Osteopontin, bone sialoprotein, and bone acidic glycoprotein-75 are three acidic phosphoproteins that are isolated from the mineralized phase of bone matrix, are synthesized by osteoblastic cells, and are generally restricted in their distribution to calcified tissues. Although each is a distinct gene product, these proteins share aspartic/glutamic acid contents of 30-36% and each contains multiple phosphoryl and sialyl groups. These properties, plus a strict relationship of acidic macromolecules with cell-controlled mineralization throughout nature, suggest functions in calcium binding and nucleation of calcium hydroxyapatite crystal formation. However, direct proof for such roles is still largely indirect in nature. The purpose of this review is to present two speculative hypotheses regarding acidic phosphoprotein function. The goal was to use new sequence information along with database comparisons to develop a structural rationalization of how these proteins may function in calcium handling by bone. For example, our analysis has identified a conserved polyacidic stretch in all three phosphoproteins which we propose mediates metal binding. Also, conserved motifs were identified that are analogous with those for casein kinase II phosphorylation sites and whose number correlates well with that of phosphoryl groups/protein. A two-state conformational model of calcium binding by bone matrix acidic phosphoproteins is described which incorporates these findings.

Four sialic acid-rich (SA-rich) proteins found in bone and dentin, osteopontin (OPN), bone sialoprotein (BSP), bone acidic glycoprotein-75 (BAG-75), and dentin matrix protein 1 (DMP1), share some common features. We used SDS-PAGE and Western immunoblots to analyze and compare SA-rich proteins in bone and dentin extracts from rats with a single chromatographic procedure. OPN was detected in dentin extracts, with a relative level less than one-seventieth of that in bone. Both bone and dentin BSP demonstrated an extremely broad distribution pattern, probably due to a high degree of heterogeneity in post-translational modifications. BAG-75 in both bone and dentin was detected as an 83 kDa band, dramatically distinct from that of DMPI. Using a polyclonal antibody raised against a purified bone 57 kDa protein (a portion of DMPI), we detected 150 kDa protein bands in bone fraction; the same bands were recognized by antirecombinant rat DMPI antibody. Bands from dentin migrating at about 150 kDa in earlier fractions and progressing to 200 kDa in later fractions showed a clear immunoreactivity to the anti-57 kDa antibody. We conclude that the majority of DMPI in rat bone is processed into fragments, whereas that in dentin remains intact.

The purpose of this review is to summarize recent functional and structural findings regarding non-collagenous matrix proteins in bone and teeth, to compare gene locations for bone and tooth matrix proteins with loci for hereditary skeletal diseases, and to present several provocative hypotheses which integrate this new information into a physiological context. Hypothesis 1 proposes that the molecular composition of rapidly deposited and mineralized woven bone, as well as the responsiveness of cells synthesizing woven bone to stimuli, is different from that for more slowly synthesized lamellar bone, implying the existence of distinctive osteogenic mechanisms. This review of recent research strongly supports this proposal. Briefly, the protein composition of woven bone matrix is enriched in acidic phosphoproteins BAG-75 and BSP, which are not expressed in lamellar bone, which is itself enriched in osteocalcin. De novo deposition and mineralization of woven bone occurs faster than in lamellar bone by means of a matrix-vesicle-assisted mechanism. Deposition of woven bone occurs at sites experiencing biomechanical strains higher than those experienced by lamellar bone. In addition, woven bone in metaphyseal regions is more susceptible to osteoclastic resorption after space flight, ovariectomy, and loss of weightbearing than is lamellar bone. Finally, osteoprogenitor cells responsive to parathyroid hormone reside in the metaphyseal region of long bones. Taken together, these findings suggest that Hypothesis 1 represents a useful paradigm for future studies.Specific functions mediated by most individual bone and tooth matrix proteins remain uncertain. A review of current literature suggests that the functionality of skeletal matrix proteins is expressed through specific binding sites composed of particular species-conserved structural motifs (Hypothesis 2). Examples include the previously recognized Asp-Ser-Ser motif of dentin phosphophoryns and the gamma-carboxyglutamic acid motif of matrix GLA protein and osteocalcin. A new polyacidic amino acid motif composed of consecutive Asp and Glu residues (n > 7) was defined in extracellular matrix components osteopontin, bone sialoprotein, and bone acidic glycoprotein-75 on the basis of strong functional analogies with similar polyacidic stretches in divalent metal storage proteins of the endoplasmic reticulum and sarcoplasmic reticulum. These structural motifs represent prime targets for future structure-function studies in vivo and in vitro.

Bone mineral density is known to be a heritable, polygenic trait whereas genetic variants contributing to lean mass variation remain largely unknown. We estimated the shared SNP heritability and performed a bivariate GWAS meta-analysis of total-body lean mass (TB-LM) and total-body less head bone mineral density (TBLH-BMD) regions in 10,414 children. The estimated SNP heritability is 43% (95% CI: 34-52%) for TBLH-BMD, and 39% (95% CI: 30-48%) for TB-LM, with a shared genetic component of 43% (95% CI: 29-56%). We identify variants with pleiotropic effects in eight loci, including seven established bone mineral density loci: WNT4, GALNT3, MEPE, CPED1/WNT16, TNFSF11, RIN3, and PPP6R3/LRP5. Variants in the TOM1L2/SREBF1 locus exert opposing effects TB-LM and TBLH-BMD, and have a stronger association with the former trait. We show that SREBF1 is expressed in murine and human osteoblasts, as well as in human muscle tissue. This is the first bivariate GWAS meta-analysis to demonstrate genetic factors with pleiotropic effects on bone mineral density and lean mass.

To define the matrix composition and architecture of canine flexor tendon, and to correlate tissue structure with applied mechanical loading, five anatomical regions of flexor tendon were studied. Histologically, two prominent fibrocartilaginous areas were observed on concave aspects of the tendon. The location of the major fibrocartilaginous area at the metacarpophalangeal joint correlated well with the region predicted by biomechanical modeling to be under greatest compressive loads during standing and claw movement. Comparative biochemical analysis showed an elevated water content, a five-fold higher hexuronic acid content, and a larger hydroxylysine/hydroxyproline ratio in this region relative to that for more tendinous areas. The major glycosaminoglycan component of fibrocartilaginous areas was chondroitin sulfate, whereas in other areas dermatan sulfate and hyaluronic acid dominated. Cell density and DNA analyses indicated a slightly higher cellularity for fibrocartilaginous areas and the region of vinculum insertion. These data document the existence of discrete areas of specialization within the flexor tendon that appear to be an adaptation to nutritional and mechanical factors.

The correlation of carbonate content with enamel microstructure (chemical and crystal structure) and mechanical properties was evaluated via linear mapping analyses using Raman microspectroscopy and nanoindentation. Mappings started at the outer enamel surface and ended in the inner enamel near the dentin-enamel junction (DEJ) in lingual and buccal cervical and cuspal regions. The carbonate peak intensity at 1070 cm−1 gradually increased from outer to inner enamel. Moreover, the phosphate peak width, as measured by the full width at half maximum (FWHM) of the peak at 960 cm−1, also increased, going from ~9 cm−1 in outer enamel to ~13 cm−1 in enamel adjacent to the DEJ, indicating a decrease in the degree of crystallinity of hydroxyapatite from outer to inner enamel. In contrast, Young’s modulus decreased from 119±12 to 80±19 GPa across outer to inner enamel with a concomitant decrease in enamel hardness from 5.9±1.4 to 3.5±1.3 GPa. There were also significant correlations between carbonate content and associated crystallinity with mechanical properties. As carbonate content increased, there was an associated decrease in crystallinity and both of these changes correlated with decreased modulus and hardness. Collectively, these results suggest that enamel carbonate content and the associated change in the crystal structure of hydroxyapatite, i.e. degree of crystallinity, may have a direct effect on enamel mechanical properties. The combination of Raman microspectroscopy and nanoindentation proved to be an effective approach for evaluating the microstructure of enamel and its associated properties.

The impact of genetics has dramatically affected our understanding of the functions of non-collagenous proteins. Specifically, mutations and knockouts have defined their cellular spectrum of actions. However, the biochemical mechanisms mediated by non-collagenous proteins in biomineralization remain elusive. It is likely that this understanding will require more focused functional testing at the protein, cell, and tissue level. Although initially viewed as rather redundant and static acidic calcium binding proteins, it is now clear that non-collagenous proteins in mineralizing tissues represent diverse entities capable of forming multiple protein-protein nteractions which act in positive and negative ways to regulate the process of bone mineralization. Several new examples from the author’s laboratory are provided which illustrate this theme including an apparent activating effect of hydroxyapatite crystals on metalloproteinases. This review emphasizes the view that secreted non-collagenous proteins in mineralizing bone actively participate in the mineralization process and ultimately control where and how much mineral crystal is deposited, as well as determining the quality and biomechanical properties of the mineralized matrix produced.

The activation peptide C4a was isolated from CIS-cleaved C4, the fourth component of complement. The peptide appeared to be homogeneous by electrophoresis on cellulose acetate and by polyacrylamide gel electrophoresis. C4a has a molecular weight of 8650 and an electrophoretic mobility at pH 8.6 of +2.1 X 10-5 cm2 V-1 sec-t. Carboxypeptidase B released approximately 1 mol of arginine per mol of C4a. The partial COOH-terminal sequence was determined to be LeuGln-Arg-COOH. The isolated C4a was spasmogenic for guinea pig ileum at a concentration of 1 AM and it desensitized the muscle (i.e., produced tachyphylaxis) with respect to human C3a anaphylatoxin (at 0.33 AiM) but not with respect to human C5a anaphylatoxin. Increased vascular permeability was observed in human skin after intradermal injection of 1 nmol of C4a, as evidenced by immediate erythema and edema formation. The spasmogenic, tachyphylactic, and vascular activities of C4a were abrogated by removal of the COOH-terminal arginine, a property that is characteristic also of the C3a and C5a ana hylatoxins. Contamination of C4a with either C3a or C5a has teen ruled out by using radioimmunoassays for these peptides. Although C4a is considerably less active than are C3a and C5a, the present observations suggest that C4a constitutes a heretofore unrecognized anaphylatoxin that is related biologically and chemically to the activation peptides of C3 and C5.The two known anaphylatoxins, C3a and C5a, are activation peptides of the complement proteins C3 and C5 (1). In the course of complement activation, the peptides are released from their respective precursors by specific complement enzymes. Both peptides induce histamine release from mast cells (2), release hydrolytic enzymes from neutrophils (3), and cause contraction of smooth muscle and increase vascular permeability (1). C5a, but not C3a, also effects directed migration of polymorphonuclear leukocytes (4) and monocytes (5). The complete amino acid sequences of human C3a and C5a have recently been determined (6, 7) and 36% homology in primary structure has been noted (8). Except for chemotactic activity, expression of the various biological activities depends on the presence of the COOH-terminal arginine residue of both peptides.The NH2-terminal sequence of C4a (9) suggests that a limited structural homology exists between C4a and both C3a and C5a. In view of these apparent structural similarities, a search was conducted for possible biological activities of C4a. The results show that C4a possesses activities that qualitatively resemble those of the two known anaphylatoxins.MATERIALS AND METHODS Purification of C4a. The method of purification will be described in detail elsewhere (10). Briefly, 450 mg of C4, isolated as described (11), was subjected to treatment with 4.5 mg of C1s (12) Fernandez and Hugh (14).COOH-Terminal Analysis. Approximately 50 ,ug of C4a (6 nmol) was boiled in 0.1 M N-ethylmorpholine buffer (pH 8.5) for 10 min. Carboxypeptidase B, 1% (wt/wt), was added and the mixture was ...