Basal Lamina Proteins_journée scientifique_2_pdf

!!

Caractérisa*on!structurale!et!fonc*onnelle!de!trois!

nouvelles!composantes!de!l’épithélium!de!jonc*on!!

!!Sofia!Kholmogorova!

Laboratoire!de!recherche!sur!!les!*ssus!calcifiés!et!les!biomatériaux!!Directeur!de!recherche:!Dr.!A.!Nanci!

Faculté!de!médecine!dentaire!!

L’aEache!épithéliale!!!!!!!!!!!!!!!!!!!!!!!!!à!l’échelle!du!nanomètre

!!L’épithélium!de!jonc*on:!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!la!première!ligne!de!défense!

-  Scellement du parodonte de l’environnement agressif de la cavité buccale

-  Atteinte de la jonction

épithéliale initie la perte d’attache et la récession gingivale

Image: p://stevenleedds.com/?Periodontal_Disease

La!membrane!basale!interne!!!!!!!!!!!!!!!!!!!!une!structure!atypique!



Image: http://www.dental.pitt.edu/informatics/periohistology/en/gu0303.htm

N.B.: l’épithélium de jonction dérive de l’organe de l’émail


Image: http://www.dental.pitt.edu/informatics/periohistology/en/gu0303.htm

N.B.: l’épithélium de jonction dérive de l’organe de l’émail

Both odontogenic ameloblast-associated protein(29, 30) and amelotin (21, 29, 31) are part of thesecretory calcium-binding phosphoprotein genecluster (20, 24, 25). This cluster contains a numberof genes encoding proteins that stabilize calciumand phosphate ions in body fluids and/or guidecalcium phosphate deposition into receptive extra-cellular matrices (20, 22). Broad expression-profilinginterestingly revealed that both odontogenic amelo-blast-associated protein and amelotin are foundnot only in the maturation stage of amelogenesis(Fig. 3) but are also strongly expressed in the junc-tional epithelium (Fig. 4) (29). As such, and con-trary to a recent report on the evolutionaryanalysis of amelotin (15), these two proteins cannotbe regarded as ‘enamel specific’.

The immunolabeling pattern of odontogenic ame-loblast-associated protein is distinct from that ofamelotin, both with respect to ameloblasts and in thejunctional epithelium (30, 31). While signals for bothproteins first become apparent at the start of the mat-uration-stage amelogenesis, the staining observedalong the apical surfaces of ameloblasts differs. Label-ing for amelotin appears as a well-defined line at thecell–enamel interface, but the apical odontogenicameloblast-associated protein signal is broader(Fig. 3). Thus, the distribution of both proteins, whenobserved by light microscopy, does not coincide pre-cisely but there is probably some overlap. Quantita-tive immunogold labeling analyses from the early tomid- and late-maturation stages of amelogenesisindicate that at the beginning of maturation, odonto-genic ameloblast-associated protein is concentratedon the cell side of the basal lamina, whereas amelotinappears to be concentrated more on the enamel side(Fig. 5). In the late-maturation stage, this differentialdistribution is no longer apparent (13).

The distinctiveness between odontogenic amelo-blast-associated protein and amelotin is particularly

Fig. 2. Electron micrograph illustrating the epithelialattachment apparatus responsible for adhesion of thejunctional epithelium to the tooth surface. It consists of a(inner) basal lamina and hemidesmosomes.

A B

Fig. 3. Immunofluorescence preparations for odontogenicameloblast-associated protein and amelotin. In thematuration stage of enamel formation (A), odontogenicameloblast-associated protein localizes at the ameloblast–enamel interface where there is an atypical basal lamina

and also extends along the apical portion of the cells(arrowheads). (B) Labeling for amelotin is discrete andrestricted to the interface. AMTN, amelotin; ODAM, odon-togenic ameloblast-associated protein.

Fig. 1. Scanning electron micrograph of a semi-thin sec-tion showing the histological organization of the dentogin-gival junction. CT, connective tissue; IBL, inner basallamina; JE, junctional epithelium; OBL, outer basal lam-ina; SE, sulcular epithelium.

Nishio et al.

60

ODAM, AMTN et SCPPPQ1 = phosphoprotéines sécrétées liant le calcium

2010

Historique!du!projet!ODAM: Odontogenic Ameloblast Associated AMTN: Amelotin SCPPPQ1: Secretory Calcium Binding Phosphoprotein Proline Glutamine-Rich 1

2006

Moffatt et al, (2006) European Journal of Oral Sciences 114: 139-146.

ODAM, AMTN et SCPPPQ1 = phosphoprotéines sécrétées liant le calcium

2010

Localisation d’AMTN et d’ODAM dans l’organe de l’émail et la jonction épithéliale

Historique!du!projet!ODAM: Odontogenic Ameloblast Associated AMTN: Amelotin SCPPPQ1: Secretory Calcium Binding Phosphoprotein Proline Glutamine-Rich 1

2006

Nishio, et al. (2010). European cells and Materials 20: 393-402.

Moffatt et al, (2006) European Journal of Oral Sciences 114: 139-146.

ImmunoLcolocalisa*on!des!trois!protéines!dans!la!membrane!basale!atypique!ODAM AMTN SCPPPQ1

Fluo

resc

ence

O

r col

loïd

al

2006

AMTN, ODAM et SCPPPQ1 = phosphoprotéines sécrétoires liant le calcium

2010

Localisation de AMTN et ODAM dans l’organe de l’émail et la jonction épithéliale

2012

Hypothèse!de!travail!Comment!ces!molécules!uniques!interagissent!pour!former!le!réseau!macromoléculaire!de!la!membrane!basale!atypique?!

Approche!expérimentale!

-  Production de protéines -  Purification de protéines -  Caractérisation structurale des protéines

ODAM

AMTN SCPPPQ1

Production de protéines recombinantes!

1. Production du vecteur d’ADN avec le gène d’intérêt



2. Insertion du vecteur dans une souche de E. coli




3. Culture du E. coli contenant le gène




3. Culture du E. coli contenant le gène

4. Induction de la surexpression du gène et la production de la protéine

16

Purification de la protéine Supernatant

Elution fractions

17

Évaluation de la pureté de la protéine

- AMTN 21.8 kDa

Supernatant

Elution fractions

Caractérisa*on!structurale!des!protéines!

•  Microscope électronique à transmission •  Microscope électronique à balayage •  La cristallographie aux rayons X •  Résonnance magnétique nucléaire •  Diffusion des rayons X aux petits angles

•  Microscope à force atomique •  Images en 2D et 3D au niveau atomique •  Permet de travailler dans des conditions natives •  Adapté à la visualisation des complexes de protéines

Microscope à force atomique!

http://scixchange.missouri.edu/blog-post/afm-an-introduction-part-iii/

Jamais&visualisées&auparavant&Ces!protéines!adoptent!des!conforma*ons!dis*nctes!

0.05ug/mL

Surface HOPG 0.05ug/mL

Surface HOPG

5ug/mL

Surface HOPG

ODAM AMTN SCPPPQ1

Highly Ordered Pyrolitic Graphite = HOPG

SCPPPQ1 +

AMTN

Caractérisation des interactions

SCPPPQ1 +

AMTN

Caractérisation des interactions

SCPPPQ1 + AMTN

km

km

km

HOPG Dent

Perspectives Court terme -  Vérifier l’attachement des protéines

au minéral de la dent

Perspectives Court terme -  Validation dans des conditions plus

physiologiques -  Étude structurale approfondie des

protéines -  Confirmation des interactions




Long terme -  Explorer les avenues

thérapeutiques possibles -  Diagnostic -  Prévention -  Traitement




Long terme -  Explorer les avenues

thérapeutiques possibles -  Diagnostic -  Prévention -  Traitement

Un grand merci à …

Dr. Antonio Nanci Dr. Rima Wazen Katia J. Ponce Cynthia Torok Dr. Marianne Ariganello Aurélien Fouillen Dr. Luciana Maia Dr. Candida Parisi Shayan Sadeghi Jean-Daniel Castonguay Daniel Yi

Dr. Pierre Moffatt Hélène Gaumond

Tip!d’AFM!

30

Échantillon

L’organe!de!l’émail!devient!l’épithélium!de!jonc*on!

http://www.dental.pitt.edu/informatics/periohistology/en/dev_images/histo55a2.htm

Les!cellules!de!l’épithélium!se!lient!à!la!lame!basale!par!des!hémidesmosomes!

Both odontogenic ameloblast-associated protein(29, 30) and amelotin (21, 29, 31) are part of thesecretory calcium-binding phosphoprotein genecluster (20, 24, 25). This cluster contains a numberof genes encoding proteins that stabilize calciumand phosphate ions in body fluids and/or guidecalcium phosphate deposition into receptive extra-cellular matrices (20, 22). Broad expression-profilinginterestingly revealed that both odontogenic amelo-blast-associated protein and amelotin are foundnot only in the maturation stage of amelogenesis(Fig. 3) but are also strongly expressed in the junc-tional epithelium (Fig. 4) (29). As such, and con-trary to a recent report on the evolutionaryanalysis of amelotin (15), these two proteins cannotbe regarded as ‘enamel specific’.

The immunolabeling pattern of odontogenic ame-loblast-associated protein is distinct from that ofamelotin, both with respect to ameloblasts and in thejunctional epithelium (30, 31). While signals for bothproteins first become apparent at the start of the mat-uration-stage amelogenesis, the staining observedalong the apical surfaces of ameloblasts differs. Label-ing for amelotin appears as a well-defined line at thecell–enamel interface, but the apical odontogenicameloblast-associated protein signal is broader(Fig. 3). Thus, the distribution of both proteins, whenobserved by light microscopy, does not coincide pre-cisely but there is probably some overlap. Quantita-tive immunogold labeling analyses from the early tomid- and late-maturation stages of amelogenesisindicate that at the beginning of maturation, odonto-genic ameloblast-associated protein is concentratedon the cell side of the basal lamina, whereas amelotinappears to be concentrated more on the enamel side(Fig. 5). In the late-maturation stage, this differentialdistribution is no longer apparent (13).

The distinctiveness between odontogenic amelo-blast-associated protein and amelotin is particularly

Fig. 2. Electron micrograph illustrating the epithelialattachment apparatus responsible for adhesion of thejunctional epithelium to the tooth surface. It consists of a(inner) basal lamina and hemidesmosomes.

A B

Fig. 3. Immunofluorescence preparations for odontogenicameloblast-associated protein and amelotin. In thematuration stage of enamel formation (A), odontogenicameloblast-associated protein localizes at the ameloblast–enamel interface where there is an atypical basal lamina

and also extends along the apical portion of the cells(arrowheads). (B) Labeling for amelotin is discrete andrestricted to the interface. AMTN, amelotin; ODAM, odon-togenic ameloblast-associated protein.

Fig. 1. Scanning electron micrograph of a semi-thin sec-tion showing the histological organization of the dentogin-gival junction. CT, connective tissue; IBL, inner basallamina; JE, junctional epithelium; OBL, outer basal lam-ina; SE, sulcular epithelium.

Nishio et al.

60

Image: Nishio et al 2013

Les!trois!protéines!sont!sécrétées!par!les!améloblastes!au!stade!de!matura*on!!

ODAM SCPPPQ1

AMTN SCPPPQ1

Principe!de!l’immunomarcage!

Approche expérimentale

• Evaluation de la surexpression des protéines – Cellules mammaliennes

• Immunofluorescence

Visualisation d’ODAM en GFP dans les cellules HaCaT

anticorps

antigène

Florochrome ou Bille d’or

Localisa*on!de!ODAM!et!AMTN!dans!l’OE!

time with a secondary junctional epithelium derivedfrom cells of the oral epithelium (1, 8, 35, 37, 39).While it is known that the structure and the functionof the junctional epithelium are influenced by theunderlying connective tissue (28) and by contact witha solid substratum, such as enamel, dentin or cemen-tum (1, 37), the exact mechanisms that lead to theformation and regeneration of the junctional epithe-lium remain unclear. Therefore, we investigated theexpression of odontogenic ameloblast-associatedprotein and amelotin throughout primary junctionalepithelium formation and during regeneration fol-lowing gingivectomy (33, 33). During tooth eruption,labeling for amelotin is detected as a discrete line atthe interface between reducing ameloblasts and theenamel surface. Odontogenic ameloblast-associatedprotein is evident in the supranuclear compartmentof these cells where the Golgi apparatus is found and

shows a more diffuse labeling pattern at the interface(Fig. 7). The prominent Golgi immunoreaction sug-gests that odontogenic ameloblast-associated proteinis continuously produced, possibly to compensate forturnover of the protein. As the erupting toothapproaches the oral surface, strong immunoreactivityfor odontogenic ameloblast-associated protein, butnot for amelotin, is observed in cell clusters situatedbetween the reducing enamel organ and the oral epi-thelium (Fig. 7, inset).

Interestingly, the expression of both odontogenicameloblast-associated protein and amelotin is alsodistinctive during junctional epithelium regenera-tion. Immunolabeling for odontogenic ameloblast-associated protein occurs at an early time intervalfollowing gingivectomy in association with clustersof presumably migrating cells at the leading edge ofthe gingival wound. The presence of cell clusters

A B

Fig. 6. Immunogold preparations showing the presence of (A) odontogenic ameloblast-associated protein (ODAM) and (B)amelotin (AMTN) in the inner basal lamina of the junctional epithelium.

A B

Fig. 7. Immunolabeling for odontogenic ameloblast-asso-ciated protein and amelotin in an erupting tooth. (A)Odontogenic ameloblast-associated protein is detected incell clusters situated between the reducing enamel organand the oral epithelium (inset). (B) Amelotin is not

detected in these clusters, but is stained as a discrete lineat the interface with the enamel surface (arrowheads).AMTN, amelotin; ODAM, odontogenic ameloblast-associ-ated protein.

Nishio et al.

62

Image: Nishio et al, 2013

time with a secondary junctional epithelium derivedfrom cells of the oral epithelium (1, 8, 35, 37, 39).While it is known that the structure and the functionof the junctional epithelium are influenced by theunderlying connective tissue (28) and by contact witha solid substratum, such as enamel, dentin or cemen-tum (1, 37), the exact mechanisms that lead to theformation and regeneration of the junctional epithe-lium remain unclear. Therefore, we investigated theexpression of odontogenic ameloblast-associatedprotein and amelotin throughout primary junctionalepithelium formation and during regeneration fol-lowing gingivectomy (33, 33). During tooth eruption,labeling for amelotin is detected as a discrete line atthe interface between reducing ameloblasts and theenamel surface. Odontogenic ameloblast-associatedprotein is evident in the supranuclear compartmentof these cells where the Golgi apparatus is found and

shows a more diffuse labeling pattern at the interface(Fig. 7). The prominent Golgi immunoreaction sug-gests that odontogenic ameloblast-associated proteinis continuously produced, possibly to compensate forturnover of the protein. As the erupting toothapproaches the oral surface, strong immunoreactivityfor odontogenic ameloblast-associated protein, butnot for amelotin, is observed in cell clusters situatedbetween the reducing enamel organ and the oral epi-thelium (Fig. 7, inset).

Interestingly, the expression of both odontogenicameloblast-associated protein and amelotin is alsodistinctive during junctional epithelium regenera-tion. Immunolabeling for odontogenic ameloblast-associated protein occurs at an early time intervalfollowing gingivectomy in association with clustersof presumably migrating cells at the leading edge ofthe gingival wound. The presence of cell clusters

A B

Fig. 6. Immunogold preparations showing the presence of (A) odontogenic ameloblast-associated protein (ODAM) and (B)amelotin (AMTN) in the inner basal lamina of the junctional epithelium.

A B

Fig. 7. Immunolabeling for odontogenic ameloblast-asso-ciated protein and amelotin in an erupting tooth. (A)Odontogenic ameloblast-associated protein is detected incell clusters situated between the reducing enamel organand the oral epithelium (inset). (B) Amelotin is not

detected in these clusters, but is stained as a discrete lineat the interface with the enamel surface (arrowheads).AMTN, amelotin; ODAM, odontogenic ameloblast-associ-ated protein.

Nishio et al.

62

evident in the junctional epithelium, where stainingfor odontogenic ameloblast-associated proteinextends from the surface near the tooth across all celllayers of this stratified epithelium (pericellular and/orintracellular labeling), whereas that for amelotin isrestricted to the very outer edge of the flattened squa-mous cells abutting the enamel surface where theinner basal lamina is found (Fig. 4). Immunogoldlabeling confirms the association of both proteinsalso with this basal lamina in the junctional epithe-lium (Fig. 6).

As both the enamel organ and the junctional epi-thelium are tightly bound to the tooth surfacethrough a basal lamina, a putative participation ofodontogenic ameloblast-associated protein and ame-lotin to the adhesive mechanism has been inferred.Using a yeast two-hybrid screen and test-tube assays,Holcroft & Ganss (18) showed that amelotin andodontogenic ameloblast-associated protein can inter-act, possibly to form homo- or heteromultimers.However, it remains to be demonstrated whetherthey actually interact in vivo to form complexes in thebasal lamina. The apparent discrete subcompartmen-talization of amelotin and odontogenic ameloblast-

associated protein within the basal lamina associatedwith maturation-stage ameloblasts, and their redistri-bution as maturation proceeds, are not consistentwith the occurrence of stable heteromeric interaction(13). Moreover, the association of odontogenic ame-loblast-associated protein also with cells of the junc-tional epithelium, forebodes additional functions. Wehypothesize that odontogenic ameloblast-associatedprotein is a multifunctional protein that may be partof the molecular mechanisms which allow the junc-tional epithelium to adhere to the tooth surface,maintain its integrity and acquire its incompletely dif-ferentiated cellular status.

Expression of odontogenicameloblast-associated protein andamelotin during junctionalepithelium formation andregeneration

As discussed earlier, during tooth eruption the pri-mary junctional epithelium is eventually replaced in

A B

Fig. 4. Immunofluorescence preparations showing theexpression of odontogenic ameloblast-associated proteinand amelotin in the junctional epithelium. (A) Labeling forodontogenic ameloblast-associated protein is visible at thejunctional epithelium–tooth interface and among cells of

the junctional epithelium. (B) Labeling for amelotin isrestricted to the interface where the inner basal lamina isfound. AMTN, amelotin; JE, junctional epithelium; ODAM,odontogenic ameloblast-associated protein.

BA

Fig. 5. Colloidal gold immunolabeling reveals that both (A) odontogenic ameloblast-associated protein (ODAM) and (B)amelotin (AMTN) localize to the basal lamina associated with maturation-stage ameloblasts.

Novel junctional epithelium proteins

61

Localisa*on!de!ODAM!et!AMTN!dans!l’EJ!

evident in the junctional epithelium, where stainingfor odontogenic ameloblast-associated proteinextends from the surface near the tooth across all celllayers of this stratified epithelium (pericellular and/orintracellular labeling), whereas that for amelotin isrestricted to the very outer edge of the flattened squa-mous cells abutting the enamel surface where theinner basal lamina is found (Fig. 4). Immunogoldlabeling confirms the association of both proteinsalso with this basal lamina in the junctional epithe-lium (Fig. 6).

As both the enamel organ and the junctional epi-thelium are tightly bound to the tooth surfacethrough a basal lamina, a putative participation ofodontogenic ameloblast-associated protein and ame-lotin to the adhesive mechanism has been inferred.Using a yeast two-hybrid screen and test-tube assays,Holcroft & Ganss (18) showed that amelotin andodontogenic ameloblast-associated protein can inter-act, possibly to form homo- or heteromultimers.However, it remains to be demonstrated whetherthey actually interact in vivo to form complexes in thebasal lamina. The apparent discrete subcompartmen-talization of amelotin and odontogenic ameloblast-

associated protein within the basal lamina associatedwith maturation-stage ameloblasts, and their redistri-bution as maturation proceeds, are not consistentwith the occurrence of stable heteromeric interaction(13). Moreover, the association of odontogenic ame-loblast-associated protein also with cells of the junc-tional epithelium, forebodes additional functions. Wehypothesize that odontogenic ameloblast-associatedprotein is a multifunctional protein that may be partof the molecular mechanisms which allow the junc-tional epithelium to adhere to the tooth surface,maintain its integrity and acquire its incompletely dif-ferentiated cellular status.

Expression of odontogenicameloblast-associated protein andamelotin during junctionalepithelium formation andregeneration

As discussed earlier, during tooth eruption the pri-mary junctional epithelium is eventually replaced in

A B

Fig. 4. Immunofluorescence preparations showing theexpression of odontogenic ameloblast-associated proteinand amelotin in the junctional epithelium. (A) Labeling forodontogenic ameloblast-associated protein is visible at thejunctional epithelium–tooth interface and among cells of

the junctional epithelium. (B) Labeling for amelotin isrestricted to the interface where the inner basal lamina isfound. AMTN, amelotin; JE, junctional epithelium; ODAM,odontogenic ameloblast-associated protein.

BA

Fig. 5. Colloidal gold immunolabeling reveals that both (A) odontogenic ameloblast-associated protein (ODAM) and (B)amelotin (AMTN) localize to the basal lamina associated with maturation-stage ameloblasts.

Novel junctional epithelium proteins

61

EO-463

+

AMTN

+

ODAM

EOL463!concentra*on!

5 ug/mL 0.5 ug/mL 0.1 ug/mL 0.005 ug/mL

EOL463!in!different!buffer!pH!

PBS pH 7.5 Tris HCl pH 8

EOL463!on!different!substrates!

0.1ug/mL on Mica 0.1 ug/mL on HOPG

Incuba*on!*me!simulates!concentra*on!effect!

0.1 ug/mL in PBS pH7.5

1 minute 0.1 ug/mL in PBS pH7.5

3 hours

L’aEache!épithéliale!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!à!l’échelle!du!nanomètre!

!Caractérisa*on!structurale!et!fonc*onnelle!de!trois!nouvelles!

composantes!de!l’épithélium!de!jonc*on!!!Sofia Kholmogorova

Faculté de médecine dentaire

!Laboratoire!de!recherche!sur!

!les!*ssus!calcifiés!et!les!biomatériaux!!Dr.!A.!Nanci!

Faculté!de!stomatologie!!

2006!AMTN,!ODAM!et!SCPPPQ1!

=!phosphoprotéines!sécrétées!liant!le!calcium!

2010!Localisa*on!d’AMTN!et!

d’ODAM!dans!l’organe!de!l’émail!et!la!jonc*on!

épithéliale

L’aEache!épithéliale!à!l’échelle!du!nanomètre!

!Caractérisa*on!structurale!et!fonc*onnelle!de!trois!nouvelles!composantes!de!l’épithélium!de!

jonc*on!!!

!Laboratoire!de!recherche!sur!

!les!*ssus!calcifiés!et!les!biomatériaux!!Dr.!A.!Nanci!

Faculté!de!stomatologie!!

Sofia Kholmogorova Faculté de médecine dentaire

Documents

Basal Lamina Proteins_journée scientifique_2_pdf