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The Structural Biology of the Developing Dental Enamel Matrix

A. G. Fincham ,* J . Mora dian -Oldak ,* an d J . P. Simmer

*Center for Craniofacial Molecular Biology, School of Dentistry, University of Southern California, Los Angeles, California 90089;

an d University of Texas S chool of Dentistry, Health S cience Center at S an Antonio, Department of Pediatric Dentistry, S an Ant onio, Texas

Received Ja nua ry 21, 1999, and in r evised form April 26, 1999

T h e b i o m i n e ra l iz a ti o n o f t h e d e n t a l e n a m e l m a -

tr i x w i th a c a r b o n a te d h y d r o x y a p a ti te m i n e r a l g e n -

e r a t e s o n e o f t h e m o s t r e m a r k a b l e e x a m p l e s o f a

v e r t e b r a t e m i n e r a l i z e d t i s s u e . R e c e n t a d v a n c e s i n

t h e m o l e c u l a r b i o l o g y o f a m e l o b l a s t g e n e p r o d u c t s

h a v e n o w r e ve a l e d t h e p r im a r y s t ru c t u re s o f t h e

p ri n c i pa l p r ot e i n s i n v o lv e d i n t h i s e x t ra c e ll u la rm i n e r a l i zi n g s y s t e m , a m e l o g e n i n s , t u f t e li n s , a m e l o -

b l a s t in s , e n a m e l i n s , a n d p r o t e i n a s e s , b u t d e t a i l s o f

t h e i r s e c o n d a ry, t e r ti a ry, a n d q u a t e rn a r y s t ru c -

t u re s , t h e i r i n t e r ac t i on s w i t h o t h e r m a t r ix a n d o r

c e l l s u r f a c e p r o t e i n s , a n d t h e i r f u n c t i o n a l r o l e i n

d e n ta l e n a m e l m a tr i x m i n e r a l i z a ti o n a r e sti l l l a r g e l y

u n k n o w n . T h i s p a p e r r e v i e w s o u r c u r r e n t k n o w l -

e d g e o f t h e s e m o l e cu l e s , t h e p r ob a bl e m o l e cu l a r

s t r u c t u r e o f t h e e n a m e l m a t r i x , a n d t h e f u n c t i o n a l

r o le o f t h e s e e x t r a c e ll u l a r m a t r i x p r o t e i n s . R e c e n t

s t u d i e s o n t h e m a j o r s t r u c t u r a l r o l e p l a y e d b y t h e

a m e l og e n i n p r o t e in s a re d i s c u ss e d , a n d s o m e n e w

d at a o n s yn th e ti c a m e lo ge n in m a t ri ce s a re

r e v i e w e d . 1 9 9 9 Ac a d e m i c P r e s s

K ey Wor d s : a m e l o g e n e s i s ; b i o m i n e r a l i z a t i on ; d e n -

ta l e n a m e l p r o te i n s;d e v e l o p m e n t;fu n c ti o n ;h y d r o x y -

a p a t i t e .

INTRODUCTION

. . . It seems t o me th at m uch which is par adoxical in enam elis tied up with the par ticular an d perhaps unu sual relation-ship which exists bet ween its organ ic and inorganic compo-nent s. The organic matr ix seems to specifically exist for t he

eventual creation of a structure which is quantitativelyalmost completely inorganic and yet possesses the largesthydroxyapatite crystals consistent with, firstly, their abilityto be nucleated and to grow in a biological system and,secondly, maintenance of sufficient elasticity in the enamelsystem as a whole to prevent its har d stru cture from beingtoo britt le. (J ohn E. Ea stoe, 1971)

Dental enamel is the most highly mineral izeds t ructure in the ver tebra te body and is formedwithin a u nique, extr acellular ma tr ix derived throughth e synth esis and secret ion of proteins by th e am elo-blast cells of the inner enamel epithelium. Maturedental enamel h as a complex form, providing a

striking example of a highly mineralized structureexquisitely adapted to absorb essential mechanicalan d abr asive stresses thr oughout t he lifetime of theorganism (Fig. 1). Recent studies have shown thatsecretory stage a meloblasts secrete a number ofproteins, some of which appear to be unique t o thedental enamel matr ix. Dental enamel differs from

other vert ebra te miner alized tissues (e.g., bone, car -tilage, an d dent ine) in tha t it is n oncollagenous, ofepithelial origin, and does not undergo resorptionand remodeling. Recent reviews of dental enamelstructure and ameloblast biology include those bySimmer and Fincham (1995), Thesleff (1995), Skobeet al. (1995), Shore et al. (1995a,b), Robinson et al.(1995a), Thesleff and berg (1997), Boyde (1997),Sasaki et al. (1997), an d Smit h (1998).

This a rt icle will consider curr ent knowledge of thestr uctu re a nd m olecula r composition of th e extra cel-lular ma tr ix of th e developing dent al ena mel, viewed

as a biominera lizing system. F irst, we consider t herequ iremen t for an effective extr acellula r min era liz-ing matrix and comparisons of the dental enamelmatrix with other biomineralizing systems. Second,we review, in some detail, current knowledge of themolecular composition of the enamel matrix. Third,we consider the structure and putative function ofthe mat r ix and the in ter re la t ions of the mat r ixproteins with t he miner al pha se. Four th, we brieflydiscuss t he pr operties of the enam el mat rix protein-ases a s th ey relate t o th e dynam ics of am elogenesis.Finally, we consider outstanding issues regarding

the dent al enam el mat rix and its components.1. ENAMEL AND ENAMELOID

. . . I think it quite reasonable to assume t hat enameloid isthe ancestral tooth crown covering . . . (R.W. Fearnhead, 1979)

Ena mel an d enameloid form the highly mineral-ized outer la yer of th e vertebr at e tooth , providing thehard abrasion-resis tant surface essent ial for theanimal to capture and consume its food. While thegross tooth form varies according to function be-tween both order an d species, the m ineral pha se ofboth of th ese tooth str uctu res is conser ved.

J ourna l of Str uctur al Biology 126, 270299 (1999)Article ID jsbi.1999.4130, available online at http://www.idealibrary.com on

27 01047-8477/99 $30.00Copyright 1999 by Academ ic PressAll rights of reproduction in any form reserved.


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A carbona ted calcium apa tite comprises the biomin-eral phase of both ma tur e enamel and mat ur e ena m-eloid str uctures in vertebrates, where i t occurs asoriented crystallites formed within a secreted extra-cellular protein matrix. In mammalian enamel, themineral is present a s str uctur ed arr ays of crystalswhose bounda ries a re defined as ena mel pr ismsorpr ism sh ea th es(see Fig. 1). In t he en am eloid of th eChondrichthyes (sharks and rays), Osteichthyes(bony fishes), an d in some Amphibia (frogs, t oads,and newts), a prismatic enamel stru cture is gener-ally absent an d t his apr ismatic stru ctur e a ppearsas one crite riu m for dist ingu ishin g en am eloidfrommammalian enamel.However, enamel and enam-eloid structures are functionally comparable andspecies-specific in form . Ena meloid str uctu re a nd itscomparative histology have been the subject of muchstu dy (Poole, 1967; Shellis, 1975; Shellis a nd Miles,1976; Meinke, 1982; N an ci et al., 1983; Kawasakiand Fear nh ead, 1983; Clement, 1984; Kemp, 1985;Uehar a a nd Miyoshi, 1987; Sasagawa an d Ishiyama,

1988; Pr ostak a nd Skobe, 1988; Pr osta k et al., 1989;Sasa gawa, 1989; Miake et al., 1991).

If both enamel and enameloid are functionallycompa ra ble an d ar e also miner alized with a comm onapa titic biominera l phase, how then m ay these stru c-tur es be distinguished? P oole (1967) distinguishedena meloid from ena mel on th e basis of its cont ent ofcollagen, which is not seen as a component of theena mel protein of th e higher vert ebra tes. Beyond thepresen ce of collagens (prin cipally Type I) a nd otherma tr ix proteins t ent at ively identified a s ena melins(Graham, 1985; Deutsch et al., 1991), little inform a-tion exists regarding tissue-specific enameloid m a-trix components. An apparent difference betweenenameloid matrices and the enamel extracellularmatrix is the presence of matrix vesicles in theformer, structures which have never been reportedfor th e ena mel m at rix. Also, a nota ble difference lieswithin the mineral phases, which in some species ofshar ks an d other fishes cont ain a high proport ion ofa carbonated fluorapatite (Suga et al., 1978, 1981).

FIG. 1. The organization of dental enamel. Scanning electron micrograph of the surface of an acid-etched ground section of maturemouse incisal dent al ena mel. Ordered ar rays of enamel prisms a re ea ch constr ucted of parallel bundles of carbonated h ydroxyapat ite

enamel crystallites. Ea ch prism st ructure is considered to ar ise as the product of a single secretory ameloblast cell of the inn er ena melepithelium . (Inset) An enlar gement of th e center s ection of the ima ges as in dicated. [Electr on micrograph court esy of Dr. W. Luo.]

271DENTAL ENAMEL MATRIX BIOMINERALIZATION


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Beyond these differences, however, a key issue ap-pears to be the presence, or absence, of the amelo-genin proteins in the enam eloid tooth m at rix.

Herold et al. (1980), employing immunofluorescenttechniques with antisera obtained from prepara-tions of bovine enamel matrix,reported the pres-ence of amelogenins in the enameloid of teeth anddermal denticles of Chondrichthyes, in the enam-

eloid of Teleostei an d Amph ibia, an d in th e ena mel ofReptilia. However, t he nonspecific char acter of t heantisera employed in this study provides cause fordoubt as to th e significance of these findings. Ka-wasaki et al. (1980), in an exhaustive biochemicals tudy of the proteins extracted from the dermaldenticles of the blue sh ar k (Prionace glauca), failedto identify a ny m ater ial chemically resembling a m-elogenins. More recently, Slavkin an d Diekwisch(1997), employing RT-PCR techniques, have re-ported evidence for the expression of amelogeninproteins in t he nonminera lized kerat inized teeth of

the agnathan, Pacific hagfish, although the validityof these observat ions h ave been cha llenged by morerecent st udies (Girondot and Sire, 1998; Ishiyam a etal., 1998).

Thus, while differences in both the extracellulartooth mat rices an d th e minera l phases of enam eloidand enamel have been described, the details andsignifican ce of these observat ions r ema in cont rover-sial. (For discussions of the evolutionary origins ofenam el, ena meloid, and the amelogenins, see Huys-seune a nd Sire, 1998; Smith an d Coat es, 1998; an dGirondot a nd Sire, 1998.) In th is paper our furt her

t reatment is limited to the biomineral izat ion of denta l enam el.

2. ENAMEL AS A BIOMINERALIZING MATRIX

It would seem to be an eart h un ited with a portion of an imalsubstance. (John Hunter, 1771)

As Ea stoe has n oted (East oe, 1979), J ohn Hun ter,the En glish surgeon an d ana tomist, was perha ps thefirst person to recognize that dental enamel con-tained ma terials oth er tha n solely minera l material.Much later it was established that immature dental

enamel conta ined a substantial proportion (circa19% by weight) of organ ic mat eria l (Deakins, 1942),establishing this str ucture a s a biominera lizing ma -trix. Pa rtial am ino acid ana lyses of matu re enamel(Block an d Bolling, 1952) led to th e su ggestion th atthe enam el mat rix protein was probably a eukera -tin, a concept which a ppeared reasonable a t thetime in view of the epithelial origins of the struc-ture or, alternatively, by analogy with bone anddentine, possibly a collagen.

Lowenst am an d Weiner (1989) ha ve described th eprocesses under which organisms form biominerals

in the following steps: (i) delineation of space; (ii)existen ce of a pr eform ed organ ic ma tr ix; (iii) crea tionof a saturated ion solution; (iv) control over crystalnucleation; (v) control over crystal growth; and,fina lly, (vi) cessation of cryst al growth. Th ese s tepsresult in the formation of biological crystals withunique morphologies and highly ordered structures.Enamel biomineralization also involves these pro-

cesses, but in a unique ma nner compared to someother mineralizing systems. In enamel, the matr ixfor biomineralization is not preformedbut is con-tinuously secreted and assembled by the amelo-blasts, which play active roles in protein synthesis,secretion, ion tra nsport, an d eventua lly protein ma-trix resorption. In enamel, we envisage these pro-cesses a s follows: (i) Secret ory a meloblast s (Tomesprocesses) and the DE J delineat e the en amel space.(ii) Secreted a melogenin proteins a ssemble, forminga supramolecular structural framework. (i i i) Cal-cium and phosphate ions are transported into theextracellular matrix by the ameloblasts, resulting ina supersaturated solut ion. (iv) The crystals arenucleated either on preexisting dentin m at rix or byother nonamelogenin ma tr ix molecules. (v) Crystalgrowth, morphology, and orientation are controlledby th e m at rix (pr obably am elogenin na nosph ere s).(vi) Cessation of initial crystal growth is indirectlydetermined by the eventual degradat ion and re-moval of the m at rix. (vii) Finally, in ena mel, th ere isa ma tu ra tionstep du ring which a ph ysical ha rden -ing occur s th rough r apid crysta l growth concomita nt

with cont rolled protein pr ocessing, degra dat ion, an dloss. This latter stage is perhaps unique to dentalenamel.

In enamel, the proteins form an organic matrixframeworkmost of which disappears after it com-pletes its function, leaving behind the highly ori-ented, largely inorganic, unique enamel structure.The orga nic ma tr ix appea rs to self-assemble thr ougha relat ively low-ener gy mechan ism (hydrophobic in-tera ctions between am elogenin molecules) whichdoes not involve proteinprotein covalent cross-linking reactions as occur in bone collagen. More-

over, this self-assem bly process result s in t he form a-tion of thermodynamically stable structures (i .e.,spheres), which appear to be the basic buildingblocks of the enamel extracellular matrix frame-work. It is the pr imary st ructur e of the am elogeninmolecule th at allows th e ma tr ix assem bly to occur insuch an intr iguing an d simple man ner. The sequenceof th e am elogenin m olecule is char acter ized as beingbipolar with two distinct regions. The carboxy-terminal telopeptide (1315 amino acid residues) ishighly charged, having an isoelectric point (pI) ofabout 4.2, compa red t o the bulk of the m olecule with

272 FINCH AM, MORADIAN-OLDAK, AND SIMMER


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a pI of 8.0. This hydrophilic carboxy-terminal isprobably external ized on the amelogenin nano-sphere surface creating negatively charged struc-tures which will interact with the forming enam elapat i te crystals at a very early s tage of enamelformation. Whether the enamel extracellular ma-tr ix, as a m iner alizing tissu e, ne eds th e ma jor a cidicma cromolecules (Lowensta m an d Weiner, 1989,

Cha p. 2, p. 22) for cont rolling crysta l nu cleat ion a ndgrowth, or whether amelogenin uniquely providesboth an acidic and a hydrophobic structure withinthe same molecule is a question requiring furtherexamination.

3. COMPONENTS OF THE MATRIX

The enam el component of teeth is such a beautifully orga-nized system of orientat ed hydroxyapat ite crystallites tha tone must s urely expect th at h ere, par excellence, is the placeto tra ce out th e part played by collagen, if th e enam el mat rixreally is collagen. That is th e question, th ough, for enam el is

very compact and there is compara tively little pr otein leftonce th e inorganic salt s h ave been r emoved, . . . (William T.Astbu ry, 1961)

Piez (1960) obta ined t he fir st complete a mino acidanalysis of the matrix protein of developing enamelfrom an unerupted human third molar tooth andconcluded that the protein was nearly all soluble inth e decalcifying solu tion . . .an d it . . . conta ined nohydr oxyproline or hydr oxylysine . . . an d . . . wasun like any oth er kn own protein.These ea rly ana ly-ses were further refined by Ea stoe (1963), whoreported that foetal enamel protein had a unique

composition with several characteristic features . . .distinguishing it from collagen, epiderm is, otherkeratins and proteins from matu re enamel. Thisun ique compositionwa s cha ra cter ized by high pr o-portions of proline, glutamyl, histidine, and leucineresidues, and subsequently became recognized asthat of the amelogeninprotein, which is the domi-nant component ofthe early (secretory-stage)enamelma tr ix (Ea stoe, 1965).

3.1. Isolation of the Matrix Proteins

It can be assum ed that the embryonic enamelin in deaggre-gating solvents constitutes a reassociating system of pro-teins centered around four distinct molecular weight aggre-gates which are multicomponent. These multicomponentaggregates are also in dynamic equilibrium with high andlow molecular weight ma ter ial. (Gera ld Mecha nic, 1971)

Further studies established that the developingena mel ma tr ix cont ain ed a complex of closely relat edamelogenin proteins ra nging in appa rent molecularmass from some 527 kDa (Seyer and Glimcher,1977a; F incha m et al., 1983). From the mid-1960sthrough the 1970s, several groups of researcherssought to isolate and further characterize these

am elogenin pr oteins (Glimcher et al., 1964; Burgessan d MacLaren , 1965; Nikiforuk a nd Simm ons, 1965;Mechanic, 1971; Eggert et al., 1973; Papas et al.,1977; Seyer and Glimcher, 1977a; Fincham, 1979;Fukae et al ., 1979). Generally, standard proteinchemistry procedures (size exclusion, ion-exchangechromatography, and preparative electrophoresis)were applied to the problem of fract ionat ion of

preparat ions of developing enamel proteins ex-tracted from porcine or bovine sources. Frequently,these s tudies led to incomplete separat ions andconfusing dat a (see r eview by Ea stoe, 1979). Quiteearly in this research, evidence suggest ing thatproteinprotein interactions (aggregation effects)were t he confounding factor (Katz et al., 1965), evenin the presence of dissociative reagents (Mechanic,1971).

Termine et al . (1980) introduced an importanttwo-step sequ ent ial d issociat ivemodifica tion to t he

usua l demineralizing extra ction procedures. In th isprocedure, the immatu re enam el ma trix prepara tionwas initially extracted into a buffered guanidinehydrochloride solution, followed by extraction in thesa me dissociat ing medium , but n ow cont ain ing EDTAas a demineralizing agent. Termine et al . (1980)showed that the initial guanidine extraction yieldedthe bulk of the m atr ix protein (90% by weight)with an amino acid composition characteristic ofamelogenin, while the second stage, demineralizingextraction, pr oduced a prepar at ion with a composi-tion typically enriched in a spart yl, serine, a nd gly-

cine am ino acid residues which was termed enam-elin, again after the suggestion of Eastoe (1979).SDSPAGE demonstrated that both of these ex-tr acts were heterogenous, with the ena melin prepa -ra tion being predomina nt ly composed of proteins inthe range of 4070 kDa, compared to the 5- to27-kDa-sized amelogenin fraction. This work is im-portant from two aspects. First, it provided a rela-tively simple, defined, and reproducible procedurefor the separation of the amelogenin fraction fromthe more acidic (mineral bound) enamelins of t he

matrix and has subsequently been extensively em-ployed for this pu rp ose. Second , however, th e separ a-tion obtained by t his procedure is certainly notquantitative or completely selective (Fig. 2), whichha s led to difficulties of dat a int erpr eta tion in experi-ment s where the separa tion ha s been assu med to bedefinitive. Cur ren t experience suggests th at dissocia-tively extra cted a melogenins will certa inly includematrix proteins such as the ameloblastins (see be-low), while comparable enamelin preparations arecerta inly heter ogenous. In a ddition, th e term enam -elin, as applied to the dissociatively extra cted prepa -



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ra tion, mu st now be clearly distinguished from thematrix protein of that name (see below). In thesections which follow, an outline of the history andcharacterist ics of the several matrix proteins ispresented.

3.2. The Am elogenins

Hence, the intact extracellular murine amelogenin is 180am ino acid residu es long with a calculat ed molecular weightof 23,532 Da. (Snea d et al., 1985)

Amelogenin has been shown to be crit ical fornorma l ena mel forma tion. The huma n amelogeningene is express ed prima rily from a sin gle gene on t heX chr omosome, a lthough minor expression (10%)from t he Y-chr omosoma l copy of th e a melogenin geneha s been detected (Lau et al., 1989; Fincham et al.,1991a; Salido et al., 1992). The X-chromosoma l copyof the human amelogenin gene has been linked toam elogenesis im perfecta or AI (Lager st rom et al.,1990), a disease characterized by inherited enameldefects. E ight different am elogenin mut at ions havebeen characterized in kindreds suffering from AI(Lager st r om et al., 1990; Aldred et al., 1992; Lenchan d Brook, 1997; Lagerst rom-Fer mer et al., 1995;Lench and Winter, 1995; Collier et al., 1997). Inrodent s, am elogenin expression ha s been specificallyand art ificially interrupted using both ant isenseoligonucleotides (Diekwisch et al., 1993) and ribo-zyme approaches (Lyngstadaas et al., 1995). In bothcases knockout of amelogenin resulted in defectiveenamel formation. These data strongly support aspecific an d highly conserved functiona l role foram elogenins in enam el biominer alization.

3.2.1. S equences. Amino acid sequen ces of amelo-genins were initia lly deter mined by Edm an sequ enc-ing of the purified proteins and later derived fromDNA sequences of a melogenin cDNAs. Protein se-quen cing ha s been report ed for a melogenins isolatedfrom pigs (Fukae et al., 1979, 1980; Fukae andShimizu, 1983;Yam ak oshi et al., 1989), cat tle (Seyer,1972; Zalu t et al., 1980; Fincha m et al., 1981; Takagiet al., 1984; Shimokawa et al., 1987), hu ma ns (Fin-cham et al., 1983; Cata lano-Sherma n et al., 1994),ra bbits (Zeichn er-David et al., 1988), ham ster s (Lya-ruu et al., 1998), opossu ms (H u et al., 1996a), guineapigs (Cerny an d Ha mmer strom, 1998, 1999), an d,most recently, platypuses, echidnas, caimans, andtoads (Toyasa wa et al ., 1998). These data haverevealed a striking level of homology of sequencebetween species (Simmer and Snead, 1995), espe-cially in the amino- and carboxy-terminal regions(see Fig. 3). Further, the amelogenins have beenshown t o be completely tissu e specific to the am elo-blast cells of th e denta l enam el orga n.

Compar ison of th e k nown a melogenin sequenceshas shown that, while variations in sequence arefound in the more centr al region (core) of themolecule, motifs of th e prima ry str uctu re a re a lmostcompletely conserved at both the carboxy- and theam ino-ter mina ls, suggesting th e conservat ion of spe-cific functional motifs. Indeed, a recent study of acase of hu ma n X-linked amelogenesis imperfecta has

FIG. 2. Sequential dissociative extraction of bovine enamelma tr ix. Fetal bovine ena mel scrapin gs (8.2 g, moist weight ; circa 6months in utero) were sequentially extracted according to theprotocol of Termine et al. (1980). Extracts were desalted (Biogel P2column ), lyophilized, and weighed. Assuming 16% wat er (based ondry-weight estimation) and 70% inorganic content (based onash-weight determinations), total matrix protein equals 2066 mg.Recovery of prot ein , 1383 m g (67%). Amelogen in fr act ion,97.2%.(A) Recoveries of sequent ial extr acts (90% in extr act 1). Tra nsi-

tion from guanidine HCl t o guanidine HCl EDTA is betweenextracts 5 and 6 (double arrow). (B) Fifteen percent SDSPAGE ofselected extra cts from the sepa ra tion shown in A (silver sta ining).Note the increase in t he pr oportion of higher molecular weightprotein (enamelin fraction) in extracts 6 and 8. Also note theapparent presence of amelogenin-sized (26-kDa marker) materialstill present in the demineralized extracts.



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revealed th e molecular lesion a s being a point m ut a-tion in the amelogenin Xgen e leading to th e tr an sfor-ma tion of proline-40 (Hu ma n AMGX) to a th reonineres idue (Collier et al., 1997).

The principal opossum amelogenin isoform of202 residues (Hu et al., 1996a) includes a strikingprol ine-r ich repet ive sequence of 27 res idues :-QPIQPIQPIQPIQPMQPMQPMQPMQPM- withinexon 6, an equivalent of which is n ot seen in mouse,rat, human, pig, or hamster amelogenins, although

similar m otifs are pr esent in both bovine an d guineapig a melogenins. Su ch repetitive pr oline-rich se-quences are suggested to function in reversible pro-teinprotein interactions (for a review of this topicsee Williamson, 1994). The rabbit (Lagomorpha)appea rs t o express an am elogenin (Zeichn er-David etal., 1988) which m ay differ significan tly from other sso far examined in conta ining elevated levels ofalanine a nd a reduced conten t of gluta myl residues(Levine et al ., 1967; Wright and Eastoe, 1989),although definitive amino acid sequence data haveyet t o be obta ined (see F ig. 3).

3.2.2. Alternative splicing. The cha ra cterizationof am elogenins isolated from developing ena mel h asrevealed tha t am elogenins ar e the tra nslation prod-ucts of alter na tively spliced RNA messa ges and t ha tmost amelogenin polypeptides have been processedby proteinases. The alternative splicing of amelo-genins wa s first proposed to explain th e origin of theleucine-rich am elogenin polypeptide (LRAP) whichwas identical t o the ma jor am elogenin protein at itsamino a nd car boxyl-term ini but lacked a large seg-

ment from th e center of the protein (Fincha m et al.,1981; Sasa ki et al., 1984).

The acronym LRAP was first used to describetwo leucine-rich amelogenin polypeptides (LRAP-1and LRAP-2)isolated from developing bovine enamel(Fincham et al., 1981). These LRAP molecules weresequenced and found to have the same sequence of42 and 46 amino acid residues (LRAP-2 having 4additiona l residues a t its carboxytermina l end). Ini-tially, it was presumed that these peptides, as wasthe case for TRAP(the amino-terminal 44/45 resi-due sequ ence of the pr incipal a melogenin), were th e

FIG. 3. Comparison of amino acid sequences. The first 50 residues of the N-terminal region of amelogenins are shown with thecons erved TRAP sequen ces (res idues 145) and t he cons erved pr oteolytic TRAP cleava ge locus -GW-. The six cons erved t yrosine re sidu es ofTRAP a re sh own in bold. Sour ces of data ar e as follows: cattle (Seyer, 1972; Zalut et al., 1980; Fincham et al., 1981; Takagi et al., 1984;Gibson et al., 1991); pig (Fukae et al., 1979; 1980; Fu kae an d Shim izu, 1983; Yam akoshi et al., 1989; Hu et al., 1996b); huma n (Fincham etal., 1983; Salido et al., 1992; Catalano-Sherman et al., 1994); rabbit (Zeichner-David et al., 1988; Fincha m, 1999, unp ublished dat a); mouse(Lau et al., 1992; Simmer et al., 1994a; Simmer, 1995; Hu et al., 1997a); ra t (Bonass et al., 1994a,b; Li et al., 1995); opossum (Hu et al.,1996a); ha mst er (Lyaru u et al., 1998); platypus, echidna , caiman, a nd t oads (Toyasa wa et al., 1998); guin ea pig (Cern y and Ha mm ers tr om ,1998, 1999).



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amino-terminal fragments of a larger (20 kDa)unidentified amelogenin. However, the later studyby Gibson et al. (1991) showed that LRAP in fact istra nslated from a shorter mRNA tha t has th e codingregions from exons 4, 5, and pa rt of 6 deleted dur ingsplicing (Bonass et al., 1994a). The sequence deter-min ed by Gibson et al. (1991) codes for 59 amino acidresidues, including th e conserved car boxy-term inal

13 r esid ues: -WPATDKTKREEVD. Thu s, LRAP,a soriginally described, is th e proteolytically pr ocessedproduct of th e alter na tively spliced am elogenin iden-tified b y Gibson et al. (1991). For th e pur poses of th isreview, LRAP is now used to r efer to th e full-length(59-residu e) a melogenin.

Alterna tively spliced amelogenin mRNAs ha venow been isolated from mouse (Lau et al., 1992;Simmer et al., 1994a, 1995; Hu et al., 1997a), rat(Bonass et al., 1994a,b; Li et al., 1995), hamster(Lyaruu et al., 1998), guinea pig (Cern y and H am ma r-str om, 1998, 1999), pig (Hu et al., 1996b), cattle(Gibson et al., 1991), opossum (Hu et al., 1996a), an dhuman (Salido et al., 1992). For reviews see Sasakiand Shimokawa (1995), Simmer and Snead (1995),Brookes et al. (1995) an d Gibson et al. (1998).

In ea ch species investigated th ere a ppears to be ama jor expr essed am elogenin isoform ra nging in sizefrom 173 (pig) to 210 am ino acids (guinea pig) (Cern yan d H am ma rst rom, 1998, 1999). At t he m RNA level,the LRAP message is about 10% as abu nda nt a s themessa ge for th e ma jor a melogenin isoform , which isprobably also a good estimate for the relative abun-

dance of the expressed protein (Yua n et al., 1996).Messages encoding th e ma jor a melogenin a nd LRAPisoforms have been cloned from every organismunder investigation, with the exception of LRAP inhum an s, alth ough the cloning of amelogenin cDNAin humans used a s t ra tegy that would not havedetected the LRAP message (Salido et al., 1992).Most amelogenins differ from the major expressedamelogenin isoform by lacking an internal (core)region. Th e m ost noteworth y exceptions to th is ar eisoforms containing a unique hydrophilic 14-aminoacid segment (-KSHSQAINTDRTAL-) encoded by

mouse exon 4. Amelogenins containing the exon4-encoded segmen t h ave been d etected in developingenamel by immunohistochemistry (Simmer et al.,1994a) and Western blot analyses (Simmer et al.,1994b), although they represent a small fraction(roughly 1%) of total expressed amelogenin protein(Ryu et al., 1996). Other amelogenin isoforms havebeen detected using a ntipeptide ant ibodies, and theexpression of some a melogenins appea rs to be devel-opmentally regulated (Gibson et al., 1995). Recentstu dies (Li et al., 1998) ha ve report ed th e presen ce oftwo additional 3 amelogenin exons. The functional

significance of these multiple amelogenin isoformsrem ains a complete myst ery.

3.2.3. Posttranslational modifications. (i) Phos-phorylation. Takagi et al. (1984) reported that thebovine amelogenin contained a single phosphory-lated residue: serine-16. Takagi et al. measured thephosphate conten t in th e inta ct bovine a melogenin,in ea ch tr yptic fra gment, a nd found 1 mol of phos-

phate in th e intact m olecule and in a tryptic frag-ment comprising the first 24 amino acid residues(Takagi et al., 1984). This observat ion a ppear ed to beat variance with other early reports , which sug-gested th at th e am elogenin polypeptides (E3 an dE4) each contained three phosphorylated residues(Seyer and Glimcher, 1977b; Papas et al., 1977).Later studies (Fincham et al., 1981) identified thebovine peptide E4 as being TRAP, the amino termi-nal section of th e principal (Mr, 20 kDa) bovineamelogenin, which m ight then be presumed t o alsocontain at least three phosphorylated residues. At-

tem pts t o verify this finding using direct sequen cingmeth ods (Fincha m et al., 1992) were n ot successfuland the enigma remained unt il mass spectrometricmeth ods becam e available to the protein chemistryworld. Fincham and Moradian-Oldak (1993) usedHPLC methods to isolate the TRAP and LRAPpolypeptides from both bovine and porcine enamelmat rices and deter mined their m asses using electro-spra y ionizat ion. It was foun d tha t, in each case, th eexperimental mass could only be correlated (2.0Da) with the computed mass, based on th e knownsequence if a single phosphorylated residue was

assum ed. Furt her, mass a na lysis of tryptic peptidesderived from cleavage of the bovine LRAP alsoidentified this phosphorylated residue as being con-ta ined within t he (24-residue) amino-term inal t ryp-tic peptide (MPLPPHP GHPGYINFSYEVLTPLK-),while a fast-atom bombar dment ma ss spectrometricanalysis of this peptide identified fragments consis-tent with the pr esence of a phosphorylated serine a tposition 16, observations wh ich were in accord withth e earlier report of Tak agi et al. (1984).

More r ecently, multiple ma ss spectrometr ic an aly-ses of amelogenins from many species have consis-

ten tly given ma ss values ma tching a lar ge nu mber ofthe alternatively spliced amelogenin isoforms andtheir cleavage products, but only after adding themass of a single phosphate group (80 Da) to themolecular m as s of am elogenin de rived from its cDNAsequen ce (Fincha m an d Mora dian -Oldak , 1993, 1996;Fincham et al., 1994a; Hu et al., 1996a; Lyaru u et al.,1998; Ryu et al., 1998a).

Mamm ar y glan d casein kinase r ecognizes th e se-quen ce motif xS xEx (Kemp an d Pea rson, 1990) andmight t hus be expected t o phosphorylate the amelo-genin serin e-16 (. . . NFSYEK . . .). H owever, recent



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studies have indicated that this amelogenin phos-phorylat ion is affected by a specific enam el ma tr ixprotein kinase (Salaih et al., 1998).

(ii) Glycosylation. Ea rlier st udies of enam el ma tr ixamelogenin preparations obtained using the guani-dine extr action procedur e (Term ine et al., 1980) ha vebeen shown to include glycosylated proteins (Nanciet al., 1989). H owever, as noted above, th is opera-

tional procedur e is not exclusive for am elogeninextra ction a nd, while the pr imar y sequence of am elo-genin displays potential targets sites for both N-linked a nd O-linked glycosylations (Asn-14 an d Ser -16), studies employing digoxigenin immuno-glycanlabeling (Fincham et al., 1991b) failed t o detect an yglycosylat ions of eith er m ur ine or bovine am elogeninprepar at ions. Akita et al. (1992) emp loyed a r an ge oflectins to pr obe for glycosylation of porcine ena melproteins a nd found su ch modifications to be confin edto the nonamelogenin matr ix proteins. Furth er, a snoted a bove, multiple mass spectr ometr ic ana lyses

of amelogenins retu rn exact m ass n umbers with outassu ming an y glycosylations (Fincham et al., 1994a).3.2.4. Am elogenin secondary and tertiary struc-

tures. Since the discovery (Piez, 1960; Ea stoe, 1960)tha t t he developing dental ena mel mat rix conta inedan apparently unique proline-rich protein (amelo-genin), attempts have been made to elicit secondaryand t ertiar y structur al inform at ion in th e belief tha tthis protein was involved in the enamel biomineral-ization process, either as a primary nucleator ofminera l or as a st ru ctur al entity. Attem pts ha ve beenmade to obtain secondary and tert iary s t ructural

information for amelogenin proteins using classicals t ructural analysis techniques, and, as early as1965, poorly resolved X-ra y diffra ction images ofdeveloping human tooth enamel matrices were ob-ta ined sh owing diffuse, nonorient at ed possible - orcross--pat tern s (Paut ar d, 1961, 1963; Bonar et al.,1965; Fincham et al., 1965). CD and NMR studieshave led to suggestions that the molecule containsboth -structures and -turns (Goto et al., 1993;Zheng et al., 1987; Renugopalakr ishnan et al., 1989).Jodaikin et al. (1986, 1987) in an X-ray diffractionstudy, considered that the -sheet reflections ob-

ta ined from ena mel mat rix proteins were more likelyderived from the nonamelogenin (enamelin) compo-nent s than t he amelogenins.

Renugopalakrishnan et al. (1989) proposed thatthe -spiral structure computed for the remarkable(Pro-Gln-X)n repetit ive sequence identified in thebovine amelogenin (Takagi et al., 1984) might beinvolved in Ca 2 transport. However, subsequentamelogenin sequence determinations have shownthis s t ructural feature to be absent from human,mouse, rat, h amst er, and pig amelogenins, althougha similar, even more str iking, repetitive featur e ha s

recent ly been identified in th e opossum an d guineapig amelogenins (Hu et al., 1996a; Cerny and Ham-ma r st rom , 1998, 1999). Term ine a nd Torchia (1980),in a solid-sta te N MR study of a sa mple of man ua llydissected bovine developing ena mel ma tr ix, report eda high (70%) level of rapid a nd un rest ricted m obil-ity. Aoba et al. (1990), in a 1H -NMR stu dy of porcineamelogenins in solution, showed t hat structural

changes appear t o accompa ny the proteolytic process-ing t ra nsform at ions from th e init ial full-length 25-kDa (173 r esidue) molecule t o th e lower m olecula rweight products . Further, they reported that thecar boxy-term inal Trp-161 an d some of th e tyrosineresidues of the amino-term inal TRAP sequence ar eexposed a t t he m olecule sur face. Recent applicationof sma ll-an gle X-ray scatt ering techn iques ha ve sug-gested that the porcine 20-kDa amelogenin mayexist in solution as an elongated bundlestructure(Matsushima et al., 1998).

3.2.5. Localization of am elogenin expression. Am-

elogenin is an enamel-specific protein expressed bycells of th e am eloblast lineage beginning just beforethe initial mineralization of dentin (Inai et al., 1991;Bronckers et al., 1993) an d termina ting in th e earlymat ur at ion st age (Wakida et al., 1999). The expres-sion of amelogenin during enamel formation hasbeen extensively report ed using in situ hybridization(Snead et al., 1988; Wur tz et al., 1996; Wa kida et al.,1999) and immun ohistochemistr y (Inai et al., 1991;Uchida et al., 1991a; Fuka e et al., 1993; Nanci et al.,1994, 1998; Simmer et al., 1994a,b; Gibson et al.,1995). This latter study showed that amelogenin

signals were generally weak and located close tosecretory cell surfaces, while ameloblastin signals(see section 3.3.4) sh owed the inver se effect, per ha pssuggesting (i) th at na scent am elogenins ar e occludedin some ma nner from immunoprobes an d (ii) tha tam eloblastin m olecules ar e ra pidly processed to frag-ment s within a short distance (1.25 m ) of the cellsurfaces.

3.2.6. Lect i n-l i ke pr opert i es . A r e ce n t s t u d y(Ravindranath et al., 1999) has shown that bothnative and recombinant amelogenins hemaggluti-na te (HA) mous e red blood cells. This HA is inh ibited

by monomers, dimers , and tet ramers of GlcNAc(N-acetylglucosamine) but not by N-acetylgalactos-amine or r elated sugar s. This activity is reta ined bythe TRAP molecule but not by LRAP (which shares33 N-terminal amino a cids of t he 45-residue con-s er ve d T RAP s equ e nce ). I t w a s s h ow n t h a t[14C]GlcNAc binds to the N-terminal sequence ofTRAP (-PYPSYGYEPMGGW)but not when the threetyrosyl residues are su bstituted with ph enylalan ineor if the th ird proline is substituted with th reonine.Significan tly, th is latt er m odificat ion m imics a pointmutat ion recent ly ident ified in a case of human



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X-linked am elogenesis im perfecta (Collier et al., 1997).It a ppear s possible tha t t his lectin-like activity of th eTRAP motif of amelogenins may be functionallyinvolved in int era ctions with en am el mat rix glycopro-teins (enam elin, t uftelin, or a meloblastin), perh apspromoting stru ctur al sta bility of th e mat rix or, alter -natively, functioning in a signaling role throughrecognition by cell su rfa ce glycopr oteins (Akita et al.,

1992). Further studies of the significance of theseinteresting observat ions to enam el mat rix biominer-alization are needed.

3.2.7. Physicochem ical properties of am elogenin s.

The development of expression systems for recombi-nant amelogenins (Simmer et al ., 1994a,b), ha sgreatly a dvanced our ability t o not only investigat esome str uctur al feat ur es, such as the puta tive am elo-genin molecular self-assembly process (Moradian-Oldak et al., 1994b, 1998c); specific cleava ge sit es onamelogenin molecules (Moradian-Oldak et al., 1994a;Ryu et al., 1999), and mineral binding properties

(Simmer et al., 1994b; Moradian -Oldak et al., 1998a;Hunter et al., in pr ess; Ryu et al., 1998b), but also toexamine the physico-chemical properties of the am-elogenin molecule and those of i ts partially pro-cessed products. Tan et al. (1998) made a detailedstudy of the solubility properties of both recombi-nant and in vivo amelogenins at a range of solutionpHs. These studies were directed at providing base-line informa tion for th e fur th er st udy of am elogeninstructure, crystallization, and proteinmineral inter-actions. The solubility of amelogenins was found tobe strongly dependent on t he protein primar y stru c-

ture, solution pH, a nd ionic strength and, a lso, tosome exten t, on th e bu ffer composition. Th e solubil-ity of th e full-length r ecombina nt a melogenin (rM179)and that of the modified amelogenin (rM166) wassimilar in acidic solutions, but the latter was m uchless soluble in ba sic solut ions (Fig. 4). This beh a viorhas been attributed to the lack of the hydrophiliccarboxy-terminal sequence in the rM166 protein(Moradian-Oldak et al., 1994b). The native porcine25-kDa am elogenin solubility showed a behavior as afunction of pH similar to th at seen for rM179. TheTRAP wa s found t o be very ins oluble at a ll pH r an ges

e xa m i n ed , a s p r ev iou s ly r e p or t e d (S ey er a n dGlimcher, 1977b), while the LRAP molecule waseven more soluble than rM179. Taking into accountth e local pH cha nges report ed to occur in th e enam elextracellular microenvironment (Sasaki et al., 1991a),the str ong dependence of a melogenin solubility onpH ma y ha ve significan ce to the following extr acellu-lar ma trix event s: (i) pr oteolytic pr ocessing a ctivi-ties, (ii) am elogenin qua ter na ry str uctu res, (iii) asso-ciation of the proteins with the mineral phase, and(iv) the kinetics of enamel biomineralization. Inan other s tu dy, Ryu et al. (1998b) have reported t ha t

amelogenins in solution may also act as a protonsink,promoting na scent OCP m inera l convers ion t oapatite.

3.3. The Ameloblastins (Amelin/ Sheathlin)

. . . we r eport on one of th ese (mRNA) sequences, which wefoun d highly expressed in a meloblast s. . . . The sequ ence is

represented by two mRNA variants with the potential tocode for proteins which we have named amelins (amelo-blastin s). The am elins ar e likely to const itut e component s ofthe extracellular ena mel mat rix. (Cerny et al., 1996)

3.3.1. S equences. The a mino an d carboxyl ends ofthe protein th at would become called am eloblast inar e different biochemically an d were discovered sepa -ra tely dur ing investigations of pig enamel proteins.The car boxyl end was represent ed by two polypep-tides with appar ent molecular weights of 27 and 29kDa, isolated dur ing a search for enamel proteinsth at boun d calcium by monitoring fra ctions du ring aprogressive purificat ion process for bands tha ts tained blue with the Stains-All dye (Fukae andTan abe, 1987a). The am ino-term inal end of am elo-blastin was discovered during a search for theexistence of nonamelogenins like enamelin(Fukaeand Tanabe, 1985, 1987b). The amino-terminus ofameloblastin is represented in th e enamel ma tr ix bya gr oup of low molecula r weight cleavage pr oducts inthe range 1317 kDa with aggregative properties(Fuka e and Tana be, 1987b).

The amino acid composition of ameloblastin wasdistinctive and suggested that the protein was nei-ther amelogenin nor enamelin (Fukae and Tanabe,

FIG. 4. Solubility properties of amelogenins. Comparison ofrM179 an d rM166 solubility with n at ive porcine (25-kDa) am elo-genin, in 0.05 pota ssium phospha te buffer from pH 4 to 9, IS 0.15M, 25C. The solubility of all the amelogenins examined wasminimum at a pH near their isoelectr ic point and increased

ra pidly below and a bove. The recombina nt am elogenin rM166 wasfound to be less soluble than rM179 at pH 8. The solubili tybehavior of amelogenins was found to be independent of thepresen ce of divalent met al ions su ch as calcium an d magnes ium inth e buffer (Tan et al., 1998).



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1987b). Polyclona l an tibodies ra ised aga inst th e 13-to 17-kDa nona melogeninsga ve a u nique p rofile inboth Western blot and immunohistochemical analy-ses (Uchida et al., 1991a), immun ostaining an a rr ayof enamel proteins in the ranges 1020 and 3040kDa (Uchida et al., 1991b). Especially interestingwas that the protein immunolocalized throughoutthe entire thickness of the developing enam el layer

an d, except for t he m ost super ficial ena mel (roughly30 m beneath the ameloblasts), showed a honey-combstaining pattern (Uchida et al., 1991a; Fuka eet al., 1993). This honeycomb pat ter n wa s due to theconcentration of ameloblastin cleavage products inthe sheath(or interprismatic) space that partiallyseparates rod and interrod enamel. Lectin bindingstudies indicat ed th at the 13- to 17-kDa nonamelo-genins specifically bound MPA (M aclu ra pom if era )lectin, and histochemistry using fluorescent-labeledMPA also showed a honeycomb patt ern thr oughoutthe developing enamel layer (Akita et al., 1992). In

adsorpt ion experimen ts t he 13- to 17-kDa n onam elo-genins showed low hydroxyapatite affinity (Akitaet al., 1992). Edman degradation revealed the firstamino acid sequence for ameloblastin, and affinity-purified antipeptide ant ibodies against the amino-terminal sequence showed the same honeycombpattern as described earlier (Uchida et al., 1995). Onthe basis of the novel immunostaining pattern, i twas proposed that these low molecular weight non-amelogenins a nd their parent protein constitute anew fam ily of enam el proteins des ignat ed a s shea thproteins(Uchida et al., 1995).

3.3.2. Cloning of ameloblastin cDNAs and genes.In 1996, two research groups, randomly screeningrat tooth-specific cDNA libra ries, independentlycloned and characterized cDNAs encoding the rathomologue of the pr otein t ha t ha d been studied forover 10 years in Japan. The protein was termedameloblastin(Krebsbach et al., 1996) and ame lin(Cerny et al., 1996). Later, the cDNA from pig wascloned an d designa ted as sheat hlin (Hu et al.,1997b). A cDNA en coding t he mous e h omologue wa scloned an d a lso called a meloblast in (Simmons et al.,1998). (Note: Ameloblastin is the name used in this

paper.)Ameloblas tin gen e loci ar e on chr omosome 5 in th e

mouse (Krebsbach et al., 1996) and chromosome4q12 in huma ns (MacDougall et al., 1997). Thehu ma n gene is within a r egion pr eviously link ed to alocal hypoplastic form ofam elogenesis im perfecta inthr ee Swedish families (Forsma n et al., 1994). Twoameloblastin isoforms are synthesized from alterna-tively spliced mRNA transcripts and differ by thedeletion or inclusion of a 15 amino acid segment thatis absolutely conserved between th e pig and t he r at(Hu et al., 1997b).

3.3.3. Bipolarity. Based upon t he pig am eloblas-tin cDNA sequence, the first 129 residues of theprotein, which correspond to a 15-kDa cleavageproduct, have a calculated pI of 10.6, wh ile the 66residues at the carboxyl-terminus have a calcu-lated pI of 4.5 (Hu et al., 1997a). The 95 residuesat the carboxyl-terminus of ameloblastin corres-pond to the 27- and 29-kDa calcium binding pro-

teins (Murakam i et al., 1997). Based upon a loss ofimmun oreactivity in t he Golgi when using an an ti-peptide antibody raised against residues 385399(EMTMDSTATPYSEHT), it has been proposed thatth is segment cont ains an O-linked glycosylation site(Uchida et al., 1998). Affinity-purified an ti-peptideantibodies specific for a segment near the carboxyl-term inus demonstr at ed that int act pig ameloblastinand cleavage products containing the carboxyl-term inus ar e highly concentra ted within 2 m of theam eloblast Tomespr ocess, and from th ere t o a dept hof about 50 m produced a reverse honeycomb

pat tern (immun osta ining the rod an d inter rod enam elbut n ot th e sheat h spa ce), an d showed no staining inthe deeper enamel (Mura kami et al., 1997). There-fore, the amino- and carboxyl-termini of ameloblas-tin show differences in charge (based upon theirisoelectric points) and locat ion in th e developingenamel matrix.

3.3.4. Localization of am eloblastin expression.

Ameloblast in is a tooth -specific pr otein. It is ex-pressed by cells derived from the inner enamelepithelium, including cells of the epithelial rootsheat h, presecretory, secretory, an d m atu ra tion am -

eloblasts, as well as tr an sient ly in pr eodont oblasts(Cerny et al ., 1996; Fong et al ., 1996a,b, 1998;Krebsbach et al., 1996; Lee et al., 1996; Hu et al.,1997a; Uchida et al., 1997, 1998; Begu e-Kirn et al.,1998; Nanci et al., 1998).

3.4. Th e Enam elins

We can t herefore describe non-am elogenins only in t erm s ofmolecular size, am ino acid composition, modificat ions (e.g.phosphorylation), immun oreactivity a nd to s ome extentlocalization within the tissue. In these terms non-amelo-genin s var y consider ably. (Robins on et al., 1989a)

En amelin was the n ame u sed to designate a classof n onamelogenin enamel proteins tha t stronglyadsorbed to enamel crystals and were not releasedfrom th e mat rix unt il th e cryst allites were dissolved(Termine et al ., 1980). [Note: In earlier studies(Mechanic, 1971) the term enamelin was appliedgenerally to any developing enam el ma tr ix protein,but this u sage was not subsequently retained (seeEa stoe, 1979).] The term ha s since come to be u sedfor a specific enam el protein which, based upon itsimmunolocalization in developing enamel, has thecrystal bindin g properties originally associated with



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th e enam elin class . Ena melin is not tu ftelin, it is notserum a lbumin, an d it is not a proteinase. Enam elinis a novel protein with an amino acid sequenceunrelated to an y other in the databases . I t is thelargest kn own ena mel protein, an d its expression ishighly restricted to developing teeth. In the pig,inta ct enam elin imm un olocalizes within 2 m of th eameloblast Tomes process and has an apparent

molecular mass of 186 kDa (Hu et al ., 1997c).En am elin cDNAs have now been cloned and cha ra c-ter ized from pigs (Hu et al., 1997c), m ice (Hu et al.,1998a), and h uma ns (Hu et al., 1998b). Un like otherenamel proteins , no enamelin isoforms that aretranslated from al ternat ively spliced RNA tran-scripts ha ve been observed. Minus th e signa l peptideand any posttranslational modifications, pig enam-elin ha s 1104 am ino acids, a molecular ma ss of 124.3k Da , a n d a pI of 6.5. Mouse enamelin has 1236amino acids, a m olecular ma ss of 137 kDa, and a pIof 9.4. Mouse enamelin is dis t inct ive in that i tcontains 14 copies of an 11 amino acid segmenttan dem repeat ed in the protein. This segment is notrepeated in the pig an d huma n proteins.

3.4.1. Processing. As is true of all enamel pro-teins, ena melin is processed by proteinases sh ortlyafter being secret ed. Only its cleavage products ha vebeen isolated a nd cha ra cterized. Enam elin cleavageproducts were first isolated an d chara cterized in thesame investigat ion t ha t yielded the a mino-term inusof ameloblastin (Fukae and Tanabe, 1987b). Likeam elogenins, en am elin is processed from its car boxyl-

terminus. Large enamelin cleavage products of 155,142, and 89 kDa apparent molecular weight havebeen chara cterized which r etain the original a mino-terminus (Fukae et al., 1996). The 89-kDa en am elinaccumu lates an d is fur ther processed to generat e the32- an d 25-kDa ena melins (Fu ka e et al., 1996).

The best-studied enam elin cleavage product has a nappar ent molecular mass of 32 kDa on SDSPAGE(Tanabe et al., 1990; Uchida et al., 1991a; Yamako-shi, 1995; Yam ak oshi et al., 1998). The pig 32-kDaenamelin has 106 amino acids (residues 174279),which include two phosphoserines and three glyco-

sylated asparagines (Yamakoshi, 1995; Fukae et al.,1996; Yamakoshi et al., 1998). In the absence ofglycosylat ions, the 32-kDa phosphoprotein ha s aderived molecular mass of 11.8 kDa and pI of 6.4.Although th e protein shows a single ban d on SDSPAGE (which st ain s blue with Sta ins-All dye), two-dimensional gel electr ophoresis detects seven differ-en t cons t ituents in th is band, having pI valuesranging from 3 to 4.5 (Tanabe et al., 1990). Thishet erogeneity is att ribut ed to variable glycosylation.

The 32-kDa enamelin is easily released from secre-tory stage ena mel by extra ction in 50 mM phospha te

(pH 7 .4), and may represent about 1% of tota lenamel protein (Tanabe et al., 1990).

3.4.2. Immunolocalization. Immunostaining t hedeveloping enamel matrix with antibodies raisedagainst purified 89- an d 32-kDa enam elins, as wellas affinity-purified anti-peptide antibodies againstthe amino-terminus of the 32-kDa enamelin, showthat enamelin specifically localizes in secretory stage

enamel from the dentino-enamel junction (DEJ) tothe surface of t he tooth and rapidly disappearsduring the early maturation stage (Uchida et al.,1991a,b; Fuka e et al., 1993). Based u pon am ino acidcomposition analyses, the finding that enamelin isfully degraded dur ing early matu ra tion casts doubtupon the earlier inter preta tion th at t he amelogeninsare select ively degraded and that enamelins areretained in the maturation enamel. The pattern ofimmunostaining in secretory stage enamel was areverse honeycomb; that is, enamelin was restrictedto the rod and interr od enam el and absent from the

sheat h space. This patt ern suggests tha t th ese enam -elin cleavage products bind enamel crystallites invivo. This finding is supported by hydroxyapatitebinding studies in vitro, but th e binding studies arehard to extrapolate back to the i n v iv o situationbecau se th ey were perform ed in buffers cont ain ing 4M guanidine (Tanabe et al., 1990). On the otherha nd, the observation th at t he 32-kDa ena melin canbe extracted with neutral 50 mM phosphate buffermay suggest weak hydroxyapatite binding in vivo.

3.5. Tuftelin

. . . a meloblast s, being ectoderma l cells, might be expected t oproduce an epithelium -like protein. Tuft protein might th ere-fore be a primitive epithelial component of a more special-ized enam el ma tr ix. (Robinson, Lowe an d Weather ell, 1975)

Ena mel crystals originate at the DEJ and a ppearto extend uninterrupted to the surface of the tooth.This means that if enamel crystals nucleate onmacromolecules and there are grounds to considerthe alternative mechanism of homogenous nucle-ation as being unlikely (Mann, 1989; Simmer andFincham, 1995), then the enamel crystal nucleatorshould be found a t t he DEJ . The requirement for a nena mel-specific nu cleat or is not cert ain, h owever, asit is presently unresolved whether enamel crystal-lites are independently nucleated (Diekwisch et al.,1994) or if they extend from preexisting dentincrystals (Arsenault and Robinson, 1989). Extendingfrom the DEJ into the overlying enamel layer inmature teeth are two types of s t ructures: enamelspindles, which a re extensions of odontoblast ic pro-cesses, and enamel tufts (Weatherell et al., 1968),which ma y represent residual enam el ma trices tha tinclude th e nu cleator of enam el crystals (Palam ar aet al., 1989; Robinson et al., 1989b). Some data



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suggested what the enamel nucleator should looklike. For insta nce, the a min o a cid compositions of th etr ichlora cetic acid (TCA)-soluble and -insoluble tu ftprotein (total protein from the DEJ) of ma turebovine an d hum an teeth were kn own. Assuming th etuft is essentially m ade up of a single protein, i tsamino acid composition was known (Robinson et al.,1975). The nucleator might also be expected to

display acidic or phosphorylated amino acid sidechains in a region of-sheet seconda ry str ucture t oregister with the arr ay of phosphate groups on t heincipient crystal (Addadi and Weiner, 1985, 1989;Jodaikin et al., 1986, 1987; Addadi et al., 1989, 1992;Moradian-Oldak et al., 1992).

3.5.1. St ructure and fu nction. A cDNAen coding aprotein called t uftelin tha t may or may n ot play arole in th e nu cleat ion of ena mel cryst allites ha s beencharacterized (Deutsch et al., 1989, 1991). The tufte-lin clone was isolated from a cDNA expressionlibra ry u sing a n affinity-purified polyclona l an tibody

raised a gainst the major 66-kDa protein in thegua nidine/EDTAfra ction of developing bovine enam el(Deutsch et al ., 1987). The deduced amino acidsequence of tuftelin fits the expected profile for tuftprotein. I t was somewhat acidic, and secondarystructure predictions suggested that i t contained-sheet . I t was novel and showed no s ignificanthomology to an y previously char acter ized protein. Itha d an am ino acid composition th at a greed with t ha tdetermined for TCA-insoluble tuft protein. Its pre-dicted molecular mass of 43.8 kDa (given the pres-ence of a sin gle pote nt ial N-linked glycosylat ion sit e)

was consistent with that of a cDNA encoding theoriginal 66-kDa tar geted protein, and affini ty-purified ant i-peptide antibodies made against t hreeregions of the deduced tuftelin sequence selectivelyimmu nosta ined th e DEJ a nd proteins in a guan idine/EDTA extract of enamel proteins (Deutsch et al.,1991, 1995, 1998; Zeichner -David et al., 1997). Giventh ese findings, tuftelin has been advan ced as a prim ecandidat e pr otein for the nucleat or of enam el crys-ta ls (Deut sch et al., 1989, 1991). The hum an tu ftelingene h as bee n cloned a nd loca lized to chr omosome 1,and efforts are underway to screen pedigrees with

inherited en am el defects for linka ge to the tuftelingene (Deutsch et al., 1994).

However, additiona l da ta concerning t he tuftelingene and protein (Bashir et al., 1998) have notpointed in the same direction as that of the earlyfindings. The bovine tuftelin gene was cloned andchar acterized an d showed th at the origina l tuftelincDNA was m issing a G, which cha nged th e r eadingfram e for t he dedu ced protein. The shift rem oved theC-term inal 92 am ino acids and replaced th em withonly 42 (Bashir et al., 1997). The revised readingfram e was also foun d in mouse a nd pig tu ftelin cDNA

(MacDougall et al ., 1998b). I t is clear tha t theoriginal bovine tuftelin cDNA clone was sequencedcorrectly (Deutsch et al., 1998), but th e origin of thedeletion is unclear. Was a nucleotide deleted duringreverse transcription when making the expressionlibrary, or could this nucleotide be deleted duringnormal tr an scription an d lead to A an d B isoforms oftu ftelin? The original a ssignmen t of th e bovine tufte-

lin start codon was tentatively made, recognizingth at other st ar t codons were possible (Deut sch et al.,1989). In forma tion gained from t he mouse t uftelincDNA makes the original start codon assignmentappea r un likely. Revising th e star t codon to a more 5position adds 52 am ino acids t o the tu ftelin amino-terminus (MacDougall et al., 1998b). Chan ges in t hededuced amino acid sequence of tuftelin makes itmore dissimilar to th e deter mined tu ft protein amin oacid composition. Ther e is no obvious signal peptideat the amino-terminus of tuftelin (regardless of which start codon you assign), and Northern blot

analyses have detected tuftelin mRNA in manynonmineralizing tissues (MacDougall et al., 1998b).To resolve t hese contr oversies surr ounding tufte-

lin and its role in enamel biomineralization, thetuftelin protein should be isolated and character-ized. This may not be as easy as i t would seem.Tuftelin has never been isolated from the enamelmatrix. It is apparent that the protein encoded byth e tu ftelin cDNA is not th e ma jor 66-kDa ena melintha t was originally used to mak e an tibodies (Deut-sch et al ., 1987). Ant i-peptide an tibodies ra isedagainst the deduced tu ftelin sequence immun osta in

primarily the DEJ and only show faint staining ofproteins in enamelin fractions on Western blots(Deutsch et al., 1991; Zeichne r-David et al., 1997).

3.6. Tuft Protein

Judging from amino acid composition, the so-called tuftprotein . . . seem s to ha ve been present in every type of toothexamined. . . . (Robinson et al., 1975)

Enamel tufts are a histological feature whichappear as sinuous bundles of fibersat the DEJ inground sections of mature human enamel (Osborn,1969). Weatherell et al . (1968) demonstrated thepresence of an insoluble proteinaceous material atthe tooth DEJ. Robinson et al. (1975) have shownthat complete demineralization of mature human(and bovine) enamel yields an insoluble tuft-likeprotein r esidue which is identified with the struc-tures observed histologically and may account forsome 0.010.06% by weight of the mature enamel.Amino acid analyses of this material showed it toha ve a relat ionsh ip t o the nona melogeninpr oteins(Robinson et al., 1989a). In ter estin gly, imm un o-cross-reactivity was shown to exist between the tuft pr o-tein a nd a polyclona l ant ikera tin a nt ibody (Robinson



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et al., 1975, 1989c). Amizuka an d Ozawa (1989) andAmizuka et al. (1992) sh owed th at polyclona l an tibod-ies raised to an enamelin preparation also cross-reacted with tuft proteins.

Most recent ly, Robins on et al. (1998a), using demin -era lized hu ma n molar s, ha ve shown tha t tuft proteinconta ins partial sequences of ameloblastin. Cur-rent ly, the r elationship between th e ra th er insoluble

tuft protein and other nonamelogenin proteinstu ftelin, a meloblast in, or en am elinsis obscure.

3.7. T he S ulfated Proteins

It is very likely that sulfated ena mel proteins . . . ha ve beenpart of certain fractions previously classified as enamelins(non-amelogenins). (Smith et al., 1995a)

Employing 35S in vivo labeling, Smith et al. (1995a)have demonstra ted t he pr esence of a group of short-l ived sulfated matr ix proteins in developing r atenamel . These components (49 and 25 kDa) areapparently proteolytically generated from a 65-kDa

precursor pr otein a nd r apidly degraded a fter secre-tion. Smith et al. (1995a) have suggested tha t t hesesulfated en am el glycoproteins m ay inter act function-ally with na scent am elogenins, a suggestion whichma y have grea ter significan ce in view of th e recentlyidentified, lectin-like properties of the amelogenins(section 3.2.6). However, th e function of these tr an -sient sulfated proteins in ena mel biominera lizationis presently unkn own.

3.8. The Proteinases

This met hod showed that th e whole layer of form ing enam elmatrix was associated with a well defined clear area pro-duced by th e proteolytic digestion of gelatin substra te . . .(Suga, 1970)

The n at ur e a nd sequence of proteolytic activitiesduring enamel biomineralization are crit ical andsomehow unique to this mineralizing tissue. First,these act ivit ies cause changes in s t ructural andphysicochemical properties of the parent enamelmatrix proteins (i .e., cleavage of the hydrophiliccarboxy-ter mina l of am elogenin (Mora dian -Oldak etal., 1994a; Tanabe et al., 1992). Second, the harden-ing of enamel, resulting from rapid crystal growthduring the m atu ration stage, appears to be depen-dent on th e complete degrada tion of the extracellu-lar organic ma trix (Eastoe, 1979; Robinson andKirkh am , 1984, 1985). Meta lloproteina ses a nd ser-ine proteinase activities have been detected in theena mel extr acellula r ma tr ix by var ious investigators(Moe an d Birkeda l-Ha nsen , 1979; Overall an d Lime-back, 1988; Smit h et al., 1989a, 1995b, 1996a; Sasa kiet al., 1991b; Tanabe et al., 1992, 1994; Moradian-Oldak et al., 1994a, 1996; Fincham and Moradian-Oldak, 1996; DenBesten et al., 1998). While thepresence of proteinases within the extra cellular

enam el matr ix has been well known from t he ear lyobservations ofSuga (1970), the isolation and charac-ter ization of th ese enzymes from th e mat rix has beenelusive. Recent cloning and determination of theexpression patt ern of th e meta lloproteina se ena mely-sin (MMP-20) (Bartlett et al., 1996; Llano et al.,1997) an d of the serin e proteina se EMSP -1 (Simm eret al., 1998) have helped to clarify the confusing

picture of the enamel proteinase occurrence andfunction. Nort her n an alysis ha s shown tha t enam ely-sin m RNA displays a development ally defined pat-tern of expression in the enamel organ when themessa ge is expressed at r elatively high levels dur ingth e presecretory and ear ly tr an sition sta ges of devel-opment (Bart lett et al., 1998). The messa ge is, how-ever, reduced during the matu rat ion stage. On theother hand , the amount of EMS P -1 mR NA wasdemonstr at ed to shar ply increase dur ing the tr an si-tion an d early ma tu ra tion sta ges (Scully et al., 1998).Incubation of a recombinant cata lytic domain of

enamelysin (rpMMP-20) with recombinant amelo-genin has shown that this metalloproteinase notonly processes the full-length amelogenin at i tscarboxy-terminal side at least six different cleavagesites, but it a lso cleaves th e TRAP polypeptide (Ryuet al., 1999). In vitro incubation experiments using afraction rich in EMSP-1 activity have demonstratedcomplete degradat ion of the recombinan t amelo-genin subst ra te to peptides as sma ll as 147 Da by th eserine proteinase (Moradian-Oldak et al., 1998b).Both enamelysin and EMSP-1 are thought to beactive against other en amel proteins such a s enam -

elins, ameloblastin, and the dentin sialo-phosphoprotein. Experimenta l evidence for such activities,however, is still lacking. Based on the above evi-dence, i t can be assumed that enamelysin is theproteinase responsible for the gradual processing ofamelogenin from the carboxy-terminal, resulting incritical st ructur al chan ges of the en am el extra cellu-lar m at rix and gener at ing the TRAP molecule, whichby itself may have an important function duringenamel development (Ravindran ath et al., 1999).The serine proteinase, on the other hand, has adigestive function, an activity which allows the

hydroxyapatite crystals to grow rapidly during thematuration stage. For more details regarding theenamel proteinases, see Bartlett and Simmer (inpress).

3.9. Other Ma trix Components

The abili ty of [125 I]-calcitonin and [12 5I]-insulin to ent erenamel matr ix adjacent to smooth-ended ameloblasts asearly as 10 min after injection shows that their intercellular

ju n ct ion s a re pe rmea ble to pr otein s with molecula r weigh tas great as appr ox. 5700. (McKee et al., 1986)

3.9.1. Serum albumin. Albumin h as been foundto be a ma jor componen t of th e deminer alized (gua ni-



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dine/ED TA) fra ction of both porcine (Limeba ck et al.,1989; Limeback a nd Simic, 1989) an d bovine en am elmat rix prepar at ions (Stra wich an d Glimcher, 1989,1990; Stra wich et al., 1993). In a patt ern typical ofvirtu ally all ena mel proteins, albumin was detectedin developing rat enamel throughout the secretoryand t ransi t ion s tages, but was lost during earlymat ura tion (Robinson et al., 1996). Although albu-

min has affinity for enamel crystallites (Menanteauet al., 1987), it a ppear s t o be blocked from bindin g tocrystallites in vivo by the pr esence of inta ct (21 kDa)am elogenins (Robinson et al., 1996). Albumin wasalso detected by immunohistochemistry during thesecretory stage, at the enamel surface, but not indeeper layer s (Okamu ra , 1983). However, albumin isnot synthesized or secreted by ameloblasts (Yuan etal., 1996), and radiolabeled serum albumin injectedinto rabbi ts did not incorporate into the enamellayer, suggesting a ph ysiological bar rier bet ween th eext ravascular fluid and the enamel mat r ix (Ki-

noshita , 1979). Furt her more, ingres s of album in intoenamel from dentin is restricted, particularly duringthe secretory stage (Shore et al., 1995a). How doesalbumin get into the enamel ma trix? Kinoshita(1979) injected 12 5I-labeled serum albumin into ra b-bits and demonstrated, by autoradiography, thatwhile th e isotope was present with in the dent ine andpredent ine, none was present within the enamelmatrix. Shapiro and Amdur (1965) demonstratedth at r ed pigmen ta tion observed in extra cted develop-ing bovine teeth (6 months in utero) was probablyder ived from hemoglobin ads orbed post mort em from

the denta l sac fluid, an observation which suggeststhat albumin would be l ikely to exhibit the samebehavior.

Robinson et al. (1992) have suggested that albu-min inclusion int o th e developing ma tr ix ma y resultin a localized defect of enamel mineralization, per-ha ps r esu ltin g in a whit e spotlesion. However, thepar ticipat ion of album in in denta l ena mel form at ion,as distinct from being derived solely by post mortuminfusion of the protein into the matrix, remains anopen qu estion.

3.9.2. Lipids. Wuthier and Irving (1964) sug-

gested tha t th e intense suda nophilia (Irving, 1963a,b)demonstr ated in t he developing enamel mat rix wasdue t o acidic lipid component s. Sha piro et al. (1966)ana lyzed ma trix ph ospholipids of both dentine an denamel matrix and reported a total phospholipidcont ent of 0.06% by weight of the tissu e. Subsequen tstudies (Fincham et al., 1972) demonstr at ed that t heneutral lipids of bovine enamel matrix representedsome 0.1% of th e (lyophilized) sample weight . Th us,the total lipid content (neutral acidic) of develop-ing enamel matrix is some 0.2% by weight , notdissimilar to levels reported for other mineralized

tissu es. Is th is lipid componen t (phospholipids, cho-lesterol, mono- and diglycerides, cholesteryl esters,and free fatty acids) functionally involved in themineralization process or in enamel m atr ix struc-ture?

Perhaps the only pointer of significance is theobserva tion (Sha piro et al., 1966) that only a fraction(40%) of th e ph ospholipid componen t is extr acta ble

without prior demineralization. Again, Goldberg etal., (1984, 1998) have explored the possibility thatlipid-rich membrane fragments of the Tomes pro-cesses may become entombed within the matrix,cont ribut ing to the overall lipid cont ent .

Beyond th e issu e of a functional r ole for lipids inthe en amel ma tr ix, it is importan t t o recognize tha tsome of the amelogenin proteins themselves, as aresu lt of th eir hydr ophobic na tu re, exhibit lipophilicproperties, such as being soluble in typical l ipidsolvents (e.g., chloroform met ha nol, 2:1, v/v) (Bur -gess and Ma cLar en, 1965; Fincha m et al., 1972).

4. ENAMEL MATRIX STRUCTURE AND FUN CTION

Its ma in functions appear to be firstly to nurtu re the r apidgrowth of the large apatite crystals, by some mechanismswhere growth in specific directions is either directed, ormore probably, as sisted by t he mat rix, and secondly to bereadily removed during enamel matura tion to permit t heultimate formation of a t issue which is a lmost entirelyinorganic. (J ohn E. Ea stoe, 1965)

As Eastoe has noted the matr ix nurt ures thegrowth of the enam el crysta llites, allowing them todevelop as an oriented array of extremely longstru ctur es, perha ps extending thr ough th e full thick-ness of th e secret ed ma tr ix. It is comm only acceptedthat these early crystallites arise at the developingDEJ an d then extend thr ough the ma tr ix by progres-sive accret ion of ions, tr an sported by th e am eloblast,onto the (001) face of the crystalli tes. How theseenamel crystall i tes may be nucleated is presentlycont roversial. One view is tha t n ucleation occurs onpreexisting dentine mineral (Arsenault and Robin-son, 1989) or al ternat ively that the crystals arenucleated de novo by acidic m at r ix pr oteinswit hinthe ena mel matrix and are n ot continuous with thedent ine miner al (Diekwisch et al., 1994). Ena melins,tuftelin, amelogenins, ameloblastin, and, most re-cently, d ent ine sialophosphoprotein (MacDouga ll etal., 1998a) have all been cited as candidate proteinsto act as mineral nucleators (Deutsch et al., 1998,1991; Zeichner-David et al., 1997; Robinson et al.,1998b; Termine et al., 1980; Fong et al., 1996a;Krebsbach et al., 1996). Convincing evidence of anu cleat ing role for an y of these proteins is, however,current ly lacking. I t i s of interest to note that ,depending on the an gle of the section, h igh resolu-tion electr on microscopic ima ges of early-stageenamel matrices do a ppear to show crystal lites



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impinging very closely to the ameloblast Tomesprocess cell membr an es (Diekwisch, persona l commu -nication), prompting the question as to whethernucleation might be ameloblast-membrane initiated.

Also associated with the ameloblast membranear e str uctu res commonly ident ified as stippled ma -ter ialcons istin g of fields of par ticles some 1020 n min diameter (Watson, 1960). These structures are

also seen within ameloblast secretory vesicles, sug-gesting tha t th ey ar e an ear ly sta ge of ma tr ix proteinsecretion. It has been suggested (Fincham et al.,1994b) tha t th e stippled mater ial may represent th einitial stage of amelogenin nanosphere assembliesas discussed below. A recent review of the enamelmatr ix properties a nd the initiation of the mineralpha se ha s been pr ovided by Robinson et al. (1998b).

4.1. Gross Matrix Composition

Both a melogenins a nd ena melins were foun d at a ll stages ofenamel development, with higher levels of enamelins being

present both at later stages where t issue maturation be-comes advanced and at early stages where a ctive matr ixsecretion is predominant . (Termin e et al. 1980)

As has already been noted, Termine et al. (1980)reported the use of a dissociative extra ction proce-dur e for isolating ena mel mat rix proteins from prepa -rations of the soft cheese-like scrapings obtainedfrom developing bovine teeth. This procedure re-sulted in th e fra ctiona tion of th e total ma tr ix proteininto t wo classes: amelogenins a nd nonamelogenins(or enam elins). On the basis of this pr ocedure Ter-mine et al. (1980) showed that in bovine enamel theproport ion of amelogenin in the mat rix varied withfeta l age, rising from some 70% in th e youn gest (24month s) to 8590% at 56 mont hs a nd t hen falling tosome 50% as mat ur at ion of the enam el takes place.By implication, the difference of these values wasmade up by the enam elin (or nonamelogenin) fra c-tion. Thu s, at th e maximu m of th e secret ory sta ge ofmatrix deposition some 10% was accounted for asnonamelogenins. This fraction would now appear tocomprise the ameloblastins, tuftelins, and enam-elins. H ow rea listic is th is pictu re?

Other data (Fincham, unpublished observations)obta ined by em ploying th e dissociative extr action of8 g of developing bovine en am el ma tr ix scra pings,using five sequential steps of guanidine extractionbefore using the guan idineEDTAst ep to deminer al-ize th e matr ix, suggest tha t (i) secretory sta ge ena melma tr ix may cont ain only some 3% or less of nona m-elogenins, and (ii), unsurprisingly, the extractionprocedure is not quan titat ive, the guanidineEDTAextract (23%oftotal protein)still apparently includ-ing some a melogenin (see F ig. 2).

This issue a ppears importa nt in view of the puta -tive r ole of th e n onam elogenin proteins (e.g., am elo-

blastin) as structural components of the matrix(Uchida et al., 1995). Based on the data of Fig. 2, itappea rs th at t he amelogenin cont ent of th e secret orystage ma trix may in fact be greater tha n pr eviouslysuggested (Termine et al., 1980). However, this isdifficult to substa ntiat e as qua ntita tive data for t heproport ions of nonamelogenin proteins (amelo-blastins, enamelins), based on direct mea surem ent,

is presen tly lacking, the pr esence of these m oleculeswithin the matrix being generally inferred eitherfrom mRNA levels or by imm un ohistological observa -tions.

The difficulty of quan titat ively identifying theproportions of ma tr ix proteins is fur th er complicat edby the temporal changes in both protein secretionand protein r esorption, which occur as the matr ixmineralizes. These changes in gross composition ofthe enam el mat rix ar e complex functions, compris-ing sequential cha nges in total protein, mineral, and

aqueous compartments. Such changes have beendeter mined by Robinson et al. (1988) on a volum etr icbasis, and th e dat a h ave recently been reviewed bySmith (1998).

4.2. Spherical Substructures

Globular structures or spheres of 300500 in diameterseem to have occupied virtually the entire volume of thetissue. (Robinson et al., 1981)

A number of earl ier s tudies of enamel matrixultrastructure suggested that a periodic substruc-

ture existed either between or around the earliestenam el crystallites (Ronn holm, 1962; Tra vis andGlimcher, 1964). In contr as t, Ea st oe (1963) pr oposedthat the matrix was essentially an unstructuredgel with thixotropic properties (i.e., a matrix whichmodified its str ucture in r esponse t o local pr essuresexerted by t he developing miner al crysta llites), whileWarshawsky (1985) took the view that the amelo-genin pr otein a cted to . . . separ at e t he cryst allitesto keep them from fusing and provide an extracellu-lar en vironm ent for r apid diffusion of miner als t o th ecalcifying sites on growing crystallites. In 1975,

Smales, working with developing rat enamel, de-scribed the presence of helical structures showing apitch varying from 30 to 90 nm. Robinson et al.(1981), in a scanning electron microscope freezefracture study of developing rat enamel, demon-st ra te d colinea r sph ere s of organ ic ma ter ial. . .an dsuggested tha t they might be miner alprotein aggre-gates. Bai and Warshawsky (1985) noted globularparticles in freezefracture replicas of fresh andfixed enam el sam ples which wer e rem oved by 4.0 Mguanidine extraction. They concluded that amelo-genins were th e nonstr uctur al, heter odispersed pa r-



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ticulate material in the intercrystallite space, al-th ough Warsh awsky (1985) considered t hese globularpar ticles to be ar tifactua l.

4.2.1. N anospheres. The recent availability ofrecombinant amelogenin together with the develop-ment of HP LC chromatographic pur ification pr oce-dures for i n vi vo amelogenin proteins has greatlyfacilitated studies of this principal matrix compo-

nent (Simmer et al., 1994b). In 1994, we reportedthat high resolution transmission electron micro-graphs showed evidence for beaded rowsof struc-tures (some 20 nm in diameter) aligned with andbetween th e initial-sta ge developing enam el cryst al-lites (Mora dian -Oldak , 1994b, 1995; Fincham et al.,1994b, 1995). It was also demonst ra ted th at r ecombi-nant mouse amelogenin protein (rM179) spontane-ously formed such structures when a solution wassprayed ont o electr on-microscope grids or altern a-tively adsorbed onto mica and imaged by AFM. Itwas proposed tha t am elogenin molecules und erwen t

a sponta neous self-assembly process genera tingnanosphere structures which, i t was argued, func-tioned t o organ ize th e microstru ctur e of the mat rix,spacing the initial crystallites at some 20-nm dis-tances and inhibiting early crystalcrystal lateralfusions (Fincham et al., 1995; Fincham and Simmer,1997). Fu rt her, current studies (Mora dian-Oldak etal., 1998a) have shown that amelogenin nano-spheres can act to adhere synthetic apatite crystalsth rough a polymer -bridgingmecha nism (Nan collasan d Bud z, 1990) (see Fig. 4).

Studies in this laboratory employing dynamic

light scattering and imaging techniques with bothrecombinant and i n v iv o amelogenins have nowfirmly established t ha t amelogenins under go a self-assembly process to generate spherical structuresvar iously ran ging in size from 5 to 100 nm, depend-ing on the local conditions (Moradian-Oldak et al.,1994b, 1998c; Fincham et al., 1998). Landis et al.(1998) have used 3D-TEM tomographic reconstruc-tion techniques t o image fixed prepa ra tions of devel-oping mouse enamel matr ix and observed r ows ofnan ospheres appar ently spira ling around th e initialmineral crystallites. These observations have been

independently confirm ed using cryo-tra nsmissionelectron microscopy, a direct imaging technique re-quiring n o prior t issue fixat ion (Leytin et al., 1998).

4.3. Th e Matrix Assembly Process

The precipitate, when separated from the supernatant bycentrifugation a nd st ored at 4C, formed a clear gel, which,when heated to 18C, reverted to a white opaque mass.(Nikiforuk a nd Sim mons, 1965)

Beyond studies of am elogenin seconda ry a nd ter-tiary st ru ctur es (noted above), it is pertinent here t onote the early work of Nikiforuk and Simmons

(1965), who first demonstra ted that amelogeninpreparat ions undergo a reversible temperature-sen sitive h ydroph obic aggr egat ion (coacer vat ion ef-fect) passing from a translucent gel at 4C to anopaque material at room temperature. Recent stud-ies (Wen et al., 1999) in wh ich t hese ear ly tempera-ture-sensitive matrix properties were further ex-plored ha ve sh own tha t a synt het icmat rix prepa red

from porcine am elogenins an d r endered t ra nslucentat 4C may be fixed in this condition using conven-tional glutaraldehyde (Karnovsky) fixation. Like-w is e, a p r ep a r a t ion h e ld a t r oom t e m pe r a t u r e(opaqu e) is fixed in t his opaque condit ion. Scann ingelectr on microgra phs of th ese prepa ra tions have nowshown th at the differing optical properties (opaqueto clear) are appa rent ly due to the r oom-temperat urepreparations containing numerous globular voids,compa red t o th e 4C prepa ra tions (Wen et al., 1999).Figur e 5 shows exam ples of th e str uctu re of a porcine

amelogenin m atr ix prepared at room temperatur e,and F ig . 6 uses AF M imaging to i l lus t ra te thedifference in the surface features of recombinant(rM179) am elogenin m at rix gels formed both a t roomtempera tur e an d at 4C.

While these experiments provide some explana-tion of the phenomenon originally described by Niki-foruk and Simmons (1965), they have also showntha t these synthetic mat rices are composed of assem-blies of spherical st ructur es (nan ospheres) ra ngingin size from 20 to 200 nm in diameter (Fig. 5). Thechange in the distribution of these structures with

tempera tur e is interpr eted to result from a changeddistribution of hydrophobic intera ction forces be-tween these nanospheres, as temperature is varied.

Also addressing the problem of amelogeninamelogenin interactions, Paine and Snead (1997),employing the yeast two-hybrid system (Fields andSong, 1989), have found that amelogenin proteinsself-associate and that this process appears to de-pend on the amino-term inal 42 residues interactingwith a 17-residue domain in the carboxyl region ofth e protein. This twin locus inter action provides

the potent ial for am elogeninam elogenin int erac-t ions to generate the supramolecular s t ructureswhich we have described. However, the situation isalmost certainly more complex, since AFM imagingstudies of a synthet ic TRAP (the N-terminal 45residues of murine amelogenin; M180) show thatth is molecule alone ha s th e ability to form supr am o-lecular structures (unpublished observation). Fur-ther, it ha s been shown th at t he tu ftelin protein alsohas a self-assembly potential, which is, however,apparently n ot manifest in ameloblastin (Paine etal., 1998a,b).



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4.4. Am elogenin Min eral I nteractions

Chan ges in th e secondary str uctur e upon adsorption, as wellas transfer of water from the molecule and the adsorbentsurface to the bulk solution, may provide the increase inentropy required for adsorption to take place. . . . Unfortu-na tely, ther e is insufficient in form at ion a bout th e molecularconform at ion of amelogenins in solution and in th e adsorbedst at e, . . . (Aoba et al., 1989)

Inh ibition of cryst al gr owth by ma cromolecules insolution is a general phenomenon which may not bedirectly related to the function of the investigated

macromolecule but determines whether the macro-molecules interact with the crystals. The adsorptionbeha vior of am elogenins in solution h as been r elatedto their inhibitory activity in crystal growth experi-ments (Doi et al., 1984; Aoba et al., 1987a,b). Theapa tite binding properties of am elogenins ha ve pre-viously been investigated , mainly in ter ms of ads orp-tion para met ers (Aoba et al., 1987a; Aoba an d Moren o,1989). Kinetic studies a lso report ed th e inhibition ofapatite crystal growth by amelogenins in solution

FIG. 5. Str uctur e of a synt hetic amelogenin gel ma tr ix. Scann ing electr on micrographs of feat ur es of a synt hetic amelogenin gel ma tr ixfixed a t room temperat ure (Wen et al., 1999). Porcine a melogenins (principally composed of 7% P173 an d 40% P148 amelogenins) wereprecipitated and sedimented by centrifugation. The resulting gel matrix was fixed with glutaraldehyde and imaged by scanning EM(original ma gnificat ion, 5000). (A) Surface featu re of th e ma tr ix shows m ultiple globular pitsdistr ibuted over t he s ur face. Compara blematrices fixed at 4C (data not shown) show an essentially pit-free surface. (B and C) Details of individual pits (rotated 90 for convenience)show t hem to be na nosphere-linedstr uctur es (origina l ma gnification,30 000). See text for discussion.



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(Doi et al., 1984). The aggregation properties ofamelogenins in solution have limited the conditionsunder which such experiments may be performed.Aoba et al. (1989) an d Simm er et al. (1994b) showedth at cleava ge of the h ydrophilic car boxy-ter mina l ofam elogenin decrea ses its a pat ite binding a ffinity. Inanother recent study, purified recombinant amelo-genin rM179 did n ot ha ve an y significan t effect on

the nucleation of hydroxyapatite crystals (Hunter etal., in press).

Our current studies have shown that cleavage ofthe amelogenin carboxy-terminal motif lowers theinhibitory potentia l of the m olecule to apat ite growthkinetics, compar ed t o the intact rM179 am elogenin(Moradian-Oldak et al., 1998a). In an at tempt toinvestigate intera ctions of am elogenin na nospheres

FIG. 6. Tapping mode, atomic force microgra phs of the su rface feat ur es of a synt het ic ena mel ma tr ix formed by purified r ecombina ntam elogenin (rM179). The purified a melogenin wa s dissolved in dilute form ic acid, and th e pH wa s adjust ed to 6.8 with N aOH addit ions.The precipitated protein was pelleted by centr ifugation, creating a n amelogenin gel mat rix which was found to exhibit a temperat ure-sensit ive reversible coacervation (clear & opaque) as originally described (Nikiforuk and Simmons, 1965). Samples of this matrix werefixed with glutaraldehyde as for conventional electron microscopy. (A) Matrix sample fixed at room temperature (23C). A field ofessent ially uniform -sized nan ospheres (4050 nm diam eter) cont ains m ajor irregular ities an d pits. (Compare F ig. 5A.) (B) Mat rix sample

fixed at 4C. Nanospheres of sizes similar t o those seen in A form a fairly uniform sur face without ma jor pit s tr uctur es. We speculate t ha tthis effect results from the increased hydrophobic interactions, generated by the breakdown of hydration shells in the room-temperaturesam ple, cau sing distortion of

Desarrollo Matriz Esmalte

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