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J Oral Biosci. 2011 August 1; 53(3): 257–266.

Amelogenins: Multi-Functional Enamel Matrix Proteins and Their

Binding Partners
Naoto Haruyama1,2, Junko Hatakeyama3, Keiji Moriyama1,2, and Ashok B. Kulkarni4
1GCOE Program, International Research Center for Molecular Science in Tooth and Bone

Diseases Tokyo Medical and Dental University, 1-5-45, Yushima, Bunkyo-ku, Tokyo 113-8549,
Japan
2Maxillofacial Orthognathics, Department of Maxillofacial Reconstruction and Function Division of
Maxillofacial/Neck Reconstruction, Graduate School of Medical and Dental Sciences, Tokyo
Medical and Dental University, 1-5-45, Yushima, Bunkyo-ku, Tokyo 113-8549, Japan
3FunctionalStructures Section, Department of Morphological Biology, Division of Biomedical
Sciences, Fukuoka Dental College, 2-15-1, Tamura, Sawara-ku, Fukuoka, 814-0193, Japan
4Laboratoryof Cell and Developmental Biology, National Institute of Dental and Craniofacial
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Research, National Institutes of Health, Bldg. 30, Convent Dr., Bethesda, MD, 20892-4395, USA

Abstract
Amelogenins are the most abundant extracellular matrix proteins secreted by ameloblasts during
tooth development and are important for enamel formation. Recently, amelogenins have been
detected not only in ameloblasts, which are differentiated from the epithelial cell lineage, but also
in other tissues, including mesenchymal tissues at low levels, suggesting that amelogenins possess
other functions in these tissues. The therapeutic application of an enamel matrix derivative rich in
amelogenins resulted in the regeneration of cementum, alveolar bone, and periodontal ligament
(PDL) in the treatment of experimental or human periodontitis, indicating the attractive potential
of amelogenin in hard tissue formation. In addition, a full-length amelogenin (M180) and leucine-
rich amelogenin peptide (LRAP) regulate cementoblast/PDL cell proliferation and migration in
vitro. Interestingly, amelogenin null mice show increased osteoclastogenesis and root resorption in
periodontal tissues. Recombinant amelogenin proteins suppress osteoclastogenesis in vivo and in
vitro, suggesting that amelogenin is involved in preventing idiopathic root resorption.
Amelogenins are implicated in tissue-specific epithelial-mesenchymal or mesenchymal-
mesenchymal signaling; however, the precise molecular mechanism has not been characterized.
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In this review, we first discuss the emerging evidence for the additional roles of M180 and LRAP
as signaling molecules in mesenchymal cells. Next, we show the results of a yeast two-hybrid
assay aimed at identifying protein-binding partners for LRAP. We believe that gaining further
insights into the signaling pathway modulated by the multifunctional amelogenin proteins will
lead to the development of new therapeutic approaches for treating dental diseases and disorders.

Keywords
amelogenin; yeast two-hybrid; M180; leucine-rich amelogenin peptide(LRAP); signaling
molecules

Amelogenin Gene Structure, Splice Isoform, and Proteins

Amelogenins are the most abundant extracellular matrix proteins secreted by ameloblasts1),
and are believed to play critical roles in enamel formation and mineralization. Human
 Haruyama et al. Page 2

chromosome 4 contains a cluster of enamel matrix genes that are located immediately
proximal to a cluster belonging to the dentin-or bone-related SIBLING genes. By contrast,
amelogenin genes are located on the sex chromosomes, and are highly conserved in at least
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26 mammalian species2). Amelogenin genes probably migrated from another locus to

preserve their function in an ancient evolutionary process. Amelogenin genes in some
species, including bovine and human, are located on both X (Amelx) and Y (Amely)
chromosomes. In humans, the number of protein transcripts from AMELY is about 10% of
the amount of AMELX3). Mice have only a single Amelx gene and no corresponding gene
on the Y chromosome4). The Amelx gene contains seven exons, although exon 4 is not
found in some species. In humans and rodents, additional exons 8 and 9 were recently
identified (Fig. 1) 5). At least 16 mRNAs are generated from the mouse Amelx gene in
ameloblasts via alternate splicing6–8). Exon 6 is the largest and it contains three internal
splice acceptor sites (Fig. 1A; a–c in exon 6). The most abundant amelogenin isoform in
mice is the product of exons 2, 3, 5, 6, and 7, which is named M180 and consists of 180
amino acids9). Leucine- rich amelogenin peptide (LRAP), also known as M59 or A-49), is
the second most abundant isoform, which results from a major cryptic splice deletion at the
5’-end of exon 6 (Fig. 1B and C) 10). Amelogenin proteins undergo sequential proteolysis by
matrix metalloproteinase 20 (MMP20) and kallikrein 4 (KLK4) during enamel secretion and
maturation11–13), resulting in highly heterogeneous proteins in the enamel matrix. The N-
terminal region includes a phosphorylation site at serine 1614), and a tyrosyl motif that binds
N-acetylglucosamine and keratins15–17), which may alter nanosphere formation and the
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biological functions of amelogenin as a matrix or enamel mineralization (Fig. 1C) 18).

The Role of Amelogenin in Periodontal Tissues: Emerging Evidence for the

Additional Roles of M180 and LRAP
Although many reports have suggested that amelogenins play critical roles in proper enamel
formation and are responsible for human amelogenesis imperfecta (AI), the functions of
each amelogenin isoform have not been clearly delineated because of their heterogeneity. In
order to elucidate the functions of amelogenin, Gibson et al. generated amelogenin null mice
with a targeted deletion in the Amelx gene19). The teeth from these null mice show a chalky-
white discoloration, resembling human hypoplastic AI. To understand the role of individual
amelogenin isoforms in enamel formation, transgenic mouse lines were generated that
expressed a single amelogenin, e.g., M180 or LRAP, under the control of the bovine
amelogenin promoter. These were subsequently mated with amelogenin null mice to
reintroduce the specific isoform into ameloblasts. The report indicated that M180 could
partially rescue the hypoplastic phenotype seen in amelogenin null mice, but that LRAP
could not20) (see Gibson’s review in this volume). The amelogenin splicing isoforms may
play distinct roles in enamel formation. Therapeutic applications of an enamel matrix
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derivative (Emdogain; Straumann, Switzerland) rich in amelogenins have been reported to

result in the regeneration of cementum, alveolar bone, and periodontal ligament (PDL) in the
treatment of experimental or human periodontitis21–24), indicating the attractive potential of
amelogenin in hard tissue formation. Interestingly, amelogenin expression outside of enamel
tissues, such as in periodontal tissues, cementoblasts, PDL cells, or Hertwig’s epithelial root
sheath (HERS), has been reported in studies utilizing immunohistochemistry or in situ
hybridization25–28). Using reverse transcription-polymerase chain reaction (RTPCR), we
also detected two amelogenin alternative splice isoforms, M180 and LRAP, in molar
cementoblast/PDL (CM/PDL) cells from wild-type mice29). These expression patterns imply
that amelogenin is involved in cementum formation and/or in the homeostasis of periodontal
tissue through unknown mechanisms. Therefore, we further investigated periodontal tissues
in Amelx null mice and observed the increased formation of cementicles, irregular calcified
globules on the root surface, as compared to the wild type. Most surprisingly, the area of

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root resorption and number of osteoclasts (odontoclasts) on the root surface were increased
significantly in 6–12-monthold mice in the Amelx null mouse tooth root. The loss of
amelogenin from the cementoblasts or epithelial cell rests of Malassez in periodontal tissues
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may lead to cementum defects and increased numbers of osteoclasts by changing the
RANK-RANKL pathway in Amelx null mice29). Moreover, the co-culture of wildtype
osteoclast progenitors with Amelx null CM/PDL cells resulted in increased tartrate-resistant
acid phosphatase (TRAP)-positive cell formation30). This was abolished by adding
recombinant porcine LRAP to the medium, but not by adding P172 (porcine analogue of
M180). In a rat root resorption model, the application of P173 (P172 plus an initial
methionine), LRAP, and enamel matrix derivative to tooth roots markedly reduced the
number of odontoclasts on root surfaces and inhibited cementum and root dentin
resorption31). In addition, LRAP mediated the induction of the migration and proliferation of
CM/PDL cells30). Although P172 also induced migration and proliferation, it had a less
significant effect than LRAP. These findings suggest that amelogenin protects the cementum
from abnormal osteoclastogenesis, and that LRAP, and to some extent full-length
amelogenin, is important for the development and maintenance of periodontal tissues.

Screening Potential Protein-Binding Partners for LRAP

As reviewed above, amelogenins, especially LRAP, may possess cell signaling roles in
several types of cell; however, it remains unclear how amelogenins affect cell signaling. To
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determine whether LRAP has unknown receptors or binding proteins that are involved in the
regulation of osteoclastogenesis and bone mineralization as potential signaling transducers,
we performed large-scale screening using a yeast two-hybrid system (Matchmaker GAL4
Two- Hybrid System 3; Clontech, CA, USA).

In experiments, two different cDNA libraries were screened as potential sources: a human
bone marrow (hBone marrow) premade cDNA library (Clontech) and a mouse CM/PDL cell
custom cDNA library. For the hBone marrow library screening, the AH109 yeast strain
transformed by a pGBKT7-mLRAP bait vector, which expresses mouse LRAP protein in
yeast, was mated with a Y187 yeast strain that had been transformed with the hBone marrow
cDNA library. The CM/PDL cell cDNA library was constructed from cultured primary
mouse CM/PDL cells according to the manufacturer’s protocol (Matchmaker SMART
cDNA Library Construction & Screening Kits; Clontech). The synthesized first-strand
cDNA was amplified by long-distance PCR in order to generate sufficient double-stranded
cDNA. Then, the double-stranded cDNA, pGADT7-Rec prey vector, and pGBKT7-mLRAP
bait vector were co-transformed into the AH109 yeast strain so that in vivo homologous
recombination generated pGADT7-Rec-cDNA library clones in the yeast and the screening
could be performed simultaneously in one step. The mated mixture for hBone marrow or the
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cotransfected mixture for mCM/PDL was plated on 50 150-mm ager plates with nutritional
selection medium (SD/ -Ade/ -His/ -Leu/ -Trp), respectively, followed by stringent
screening for transformants expressing interacting proteins. After 7 days, the growing
colonies were inoculated on fresh SD/-Ade/-His/-Leu/-Trp/X-α -Gal master plates and
cultured for an additional 4–7 days to identify the positive interacting protein pairs that
activated the MEL1 reporter gene, which encodes α-galactosidase.

In hBone marrow and mCM/PDL screening, 500 and 213 blue colonies were picked up from
each master plate, respectively. We amplified the library inserts by PCR and analyzed the
PCR products and their fragment sizes when digested with the four-cutter restriction enzyme
AluI. Agarose gel electrophoresis revealed that 240 and 180 clones were unique clones in
each respective library. The plasmids containing cDNA from the library were recovered and
sequenced. Among the unique clones in each library, ~40 and ~50 colonies, respectively,
contained an open reading frame fused to the activation domain sequence, indicating that the

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proper fusion proteins were made in these clones. The number of clones screened was
estimated by counting the number of growing colonies on an SD/-Leu/-Trp test plate. We
screened 7.0×106 and 4.2×106 clones from two rounds of hBone marrow library and mCM/
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PDL screenings, respectively, which should be sufficient to represent every gene for most
organisms.

LRAP Interacting Proteins in the Bone Marrow and CM/PDL

Based on the results of the yeast two-hybrid screening, potential candidates for binding
partners of LRAP were identified, including cell membrane proteins, enzymes, and
extracellular matrix. The interacting proteins that fit our search criteria are listed in Tables 1
and 2. From hBone marrow library screening (Table 1), we found that LRAP bound to an
immunoglobulin (Ig)-like domain in several different molecules, which may be involved in
the immune system, oxidase-like family were identified as common candidates in both the
hBone and mCM/PDL libraries. Here, we will discuss a few promising candidates.

The carcinoembryonic antigen (CEA)-related cell adhesion molecule 6 (CEACAM6, also

known as CD66c), a human homologue of mouse CEACAM1 isoform 2, is a gene located
on human chromosome 19q13.2. The glycosylphosphatidylinositol (GPI)- anchored
membrane protein CEACAM6 belongs to the Ig superfamily and appears as a highly
glycosylated protein with the typical N-terminal variable Ig like domain followed by 0 to 6
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constant Ig-like domains. CEACAMs are expressed in normal epithelium, angiogenically

activated endothelium, and hematopoietic cells32,33) The cytoplasmic domain of
CEACAM1-3/4 L possesses an immunoreceptor tyrosine-based inhibition motif (ITIM),
which contains two tyrosine residues that can recruit and activate SH2 domain-containing
tyrosine kinases and tyrosine phosphatases34). Interestingly, there are now convincing data
showing that CEACAMs and several of the human GPI-linked molecules participate in
signal transduction; however, no GPI-linked proteins belonging to the CEA family are
expressed in rodents32,35), leading to difficulty in determining the functions of GPI-linked
CEA family members. Possible functions of CEACAM family members are cell adhesion,
tumor suppression, regulation of lymphocyte and dendritic cell activation, and acting as a
receptor of bacteria, including Neisseria species.

Leukocyte immunoglobulin-like receptor, subfamily B (LILR-B) is a mouse homologue of

paired immunoglobulin-like receptor B (Pir-b), located on human chromosome 19q13.4.
PIR-B and four isoforms of LILR-B, harboring ITIM inhibitory domains, constitutively
recruit SHP-1 in the presence of RANKL and M-CSF on cultured osteoclast precursor cells,
suggesting that some can suppress osteoclast development in vivo and in vitro36). Indeed,
co-stimulatory signals mediated by the ITAM (immunoreceptor tyrosine-based activation
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motif) cooperate with RANKL to achieve bone homeostasis. FcRγ and DAP12, both of
which harbor an ITAM, regulate the development of functional osteoclasts via Syk tyrosine
kinase37). Consequently, dual regulation of intracellular signals through the ITAM and ITIM
may be crucial for maintaining a balance of osteoclastogenesis. The binding of LRAP and Ig
receptors should be investigated to elucidate whether amelogenin can regulate
osteoclastogenesis by changing the kinase activities in osteoclast precursors, although
reports have suggested that regulating the RANK-RANKL signaling pathway reduced
osteoclastogenesis in mesenchymal cells.

ITM2A and Itm2b, members of the integral membrane protein 2 (ITM2) family (also
referred to as the BRI2 family), were identified from each cDNA library independently. This
gene family encodes a type II membrane protein, possesses a BRICHOS domain, which is a
conserved motif common to family members, and is thought to play a role in the targeting of
proteins to the secretory pathway or to intracellular processing38). Proteins sharing the

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BRICHOS motif are associated with diverse phenotypes, such as dementia, cancer, and
respiratory distress. In particular, recent studies have focused on the involvement of ITM2B,
which shows high homology to ITM2A, in dementia observed in a patient with ITM2B gene
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mutations39,40). In the early stages of differentiation, ITM2A may be associated with the
inhibition of chondrogenesis initiation, and elevated expression of ITM2A in adipose tissues
may be linked to the poorer chondrogenic differentiation potential of these cells41). Several
reports suggest that LRAP influences chondrocytic differentiation and proliferation in
mesenchymal cells42–44). We are currently investigating the effects of LRAP on
chondrocytic cell differentiation in the context of ITM2A involvement.

Lysyl oxidase-like 3 and 1 were independently identified from the library screening of
hBone marrow and mouse CM/PDL cells, respectively. The lysyl oxidase (LOX) family is
an extracellular copper-dependent amine oxidase that is critical for the biogenesis of
connective tissue matrices through the crosslinking of collagens and elastin. A highly
conserved amino acid sequence at the C-terminal end of the family members appears to be
sufficient to retain enzymatic function45). In addition to its enzymatic role, the
phosphorylation of MAPK, ERK1, and ERK2 induced by fibroblast growth factor (FGF)-2
was significantly reduced by the addition of LOX in vitro46). A recent report indicated that
LOX propeptide in proLOX inhibited FGF-induced signaling and osteoblast proliferation47).
Moreover, fibronectin, which is critical for the proteolytic activation of proLOX48), was
downregulated via unknown mechanisms on adding amelogenin (M180) to osteoblasts49).
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Collectively, these experiments suggest that LRAP is also involved in the MAPK-ERK
pathway, possibly by regulating LOX function directly or indirectly. LOX activity and
proLOX cleavage in the presence or absence of LRAP are worthy of further investigation.
Two splice products of amelogenin [M73 (LRAP+exon4) and M59 (LRAP)] have been
shown to bind to a 95-kDa transmembrane protein identified as lysosomal membrane
protein-1 (LAMP-1)50,51). Furthermore, the amelogenins also bound to LAMP-2 and
LAMP-3/CD63 using a yeast two-hybrid system50,52), although no LAMPs were identified
in our screening. LAMPs are highly glycosylated proteins, associated with vesicular
structures of the endocytic pathway, and are implicated as binding proteins that function in a
scavenger or protein uptake process after the degradation of amelogenin protein in tooth
development53); however, a recent study suggested that H174 (human homologue of M180)
increased the proliferation of mesenchymal stem cells by interacting with LAMP-1 via the
MAPK-ERK signaling pathway54). Although the exact mechanisms mediated by LAMPs
remain to be elucidated, LAMPs are the most thoroughly investigated binding proteins of
amelogenins to date.

Concluding Remarks
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Emerging evidence suggests that amelogenins possess multiple functions, not only as an
extracellular matrix for enamel mineralization, but also as signaling molecules in
mesenchymal cells including periodontal tissues and in osteoclastogenesis. In addition to the
MEK-ERK pathway, recent publications suggested that amelogenins including full-length
amelogenin and LRAP also regulate the Wnt/β-catenin pathway55,56). Further
comprehensive insights into the signaling pathway modulated by the amelogenin proteins
will lead to the development of new therapeutic approaches for treating dental diseases and
disorders such as idiopathic root resorption and periodontitis.

Acknowledgments
We would like to acknowledge the great help provided by the NIDCR DNA Sequencing Core. We would also like
to thank Dr. Shin-ichi Harashima and Kiyoyuki Torigoe for their useful discussion and technical advice regarding
yeast screening.

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 Haruyama et al. Page 6

We would like to thank Taduru Sreenath for helpful discussions and Shelagh Powers for expert editorial assistance.
These studies were supported by the Division of Intramural Research, NIDCR, NIH. We are also grateful for the
grant given to us by the Japanese Ministry of Education, Global Center of Excellence (GCOE) Program, which
supported our research.
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Fig. 1.
Amelogenin gene architecture, splice isoforms, and proteins in the mouse. (A) The
amelogenin gene (Amelx) consists of nine exons. There are three alternative acceptor sites
(arrowheads) in exon 6. (B) The two major splicing isoforms of the amelogenin gene: M180
and LRAP. Most transcripts are transcribed from exons 1 to 7 and skip exons 4, 8, and 9.
Exon 2 consists of a Kozak consensus sequence in addition to an ATG start codon. A stop
codon is located as the second codon of exon 7. (C) M180 and LRAP (M59) amelogenin
proteins. The initial 16 amino acids (AAs) at the N-terminus are signal peptides and are
removed before secretion.

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Mature amelogenins retain only two AAs from exon 2 and one AA from exon 7. The serine
at position 16 from the N terminus is phosphorylated. LRAP lacks a tyrosyl motif and the
central hydrophobic part in M180, which are translated from exon 6a, b, and c. The
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terminology for exon 6 splicing has been published previously. 6,9)

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Table 1
LRAP interacting proteins in human bone marrow.
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Gene accession Abbreviation Details

no.

NM_002483 CEACAM6 Carcinoembryonic antigen-related cell adhesion molecule 6. Seven clones: AA135–344 (stop), 157–344
(stop) and full clones incl. immunoglobulin domains and asparagine-linked glycosylation sites.

NM_006864 LILRB3 Leukocyte immunoglobulin-like receptor, subfamily B (with TM and ITIM domains), member 3
(LILRB3), transcript variant 2 (mouse homologue of PIR-B). Two clones: AA 256–631 (stop) incl.
immunoglobulin domains.

NM_032603 LOXL3 Lysyl oxidase like-3. AA 365–3121 (stop) incl. scavenger receptor cysteine-rich (a family reminiscent of
the immunoglobulin superfamily) domain.

NM_004867 ITM2A Integral membrane protein 2A. Full clone incl. BRICHOS domain.

NM_002778 PSAP Prosaposin. AA 422–526 (stop) incl. saposin-like type A and B region.

NM_002205 ITGA5 Integrin alpha 5 (fibronectin receptor, alpha polypeptide). Full clone.

NM_001909 CTSD Cathepsin D. AA 295–412(stop).

List of LRAP interacting proteins in human bone marrow.

NCBI (http//www.ncbi.nlm.nih.gov/nuccore) gene accession number, abbreviations of the gene name, and clone details including the protein
portion isolated in yeast two-hybrid screening are listed. The clones in bold are discussed in this manuscript. A full clone includes an entire open
reading frame. stop, stop codon: AA, amino acid.
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Table 2
LRAP interacting proteins in mouse CM/PDL.
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Gene accession Abbreviation Details

no.

NM_010729 Loxl1 Lysyl oxidase-like 1. Seven clones: AA 259–607 (stop), 430–607 (stop), and 496–607 (stop) incl. lysyl-
oxidase superfamily domain.

NM_007743 Col1a2 Collagen, type I, alpha 2. Two clones: AA 576–1372 (stop) 1178–1372 (stop) incl. fibrillar collagen C-
terminal domain.

NM_007737 Col5a2 Collagen, type V, alpha 2. Two clones: AA 935–1497 (stop) and full clone.

NM_019986 Habp4 Hyaluronic acid binding protein 4. Full clone. Also known as Ki-1/57, probably involved in cellular
events, such as pre-mRNA splicing.

NM_008410 Itm2b Integral membrane protein 2B. AA 105–206 (stop) incl. BRICHOS domain.

NM_010227 Flna Filamin alpha. AA 2560–2639 (stop) of filamin alpha transcript variant 1 incl. filamin tandem sequence
repeats that are predicted to have immunoglobulin-like folds.

NM_010860 Myl6 Myosin light polypeptide 6. Full clone incl. EF-hand, calcium binding motif.

NM_001164352 Efemp2 EGF-containing fibulin-like extracellular matrix protein 2 isoform 2. AA 140–370 (stop) incl. calcium-
binding EGF-like domain and Von Willebrand factor type A (vWA) domain.

List of LRAP interacting proteins in mouse CM/PDL.

NCBI (http://www.ncbi.nlm.nih.gov/nuccore) gene accession number, abbreviations of gene name, and clone details including the protein portion
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isolated in the yeast two-hybrid screening are listed. The clones in bold are discussed in this manuscript. Full clone includes an entire open reading
frame. stop, stop codon: AA, amino acid
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