Phosphoproteins as Mediators of Biomineralization - ACS Symposium

Chapter 1

Phosphoproteins as Mediators of Biomineralization Arthur Veis, Boris Sabsay, and Chou Bing Wu Division of Oral Biology, Northwestern University, Chicago, IL 60611 Acidic proteins, and phosphorylated acidic proteins in particular, appear to play several important roles in the mechanism of mineralization in biological systems. They may direct the placement of the mineral crystals within and upon the organic matrix of tissues such as bone and dentin. They may also regulate the crystal growth rate. Further they may limit the size of the formed crystals by surface adsorption. Dentin has been selected as a model for the study of these problems because it is a rich source of a unique phosphoprotein, phosphophoryn, which may serve all of these regulatory functions. As a mineralization mediator the phosphophoryn appears to bind to the collagen matrix and to act to complex inorganic ions to initiate calcium hydroxyapatite crystallization in an ordered fashion on the matrix. Studies on the native and dephosphorylated protein show the profound effect of the phosphate groups on the calcium binding properties of the phosphoprotein. The phosphophoryn molecule has distinct domains which may relate to the different regulatory activities postulated for this one molecule. In this paper, a general background on biomineralization is provided, defining concepts and hypotheses in their broadest terms. The dentin system is then used as one model for examination of these concepts and hypotheses. Biomineralization The presence of deposits of inorganic crystals in living organisms is very widespread and the processes of formation of the crystalline phases are grouped together under the name "biomineralization", the "bio" in the term suggesting the active involvement of the cells of the host organism in the process. The relationships between mineral and organic phases are very diverse, but a few years ago Lowenstam and Weiner (1) identified two broad categories: biologically induced mineralization and matrix-mediated mineralization. 0097-6156/91/0444-0001$06.00/0 © 1991 American Chemical Society

2

SURFACE REACTIVE PEPTIDES AND POLYMERS

In biologically induced mineralization, macromolecules of biological origin interact with the microions, usually cations, to initiate a mineral phase. The mineral phase crystals, however, adopt crystal habits similar to those they would have formed from saturated solutions of the microions in the absence of the organic macromolecules. Furthermore, the crystal deposits are essentially random within the organic matrix. That is, the crystal orientations do not appear related to the structure of the underlying matrix. There is no regulation of crystal size. In matrix-mediated mineralization one again has macromolecules of the organic matrix interacting with mineral microions (cations) to initiate the mineral phase. This is the same statement given above, but in this case the mineral phase grows within the preformed organic matrix and, most importantly, the minerals adopt some unique crystal habits with respect to either the nature of the crystal (possibly different from that produced by spontaneous crystallization from a saturated solution of the microions) or the orientation of the crystals relative to the underlying organic matrix. Matrix-mediated mineralization is further characterized by the crystals having a fairly narrow size range. All aspects of matrix-mediated mineralization appear to be highly regulated. In our own studies (2) we had also recognized the distinctions between these two types of biomineralization processes. However, because of our interest in vertebrate systems, and bone mineralization in particular, we focused our efforts on the matrix-mediated mineralization process. We postulated that the matrix within which the mineral crystals grow is itself a two component system. The first part of this system is a structural protein. The organization of this protein defines both the internal architecture of the mineralized tissue and the overall shape of the tissue. The second part of the two component system is another protein, or another macromolecule, which can interact both with the microions to be incorporated into the crystals and with the structural protein. These dual interactions direct the placement and orientation of the initial mineral crystals within the matrix. Further crystallization may develop by growth on the initial crystalline deposits, so that all of the crystallization is not confined to this first phase interaction, but it is this first interaction which establishes the order and crystal habit. Finally, it has to be made explicitly clear that the organic matrix is not homogeneous in composition and that in a mineralizing tissue there are well defined zones with different properties. All of the structure is generated by the cells of the particular tissue. In the immediate vicinity, or territorial matrix, of the cells there is generally a non-mineralized zone, or zone of crystallization inhibition (in bone this is the "osteoid", in dentin the "predentin"). Adjacent to this is a zone where crystal nucleation and growth occurs (in bone and dentin this is the "mineralization front"). Deeper within the mineralized tissue there is a final zone of crystal stabilization. A key tenet of our basic working hypothesis (2) is that specific, interactive matrix macromolecules regulate all stages of the mineralized phase initiation and maturation.

1. VEISETAL.

3

Mediators of Biomineralization

Dentin: Type I Collagen and Phosphophoryn We selected dentin as a relatively simple model for bone mineralization because only a single population of cells, the odontoblasts, is engaged in forming the matrix. No resorptive cells are present and the tissue does not remodel. Like bone, the dentin matrix has Type I collagen as the structural component. The collagen fibers which constitute the matrix are fairly uniform in size. The initial mineralization at the mineralization front, at the boundary between predentin and dentin, takes place in association with the collagen fibers. Several studies directed explicitly to the question (3) have shown that there is very little to distinguish the type I collagen of dentin from the predominant type I collagen of the soft tissues. A large number of noncollagenous proteins (NCP) are present in the dentin matrix, as illustrated in the gel electrophoretic pattern shown in Figure 1. One of these proteins, a highly phosphorylated protein which we have named phosphophoryn (4), is the most abundant of the NCP. The phosphophoryn (PP) is unique to dentin, none has been found in other tissues. However, we believe that there are functional analogs of this molecule in other mineralizing tissues. Bovine PP (bPP) is the best characterized of the phosphophoryns (5). It is a large molecule, M ~ 150,000, containing about 1130 amino acid residues, Table I. Aspartic acid accounts for about 450 residues and serine another 550. Of these serines about 90% are phosphorylated. Thus, about 950 of the 1130 total residues (84%) are potentially anionic. Moreover, since the phosphate groups have a second ionizable group with a pK of 6.8 (6), a substantial number of the phosphoserines must be doubly ionized at physiological pH and ionic strength. Thus, the net charge on the molecule in vivo is on the order of -1300, making it an extremely anionic macromolecule with a net charge per backbone residue > 1. In our very earliest experiments where free flow electrophoresis was used, this protein had the highest electrophoretic mobility of any component in the system and it was initially called the "fast" component. Molecules equivalent to bPP have been found in the dentin of every species thus far examined. The unique anionic character is well preserved, but the apparent molecular weights vary from one species to the next. In the rat incisor riPP has a Mr ~ 70,000; in human dentin the highest weight hPP has M ~ l 10,000. In both of these tissues, in contrast to the bovine case, there may be more than one class of PP, varying in both amino acid composition and the degree of phosphorylation. In the human, the variation in molecular weights may be related to an age-dependent in vivo degradation. r

r

4


Figure 1. Gel electrophoresis of total 0.6 M H Q extract of rat incisor dentin, using a 5 to 15% acrylamide gradient, 0.1% SDS, reduced with mercaptoethanol. Lane A. Molecular weight standards. Lane B. Silver stain of the proteins. Lane C. The same as lane Β but stained with Stains All after silver staining. Note that different bands are stained. Although there are obviously many protein bands, the H Q extract does not solubilize all of the matrix proteins. The band in lane C marked with the arrow is phosphophoryn. It is colored blue with the Stains All but not stained by silver.

1. VEISETAL.


5

Table I. The Amino Acid Composition of Bovine Dentin Phosphophoryn Residue

Number of residues/molecule

Lys His Arg Asp Thr Ser PSer Glu Pro Gly Ala 1/2 Cys Val Met lie Leu Tyr Phe

45 6 3 452 8 27 518 14 6 27 7 1 3 1 3 4 2 2 Total GluNH

2

1130 1

Calculated on the basis of a molecular weight of 155,000. Data of Stetler-Stevenson and Veis (5)

Phosphophoryn is a Domain Structure Protein Attempts to sequence any of the PP have not met with much success. The overriding content of just two amino acids, plus the high content of phosphoserine, makes the problem very difficult from the perspective of direct sequencing. Thus, the sequencing will probably require the cloning of the PP gene and analysis with the use of the techniques of molecular biology. Such studies are under way in our laboratory, as well as in a few others but not much information is available as yet. We have gained some useful information, however, by exarnining the aminoterminal sequences from peptides produced by limited trypsin digestion and by limited acid hydrolysis with weak acids. In this latter procedure the acid hydrolysis cleaves out the aspartic acid residues in peptide sequences. The peptides inserted between the Asp residues are stable to mild acid hydrolysis and can be collected and examined for amino acid composition and sequence. This has turned out

6


to be a formidable task since in addition to free Asp, other free amino acids and some very small peptides (di, tri) were released. However, these data clearly suggest the presence of domains with blocks of sequences such as: {Asp} χ {Asp-(P)Ser} {(P)Ser} ζ The symbol (P)Ser is used to indicate that the residue may be either serine or phosphoserine. Acid hydrolysis does not cleave out every Asp residue and some larger peptides can be recovered. Work on these peptides is in progress. The tryptic digestion studies, also still in progress, have allowed two preliminary conclusions. First, some atypical, low Ser and Asp sequences containing some of the more hydrophobic residues are in the end regions of the molecule, and one peptide with the amincMerminal sequence {Ser-PSer-PSer-Ser-PSer-PSer-Ser-Ser-Ser-} has been isolated. The presence of this peptide confirms the presence of blocks of {(P)Ser} noted above, the details of these experiments will be presented elsewhere. However, the PP molecule can apparently be modeled as comprised of: {H-domain 1} {H-domain 2} {Asp domains} {Asp-Ser domains} {(P)Ser domains} In this model "H-domains" indicate relatively hydrophobic regions with a lower content of Asp and (P)Ser. These studies are very exciting and should ultimately permit a detailed consideration of the PP properties and mechanisms of action. In the meantime these data do suggest that the molecule may be multifunctional in the sense that different domains may be involved in different aspects of the mineralization process. y

z

x

y

z

Phosphophoryn is a Calcium Ion Binding Protein Neither phosphoserine nor aspartic acid side chains in polypeptides have a particularly high binding affinity for calcium ions, yet, as shown in Figure 2, one can distinguish two clear classes of calcium ion binding affinities. The binding constants shown in Table II for the high affinity class, are really quite modest when compared to the binding affinities of intracellular proteins such as calmodulin. However, the bPP makes up for that by its enormous binding capacity. We assume that the weaker second class of binding sites reflects a typical colloidal reversal of charge phenomenon, in which the high positive surface charge brings in counter anions and a second layer of diffusely bound divalent cations. Note that in the experiments of Figure 2 and Table II the calcium ion binding was measured in the presence of 0.5 M KC1, a supporting electrolyte concentration that would swamp out nonspecific ionic interactions. The addition of even millimolar concentrations of C a to bPP in 0.5 M KC1 is sufficient to convert the bPP in dilute solution from a random chain to a more ordered structure, probably to a /J-sheet like conformation. At physiological ionic strength the calcium 2+

1. VHS ET AL.


3.5 h

l/[ca

+ 2

]

F

χ

I0"

4

Figure 2. Double reciprocal plots of the binding of calcium ion to bPP in the presence of 0.5 M KC1, 0.01 M Tris-HCl, pH 8.3. The upper plot is at a bPP concentration of 0.05 mg/ml, lower plot at 0.5 mg/ml. These differences clearly show the non-ideal, bPP concentration dependence of the binding, as well as the biphasic nature of the calcium ion-bPP interaction. Reprinted with permission from ref. 11. Copyright 1987 Springer-Verlag.

8


binding coefficient for bPP is higher than in 0.5 M KC1, on the order of 3.6 χ 10" mof (6). 4

1

Table II. Binding of Calcium Ions to Bovine Dentin Phosphoryn at the High Affinity Sites bPP, mg/ml

0.05 0.50

moles Ca/mg bPP 6

2.28 χ 10" 5.65 χ 10"

6

moles Ca/mole bPP 353 876

Κ,, moles

1

771 780

Determined in 0.5 M KC1,0.01 M Tris.HCl, pH 8.3. Data of Stetler-Stevenson and Veis (Π). Studies with model peptides suggest that repetitive carboxyl-phosphate side chain sequences strongly promote calcium binding and enhance the apparent binding constant for the carboxyl group calcium binding by at least an order of magnitude. Thus repetitive {Asp-(P)Ser}y sequence domains may have a particularly strong role in the calcium ion binding behavior of PP (7). Phosphophoryn is a Collagen Binding Protein Phosphophoryn binds directly to both monomelic collagen in solution or to the surfaces of preformed fibrils. In the in vivo situation the odontoblast secretes the collagen matrix and forms it into fibrils. The PP is secreted separately and then binds to the surfaces of the already formed fibrils (8,9). The binding of bPP to collagen is essentially electrostatic and can be reduced at high ionic strength, but it is so strong as to have many elements of specificity. In experiments in which I-bPP was added to collagen, cold bPP could displace the labeled bPP. However, phosvitin, an unrelated but equally highly phosphorylated protein required six-fold higher concentrations to displace an equivalent amount of I-bPP from collagen (10). Serum albumin and other matrix proteins were unable to displace significant quantities of bPP from collagen. 125

125

Phosphophoryn Bound to Collagen Fibrils Enhances Calcium Ion Binding of the Fibrils The uptake of calcium ion by bPP-conjugated to collagen fibril surfaces was studied in an effort to determine if the electrostatic interaction between collagen and bPP diminished the ability of the bPP to bind calcium ions in solution (9). The data on the uptake of C a onto cold bPP-collagen fibers was unequivocal in showing that collagen fibers with associated bPP interacted with calcium as avidly as did free bPP. Figure 3 compares the calcium binding to collagen fibers 45

1. vus ET AL


[ 45

4 5

2

Ca* ]

9

Added, mM

Figure 3. The uptake of C a by collagen in the presence of bPP, upper plot [ · ] , and in the presence of osteonectin from bone, lower plot [^]. The osteonectin data is essentially identical to that of collagen alone. The collagen concentrations, and the bPP and osteonectin concentrations were constant in every analysis in each assay. Reprinted with permission from ref. 10. Copyright 1986 Springer-Verlag.

10


in the presence and absence of surface associated bPP. The phosphate groups on the bPP were very important. Dephosphorylation markedly diminished both the calcium binding affinity and total binding, in accord with the peptide analog studies (7). In the studies described above in which I-bPP was added to preformed collagen fibers, the binding of the bPP to the collagen was on the fiber surface. In the bPP concentration ranges studied, binding never reached saturation and calcium binding to collagen fibers has not been studied under bPP saturation conditions. In many of the early discussions of the potential role of PP in mineralization, a prime argument raised by critics of the idea was that the molar ratio of PP to collagen was less than one to one. Since the PP binding is to the fiber surfaces, and since one molecule of PP may sequester hundreds of calcium ions, it can function readily as a nucleating agent. 125

Phosphorylation of Phosphophoryn The phosphorylation of PP is an extremely interesting problem. At the moment we have no knowledge of either the mechanism of phosphorylation nor the kinases involved. The intracellular locus of phosphorylation is not known. The probable amino acid sequences within the Ser-rich domains of the PP are not known to be substrates for any of the known kinases. We have therefore begun a study of the protein kinases in order to determine what type of kinase might be involved. We selected ROS 17/2.8 cells, an osteoblast-like tumor cell line, as an appropriate source for kinases which might phosphorylate extracellular matrix phosphoproteins. An assay system was developed which permitted the specific identification, activation or inhibition of each of the known kinases. Native riPP, without any treatment to remove phosphate groups already present, was found to be a substrate for a ROS 17/2.8 kinase. However, application of the assay system to determine the kinase responsible showed that the kinase was unique. That is, none of the messenger dependent or messenger independent kinases thus far described in the literature was responsible. The kinase, which we have named DPP-kinase, shares some features in common with casein kinase II. They are both cAMP-, cGMP-, and Ca -independent; they can both utilize ATP and GTP as phosphate donors; heparin, spermine and sodium chloride inhibit both kinases; and, they both favor acidic substrates. However, the DPP-kinase is associated with the membrane bound, cell particulate fraction whereas the casein kinase II is found in the cytosol. Moreover, the two kinases have distinctly different pH optima for maximal activity. The DPP-kinase has a higher activity on the residual serines of non-dephosphorylated phosphophoryn than casein kinase II has on partially dephosphorylated casein. Further work on this very important problem requires a better definition of the sequences which are the substrates for the DPP-kinase, and a detailed study of the rephosphorylation of the dephosphorylated PP. These studies are in progress. 2+

1. VEIS ET AL.


11

Generalizations on Biomineralization Based on Observations on the Dentin System The development of dentin does appear to be an example of matrix-mediated mineralization. Although the role of each of the components has not been determined rigorously, we can consider the dentin system in terms of our basic postulates (2). The type I collagen fiber network surely serves as the structural framework which defines the shape of the tooth, the spaces within which the crystallization proceeds, and determines the orientations of the crystallites. For all of the reasons described above, we propose that the highly anionic phosphoprotein, phosphophoryn, is the principal bifunctionally interactive protein which, by interacting with the preformed collagenfibrilsin a fairly specific manner, also determines, via its strong interaction with calcium ions, the locus of crystal nucleation within the collagenfibernetwork. The cellular control of this extracellular process of regulated crystallization residues in the repertoire of proteins produced for secretion and their delivery to the extracellular space. For example, the cellular sequestration of the collagen and phosphophoryn is such that the phosphophoryn is delivered directly to the mineralization front and deposited upon the collagen fiber network. A host of other macromolecular NCP components must also operate in regulation of the system. It is likely, for example, that one or more proteoglycans may inhibit induced mineralization within the predentin, that phosphatases present near the mineralization front may delay mineralization by dephosphorylating proteins which might otherwise induce premature mineral crystal initiation, and that other anionic components within the mineralization region might limit crystal growth by binding to growing crystal surfaces. We believe that many biomineralization systems follow this same overall strategy for regulation of matrix mediated mineralization. The specific macromolecular components may vary depending upon the use and required metabolic stability of the mineral phase, but components of corresponding function will be found in quite different systems. Where proteins are involved in the process, phosphorylated species are likely to be involved because of the advantage such groups provide relative to carboxyl groups in enhancing calcium binding affinity. Where glycosaminoglycans are involved, the degree or nature of the sulfation may play a comparable regulatory role. Literature Cited 1. Lowenstam, Η. Α.; Weiner, S. In Biomineralization and Biological Metal Accumulation, Westbroek, P.; DeJong, E. W., Eds.; D. Reidel: Doredrecht, 1983;p191. 2. Veis, Α.; Sabsay, B. In Biomineralization and Biological Metal Accumulation, Westbroek, P.; DeJong, E. W., Eds.; D. Reidel: Doredrecht, 1983;p273. 3. Volpin, D.; Veis, A. Biochemistry 1973, 12, 1452.

12 4. 5. 6. 7. 8. 9. 10. 11.


Dimuzio, M . T.; Veis, A. Calcif. Tissue Res. 1978, 25, 169. Stetler-Stevenson, W. G.; Veis, A. Biochemistry 1983, 22, 4326. Lee, S. L.; Veis, Α.; Glonek, T. Biochemistry 1977, 16, 2971. Lee, S. L.; Veis, A. J. Peptide Protein Res. 1980, 16, 231. Weinstock, M.; Leblond, C. P. J. Cell Biol. 1974, 60, 92. Maier, G. D.; Lechner, J. H.; Veis, A. J. Biol. Chem. 1983,258, 1450. Stetler-Stevenson, W. G.; Veis, A. Calcif. Tissue Int. 1986, 38, 135. Stetler-Stevenson, W. G.; Veis, A. Calcif. Tissue Int. 1987, 40, 97.

RECEIVED August 27, 1990

Phosphoproteins as Mediators of Biomineralization - ACS Symposium

Recommend Documents