high mobility group protein HMG 14 for single-stranded DNA

The effect of phosphorylation on the affinity of HMG 14 from calf thymus for single-stranded DNA (ssDNA) was studied, using a cyclic GMP-dependent protein kinase from bovine lung and a nuclear protein kinase II from rat liver. When phosphorylated by G-kinase, HMG 14 eluted at 0.27 M NaCl from the ssDNA-column, whereas the native protein eluted at 0.30 M salt concentration. In contrast, phosphorylation by nuclear protein kinase II did not alter dissociation of HMG 14 from ssDNA and the phosphoprotein consequently coeluted with the native HMG 14. Thus, addition of a negative charge by phosphorylation of the Ser-6 residue by G-kinase presumably weakens the interaction between the DNA-binding amino acids of HMG 14 and the negatively charged phosphate groups of DNA.