Characterization of the human cornea proteome with reference to ...
Characterization of the human cornea proteome with reference to ...
Characterization of the human cornea proteome with reference to ...
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<strong>Characterization</strong> <strong>of</strong> <strong>the</strong> <strong>human</strong><br />
<strong>cornea</strong> <strong>proteome</strong> <strong>with</strong> <strong>reference</strong> <strong>to</strong><br />
ocular transparency<br />
by<br />
Henrik Karring<br />
Department <strong>of</strong> Molecular Biology, Aarhus
The eye<br />
Lens<br />
Retina<br />
Pupil<br />
Cornea<br />
Optic nerve<br />
Iris<br />
Sclera<br />
Vitreous humor
The <strong>cornea</strong><br />
Pupil<br />
Cornea<br />
Iris<br />
Cornea<br />
Sclera
The <strong>cornea</strong>l stroma<br />
epi<strong>the</strong>lium<br />
Collagen<br />
lamellae<br />
Quiescent and<br />
transparent<br />
kera<strong>to</strong>cytes<br />
stroma<br />
~ 450 μm<br />
endo<strong>the</strong>lium
Transparency <strong>of</strong> <strong>the</strong> extracellular matrix<br />
Collagen lamellae<br />
kera<strong>to</strong>cyte<br />
Collagen fibers<br />
Hexagonal oriented array<br />
~30 nm diameter<br />
~60 nm regular spacing
Proteome analysis <strong>of</strong> <strong>the</strong><br />
<strong>human</strong> <strong>cornea</strong>
Materials<br />
• 16 normal <strong>human</strong> donor <strong>cornea</strong>s<br />
• Age 22-86 years<br />
• Containing all cell-layers<br />
• Lyophilized and homogenized in N 2<br />
• Fine <strong>cornea</strong>l powder
Extraction pro<strong>to</strong>cols 1 and 2<br />
• Proteins were extracted in urea or 100 mM NaCl<br />
• Separated by 2D gel electrophoresis<br />
• Proteins identified by MALDI-TOF MS
Peptide mass fingerprinting by<br />
MALDI-MS<br />
Trypsin<br />
digestion<br />
Abundance<br />
p5<br />
p3<br />
p1<br />
p4<br />
p2<br />
MALDI-MS<br />
Peptide<br />
extraction<br />
and matrix<br />
MALDI target<br />
m/z
Extraction pro<strong>to</strong>col 3<br />
• The <strong>cornea</strong>l powder was solubilized using<br />
CNBr and trypsin<br />
• The peptides were separated by <strong>of</strong>f-line strong<br />
cation exchange and identified using LC-<br />
MS/MS
Corneal<br />
powder<br />
CNBr and<br />
trypsin<br />
Abundance<br />
LC-MS/MS<br />
m/z<br />
Strong cation<br />
exchange<br />
App. 30 pools
Extraction pro<strong>to</strong>col 4<br />
• Proteins were extracted in SDS sample buffer<br />
and separated by SDS-PAGE<br />
• The gel lane was sliced in ~2-mm strips and<br />
digested <strong>with</strong> trypsin<br />
• The extracted peptides were identified by LC-<br />
MS/MS
Mw<br />
(kDa)<br />
200<br />
116<br />
97<br />
66<br />
45<br />
31<br />
22<br />
14<br />
6<br />
35 slices<br />
trypsin<br />
Abundance<br />
LC-MS/MS<br />
m/z
Results<br />
• Urea, 2D-gels, MALDI-TOF MS:<br />
• CNBr-trypsin, SCX, LC-MS/MS: 31<br />
• SDS-PAGE slices, LC-MS/MS: 103<br />
Identified<br />
proteins<br />
67 (165 spots)<br />
Total number <strong>of</strong> different proteins: 141<br />
Karring H. et al., MCP, 2005
Tissue distribution<br />
38%<br />
60%<br />
Karring H. et al., MCP, 2005
Functional groups<br />
Extracellular<br />
Intracellular<br />
39%<br />
27% 24% 28%<br />
17%<br />
Karring H. et al., MCP, 2005
Comparison <strong>of</strong> protein and gene<br />
expression in <strong>the</strong> <strong>cornea</strong><br />
61% <strong>of</strong> <strong>the</strong> identified proteins were recognized<br />
in <strong>the</strong> NEIBank gene expression libraries<br />
Structural proteins: 64%<br />
Blood/plasma proteins: 14%<br />
Metabolic proteins: 75%<br />
Immune defence proteins: 40%<br />
Redox proteins: 100%<br />
Folding and degradation: 100%<br />
O<strong>the</strong>r functions: 62%
Conclusions<br />
• 99 (70%) <strong>of</strong> <strong>the</strong> 141 identified proteins have<br />
not previously been identified in <strong>the</strong> <strong>cornea</strong>.<br />
• A significant number <strong>of</strong> protein is<strong>of</strong>orms were<br />
identified (albumin, Ig-kappa chain, TGFBIp).<br />
• Only 14% <strong>of</strong> <strong>the</strong> plasma proteins were<br />
recognized in gene expression libraries.<br />
• First part <strong>of</strong> a protein <strong>reference</strong> library for<br />
targeted studies <strong>of</strong> <strong>cornea</strong>l diseases.
Corneal TGFBIp dystrophy<br />
Normal<br />
Dystrophy
2D gels and proteomics<br />
Normal<br />
Dystrophy<br />
Hedegaard C. et al., MolVis, 2003
Proteomic analysis <strong>of</strong> <strong>the</strong><br />
soluble fraction from cultured<br />
<strong>human</strong> <strong>cornea</strong>l fibroblasts
Corneal wound-healing<br />
Hazy <strong>cornea</strong>l<br />
fibroblasts<br />
Apop<strong>to</strong>sis<br />
Proliferation<br />
and migration
Near-sightedness (Myopia)
Excimer laser<br />
2<br />
keratec<strong>to</strong>my<br />
(LASIK)<br />
Flap<br />
1<br />
3
Cellular haze after eximer laser<br />
keratec<strong>to</strong>my<br />
Normal<br />
Hazy<br />
Møller-Pedersen T. 2004
Cellular transparency<br />
• Abundant cy<strong>to</strong>plasmic water-soluble proteins (5-40%)<br />
• Normally metabolic enzymes – enzyme-crystallins<br />
• Taxon-specific in vertebrates and invertebrates<br />
(E.g. aldehyde dehydrogenase 3 or 1, and transke<strong>to</strong>lase)<br />
• May regulate <strong>the</strong> refractive index <strong>of</strong> <strong>the</strong> cy<strong>to</strong>plasm ?<br />
backscattering<br />
Knockout mice<br />
have normal<br />
<strong>cornea</strong>s
Cell culturing (Explant)<br />
10 % FBS<br />
Proteomics <strong>of</strong><br />
soluble fraction<br />
Isolate and<br />
lyse cells
No enzyme-crystallins in<br />
<strong>cornea</strong>l fibroblasts<br />
Vimentin (51 kDa is<strong>of</strong>orm) 1.1 +/-0.1%<br />
Vimentin (59 kDa is<strong>of</strong>orm) 1.5 +/-0.1%<br />
Actin 2.1 +/-0.2%<br />
Annexin A2 1.0 +/-0.2%<br />
All < 5%<br />
No enzyme-crystallins<br />
Karring H. et al., MCP, 2004
M r /kDa<br />
pH<br />
4.5 5.5<br />
64<br />
41<br />
78<br />
90<br />
5<br />
13<br />
77<br />
52<br />
72<br />
110<br />
111<br />
5.0 5.25<br />
60<br />
39<br />
134<br />
133<br />
58<br />
130<br />
127<br />
128<br />
51<br />
123<br />
122<br />
34<br />
48<br />
22<br />
21<br />
24<br />
120<br />
108<br />
119<br />
26<br />
35<br />
36<br />
135<br />
136<br />
137<br />
141<br />
142<br />
102<br />
146<br />
147<br />
149 150 151<br />
152<br />
153<br />
154<br />
155<br />
66<br />
157<br />
158<br />
159<br />
9-11<br />
8<br />
162<br />
7<br />
163 164 4<br />
166<br />
83<br />
176<br />
96<br />
29<br />
62<br />
99<br />
182 171<br />
170<br />
76<br />
14<br />
15<br />
16<br />
17<br />
174<br />
183<br />
184<br />
185<br />
186<br />
187<br />
173<br />
189<br />
192<br />
193<br />
194<br />
195<br />
196<br />
50<br />
198<br />
199<br />
201<br />
202<br />
204<br />
88<br />
31.0<br />
21.0<br />
14.4<br />
45.0<br />
66.2<br />
94.4<br />
4.75
Peptide mass fingerprinting or<br />
MALDI-MS/MS<br />
Protein Acc. # Function and FL aa /M th /pI th Gel ID. Spot ID. Covered fragment<br />
localization<br />
Actin, beta or gamma<br />
P02570 or<br />
P02571<br />
C/c 375/41.7/5.3 A/C<br />
A/C<br />
A/C<br />
A/C<br />
A<br />
A<br />
A<br />
C<br />
C<br />
C<br />
C<br />
C<br />
C<br />
14<br />
15<br />
16<br />
17<br />
25<br />
83<br />
114<br />
152<br />
153<br />
176<br />
186<br />
198<br />
199<br />
A19-K373<br />
A29-K373<br />
A19-K373<br />
A29-K373<br />
A19-K373<br />
V96-R372<br />
A29-K373<br />
A19-K336<br />
A19-K336<br />
G63-K373<br />
V96-R372<br />
I85-K336<br />
I85-K336<br />
Acyl-CoA-binding protein P07108 M/c/n 87/9.9/6.1 D 216 S2-K67<br />
Alcohol dehydrogenase [NADP + ] (Aldo-ke<strong>to</strong> reductase P14550 M/c 325/36.8/6.3 D 145 M14-K308<br />
1A1)<br />
Alpha-actinin 1 P12814 C/c 892/103.5/5.2 A 66 I656-R863<br />
Annexin A1 P04083 P/L/T/c/a/n 346/38.8/6.6 A<br />
A<br />
B<br />
Annexin A2 P07355 P/L/T/c/a 339/38.7/7.6 A<br />
A<br />
D<br />
C<br />
34<br />
48<br />
218<br />
53<br />
95<br />
121<br />
141<br />
Q10-R228<br />
G30-R228<br />
Q10-R228<br />
L11-R168<br />
A29-R205<br />
L11-R245<br />
L11-R168<br />
Annexin V P08758 P/L/T/c/a 320/35.8/5.0 A 75 G7-R285<br />
ATP synthase alpha chain, mi<strong>to</strong>chondrial precursor P25705 M/m 553/59.8/9.2 C 157 T46-K194<br />
ATP synthase beta chain, mi<strong>to</strong>chondrial precursor P06576 M/m 529/56.5/5.3 A/C 4 L95-K480<br />
ATP synthase D chain, mi<strong>to</strong>chondrial precursor O75947 M/m 161/18.5/5.2 C 120 T10-K121<br />
Beta-2-microglobulin, precursor P01884 I/s 119/13.8/6.1 D 230 V102-K111<br />
Calcyclin (S100A6) P06703 P/F/L/r/n 90/10.2/5.3 C<br />
C<br />
133<br />
134<br />
E41-R55<br />
E41-R55<br />
Calgizzarin (S100A14 or S100A11p) NP_066369 U/u 102/11.4/7.8 A 61 I4-K78<br />
Calmodulin P02593 L/S/P/c 149/16.8/4.1 A 49 E15-K149<br />
Calreticulin, precursor P27797 F/S/e 417/48.3/4.3 A 74 E25-K286<br />
Karring H. et al., MCP, 2004
Proteomic data<br />
• 254 spots identified by MALDI-MS or MS/MS<br />
• 118 distinct proteins<br />
• 1 hypo<strong>the</strong>tical protein<br />
“bone marrow stroma-derived growth fac<strong>to</strong>r”<br />
• 5 proteins classified as enzyme-crystallins in various species<br />
α-enolase<br />
non-filamen<strong>to</strong>us actin<br />
isocitrate dehydrogenase<br />
glutathione-S-transferase<br />
peptidyl prolyl cis-trans isomerase A
Protein folding and degradation: 32<br />
Cell proliferation, differentiation, and apop<strong>to</strong>sis: 24<br />
Metabolism: 23<br />
Cy<strong>to</strong>skele<strong>to</strong>n organisation and cell motility: 21<br />
Redox regulation and oxidative stress defence: 17<br />
Signal transduction: 13<br />
Secre<strong>to</strong>ry pathway: 11<br />
Indicate that oxidative stress and protein misfolding<br />
may participate in <strong>the</strong> backscattering <strong>of</strong> light from<br />
<strong>cornea</strong>l fibroblasts
Protein denaturation<br />
Fried egg<br />
Cataract<br />
Denaturation and precipitation <strong>of</strong> proteins means<br />
scatter <strong>of</strong> light and loss <strong>of</strong> transparency.
Model <strong>of</strong> <strong>cornea</strong>l cellular haze<br />
Irreversible oxidation<br />
Unfolding<br />
Reactive oxygen species (ROS)<br />
High molecular<br />
weight aggregates<br />
Protein degradation
Acknowledgements<br />
Department <strong>of</strong> Molecular Biology<br />
Aarhus University<br />
Jan J. Enghild, PhD, Associate Pr<strong>of</strong>essor<br />
Ida B. Thøgersen, Technician<br />
Department <strong>of</strong> Ophthalmology<br />
Aarhus University Hospital<br />
Torben Møller-Pedersen, MD, DMSc<br />
Foundations<br />
Danish Medical Research Council<br />
The Danish Association for Prevention<br />
<strong>of</strong> Eye Diseases and Blindness<br />
The Novo Nordic Foundation<br />
The Synoptik Foundation<br />
National Institute <strong>of</strong> Health<br />
Duke University Medical Center,<br />
North Carolina<br />
Gordon K. Klintworth, Pr<strong>of</strong>essor
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