Glucocorticoids induce glutamine synthetase expression in human osteoblastic cells: a novel observation in bone.

Glucocorticoids have marked effects on bone metabolism, and continued exposure of skeletal tissue to excessive amounts of these steroids results in osteoporosis. Therefore, in the present proteomic study, we characterized the potential effects of glucocorticoids on protein expression in human osteoblastic cells. Using two-dimensional gel electrophoresis and mass spectrometry, we identified an increased expression of glutamine synthetase (GS) in dexamethasone (Dex)-treated human MG-63 osteosarcoma cells. GS is an enzyme catalyzing the conversion of glutamate and ammonia to glutamine. Intracellular and extracellular glutamate levels may be important in cell signalling mediated by glutamate transporters and receptors which have recently been found in bone cells. The induction of GS protein by Dex was accompanied by an increase in mRNA level and enzyme activity. Dex induction of GS was also mediated by glucocorticoid receptors (GRs) because it was blocked by the GR antagonist RU-38486. In addition, Dex induction of GS expression was partially blocked by cyclohexamide indicating that it at least partly required new protein synthesis. GS induction by Dex was not associated with apoptosis as determined by Bax/Bcl-2 ratio and DNA staining. In addition to MG-63 cells, Dex induction of GS was also observed in human G-292 osteosarcoma cells as well as conditionally immortalized human preosteoblastic (HOB-03-C5) and mature osteoblastic (HOB-03-CE6) cells. However, in two other human osteosarcoma cell lines, SaOS-2 and U2-OS, GS expression was not affected by Dex. This observation may be explained by the lower levels of GR protein in these cells. In summary, this is the first report of the regulation of GS expression by glucocorticoids in bone cells. The role of GS in bone cell metabolism and glucocorticoid action on the skeleton is not yet known, but as a modulator of intracellular glutamate and glutamine levels, it may have an important role in these processes.

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.

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, beta-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 beta-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.

Molecular analysis of allergenic proteins in bovine dander.

An analytic procedure was established to characterize bovine dander proteins with allergenic properties. The proteins from dander extract were separated by size-exclusion gel filtration, and the fractions were studied with SDS-PAGE followed by immunoblotting. An 11-kDa allergen was found in the same gel filtration fractions as 20- and 22-kDa allergens, and this suggests that the 11-kDa allergen is a dimer in its native form. Our method also detected two separate 22-kDa allergens. The primary structure of the major bovine dander allergen (BDA20) was also studied. A protein sequencer was used to determine the amino acid sequences of enzymatically cleaved peptides. The homology searches revealed that BDA20 is not a previously known bovine protein.

cDNA cloning and protein analysis of a bovine dermal allergen with homology to psoriasin.

Immunoscreening of a cDNA library from bovine skin led to isolation of clones coding for an allergen named BDA11. Sequence analysis of the clones revealed that they can encode a protein of 11.6 kDa with a predicted pI of 5.19. Allergenicity of BDA11 was verified by the IgE reactivity in cattle-allergic patients' sera with the recombinant protein produced in Escherichia coli. A biochemically purified native allergen of 11 kDa from bovine dander was identified as BDA11 by peptide sequencing. Homology comparisons showed that BDA11 had a 63.4% amino acid identity with human psoriasin. Psoriasin is a calcium-binding protein expressed in keratinocytes, and it is strongly up-regulated in psoriatic skin. BDA11 also had segments homologous with calcium-binding proteins from three other species.

P2 protamines are phosphorylated in vitro by protein kinase C, whereas P1 protamines prefer cAMP-dependent protein kinase. A comparative study of five mammalian species.

P1 protamines isolated from ejaculated human, stallion, bull, boar and ram spermatozoa and P2 protamines from human and stallion spermatozoa were subjected, after alkaline phosphatase treatment, to in vitro phosphorylation reactions using cAMP-dependent protein kinase (PKA) and protein kinase C (PKC). All P1 protamines were phosphorylated by PKA, whereas P2 protamines were phosphorylated only by PKC. In addition, human, stallion and boar, but not bull and ram, P1 protamines were phosphorylated by PKC. After phosphoamino acid analysis, the protamines showing positive signals for phosphoserine (P-Ser) were subjected to P-Ser conversion reaction and protein sequencing. Only stallion (St1) and human (HP1) P1 protamines contained P-Ser after PKA phosphorylation, located in the middle region of the molecule, i.e., at Ser29 in St1 and Ser28 in HP1. All other phosphorylated P1 protamines contained only P-Thr, which could not be further localized in the sequence with the present methods. After PKC phosphorylation, the internally located Ser residues in human (ser21) and stallion (Ser29) P1 protamines were phosphorylated and, in boar P1 protamine, only Thr43 was slightly phosphorylated. The N-terminally located Ser residues in P1 protamines, which are known to be phosphorylated in vivo, were not phosphorylated by either kinase, indicating that there must still be other types of protamine kinases in sperm cells responsible for their phosphorylation. Within P2 protamines, HP2 was equally well phosphorylated at all Ser residues in addition to some Thr phosphorylation, whereas, in St2, Ser32 was the main target for PKC phosphorylation in vitro. Collectively, PKC is a good candidate for in vivo phosphorylation of P2 protamines and PKA for phosphorylation of some hydroxyamino acid residues in P1 protamines.

Identification of phosphoseryl residues in protamines from mature mammalian spermatozoa.

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.

Partial characterization of a fraction from bull seminal vesicle fluid that potentiates the bull sperm acrosome reaction in vitro.

A fraction from bovine seminal vesicle fluid that initiated acrosome reaction of bovine epididymal spermatozoa in vitro in the presence of heparin was prepared by sequential chromatographies on heparin-Sepharose, gel filtration (Superose 12) and reversed phase chromatography (ProRPC). Sequence analysis of the separated fraction showed that it contained the major protein (PDC-109) with 100% homology. This fraction contained also Ca(2+)-dependent phospholipase A2-like activity which hydrolysed phosphatidylethanolamine and phosphatidylcholine with 14C-labelled linoleic (lino-PE, lino-PC) or arachidonic acid (ara-PE, ara-PC) at sn-2 position. This protein was not detected in N-terminal sequence analysis. Lysophosphatidylcholine, lysophosphatidylethanolamine, and p-bromophenacyl bromide (p-BPB) inhibited this lipolytic activity. Sulfoglycolipid (Seminolipid) had inhibitory effect at concentrations above 0.1 mM but activated slightly the enzyme at lower concentrations. Boiling destroyed acrosome initiating activity in the separated fraction.

An aminoterminally extended P1-protamine variant in the boar.

We have found a minor form of boar sperm P1-protamine with a six amino acid long N-terminal extension. When this sequence was compared with the recently published gene sequence of boar P1-protamine, and with DNA sequences obtained from the boars studied here, a perfect match was found with the 5'-region upstream of the ATG initiation codon of the authentic P1-protamine. Since no extra in-frame ATG initiation codons are present in the 5'-region of the gene, we conclude that the translation of boar P1-protamine variant starts with a non-AUG initiation codon, CUG, which precedes the codon of the first amino acid (serine) of the sequenced variant.

In vitro phosphorylation sites of stallion and bull P1-protamines for cyclic adenosine 3',5'-monophosphate-dependent protein kinase and protein kinase C.

Fish and mammalian protamines are phosphorylated after their synthesis during sperm cell maturation. Cyclic AMP-dependent protein kinase (PKA) and protein kinase C (PKC), both requiring basic amino acids at their recognition sites, have previously been found to phosphorylate fish protamines in vitro. In this study, these enzymes were used to phosphorylate stallion and bull sperm P1-protamines in vitro. A species-specific difference was found, since PKA was able to phosphorylate both protamines while PKC phosphorylated only stallion protamine. Thr-41, the only threonine residue in stallion P1-protamine, and most probably the homologous Thr-43 in bull P1-protamine are the sites for PKA phosphorylation in addition to an internally located Ser-29 present only in stallion protamine. This Ser residue was phosphorylated in vitro by both kinases. Protamine phosphorylation by PKA was found to be almost independent of cAMP and was inhibited only by a tenfold concentration of PKI when compared to phosphorylation of a model peptide, kemptide. Addition of calcium, phosphatidylserine, and diolein caused a twofold stimulation in phosphorylation of stallion protamine by PKC, indicating that specific cofactors of PKC may have a role in mammalian protamine phosphorylation. We suggest that PKA is a good universal candidate for in vivo phosphorylation of P1-protamines.

Highly homologous cytochromes P-450 and b5: a model to study protein-protein interactions in a reconstituted monooxygenase system.

Cytochrome b5 from mouse and rat liver formed a type I spectral complex with two murine cytochrome P-450 isozymes, the P450Coh and P450PBI. Mouse b5 stimulated the reactions catalyzed by reconstituted P450Coh and an equimolar amount of b5 to P450Coh was needed for maximal effect. In contrast, rat b5 inhibited P450Coh-mediated reactions progressively starting from 1:1 ratio of b5 to P-450. Neither b5 had any effect on reactions catalyzed by P45015 alpha, an isozyme highly homologous to P450Coh, but with a point mutation (Arg-129----Ser) at site considered important for P-450-b5 interactions. In case of P450PBI, neither b5 protein had any effect on the associated activities at b5: P-450 ratios below 1, and a progressive inhibition occurred when b5: P-450 ratio was above 1. The results were similar with either rat or mouse liver NADPH-cytochrome P-450 reductase used in reconstitution demonstrating that the critical differences take place in P-450-b5 interactions. Kinetic and spectral experiments revealed that the stimulatory and inhibitory effects of b5 on the enzymatic reactions were due to corresponding changes in the reaction velocity, and that b5 does not compete with the flavoprotein nor with the substrate for binding to P-450. These results indicate that the high spin shift of P-450 does not necessarily correlate with enhanced reaction rates. Also, the increase in the coupling efficiency of P450PBI may result from the increased affinity for substrate in the presence of b5. Sequenation of mouse b5 peptides generated with proteinases revealed three amino acid changes between the mouse and rat b5, two of which appeared at the hydrophobic domain necessary for the P-450-b5 interaction. This could explain the species specificity of b5 proteins in supporting the P-450-mediated reactions. This is the first time functionally important differences in the interaction of highly homologous cytochromes P-450 and b5 have been demonstrated. Isozymes P45015 alpha and P450Coh, and mouse and rat b5 could serve as an excellent model for further studies on the nature and significance of P-450-b5 interactions.

Identification of a low-Mr acidic nuclear protein as prothymosin alpha.

We have purified to homogeneity a 15-kDa perchloric acid (PCA)-soluble protein from rat thymus nuclei. This highly acidic protein showed a Mr of ca. 30 kDa in acetic acid/urea gels, probably due to oligomer formation. Sequence analysis of internal tryptic and thermolytic peptides revealed that the purified protein is, in fact, prothymosin alpha, a very hydrophilic polypeptide, which has been previously classified as a thymic or immunomodulating hormone. We found that prothymosin alpha 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 alpha suggest that its function is related to those of other long polyacidic regions containing nuclear proteins.

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.

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

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-Gl u-Gln. It corresponds to the C-terminal residues 90-107 of HMG-I [(1989) Mol. Cell. Biol. 9, 2114-2123]. Sequence analysis of the native peptide (90-107) after treatment, which specifically converts phosphoserine residues to S-ethylcysteine, revealed that 70-80% 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.

Comparison of partial amino acid sequences of two protamine 2 variants from stallion sperm. Structural evidence that the variants are products of different genes.

Protamine 1 and two protamine 2 variants were isolated from stallion sperm and separated by acetic acid-urea gel electrophoresis. After electroblotting onto polyvinyldifluoride filters, their amino-terminal amino acid sequences were determined by pulse-liquid peptide sequencing. The sequences of the two protamine 2 variants are homologous but slightly different in length and amino acid composition and indicate for the first time the existence of two different genes for this protamine species.

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-Mr 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-Mr HMG proteins, HMG 2. The identity of the low-Mr 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.

Phosphorylation of acid-soluble chromatin proteins from tissues of different species by purified cyclic GMP-dependent protein kinase.

1. Phosphorylation of acid-soluble chromatin proteins from thymus or liver of calf, rabbit, pig, rat, rooster and trout by purified cyclic GMP-dependent protein kinase was studied in vitro using acetic acid-urea slab gel electrophoresis and autoradiography. 2. HMG 14, histone H1 and an unknown band representing probably a proteolytic fragment of histone H1 were phosphorylated in all mammals studied. 3. In avian liver, HMG 14 showed no phosphorylation and histone H1 was replaced by a H1(0)/H5-like heavily phosphorylated protein. 4. The only 32P-acceptor in trout liver apparently belongs to the C/D-family of acid-soluble chromatin proteins. H6-protein was not phosphorylated.

Alterations in amounts and covalent modifications of low-molecular-weight chromosomal proteins in Chinese hamster ovary cells during polyamine depletion.

The effect of polyamine depletion on phosphorylation and ADP-ribosylation of low-Mr chromosomal proteins was studied in intact, mutant Chinese hamster ovary cells (CHO-P22) devoid of ornithine decarboxylase activity. When starved of polyamines for 6 days, severe polyamine deficiency develops and the cells gradually stop growing. The rate of DNA synthesis was retarded to 16% of the control value and to 29% in density-inhibited cells. The synthesis of high-mobility-group (HMG) proteins was decreased by 65% in polyamine-depleted cells and by 40% in density-inhibited cells. The synthesis of core histones was decreased by 40% both in polyamine-depleted and density-inhibited cells. In polyamine-depleted cells the molar ratio of the higher-Mr HMG proteins (HMG 1 + 2) to the lower-Mr HMG proteins (HMG 14 + P) was about one-half of that found in cells grown in the presence of putrescine or in density-inhibited cells. In contrast to HMG proteins, no major differences were found in the content of core histones in these cell populations. In the perchloric acid-soluble fraction of nuclear proteins, 32P was incorporated mainly into histone H1, HMG P and a protein migrating more slowly than HMG 1 (protein P1). Specific changes in the 32P-labeling and migration of a number of protein bands, including histone H1, was observed in polyamine-depleted cells as compared to cells grown in the presence of putrescine or to density-inhibited cells. ADP-ribosylation experiments using [3H]adenosine showed a different pattern of label distribution; the higher-Mr HMG proteins from polyamine-depleted cells contained about one-half the amount of label found in the proteins from control cells. The lower-Mr HMG proteins and histone H1 were the preferentially labeled proteins in polyamine-depleted cells. Labeling of core histones with [32P]orthophosphate or [3H]adenosine did not differ markedly in the two cell populations. The results obtained using intact polyamine auxotrophic cells indicated that polyamine depletion is connected with more severe alterations in amounts and covalent modifications (phosphorylation and ADP-ribosylation) of HMG chromosomal proteins and histone H1 than core histones.

Phosphorylation of a low Mr high mobility group protein in Chinese hamster ovary cells.

Phosphorylation of high mobility group (HMG) chromatin proteins was studied both in intact Chinese hamster ovary cells (strain CHO-P22) and in vitro conditions using isolated HMG proteins from the same cells and purified protein kinases. Prominent phosphorylation of serine in a low Mr HMG protein designated as HMG P was observed in unsynchronized cells. Of the three protein kinases tested, only nuclear type II protein kinase phosphorylated HMG P in vitro. The phosphorylated amino acid was phosphoserine. Cyclic nucleotide dependent protein kinases did not phosphorylate HMG P but phosphorylated HMG 14 with a preference for cGMP-dependent protein kinase. 32P-labeling of HMG 17 was not observed in intact cells or in vitro.