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Dynamics of amelogenin self-assembly during in vitro proteolysis by Mmp-20

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Dynamics of amelogenin self-assembly during in vitro proteolysis by Mmp-20

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DYNAMICS OF AMELOGENIN SELF-ASSEMBLY DURING IN VITRO
PROTEOLYSIS BY MMP-20

by

Ruiwen Ma

A Thesis Presented to the
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
MASTER OF SCIENCE
(CRANIOFACIAL BIOLOGY)

May 2011

Copyright 2011 Ruiwen Ma

ii
Acknowledgements
This thesis would not have been possible without the expertise and guidance from my
supervisor Dr. Janet Moradian-Oldak, who has supported me throughout my research
training and thesis editing. Her sharp insight as a scientist and kindness as a human being
have influenced me not only during my graduate study but also in my future.
I am heartily thankful to Dr. Michael Paine for his encouragement and consideration
regarding my academic and career development. I also thank Dr. Margarita Zeichner-
David for generously giving her time and advice to help me accomplish this thesis.
Thanks to all the three professors for contributing and extending their valuable assistance
in the preparation and completion of this thesis. It is an honor for me to thank Dr. Glenn
Clark for introducing me to this great program and research center.
I am indebted to everyone in my lab: Dr. Xiudong Yang, Dr. Daming Fan, Shibi Mathew-
George, Dr. Keith Bromley, Dr. Sowmya Bekshe Lokappa as well as Jaime Jimenez,
Katie Trawick for their unselfish and unfailing support. I also would like to express my
gratitude to Dr. Zhi Sun and Sha Li for their assistance.
Lastly, I offer my regards and blessings to all my family, friends at USC and in China,
who supported me in any respect during the completion of my study.

iii
Table of Contents
Acknowledgements ································ ································ ·················· ii

List of Tables ································ ································ ························· v

List of Figures ································ ································ ························ vi

Abbreviations ································ ································ ························ vii

Abstract ································ ································ ······························ viii

Chapter 1: Introduction ································ ································ ·············· 1
1.1 Background ································ ································ ·················· 1
1.2 Objectives and Scope of the Work································ ························ 4

Chapter 2: rP172 Proteolysis by rpMmp-20 and Proteolysis Mimicking at pH 8.0 ········ 6
2A rP172 Proteolysis by rpMmp-20 at pH 8.0 ································ ················· 6
2A.1 Rationale ································ ································ ··················· 6
2A.2 Materials and Methods ································ ································ ··· 6
2A.2.1 Expression and Purification of rP172 ································ ·········· 6
2A.2.2 Expression and Purification of Recombinant Porcine Mmp-20 ············· 8
2A.2.3 Analysis of rpMmp-20 by Zymography ································ ········ 8
2A.2.4 Amelogenin Proteolysis by rpMmp-20 ································ ········· 9
2A.2.5 SDS-PAGE ································ ································ ·········· 9
2A.2.6 Size Analysis by Dynamic Light Scattering (DLS) ··························· 9
2A.2.7 Transmission Electron Microscopy (TEM) ································ ···· 9
2A.3 Results ································ ································ ····················· 10
2A.3.1. Progress of Amelogenin Proteolysis Monitored by SDS-PAGE ·········· 10
2A.3.2 DLS Analysis of Particle Size Changes during Proteolysis ················· 12
2A.3.3 Proteolysis Induced Nanochain Assembly Observed by TEM ············· 14
2A.4 Discussion ································ ································ ················· 17
2B Mimicking the Proteolysis Process at pH 8.0 ································ ············· 18
2B.1 Rationale································ ································ ··················· 18
2B.2 Materials and Methods ································ ································ ·· 19
2B.2.1 Expression and Purification of Recombinant Amelogenins ················· 19
2B.2.2 Mimicking Proteolysis ································ ···························· 19
2B.2.3 Size Analysis by Dynamic Light Scattering (DLS) ·························· 20
2B.3 Results ································ ································ ····················· 20
2B.4 Discussion ································ ································ ················· 21

iv
Chapter 3: Amelogenin Proteolysis by rpMmp-20 at pH 7.4 ································ 23
3.1 Rationale ································ ································ ····················· 23
3.2 Materials and Methods ································ ································ ···· 23
3.2.1 Expression and Purification of rP172 and rpMmp-20 ························· 23
3.2.2 Amelogenin Proteolysis by rpMmp-20 ································ ·········· 23
3.2.3 SDS-PAGE ································ ································ ··········· 23
3.2.4 Size Analysis by Dynamic Light Scattering (DLS) ···························· 24
3.2.5 Transmission Electron Microscopy (TEM) ································ ······ 24
3.3 Results ································ ································ ······················ 24
3.3.1 Progress of Amelogenin Proteolysis Monitored by SDS-PAGE ············· 24
3.3.2 DLS Analysis of Particle Size Changes during Proteolysis ··················· 26
3.3.3 Proteolysis Induced Nanochain Assembly at pH 7.4 Observed by TEM ··· 28
3.4 Discussion ································ ································ ··················· 30

Chapter 4: General Discussion and Future Directions ································ ········· 32

References ································ ································ ···························· 39

v
List of Tables
Table 1: Percentage of Each Protein/peptide Band Detected by SDS-PAGE
(pH 8.0, substrate: enzyme=200:1) 11

Table 2: Isolated Amelogenin Particle Size Changes during Proteolysis
at pH 8.0, Measured in TEM Images 16

Table 3: Percentage of Each Protein/peptide Band Detected by SDS-PAGE
(pH 7.4, substrate: enzyme=200:1) 25

Table 4: Isolated Amelogenin Particle Sizes Changes during Proteolysis
at pH 7.4, Measured in TEM Images 30

vi
List of Figures
Figure 1: Amino Acid Sequences of Amelogenins 7

Figure 2: Proteolysis Process Monitored by SDS-PAGE
(pH 8.0, substrate: enzyme =200:1) 11

Figure 3: Proteolysis Process Monitored by SDS-PAGE
(pH 8.0, substrate: enzyme =100:1) 12

Figure 4: DLS Data Showing the R
H
of rP172 and Particle
Size Changes during Proteolysis at pH 8.0 13

Figure 5: TEM Images of rP172 Proteolysis at pH 8.0, 37 º C,
at Different Proteolysis Time Points 15

Figure 6: Plots of Particle Size Distribution of rP172 and
rP148 Mixtures at Different Ratios 21

Figure 7: Proteolysis Process Monitored by SDS-PAGE
(pH 7.4, substrate: enzyme =200:1) 25

Figure 8: DLS Data Showing the R
H
of rP172 and Particle
Size Changes during Proteolysis at pH 7.4 27

Figure 9: TEM Images of rP172 Proteolysis at pH 7.4, 37 º C,
at Different Proteolysis Time Points 29

vii
Abbreviations
CBB: coomassie brilliant blue
DLS: dynamic light scattering
rP172: full length recombinant porcine amelogenin
rP148: a recombinant amelogenin fragment that lacks the hydrophilic C-terminal 24
amino acids

rpMmp-20: recombinant porcine matrix metalloproteinase 20 (enamelysin)
R
H
: hydrodynamic radii
SDS-PAGE: sodium dodecyl sulfate polyacrylamide gel electrophoresis
TEM: transmission electron microscopy
TRAP: tyrosine rich amelogenin polypeptide
RP-HPLC: Reversed phase high performance liquid chromatography
MF: minifermentation number

viii
Abstract
Amelogenin self-assembly and its stepwise processing are crucial for the enamel
biomineralization. The removal of highly conserved (A- and B-) domains affects
amelogenin self-assembly, resulting in enamel defect in transgenic and knock-in mice.

We hypothesized that the amelogenin self-assembly can be altered by its stepwise
proteolysis by Mmp-20, and the changes in self-assembly will eventually affect the
structural organization of the forming enamel matrix, apatite nucleation, and
mineralization. To examine the hypothesis, the full-length recombinant pig amelogenin
(rP172), and a recombinant analogue (rP148) representing the most stable proteolytic
product (the “20k”) were used. Recombinant rpMmp-20 was used to digest rP172 at pH
8.0 and pH 7.4, at temperature 37 ° C, with substrate: enzyme ratios of 100:1 and 200:1.
The proteolysis progress and particle size distribution during proteolysis were monitored
by SDS-PAGE and dynamic light scattering.

At pH 8.0, 37º C, SDS-PAGE revealed that, a major product (2-148 segment) was
detected after 1 hour of proteolysis, and was accumulated until up to 20 hours; when the
majority of the substrate was cleaved. This major product was confirmed to be the
analogue of native “20k” amelogenin. Mass spectrometric analysis confirmed the
presence of the Tyrosine Rich Amelogenin Polypeptide (TRAP) and other small
proteolytic fragments. Analysis by dynamic light scattering revealed that rP172 possessed

ix
a distribution of particles with R
H
of 13.49 nm. During the first ten minutes of rP172
proteolysis small particles of 3.8 nm appeared in the solution. After the first hour of
rpMmp-20 action, in addition to the 3.8 nm and 19.7 nm particles, large mono-dispersed
assemblies with R
H
of 103.8 nm were also detected. Following 2 hours of proteolysis, the
sizes of the large assemblies increased to about 125.7 nm. DLS analysis of a 1:1 mixture
of recombinant rP172 and rP148 demonstrated a significant shift in particle size
distribution when compared to the individual components: rP172 and rP148 alone. Using
TEM, we detected monodispered spherical particles (~16nm diameter) formed on the
TEM grid of rP172 at pH 8.0. After rpMmp-20 digestion, aggregation of the spherical
particles was initiated around 1 hour, and chain formation was evident during 2nd to 4th
hours, chain-like structures formed around 4 hours and curled after 20 hours. A general
decrease in diameters was also observed after proteolysis. While the pure amelogenin
samples (rP172, rP148) did not show any signs of chain formation after 24 hours
incubation.

At pH 7.4, SDS-PAGE revealed that the proteolysis proceeded during the first hour and
slowed down after 2 hours comparing to that at pH 8.0. In DLS analysis, rP172 possessed
a major particle size distribution of R
H
at 28.3 nm. During the first ten minutes of
proteolysis, small particles of 5.7 nm appeared in the solution. After the first hour of
rpMmp-20 action, in addition to the 2.6 nm and 11.5 nm particles, large assemblies with
R
H
of 45.3 nm were also detected. Following 10 hours of proteolysis, the sizes of the
large assemblies increased to about 151.1 nm. Large aggregates (≥7000 nm) presented

x
throughout the proteolysis, with a gradual decrease in their percentage mass. Under TEM
observation, trends in nano-chain assembly was demonstrated similar to that mentioned at
pH 8.0 proteolysis, but the appearance of chain-assembly occurred earlier than in the case
of pH 8.0 (i.e. after 2 hours). No sign of chain formation was observed after incubating
rP172 for 4 days.

These results support the hypothesis that the stepwise cleavage of amelogenin by Mmp-
20 alters the dynamic of amelogenin self-assembly promoting the formation of chain-like
structures. And there is a co-assembly between the full-length amelogenin and its
proteolysis products, mainly the 2-148. It is proposed that such proteolysis activities will
control the structural organization of forming enamel matrix and eventually the process
of elongated apatite growth.

Key words: amelogenin, rP172, rP148, self-assembly, Mmp-20, proteolysis

1
Chapter 1: Introduction
1.1 Background
During amelogenesis, the enamel structural proteins are expressed and secreted in a
unique manner. Of these proteins, amelogenin constitutes more than 90% (weight) of the
organic matrix (Termine et al., 1988). Amelogenin is secreted by ameloblasts into the
extracellular space and immediately processed at the C-terminal by the metalloproteinase
enamelysin (Mmp-20), producing intermediate products that are stable throughout the
secretory stage of enamel formation. The N-terminal is subsequently cleaved resulting in
additional stable products (Bartlett et al., 1998). The importance of amelogenin self-
assembly and stepwise proteolytic processing for elongated crystal growth, organization
and maturation of enamel prisms has been well recognized (Warshawsky et al., 1989;
Fincham et al., 1995; Fincham et al., 1999; Moradian-Oldak et al., 1998b; Gibson et al.,
2001). The removal of highly conserved (A- and B-) domains, which affects amelogenin
self-assembly, leads to the enamel malformation in transgenic and knock-in mice
(Caterina et al., 2002; Ozdemir et al., 2005; Espí rito Santo et al., 2007; Lee et al., 2010 ).

It was suggested that, during the secretory stage of pig teeth enamel development, Mmp-
20 is the only significantly active proteinase in the enamel extracellular space (Fukae et
al., 1998). It processes amelogenins into a group of cleavage products that accumulate
and slowly further degraded by Mmp-20. Through the fluorescent peptides digestion of
P173, it was demonstrated that Mmp-20 digests amelogenin sequences at the exact sites

2
that yield all of the major amelogenin cleavage products (Ryu et al 1999; Moradian-
Oldak et al., 2001; Sun et al., 2005): the 23kDa (Met1-Pro162), 20kDa (Met1-Ser148),
13kDa (Leu46-Ser148), 11kDa (Ala63-Ser148/Leu64-Ser148), extended TRAP (Met1-
His62/Met1-Ala63), and TRAP (Met1-Trp45). Mmp-20 had limited activity for
degrading the tyrosine- and leucine-rich amelogenin peptides, which accumulated during
the proteolysis. The critical function of Mmp-20 was evident by the previous reports that
the mutation in Mmp-20 gene (Bartlett et al., 1996) leads to abnormal enamel formation
and amelogenesis imperfecta (Stephanopoulos et al., 2005). The slow proteolysis of
amelogenins by Mmp-20 is supposed to be responsible for the widening of secretory-
stage enamel crystals with depth (Daculsi and Kerebel, 1978). The importance in the
Mmp-20 processing function is sustained by the loss of prism structure and failure to
achieve the full enamel thickness in the Mmp-20 gene knockout mice; while its
degradative function is supported by the enamel protein retention and the fragile enamel
in those mice (Caterina et al., 2002). While it has been accepted that MMP-20 is
absolutely essential for normal enamel formation, detailed molecular mechanisms for its
activity and its effect on amelogenin self-assembly is still limited.

The purpose of this study was to test the hypothesis that amelogenin self-assembly will
be altered following its stepwise proteolysis by Mmp-20, and the change in self-assembly
will eventually affect the structural organization of the forming enamel matrix, apatite
nucleation, and mineralization. To prove this hypothesis, transmission electron
microscopy (TEM), dynamic light scattering (DLS) and SDS-PAGE were used to

3
monitor the changes in rP172 particle size distribution and assembly behavior, during its
proteolysis by Mmp-20 at pH 8.0 and pH 7.4. The highly purified pig recombinant
amelogenin rP172 was used, as an analogue of the natural full-length porcine amelogenin
P173 (Fig. 1). The rP172 maintains the major bio-molecular properties of nature porcine
amelogenin, even though it lacks the methionine residue at its first amino acid position
and a phosphate at Ser16. In addition, I investigated the co-assembly of recombinant full-
length rP172 and rP148 under defined ratios mimicking the proteolysis process. rP148 is
a recombinant analogue of the 20kDa amelogenin proteolytic (Yamakoshi et al., 1994;
Sun et al., 2005). The rP148 is a mutant form, which lacks the N-terminal Met and ends
at Met 149. Comparing to the rP172, rP148 demonstrates different assembly patterns. At
room temperature, rP148 molecules will assemble into monomers, dimers, oligomers, and
some nanospheres. Large aggregates were also observed in some solutions. A higher
aggregation tendency of rP148 protein also shows at pH 7.4–8.0, for its lacking of the
hydrophilic C-terminus. The absence of the hydrophilic C-terminus can have a significant
influence on the molecular configuration and protein self-assembly property. Previous
studies have demonstrated that a 16-amino-acid-length region within the C-terminus
(self-assembly „B-‟ domain) is critical for the protein-protein interaction and proper
nanosphere self-assembly (Moradian-Oldak et al., 2000). However it is still not clear
whether the domains within this C-terminus are directly involved in self-assembly.

4
1.2 Objectives and Scope of the Work
The purpose of this study is to test the hypothesis that amelogenin self-assembly will be
altered following its stepwise proteolysis by Mmp-20. We proposed that the changes in
self-assembly will eventually affect the structural organization of the forming enamel
matrix, apatite nucleation, and mineralization.

Three specific aims were proposed to examine this hypothesis in a series of in vitro
experimental model systems:
I. To monitor amelogenin assembly following proteolysis at pH 8.0 (Chapter
2A)
II. To mimic amelogenin co-assembly during proteolysis at pH 8.0 by combining
the substrate with different cleavage components (Chapter 2B)
III. To monitor amelogenin assembly following proteolysis at pH 7.4 (Chapter 3)

The rP172 and rP148 were used as the substrate and major cleavage products to analyze
the proteolytic processing of amelogenin. Recombinant porcine MMP-20 (rpMmp-20)
was used as the enzyme replicating the proteolytic events during enamel
biomineralization. Transmission electron microscopy (TEM), dynamic light scattering
(DLS) and SDS-PAGE were selected to monitor the changes in rP172 particle size
distribution and assembly behavior, during its proteolysis by Mmp-20 at pH 8.0 and pH
7.4. DLS was also used to investigate the mimicking process.

5
In order to further understand the purpose of amelogenin proteolysis by MMP-20 in
enamel formation two chapters are presented. In chapter 2, I will focus on experiments at
pH 8.0, conditions under which amelogenin solubility and nanosphere formation are
optimal. The rP172 proteolysis progress and assembly changes following rpMmp-20
action will be monitored (aim I). I will then analyze the proteolysis mimicking
experiments at pH 8. 0 in order to further demonstrate co-assembly between amelogenin
and its proteolytic products (aim II). In chapter 3, I will focus on the physiologically
relevant pH 7.4 where the assembly alterations of rP172 following proteolysis will be
examined the same way as in chapter 2.

6
Chapter 2: rP172 Proteolysis by rpMmp-20 and Proteolysis
Mimicking at pH 8.0
2A rP172 Proteolysis by rpMmp-20 at pH 8.0
2A.1 Rationale
Proteolysis was conducted at pH 8.0 primarily because of the solubility of amelogenins.
Previous studies have shown that amelogenins dissolve better in Tris buffers, and the
rP172 has a good solubility at pH 8.0 where nanosphere formation is optimal (Fincham et
al. 1994; Moradian-Oldak et al., 1994). Another reason that makes pH 8.0 ideal for our
experiments is that the formation of nano-chains by amelogenin does not occur at pH 8.0
but neutral pH (Wiedemann-Bidlack et al. 2007). Finally, pH 8.0 is within the optimal pH
ranges for Mmp-20 activity.

The substrate: enzyme ratio was selected to be 200:1 according the previous findings
(Sun, 2008). We have optimized speed and efficiency by performing a few preliminary
experiments to access rpMmp-20 activity. The standard substrate: enzyme ratio of 100:1
was also used (Moradian-Oldak et al., 2001) to explore the enzyme properties

2A.2 Materials and Methods
2A.2.1 Expression and Purification of rP172
Recombinant porcine amelogenin rP172 was expressed, purified, and characterized as
previously described (Simmer et al., 1994; Ryu et al., 1999; Sun et al., 2006). In brief,

7
rP172 was expressed in E. coli strain BL21-codon plus (DE3)-RP (Strategene, La Jolla,
CA, USA) and precipitated by ammonium sulfate. The precipitate was dissolved,
fractioned, and purified by reverse phase-high performance liquid chromatography (RP-
HPLC, C4 semi-preparative column, Jupiter, 5 μm, 10 mm x 250 mm). The homogeneity
of the protein was verified by analytical chromatography (C4 analytical column, Jupiter,
5 μm, 2 mm x 250 mm). The protein solution was lyophilized and stored at -20° C. The
rP172 batch “MF118” (minifermentation number 118) was used in proteolysis at pH 8.0
and for the mimicking experiments (see Chapter 2B for details and definition of
mimicking experiments).

Figure 1: Amino Acid Sequences of Amelogenins. A, full length porcine amelogenin (P173). B.
recombinant amelogenin rP172 and rP148: the rP172 used in this thesis lacks the first methionine and a
phosphate group on serine
16
, and the rP148 used lacks the first methionine and ends at Met
149
. “--”
represents the missing amino acid; the underlined part represents the hydrophilic C-terminal domain (the
remaining residues are primarily hydrophobic).
A. Amino Acid Sequence of Full-length Porcine Amelogenin (P173):
10 20 30 40 50
MPLPPHPGHP GYINFS
P
YEVL TPLKWYQNMI RHPYTSYGYE PMGGWLHHQI IPVVSQQTPQ
60 70 80 90 100 110
SHALQPHHHI PMVPAQQPGI PQQPMMPLPG QHSMTPTQHH QPNLPLPAQQ PFQPQPVQPQ
120 130 140 150 160 170
PHQPLQPQSP MHPIQPLLPQ PPLPPMFSMQ SLLPDLPLEA WPATDKTKRE EVD

B. Amino Acid Sequence of Recombinant Porcine Amelogenins :
rP172 10 20 30 40 50
--PLPPHPGHP GYINFSYEVL TPLKWYQNMI RHPYTSYGYE PMGGWLHHQI IPVVSQQTPQ
60 70 80 90 100 110
SHALQPHHHI PMVPAQQPGI PQQPMMPLPG QHSMTPTQHH QPNLPLPAQQ PFQPQPVQPQ
120 130 140 150 160 170
PHQPLQPQSP MHPIQPLLPQ PPLPPMFSMQ SLLPDLPLEA WPATDKTKRE EVD

rP148 10 20 30 40 50
--PLPPHPGHP GYINFSYEVL TPLKWYQNMI RHPYTSYGYE PMGGWLHHQI IPVVSQQTPQ
60 70 80 90 100 110
SHALQPHHHI PMVPAQQPGI PQQPMMPLPG QHSMTPTQHH QPNLPLPAQQ PFQPQPVQPQ
120 130 140
PHQPLQPQSP MHPIQPLLPQ PPLPPMFSM--------------------------------------------------

8
2A.2.2 Expression and Purification of Recombinant Porcine Mmp-20
The rpMmp20 was processed as previous reported (Ruy et al., 1999; Moradian-Oldak et
al., 2000; Sun et al., 2006). It was expressed from the pPROEX-1 vector (Life
Technologies, Carlsbad, CA, USA) in E. coli strain XL-1 Blue (Strategene, La Jolla, CA,
USA). The rpMMP-20 was purified by affinity column using Ni-NTA resin (Qiagen,
Valencia, CA, USA) and activated by 4-aminophenylmercuric acetate (APMA) (Ryu et al
1999). Enzyme concentration was quantified by UV adsorption at 220 nm. The enzyme
solutions were stored at -20º C.
2A.2.3 Analysis of rpMmp-20 by Zymography
The activity of rpMmp-20 was analyzed by 12% Zymogram (Casein) Gel (Novex,
Inventrogen). Corresponding HPLC fractions was desalted against Tris-HCl buffer
(50mM, pH 7.4), activated (50mM Tris-HCl + 5mM Ca
2
Cl, pH 7.4) at 37° C for 24 hours,
and centrifuged. The supernatant was kept, and incubated with rP172 for 2 hours.
Samples were mixed with loading buffer and electrophoresed on 12% SDS-
polyacrylamide gels containing 2% casein at 125V. The gel was washed and incubated in
incubation buffer (100mM Tris-HCl + 10mM Ca
2
Cl, pH 8.0) at 37 ° C for overnight.
Then the gel was stained with Coomassie Brilliant Blue R-250 (0.125% Coomassie
Brilliant Blue R-250, 0.1% amino black, 10% acetic acid, 50% methanol) for 1 hour, and
distained in distaining buffer. Active rpMmp-20 appeared as clear zones against the blue
background (22 kDa).

9
2A.2.4 Amelogenin Proteolysis by rpMmp-20
For proteolysis at pH 8.0, recombinant porcine amelogenin rP172 was dissolved in
25mM Tris-HCl (pH 8.0, 37º C) with a final rP172 concentration of 0.2mg/ml, and
incubated with rpMmp-20 at 37º C with a substrate: enzyme ratio of 200:1 or 100:1
(w:w). The proteolysis products were collected and analyzed at 0.1, 1, 2, 4 and 10 hours
respectively (Sun et al., 2008; Sun et al., 2010).
2A.2.5 SDS-PAGE
The rP172 and its proteolysis samples at pH 8.0 were loaded onto a 12% acrylamide gel
(Bio-Red, 161-0325), electrophoresed under 125V, and stained with Coomassie Brilliant
Blue R 250. To quantify each proteolysis portion and monitor the proteolysis process, gel
density was analyzed by ImageJ 1.43 software.
2A.2.6 Size Analysis by Dynamic Light Scattering (DLS)
Samples (25 µ l) from rP172 and its proteolysis at pH 8.0 were immediately measured by
DLS (Wyatt DynaPro Nanostar, Santa Barbara, CA) with a solid-state laser operating at
663 nm. The experimental temperature was set at 37 º C. Data was collected from 30
measurements and analyzed with Dynamics 7.0 software. The hydrodynamic radii (R
H
)
and mass percentage of amelogenin particles were obtained from regularization analysis
(Sun et al., 2006; Lakshminarayanan et al., 2010), with cutoff points at 1.0 and 1.0E+4.
2A.2.7 Transmission Electron Microscopy (TEM)
300 mesh carbon coated grids were merged in the samples (30 µ l) from rP172 and its
proteolysis collections at pH 8.0 for 0.5 minute, followed by 0.5 minute of 1% urinal
acetate staining and air drying. The sample grids were observed under the Jeol TEM with

10
voltage of 80 kV. Particle shown were measured with ImageJ, and their diameters were
compared and analyzed.

2A.3 Results
2A.3.1. Progress of Amelogenin Proteolysis Monitored by SDS-PAGE
During rP172 proteolysis by rpMmp-20 under 200:1 ratio, the substrate rP172 and six
proteolytic products were detected by SDS-PAGE (Figure 2A). Each product is shown by
amino acid range based on the full length porcine amelogenin (Fig.1 ). The six products
are 2-162, 2-148, 46-148 (13k), 164-148 (11k), 2-63 (extended TRAP), 2-45 (TRAP)
(Ryu et al., 1999, Sun et al., 2005).

As evaluated by their corresponding band densities on SDS-PAGE (Figure 2B, Table 1),
about 5% of rP172 (MF 118) was cleaved immediately after the addition of rpMmp-20
(within 10 min), and 50% of rP172 was digested after around 2 hours. The 2-148 segment,
which migrated around 20kDa, was the immediate and main proteolysis product without
being significantly digested by rpMmp-20 even after 20 hours (Sun, 2008). The 2-162
segment appeared throughout the proteolysis and reached a stable level after 4 hours.
Other products, 13k, 11k, extended TRAP (2-63 segment), and TRAP became detectable
after 4 hours, with a slow increase in amount with the progress of proteolysis. The rP172:
2-148 ratios achieved 3:1, 1:1, 1:2, and 1:10 around 1, 2, 4, 10 hours respectively. The
rP172 was fully cleaved after 20 hours.

11

Figure 2: Proteolysis Process Monitored by SDS-PAGE (pH 8.0, substrate: enzyme =200:1). A, SDS-
PAGE stained with Coomassie Brilliant Blue. Numbers shown on top are proteolytic time in hours. Lane
Std. shows a molecular size standard. The values on the left of each band in the standard lane are molecular
weight in kDa. The two lanes on right (rP172, rP148) were pure protein controls without rpMmp-20.
Proteolytic products were detected as dark bands. Each marked band is shown by amino acid range based
on the full length porcine amelogenin. For each band, the range is: I, 2-173 (rP172, MF 118); II, 2-162; III,
2-148; IV, 46-148 (13k); V, 164-148 (11k), VI, 2-63 (extended TRAP); VII, 2-45 (TRAP) (Ryu et al.,
1999, Sun et al., 2005). B, Gel density analysis of each band. Numbers on X axis are proteolytic time in
hours, values on Y axis are the percentage of substrate or each product detected during the proteolysis
corresponding to SDS-PAGE.

Time
(hrs)
%
0.1 1* 2 4 10 20
rP172 95.38 61.11 45.98 24.62 5.66 1.28
2-162 ---- 14.45 17.24 21.78 22.64 24.3
2-148 4.62 24.44 36.78 49.24 56.60 53.71
13k ---- ---- ---- ---- 5.66 6.39
11k ---- ---- ---- ---- 2.83 3.32
Extended TRAP ---- ---- ---- 1.52 1.89 3.84
TRAP ---- ---- ---- 2.84 4.72 7.16
rP172:2-148 ratio 21:1 3:1 1:1 1:2 1:10 1:42

Table 1: Percentage of Each Protein/peptide Band Detected by SDS-PAGE (pH 8.0, substrate:
enzyme=200:1). *: at 1 hour, amelogenin chain formation was initiated under TEM observation; ----:
product not detected.

A B

12
The proteolysis under a substrate: enzyme ratio of 100:1 (Figure 3) also showed a similar
proteolysis pattern, which may indicate that: the reaction time is more crucial for the
amelogenin proteolysis than the substrate: enzyme ratio; and the rpMmp-20 cleavage
may be time sensitive and sequence specific.

Note that in order to access changes in amelogenin self-assembly, in the following
sections the substrate: enzyme ratio of 200:1 was selected for the proteolysis
experiments.

Figure 3: Proteolysis Process Monitored by SDS-PAGE (pH 8.0, substrate: enzyme =100:1). A, SDS-
PAGE stained with Coomassie Brilliant Blue. Numbers shown on top are proteolytic time in hours. Lane
Std. shows a molecular size standard. The values on the left of each band in the standard lane are molecular
weight in kDa. Proteolytic products were detected as dark bands. Each marked band is shown by amino
acid range based on the full length porcine amelogenin. For each band, the range is: I, 2-173 (rP172, MF
118); II, 2-162; III, 2-148; IV, 46-148 (13k); V, 164-148 (11k); VII, 2-45 (TRAP) (Ryu et al., 1999, Sun et
al., 2005). B, Gel density analysis of each band. Numbers on X axis are proteolytic time in hours, values on
Y axis are the percentage of substrate or each product detected during the proteolysis corresponding to
SDS-PAGE.

2A.3.2 DLS Analysis of Particle Size Changes during Proteolysis
The substrate rP172 possessed a major distribution of particles with R
H
of 13.49 nm at
pH 8.0 (Figure 4). During the first ten minutes of proteolysis, small particles of 3.8 nm
A B

13
appeared in the solution. Such small particles may well be associated with small
proteolytic products or disassembled amelogenin fragments following the cleavage of
TRAP domain.

After the first hour of rpMmp-20 action, in addition to the 3.8 nm and 19.7 nm particles,
large mono-dispersed assemblies with R
H
of 103.8 nm were also detected. Following 2
hours of proteolysis, when the ratio of rP172 to 2-148 is 1:1, the sizes of the large
assemblies increased to about 125.7 nm. Larger particles with 5400-7800 nm R
H
were
detected throughout the whole digestion process, mainly due to the undissolved huge
aggregations formed in the substrates difficult to digest by rpMmp-20.

Figure 4: DLS Data Showing the R
H
of rP172 and Particle Size Changes during Proteolysis at pH 8.0.
rP172 rP148
0.1 hr 4 hrs

14
Figure 4 (Continued)

2A.3.3 Proteolysis Induced Nanochain Assembly Observed by TEM
The rP172 formed monodispered spherical particles (~16nm diameter) on the TEM grid
at pH 8.0 (Figure 5: rP172; Table 2). Aggregation of the spherical particles was initiated
around 1 hour of proteolysis (Figure 5: 1 hr, white arrows), and chain formation was
evident during the second to fourth hours (Figure 5: 2 and 4 hrs, white arrows), a chain-
like structures was formed around 4 hours (Figure 5: 4 hrs, white arrows) and curled after
20 hours (Figure 5: 10 hrs, white arrows). Chain formation was initiated when the ratio of
rP172: 2-148 segment reached 3:1 and longer chains were formed at the ratio of 1:1
(Table 2). However, when control samples rP172 and rP148 were incubated individually
1 hr 10 hrs
2 hrs 20 hrs

15
without any enzyme, at 37º C for 24 hours, only amorphous aggregations were observed
without any chain-like structures (Figure 5: rP172, 24 hrs; Figure 5: rP148, 24 hrs).

Particles were measured as isolated spheres without any aggregations. For the isolated
spheres, a general decrease in diameter was observed after proteolysis. Those particles
with darker background resembled the spherical particles formed by recombinant porcine
amelogenin rP148 (Figure 5: rP148, black arrows).

Figure 5: TEM Images of rP172 Proteolysis at pH 8.0, 37 º C, at Different Proteolysis Time Points.
rP172 and rP148 shown were used as control without mixing with rpMmp-20. Image of 1hour digestion
showes chain innatiation, images of 2 and 4 hours show the process of chain formation, and image of 20
hours shows chains already curled. The rP172 and rP148 on the bottom were pure amelogenin solutions
incubated in 37º C for 24 hours before sampling. The black arrows on these two images show amorphous
aggregations.

rP172 rP148

1 hr 2 hrs

16
Figure 5 (Continued)

Particle
Diameters
(nm)
Measured as
isolated
particles,
based on 50
particles of
each sample
rP172 1 hr* 2 hrs 4 hrs 20 hrs rP148
Ave. 16.821 15.560 15.030 15.392 14.095 11.307
StdDev 2.682 2.921 3.034 2.971 2.045 1.244
Min. 12.035 10.839 10.462 10.383 10.482 8.765
Max. 24.786 21.398 22.215 22.313 19.901 14.262
rP172: 2-148 ratio
(Data from SDS-PAGE)
------ 3:1 1:1 1:2 1:10 ------
Table 2: Isolated Amelogenin Particle Size Changes during Proteolysis at pH 8.0, Measured in TEM
Images. *: chain formation initiated after 1 hour of proteolysis. After 2 hours, a rP172:2-148 ratio about
1:1 was achieved (according to corresponding SDS-PAGE analysis).
4 hrs 20 hrs

rP172, 24hrs rP148, 24 hrs

17
2A.4 Discussion
At pH 8.0, rpMmp-20 was active and functioned in a time-sensitive and sequence
specific manner. According to SDS-PAGE, the proteolysis process was more dependent
upon the reaction time than the substrate: enzyme ratio. The 0 to 20 hour range was an
appropriate duration and 200:1 was a proper substrate: enzyme ratio to conduct the
proteolysis.

Following the stepwise cleavage of amelogenin by rpMmp-20 at pH 8.0, alteration in
amelogenin self-assembly and the formation of chain-like structures were evident by
DLS and TEM. It is important to note that chain formation was not observed after
incubating the rP172 or rP148 alone under the same solution conditions: amelogenin
concentration (0.2 mg/ml) at pH 8.0, 37º C, even after 24 hours. Based on the quantitative
estimation of full-length amelogenin and its main proteolytic product at different time
points during proteolysis (Table 1) we suggest that chain formation at pH 8.0 required
both the full-length and the truncated amelogenin. After the first hour of proteolysis, the
large particles detected by DLS and the chains observed in TEM are therefore suggested
to be the result of co-assembly of amelogenin with their proteolytic products. Note that
due to high hydrophobicity and tendency to attach to each other, the long chains curl
together after 24 hours when the majority of the products is the 2-148 segment. In the
presence of mineral in vivo, the situation would be different and such general aggregation
of long chains would not occur. The progressive chain formation may suggest that during
enamel formation, amelogenins is cleaved by Mmp-20 leaving spherical particles

18
composed of full length amelogenin and its proteolytic products. Such chain-like
structure may have high affinity to minerals, guiding the elongated growth of apatite
crystals. With the Mmp-20 cleavage proceeds, the chains shrink in diameter leaving
space for more minerals to deposit between the chains.

The curved chains observed after 20 hours of proteolysis under TEM seemed not to be
detected on the corresponding DLS data. However, while looking at the DLS data from
its %Intensity panel (% intensity as Y Axis) rather than the %Mass panel, as showed
above, 99.2% of intensity came from particles with 58.9 nm radius. This group of
predominant particles is actually consistent with the curved chains in 20 hours TEM
image, whose diameters were around 100 nm. This may be attributed to the effect of
small particles which sheltered the curved chains in DLS %Mass panel.

2B Mimicking the Proteolysis Process at pH 8.0
2B.1 Rationale
In order to test the hypothesis that the full-length amelogenin co-assembles with its
proteolytic products, we designed experiments in which known ratios of full-length
(rP172) and truncated amelogenins (rP148) were mixed. The proteolysis was mimicked
by combining rP172 and rP148 at defined ratios. The rP148 is an analogue of the main
cleavage product (2-148 segment) during the rP172 proteolysis by rpMmp-20 (Figure 1).
According to the SDS-PAGE analysis of the proteolysis at pH 8.0, the ratios between
rP172 and 2-148 were about 3:1, 1:1, 1:2: 1:10 after 1, 2, 4 and 20 hours of proteolysis. If

19
similar results were observed in the mimicking experiments as those obtained during
rP172 proteolysis, it may suggest that the co-assembly of amelogenin and its proteolytic
product are responsible for altering amelogenin assembly behaviors following proteolysis.

2B.2 Materials and Methods
2B.2.1 Expression and Purification of Recombinant Amelogenins
Recombinant full-length porcine amelogenin (rP172) and an engineered mutant form
(rP148) were expressed, purified, and characterized as previously described (Simmer et
al., 1994; Ryu et al., 1999; Sun et al., 2006). In brief, rP172 and rP148 were expressed in
E. coli strain BL21-codon plus (DE3)-RP (Strategene, La Jolla, CA, USA) and
precipitated by ammonium sulfate. The precipitate was dissolved, fractioned, and purified
by reverse phase-high performance liquid chromatography (RP-HPLC, C4 semi-
preparative column, Jupiter, 5 μm, 10 mm x 250 mm). The homogeneity of the proteins
was verified by analytical chromatography (C4 analytical column, Jupiter, 5 μm, 2 mm x
250 mm). And the protein solutions were lyophilized and stored at -20° C. The rP172
batch MF118 and rP148 batch MF113 were used.
2B.2.2 Mimicking Proteolysis
rP172 and rP148 were individually dissolved in 25mM Tris-HCl (pH 8.0) with a final
amelogenin concentration of 0.2mg/ml for each sample, mixed at 3:1, 1:1, 1:2, 1:10 ratios
(w:w) according to SDS-PAGE density analysis of pH 8.0 proteolysis at different
proteolysis time points. And the particle sizes of rP172, rP148 and their mixtures were
measured by DLS.

20
2B.2.3 Size Analysis by Dynamic Light Scattering (DLS)
Samples (25 µ l) of rP172, rP148 and their mixtures were incubated in 37°C for 30 min,
measured by DLS (Wyatt DynaPro Nanostar) with a solid-state laser operating at 663 nm.
The experimental temperature was set at 37 º C. Data was collected from 30
measurements and analyzed with Dynamics 7.0 software. The hydrodynamic radii (R
H
)
and mass percentage of amelogenin particles were obtained from regularization analysis
(Lakshminarayanan et al., 2010, Sun et al., 2006), with cutoff points at 1.0 and 1.0E+4.

2B.3 Results
The rP172 possessed a major distribution of particles with R
H
of 13.49 nm (mass=88.2%)
at pH 8.0 37º C (Figure 6: blue bars), which was an indication for the present of
nanospheres in solution, while 7.8% particles had a R
H
of 7446.48 nm showing large
aggregation in solution. The rP148 has a higher tendency to aggregate at pH 8.0 37º C,
which was evident by the R
H
distribution at 372 and 3018.62 nm (Figure 6: green bars).

DLS analyses of 3:1, 1:1, and 1:2 ratio (w:w) of rP172 and rP148 revealed a significant
shift in the size distribution of particles detected in the mixtures (Figure 6: yellow bars)
when compared to each individual component: rP172 and rP148 alone. 1:10 mixture also
showed the similar trend, but with about 9.8% of the mixture obtained a slightly smaller
R
H
(12.8 nm) than the smallest R
H
of the

rP172 (13.8 nm). This small portion of particles
may be the oligomers formed by the mixture of rP172 and rP148.

21

Figure 6: Plots of Particle Size Distribution of rP172 and rP148 Mixtures at Different Ratios. Blue
bars represent rP172; green bars represent rP148, and yellow bars represent the mixtures of rP172 and
rP148 at different ratios. Values on the left are percentage mass of each component; values at the bottom
are the hydrodynamic radii (R
H
) of corresponding amelogenin particles.

2B.4 Discussion
The results of mixing experiments of full-length amelogenin and its proteolytic product
suggested that there might be co-assembly between the two components (He et al., 2008),
which may result in the initiation and formation of amelogenin chain-like structure and
nanoparticles. It is important to note that the rP148 slightly differs from 2-148 (Figure 1),
and other proteolysis products were not included in this mixing system. Moreover, the
stepwise cleavage of amelogenin by Mmp-20 should induce some time-dependent events
which were not considered in this mimicking process. In addition, other techniques such
as TEM and fluorescence are needed to support our hypothesis and reach a better

22
understanding of amelogenin co-assembly in vitro. Such studies will provide insight into
amelogenin assembly and function in vivo.

23
Chapter 3: Amelogenin Proteolysis by rpMmp-20 at pH 7.4
3.1 Rationale
Since the pH of enamel matrix during the secretary stages of enamel formation is 7.0 -
7.4 (Aoba and Moreno, 1987; Smith et al., 1996; Fukae et al.1998), and the optimal pH
for Mmp-20 is around pH 7.4, the proteolysis at pH 7.4 was also conducted to investigate
and mimic what can actually happen in vivo during the stepwise cleavage of amelogenin.

3.2 Materials and Methods
3.2.1 Expression and Purification of rP172 and rpMmp-20
The rP172 and rpMmp-20 was expressed, purified, and characterized as described in
previous chapters. In this set of experiments, rP172 batch MF95 was used, and rpMmp-
20 was from the same batch as mentioned in the proteolysis at pH 8.0.
3.2.2 Amelogenin Proteolysis by rpMmp-20
For proteolysis at pH 7.4, rP72 was dissolved in 25mM Tris-HCl (pH 7.4, 37º C) to
achieve a final rP172 concentration of 0.2mg/ml. The solution was incubated with
rpMmp-20 at 37º C with a substrate: enzyme ratio of 200:1 (w:w). The proteolysis
products were collected and analyzed at 0.1, 1, 2, 4 and 10 hours respectively.
3.2.3 SDS-PAGE
The rP172 and its proteolysis samples at pH7.4 were loaded onto a 12% acrylamide gel,
electrophoresed under 125V, and stained with Coomassie Brilliant Blue R250. The gel
density was analyzed by ImageJ 1.43 software to quantify each proteolysis portion.

24
3.2.4 Size Analysis by Dynamic Light Scattering (DLS)
Samples (25 µ l) from rP172 and its proteolysis at pH 7.4 were immediately measured and
analyzed as described in previous chapters.
3.2.5 Transmission Electron Microscopy (TEM)
300 mesh carbon coated grids were merged in the samples (30 µ l) from rP172 and its
proteolysis collections at pH 7.4 for 0.5 minute, followed by 0.5 minute of 1% urinal
acetate staining and air drying. The sample grids were observed under the Jeol TEM with
voltage of 80 kV.

3.3 Results
3.3.1 Progress of Amelogenin Proteolysis Monitored by SDS-PAGE
During the proteolysis process, substrate rP172 (MF 95) and two proteolytic products 2-
162, 2-148 were detected by SDS-PAGE (Figure 7A). Unlike previous gel of rP172 (MF
118) at pH 8.0 proteolysis, other products (13k, 11k, extended TRAP and TRAP) were
not detected.

As evaluated by their corresponding band densities on SDS-PAGE (Figure 7B, Table 3),
about 15% of rP172 (MF 95) was cleaved immediately after the addition of rpMmp-20
(within 10 min), and 50% of rP172 was cleaved after around 1 hours. The 2-148 was still
the immediate and main proteolysis product. The 2-162 appeared throughout the
proteolysis process with larger amount than pH 8.0 proteolysis, accumulating in a speed
similar to the 2-148. However, other products, 13k, 11k, extended TRAP, and TRAP

25
were not detected during the whole proteolysis process. Comparing to the proteolysis at
pH 8.0, rP172 was cleavage faster at the beginning of the process (from start to 1 hour)
and the cleavage slowed down afterwards, leaving a portion of rP172 undigested even
after 20 hours (data not shown).

Figure 7: Proteolysis Process Monitored by SDS-PAGE (pH 7.4, substrate: enzyme =200:1). A, SDS-
PAGE stained with Coomassie Brilliant Blue. Numbers shown on top are proteolytic time in hours. Lane
Std. shows a molecular size standard. The values on the left of each band in the standard lane are molecular
weight in kDa. The two lanes on the right (rP172, rP148) are pure protein controls without rpMmp-20.
Proteolytic products were detected as dark bands. Each marked band is shown by amino acid range based
on the full length porcine amelogenin. For each band, the range is: I, 2-173 (rP172, MF 95); II, 2-162; III,
2-148. B, Gel density analysis of each band. Numbers on X axis are proteolytic time in hours, values on Y
axis are the percentage of substrate or each product detected during the proteolysis corresponding to SDS-
PAGE.

Table 3: Percentage of Each Protein/peptide Band Detected by SDS-PAGE (pH 7.4, substrate:
enzyme=200:1). *: at 0.1 hour, amelogenin chain formation was initiated under TEM observation; ----:
represents the product not detected.

Time
(hrs)
%
0.1* 1 2 4 10
rP172 84.48 50.91 40.63 33.73 20.37
2-162 ---- 14.54 23.43 27.11 31.48
2-148 15.52 34.55 35.94 39.16 48.15
rP172:2-148 ratio 21:1 3:2 1.1:1 1:1.1 1:2.5
A B

26
3.3.2 DLS Analysis of Particle Size Changes during Proteolysis
At pH 7.4 the substrate rP172 possessed a major particle size distribution of R
H
at 28.3
nm (mass= 82.1%, Figure 8). During the first ten minutes of proteolysis, small particles
of 5.7 nm appeared in the solution. After the first hour of rpMmp-20 action, when the
ratio of rP172:2-148 was 3:2 (Table 3) in addition to the 2.6 nm and 11.5 nm particles,
large mono-dispersed assemblies with R
H
of 45.3 nm were also detected. Following 10
hours of proteolysis, when the majority of the mass was composed of the 2-148 product,
the sizes of the large assemblies increased to about 151.1 nm. Large aggregates (≥7000
nm) were present throughout the proteolysis, with a gradually decrease in percentage
mass.

The rP148 was shown as a control for the reason that it is an analogue of the main
proteolytic product (2-148) of rP172 during the digestion by rpMmp-20 (Figure 1). It
may bear the similar properties of the 2-148, and provide some explanations for the
phenomenon observed during proteolysis. In the DLS analysis, the rP148 processed a
major distribution of particle size above 2000 nm (more than 99%) at pH 7.4, 37º C,
presenting a great aggregation tendency.

27

Figure 8: DLS Data Showing the R
H
of rP172 and Changes in Particle Size during Proteolysis at
pH7.4.
rP172 rP148
0.1 hr 4hrs
1 hr 10 hrs
2 hrs 20 hrs

28
3.3.3 Proteolysis Induced Nanochain Assembly at pH 7.4 Observed by TEM
The rP172 formed monodispered spherical particles on the TEM grid at pH 7.4 (Figure 9:
rP172, black arrows), as they did at pH 8.0 but smaller in diameter (~12 nm, Table 4)
than at pH 8.0 (~16 nm). Aggregation of the spherical particles to initiate chain formation
happened earlier than that at pH 8.0 (initiated around 1 hour), which was initiated almost
immediately after rpMmp-20 proteolysis (within 10 min) and the chain-like structure was
already formed and curled after 2 hours. As Table 4 shows, a general diameter decrease
in isolated particles was also observed after proteolysis (particles were measured as
isolated spheres without any aggregations). Note that those particles were still larger than
pure rP148 at pH 7.4 (Figure 9, rP148, black arrows). There was no sign for chain
formation even after incubation of rP172 for 4 days.

29

Figure 9: TEM Images of rP172 Proteolysis at pH 7.4, 37 º C, at Different Proteolysis Time Points.
rP172 and rP148 shown were used as control without mixing with rpMmp-20. Image of 10 minutes
digestion showes chain innatiation; images of 1 hour shows chain elongation; image of 2 hours shows
rP172 10 min
1 hr 2 hrs
10 hrs rP148
rP172, 24 hrs rP172, 4 days

30
chains already curled; and after 10 hours, more chains curled together and formed larger aggregated
particles around 200 nm in diameter.

Particle Diameters
(nm)
Measured as
isolated particles,
based on 50
particles of each
sample
rP172 10min* 1 hr 2 hrs** 10 hrs rP148
Ave. 12.128 12.208 11.605 11.139 10.911 9.610
StdDev 1.219 1.119 1.176 1.094 1.069 1.025
Min. 10.213 10.286 9.231 8.938 8.332 7.748
Max. 15.500 14.514 14.022 13.188 13.148 11.334
rP172: 2-148 ratio
(Data from SDS-PAGE)
---- 21:1 3:2 1:1 1:2.5 ----

Table 4: Isolated Amelogenin Particle Sizes Changes during Proteolysis at pH 7.4, Measured from
TEM Images. *: chain formation initiated after 10 minutes of proteolysis; **: chains became curled after 2
hours of proteolysis, achieving an rP172:2-148 ratio about 1:1 (according to corresponding SDS-PAGE
analysis). The proteolysis process slowed down after 2 hours.

3.4 Discussion
Alteration in the dynamics of amelogenin self-assembly and the formation of chain-like
structures were also evident after the stepwise cleavage of amelogenin by Mmp-20 at
physiological pH 7.4.

Comparing to the proteolysis at pH 8.0, SDS-PAGE revealed that the pH 7.4 proteolysis
proceeded faster at the beginning but slowed down after 2 hours‟ digestion. While the
chain forming behavior exhibited during proteolysis at pH 7.4 was similar to but faster
than that of proteolysis at pH 8.0, which almost completed around 2 hours under TEM

31
observation. This phenomenon may be attributed to the subtle differences in substrate
conditions or the enzyme activity decay. The former possibility was observed during
previous experiments, which trace amount of purification solvent residues in different
rP172 batches would result in slightly different pH values or protein aggregation
properties.

However, the decrease in rP172 cleavage rate may also be explained by the co-assembly
theory between the full-length amelogenin and its proteolysis products during digestion.
The rP172 may act as a nucleation core and surrounded by its digestion fragments, which
were mainly 2-148 segment. Therefore, rpMmp-20 could not penetrate into the structure
center and rP172 were protected from digestion.

32
Chapter 4: General Discussion and Future Directions
The purpose of this study was to test the hypothesis that amelogenin self-assembly will
be altered following its stepwise proteolysis by Mmp-20, and the changes in self-
assembly will eventually affect the enamel formation. For this objective, in vitro
experiments were design: 1) to monitor the changes in rP172 particle size distribution and
assembly behavior during its proteolysis by rpMmp-20 at pH 8.0; 2) to mimic the
proteolysis process by combining recombinant amelogenin and the analogue of its main
proteolytic product; 3) to monitor the changes the proteolysis and assembly alteration at
pH7.4.

In order to replicate the proteolysis during enamel biomineralization, two recombinant
amelogenins were used: rP172 was used as the substrate and rP148 as control and the
analogue of main proteolytic product. Recombinant porcine Mmp-20 was used as the
enzyme source. Transmission electron microscopy (TEM), dynamic light scattering
(DLS) and SDS-PAGE were applied for this study.

For the experiments at pH 8.0, proteolysis exhibited similar pattern and rate as observed
in SDS-PAGE regardless of the substrate: enzyme ratio. The substrate: enzyme ratio was
therefore set at 200:1 for proteolytic efficiency. The 2-148 segment was shown as the
main product and small proteolytic products such as 13k, 11k, extended TRAP and
TRAP were detected. The DLS analysis demonstrated that the full length amelogenin

33
rP172 had a R
H
values around 13 nm. The appearance of small R
H
particles (≤5 nm)
immediately after the addition of rpMmp-20, and the formation of larger particles
(~100nm) overtime was also confirmed. In accordance with previous observations from
other groups, very large aggregates were present throughout the process (He et al., 2008).
Following proteolysis, decrease in isolated particle size and the amelogenin chain
formation were apparent under TEM observation. The chain formation was initiated
around 1 hour of proteolysis, formed around 4 hours and curled into larger spheres
(diameter≈100 nm) after 20 hours. These phenomena were not observed in the pure
rP172 and rP148 samples incubated for 24 hours at 37º C pH 8.0. The chain formation
should be therefore dependent of the proteolysis, rather than the incubation time. By
performing the experiments in this section (chapter 2), a general idea was obtained, of
what would happen during the amelogenin proteolysis by Mmp-20 under optimal
experimental conditions. Those findings also support the hypothesis that amelogenin
would alter its assembly after proteolysis.

Interestingly, the proteolytic mixture at pH 8.0 at 10 hours contains: rP172, 5.66%; 2-162
segment, 22.64%; 2-148 segment, 56.6%; 13k, 5.66%; 11k, 2.83%; extended TRAP:
1.89%; and TRAP 4.72%. This composition was similar to that of amelogenins extracted
from the 2nd and 3rd molar of six month old piglets. The porcine developing enamel
matrix was analyzed to be composed of “25K,” 7.4%; “23K,” 10.7%; “20K,” 49.5%; and
smaller peptides, 32.4% (Wen et al., 1999). This provided another reason for our in vitro
mimicking experiments, especially for the 1: 10 ratio between rP172 and rP148.

34
While mimicking the proteolysis at pH 8.0, rP172 and rP148 were mixed at defined ratios
corresponding to the ratios of rP172 and 2-148 at different time points (Table 1 and 2). In
the DLS analysis, rP148 showed great tendency to form very large aggregates, while
rP172 showed a major R
H
around 13 nm. A significant shift in the particles size
distribution was detected in the mixtures when compared to each individual component.
Although the DLS data cannot determine the structural parameters of protein assemblies
precisely, it provided a support for the co-assembly theory of amelogenin and its
proteolysis products reported by others (He et al., 2008).

For the proteolysis at pH 7.4, similar results were found as those in proteolysis at pH 8.0.
The SDS-PAGE revealed that the cleavage was faster at the beginning, but slowed down
after 2 hours and the substrate rP172 was not fully cleaved as that at pH 8.0 proteolysis
even after 20 hours. The 2-148 was still the main product but smaller proteolytic products
were not detected. The DLS analysis indicated that the particle size of rP172 doubled (~
28 nm) at pH 7.4 than at pH 8.0 (~13 nm), and rP148 still tended to form he aggregation
at pH 7.4. During the first ten minutes of proteolysis, small particles of 5.7 nm appeared
in the solution. After the first hour large mono-dispersed assemblies with R
H
of 45.3 nm
were also detected. Following 10 hours of proteolysis, the sizes of the large assemblies
increased to about 151.1 nm. Large aggregations (≥7000 nm) presents throughout the
proteolysis, with a gradually decrease in percentage mass. From the TEM images, the
rP172 formed monodispered spherical particles as they did at pH 8.0 but smaller in
diameter (~12 nm) than at pH 8.0 (~16 nm). Chain formation exhibited similar pattern

35
but proceeded faster than that at pH 8.0. Chain formation was initiated almost
immediately after rpMmp-20 proteolysis (within 10 min) and the chain-like structure was
already formed and curled after 2 hours. A general diameter decrease in isolated particles
was also observed after proteolysis. There was still no sign for chain formation in the
pure amelogenin incubations. By conducting the proteolysis at pH close to physiological
conditions, a faster proteolysis and chain-like structure formation were observed. These
results were in accordance with previous findings at pH 8.0, further supporting the
hypothesis of this study.

Recent studies from different laboratories have reported that the full-length amelogenin
can further aggregates to form nanospheres chains (Moradian-Oldak et al., 2006;
Wiedemann-Bidlack et al., 2007), and its proteolysis by Mmp-20 should take place in
these spherical particles (Fukae et al., 2007; He et al., 2008). According to our TEM
observation of proteolysis at pH 8.0 and pH 7.4, it is postulated that the entire amelogenin
nanospheres would maintain its structural character after the full length amelogenin being
digested into smaller products (Fukae et al., 2007).

It has been reported that the onset and scope of amelogenin aggregation are dependent on
both pH and temperature (Moradian-Oldak et al. 1998a; Petta et al. 2006). Recent studies
have also suggested that the changes in pH play an indispensible role in regulating the
self-assembly and higher order structures formation of amelogenins in vivo (Wiedemann-
Bidlack et al., 2007; Wiedemann-Bidlack et al., 2010). The pH changes can modify the

36
side chain charges in protein residues, which in turn affect protein self-assembly. Such
changes can enhance both intra- and inter-molecular protein interactions by reducing
electrostatic repulsion (Yu et al. 2006). The estimated isoelectric point is pH 7.05 for
rP172, pH 7.94 for rP148 and the 2-148. At pH 8.0, the calculated charge is -3.2 for
rP172, –0.1 for rP148 and 2-148; at pH 7.4, the calculated charge is -1.6 for rP172 and
+1.3 for rP148 and 2-148 (http://www.scripps.edu/~cdputnam/protcalc.html). The
different charges on various amelogenin fragments in the processed amelogenin particles
could make the amelogenin particles more likely to attract to each other and form higher
hierarchical structures (He et al., 2008). Since intermolecular repulsion of proteins is
minimal at their isoelectric pH, both full-length and cleaved amelogenins will tend to
form large assemblies at pH values that are close to their calculated isoelectric points
(Wiedemann-Bidlack et al., 2007). There should be a favorable electronic attraction
between rP172 and its main proteolysis product 2-148 at pH 7.4, which explains the
faster amelogenin chain formation in TEM images. A lower pH value from 8 to 7.4 led to
an increased R
H
. The gradual increase in temperature would have the similar effect as it
results in a drop in pH and a marked increase in particle size for all proteins studied
(Wiedemann-Bidlack et al., 2007).

Under in vivo conditions amelogenins do not exist alone and the self-assembly may well
be influenced by other factors such as non-amelogenin proteins and by the initial
mineralization events and growing mineral crystals. Amelogenin self-assembly into
nanochain will provide a scaffold for apatite crystals to grow with high-aspect ratio and

37
parallel alignment (Habelitz et al., 2004; Beniash et al., 2005; Iijima et al., 2005;
Moradian-Oldak et al., 2006). The C-terminal of amelogenin has significant apatite
binding affinity and the potential to control the crystals to grow in parallel (Beniash et al.,
2005; Moradian-Oldak et al., 2002). Removal of the C-terminal results in a product that
may also specifically interact with growing calcium phosphate crystals, affecting their
shape and size (Iijima et al., 2001; Iijima et al., 2002; lijima et al., 2005; Beniash et al.,
2005; Margolis et al., 2006). Previous data together with current findings reveal a
potential for amelogenin to self-assemble into elongated structures in a hierarchical
manner, which could eventually provide guidance for apatite crystals growth in enamel.

Collagen and amelogenin, presented at the dentin-enamel boundary (DEB), are the two
major extracellular organic matrix proteins in dentin and enamel respectively. It was
indicated that collagen fibrils guide amelogenin to assemble into elongated chain or
filament-like structures oriented along the long axes of fibrils. The interactions between
collagen and amelogenin might play an important role in the formation of the DEB
providing structural continuity between dentin and enamel (Deshpande et al., 2010).

There are still many gaps between the present in vitro findings and the complete
understanding of the amelogenin assembly events in vivo. One should bear in mind that
the amelogenin concentration in this study was 0.2 mg/ml, which is much lower than the
in vivo concentration. During the secretory stage of enamel formation, the enamel matrix
proteins concentration was reported to be 200-300 mg/ml (about 90% are amelogenins)

38
(Robinson et al, 1988; Fukae et al., 2002). The findings in this study therefore
represented a diluted in vitro system for the optimal research conditions. In the future,
more detailed and systematic studies should be performed to explore the amelogenin self-
assembly alterations during proteolysis by other techniques such as atomic force
microscopy and fluorescence. Further mimicking studies could be done by combining the
full length amelogenin and various recombinant forms of amelogenin proteolytic
products, in order to get a controlled model to investigate the mechanism and amelogenin
regions responsible for the self-assembly and its alterations. With the accumulating
understanding of enamel formation, it should be promising to develop strategies for in
vitro enamel regeneration and replacement that could be applied clinically in the future.

39
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Abstract (if available)

Abstract Amelogenin self-assembly and its stepwise processing are crucial for the enamel biomineralization. The removal of highly conserved (A- and B-) domains affects amelogenin self-assembly, resulting in enamel defect in transgenic and knock-in mice. We hypothesized that the amelogenin self-assembly can be altered by its stepwise proteolysis by Mmp-20, and the changes in self-assembly will eventually affect the structural organization of the forming enamel matrix, apatite nucleation, and mineralization. To examine the hypothesis, the full-length recombinant pig amelogenin (rP172), and a recombinant analogue (rP148) representing the most stable proteolytic product (the “20k”) were used. Recombinant rpMmp-20 was used to digest rP172 at pH 8.0 and pH 7.4, at temperature 37 °C, with substrate: enzyme ratios of 100:1 and 200:1. The proteolysis progress and particle size distribution during proteolysis were monitored by SDS-PAGE and dynamic light scattering.

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Asset Metadata

Creator Ma, Ruiwen (author)

Core Title Dynamics of amelogenin self-assembly during in vitro proteolysis by Mmp-20

School School of Dentistry

Degree Master of Science

Degree Program Craniofacial Biology

Publication Date 04/11/2011

Defense Date 02/14/2011

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag amelogenin,MMP-20,OAI-PMH Harvest,proteolysis,rP148,rP172,self-assembly

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Moradian-Oldak, Janet (committee chair), Paine, Michael (committee member), Zeichner-David, Margarita (committee member)

Creator Email maruiwen0000@yahoo.com.cn,ruiwenma@usc.edu

Permanent Link (DOI) https://doi.org/10.25549/usctheses-m3717

Unique identifier UC193406

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Source University of Southern California (contributing entity), University of Southern California Dissertations and Theses (collection)

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