Proteomic Analysis of Skeletal Framework Proteins
from the Stony Coral, Stylophora pistillata
Jeana L. Drake, Tali Mass, Liti Haramaty, Ehud Zelzion, Debashish Bhattacharya, Paul Falkowski
Institute of Marine and Coastal Sciences, Rutgers University, New Brunswick, NJ
I. Research Context
Diagram of coral polyp and skeletal structure.
Veron (1986)
Decalcified skeleton; remaining biomolecules stained
with eosin. Photo: I. Brikner
Carbonate formation by biological organisms is catalyzed by a set of biomolecules including proteins that, in corals, are poorly
characterized. In an effort to understand the coral calcification process, we sequenced the Stylophora pistillata skeletal organic
matrix (SOM) protein complex by liquid chromatography-tandem mass spectrometry. Our analysis reveals several ‘framework’
proteins and two coral acid-rich proteins (CARPs; aspartic and glutamic acid content >25%). The SOM framework proteins show
sequence homology with other stony corals as well as with calcite biomineralizers. Several of these proteins exhibit calciumbinding domains, while others are likely involved in attachment of the coral calicoblastic layer to the newly formed skeleton
Stylophora pistillata
substrate. We are using antibodies to confirm the presence of selected proteins in extracted SOM by immunoblotting. This work
is the first comprehensive proteomic analysis of coral SOM. Together with our examination of other highly acidic candidate proteins (poster B53D-0704),
this work will allow us to construct a three-dimensional model of the coral biomineralization space to better understand the mechanism of aragonite
nucleation and growth.
II. Methods
1. Extracting SOM from Coral Skeletons
2. De Novo Sequencing
3. Bioinformatics of Protein Candidates
• Dissolve twice-bleached S. pistillata
skeleton in 1N HCl
• TCA-acetone protein precipitation
• Enzymatic deglycosylation
• Five bands clearly present in SDS-PAGE of
SOM (Figure 1)
• High glycosylation background prevented
N-terminal sequencing
• Trypsin/Proteinase K digestion of SOM
protein complex
• Thermo LTQ-Orbitrap-Velos ETD mass
spectrometer with Dionex U-3000 Rapid
Separation nano-LC system
• LC-MS/MS data searched against our
group’s draft gene model for S. pistillata
(see poster B53D-0704 for further details)
by X!Tandem using an in-house version of
the Global Proteome Machine (GPM USB,
Beavis Informatics Ltd, Winnipeg, Canada)
• Data filtered for hits to standard
contaminants
• Non-redundant list of 36 proteins with evalues ≤ 10-10
• NCBI BLAST of 36 top LC-MS/MS hits
• Compare top 36 LC-MS/MS proteins to related mineralizing and non-mineralizing organisms’ predicted genes:
Figure 1. Silver stained SOM
proteins from decalcified (a) 3%
bleach- and (b) 1M NaOHcleaned S. pistillata skeleton.
Arrows next to (c) the molecular
weight marker indicate protein
band sizes in kDa.
Acropora digitifera (branching
coral, Family Acroporidae)
Pocillopora damicornis (branching
coral, Family Pocilloporidae)
Favia sp. (massive coral,
Family Faviidae)
Pinctada maxima
(pearl oyster)
Strongylocentrotus purpuratus
(sea urchin)
4. Confirming Proteins in SOM
Emiliania huxleyi CCMP 1516
(coccolithiphore; draft)
Reticulomyxa filosa
(naked forminiferan)
Thallasiosira pseudonana
(centric diatom)
Homo sapiens
• Retain predicted proteins from
comparison species with
sequence similarities ≥ 35% and
Nematostella vectensis
e-values ≤ 10-10
• Immunoblotting
• Carbonic anhydrase
• Collagen
• Actin
• Proto-cadherin-like
(anemone)
III. Results and Discussion
MW
1
2
Figure 3. Western blot of an antibody
raised against carbonic anhydrase (CA)
from Thalassiosira weissflogii. Lane 1 is
the molecular weight marker. Lanes 2
and 3 are deglycosylated soluble SOM
extracted from S. pistillata skeletons
reared in aquaria and in the ocean,
respectively. The arrow denotes a 40
kDa protein. Two known S. pistillata CAs,
STPCA1 and STPCA2, are both ~35-40
kDA when deglycosylated. Protein P35’s
predicted sequence is the same as
STPCA2.
Insoluble
SOM
Soluble
SOM, 1X
Soluble
1X coral
SOM, 5X cell lysate
5X coral
cell lysate
10X coral
cell lysate
a
.
b.
c.
Figure 2. Multiple sequence alignment of S. pistillata CARPs 4 & 5 with
highly similar predicted proteins from A. digitifera, P. damicornis, and
Favia sp. Stars represent identical amino acids between all sequences
while dots represent similar amino acids. These coral-specific proteins
are highly conserved across families.
Table 1. 36 S. pistillata SOM framework proteins determined by LC-MS/MS. Highly acidic proteins
are highlighted.
This
¤ represents presence of an export signal peptide in the predicted gene.
This
n represents >70% similarity between S. pistillata predicted protein and comparison sequence.
This
★ represents the best match of all comparison species to S. pistillata predicted protein.
V. Acknowledgements
This research is funded by the National Science Foundation Grant 432835 to PGF. Co-PIs on the project with PGF are Yair Rosenthal, Oscar Schofield,
and Rob Sherrell. We are grateful to the F. Morel lab for the gift of T. weissflogii CA antibody, to J. Yaiullo and I. Brikner for S. pistillata skeletons and
nubbins, and the Biological Mass Spectrometry Facility-Rutgers/UMDNJ for SOM analyses. Collagen, actin, and protocadherin-like P1 antibodies
were purchased from Pierce Antibodies.
Figure 4. Dot blots of antibodies raised against (a) H. sapiens collagens I-VI,
(b) multiple species’ beta-actin, and (c) S. pistillata protocadherin (transcript
P1, gene g11108). SOM and cell lysates were extracted from S. pistillata
skeleton and tissue, respectively. Collagen, actin, and the protocadherin-like
P1 antibodies clearly bound cell lysate protein. Collagen and protocaderhinlike P1 antibodies bound both soluble and insoluble SOM, while the actin
antibody bound only insoluble SOM. Similar observations of these proteins’
solubility were found by LC-MS/MS (not shown).
IV. Key Findings
• Most coral SOM proteins sequenced by LC-MS/MS are ‘framework’ proteins, likely involved in
adhering calicoblastic cells to the aragonite skeleton.
• Two acidic proteins (CARPs 4 & 5) sequenced by LC-MS/MS are homologous, are likely glycosylated,
and appear to be stony coral-specific.
• STPCA2, likely glycosylated, was observed in SOM by LC-MS/MS. Previously, only STPCA1 was known
to occur in coral skeleton.
• LC-MS/MS and immunoblotting are in consensus that collagen(s) and a protocadherin are found in
both solubility fractions of SOM, while actin(s) are present only in the insoluble SOM.