Proteomic analysis of skeletal organic matrix
from the stony coral Stylophora pistillata
Jeana L. Drakea, Tali Massa, Liti Haramatya, Ehud Zelzionb, Debashish Bhattacharyaa,b, and Paul G. Falkowskia,c,1
a
Environmental Biophysics and Molecular Ecology Program, Institute of Marine and Coastal Sciences, bDepartment of Ecology, Evolution, and Natural
Resources, and cDepartment of Earth and Planetary Sciences, Rutgers University, New Brunswick, NJ 08901
acid-rich proteins
| collagen | cadherin | carbonic anhdyrase
B
iomineralizing organisms are found in all biological kingdoms
and incorporate a variety of metals, from sodium to lead, as
major components of minerals whose nucleation and growth are
under a range of biological control (1–5). The skeletal organic
matrix (SOM), occluded in the mineral, has been implicated as a
source of the increased strength of biominerals over comparable
geominerals and has long been hypothesized to aid in the stabilization, nucleation, growth, and spatial orientation of biominerals
(6–10). However, the mechanisms for the role of the SOM in coral
biomineral formation remain to be elucidated, primarily because
the organic molecules have yet to be characterized.
At present, the best-characterized SOM is that contained in
mammalian bones and teeth and is divided into structural and
highly acidic protein categories. Collagen, a well-characterized
fibrillar (i.e., framework) protein, plays a structural role in mammal
SOM (reviewed in ref. 10), and has been investigated in the structural components of several coelenterates (11, 12), of which Order
Scleractinia (i.e., stony corals) is a member. However, the greatest
emphasis for nucleation proteins rests on the highly acidic proteins, such as bone sialoproteins and dentin matrix proteins in
teeth in vertebrates (13, 14). Highly acidic proteins also have been
identified or hypothesized in mineralizing invertebrates, and several have recently been described, including the Asprich family
in the pen shell, Atrina rigida (15), Pif and Aspein in the pearl
oyster, Pinctada fucata (16, 17), and the Adi-SAPs (highly
acidic proteins proposed to be soluble or secreted) in the stony
coral Acropora digitifera (18). Sequence-based homologs of each
have been found in a variety of other invertebrates, yet these
proteins, and their consequent biomineralization reactions, appear to have originated several times independently (19, 20), and
their sequence similarity is most likely explained by convergent
evolution. Proteins containing acidic amino acids (Asp and
Glu) or phosphorylation sites (on Ser residues) are thought to
be used at various stages of aragonite and calcite mineralization to temporarily stabilize amorphous calcium carbonate or
www.pnas.org/cgi/doi/10.1073/pnas.1301419110
nucleate the mineral under appropriate conditions (reviewed in
ref. 21).
Stony corals (Class Anthozoa) are early-branching metazoans
composed of four cell layers (22). Whereas the oral endodermal
cells host endosymbiotic photosynthetic algae of the genus
Symbiodinium, the aboral ectodermal cells, or calicoblastic cells,
are the sites of biomineralization. Calicoblastic cells are thought to
secrete SOM, which adheres the cells to recently formed extracellular skeleton, and are also considered to be intimately involved
in nucleation and growth of aragonite crystals (23, 24). SOM is
retained in the coral skeleton and the amino acid composition
of the protein fraction indicates that the SOM is distinct from
cellular or mucus protein (25, 26). To date, only one coral SOM
protein, galaxin, has been fully sequenced and its role in the
biomineralization process is not well understood because it does
not bind calcium (27). Other proteins hypothesized to play a role
in the mineralizing space between the calicoblastic cells and the
skeleton include carbonic anhydrases (28), collagen (12), ion
transporters (29), cysteine-rich proteins (30), von Willebrand
factor type A domain-containing proteins and zona pellucidas
(31), and secreted acidic proteins (18, 32). Together, these proteins may represent a “biomineralization toolkit” of calcifying
proteins in corals. However, most of these proteins remain to
be independently confirmed in coral skeleton, described as
complete genes, or characterized with respect to function.
Here we use a proteomics approach to describe the SOM
proteins in the widely distributed, Pocilloporid coral, Stylophora
pistillata. Using liquid chromatography-tandem mass spectrometry (LC-MS/MS) protein sequencing, and a draft genome from
S. pistillata, we identify partial and complete sequences of proteins in the S. pistillata aragonite skeleton. Comparison of our
results with genome and transcriptome data from other mineralizers suggests that a coral skeleton contains a complex group of
proteins that guide the biomineralization process to form specific, genetically determined structures. This, a unique proteome
from a coral skeleton, identifies a biomineralization toolkit in
these key, ecologically critical organisms.
Results and Discussion
Thirty-six proteins, predicted from the draft S. pistillata genome assembly, were detected by three LC-MS/MS analyses
of SOM proteins (Table 1). This amount is similar in number,
but not sequence identity, to the 33 shell matrix proteins recently
identified by LC-MS/MS from mollusk nacre (aragonite) (33).
Twenty-five of the coral SOM protein candidates were only observed after deglycosylation (Table S1). Thirty-one of these
candidates could be observed with tryptic digestion and the
Author contributions: J.L.D., T.M., L.H., and P.G.F designed research; J.L.D., T.M., and L.H.
performed research; J.L.D., E.Z., and D.B. analyzed data; and J.L.D., T.M., L.H., E.Z., D.B.,
and P.G.F. wrote the paper.
The authors declare no conflict of interest.
Freely available online through the PNAS open access option.
Data deposition: GenBank accession numbers for genes identified in the paper can be
found in Table 1 in the text.
1
To whom correspondence should be addressed. E-mail: [email protected].
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
1073/pnas.1301419110/-/DCSupplemental.
PNAS Early Edition | 1 of 6
ENVIRONMENTAL
SCIENCES
It has long been recognized that a suite of proteins exists in coral
skeletons that is critical for the oriented precipitation of calcium
carbonate crystals, yet these proteins remain poorly characterized.
Using liquid chromatography-tandem mass spectrometry analysis
of proteins extracted from the cell-free skeleton of the hermatypic
coral, Stylophora pistillata, combined with a draft genome assembly from the cnidarian host cells of the same species, we identified
36 coral skeletal organic matrix proteins. The proteome of the
coral skeleton contains an assemblage of adhesion and structural
proteins as well as two highly acidic proteins that may constitute
a unique coral skeletal organic matrix protein subfamily. We compared the 36 skeletal organic matrix protein sequences to genome
and transcriptome data from three other corals, three additional
invertebrates, one vertebrate, and three single-celled organisms.
This work represents a unique extensive proteomic analysis of
biomineralization-related proteins in corals from which we identify a biomineralization “toolkit,” an organic scaffold upon which
aragonite crystals can be deposited in specific orientations to form
a phenotypically identifiable structure.
BIOCHEMISTRY
Contributed by Paul G. Falkowski, January 24, 2013 (sent for review November 10, 2012)
2 of 6 | www.pnas.org/cgi/doi/10.1073/pnas.1301419110
Drake et al.
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g11187
g12510
g9861
g11674
g11666
g4601
g9654
g10811
g11107
g13727
g2385
g6918
g9951
g1532
g11702
g12472
g810
g20041
g6066
g18277
g19762
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g11220
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JX891654
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Accession no.
Protocadherin fat-like
CARP8
Thrombospondin
Viral inclusion protein
Hemicentin
Actin
Actin
Major yolk protein
Protocadherin fat-like
Cadherin
Actin
—
Sushi domain-containing
Collagen - alpha
CARP9
—
Glyceraldehyde 3-phosphatase
dehydrogenase
Collagen - alpha
Contactin-associated protein
MAM domain anchor protein
Zona pellucida
—
Protocadherin
Vitellogenin
Ubiquitin
Vitellogenin
Integrin - alpha
Late embryogenesis protein
Tubulin - beta
Myosin regulatory light chain
Neurexin
Kielin/chordin llke
Flagellar associated protein
MAM/LDL receptor domain
containing protein
Carbonic anhydrase (STPCA2)
Zonadhesion-like precursor
Name
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P. damicornis A. digitifera Favia sp. N. vectensis P. maxima S. purpuratus E. huxleyii R. filosa H. sapiens T. pseudonana
Returned sequences with e-values ≤10−10 are presented in order of decreasing e-value. “Protein name” is the best BLAST hit in NCBI. “Gene” is the code number in our S. pistillata gene prediction model.
The “+” and “–” represent presence and absence, respectively, of similar sequences in comparison species.
*Sequence similarity is greater than 70%.
†
Most similar sequence by bit score.
‡
Indicates export signal.
Gene
Protein
Table 1. Thirty-six predicted proteins in S. pistillata SOM samples detected by LC-MS/MS and their bioinformatics analysis
Drake et al.
A
B
Fig. 1. Predicted structure of P12, a potential BMP inhibitor. The amino acid
sequence (A) and predicted secondary structure (B) of a unique SOM protein
after cleavage of the export signal peptide. For the sequence, peptides sequenced by LC-MS/MS are colored orange; translated sequence confirmed
from PCR amplification of S. pistillata cDNA is underlined; stop codon is
marked with an asterisk; the predicted glycosylation site, Asn79 is indicated
by a “+.” In the structure, the N-terminal region corresponding to a potential binding site with a bone morphogenic protein is shown in blue, and
Cys potentially involved in a cystine knot fold disulfide bonds between
Cys107 and Cys144, and Cys-137 and Cys181 are colored red.
PNAS Early Edition | 3 of 6
BIOCHEMISTRY
Structural Proteins. The highest scoring predicted protein, P1, is
an incomplete protocadherin fat 1-like protein that contains a
von Willebrand factor type A domain. We identified three separate PCR amplicons that map to this gene in S. pistillata cDNA,
validating its expression. These transcripts encompass 11 of the
15 MS-sequenced peptides for this candidate protein (Fig. S2).
Comparison with a very similar predicted protein from A. digitifera
and Favia sp. (Table 1) suggests that the S. pistillata protein
is incomplete and can be extended ∼750 residues toward the
N terminus, likely containing a secretory signal, and over 1,000
residues toward the C terminus (Table S1) (18, 35). Two additional proteins among the top 10 detected also show cadherin
precursor-like qualities (P9, P10) (Table 1).
Cadherins are calcium-dependent cell-cell adhesion molecules
with specificity for certain cell types, such as neurons or osteoblasts (36). Apoptotic osteoblasts and intact retinal cells can
cleave the extracellular portion of their respective N-cadherins
to produce a 90-kDa soluble protein that may have roles in cell
survival and protein expression. In addition, these cadherins have
been implicated in the aggregation of cells into which they have
been cloned (37), and could help explain the proto-polyps recently
observed in S. pistillata cell cultures (34).
Of the six detected proteins that contain von Willebrand factor
type A (an adhesion glycoprotein), four (P3, P5, P14, and P18)
show strong sequence similarity between the predicted sequences
from S. pistillata and those from A. digitifera, Favia sp., and
Pocillopora damicornis (Table 1 and Table S1) (18, 35, 38). We
compared these four predicted proteins from S. pistillata with
pre- and postsettlement genes in Acropora millepora larvae as
described by Hayward et al. (31). A. millepora genes A9, A90,
and A102 are ≥40% similar to the P5 protein found in this study,
with blastp e-values ranging from 10−15 to 10−23. All three proteins in the A. millepora expression study were up-regulated in
presettlement planulae relative to the postsettlement stage and
two, A9 and A90, were localized to the aboral region postsettlement (31). This finding suggests that A9, A90, and A102—
and therefore, possibly P5—are involved in adhesion of calicoblastic cells to the skeleton. The highly acidic mollusk nacre protein, Pif, also contains a von Willebrand factor type A, although
there is no sequence similarity between Pif and the coral proteins
described here (16).
Collagen and chitin share similar roles in biomineralizing
organisms (39), although chitin would not be detectable by the
procedures we used here. Two α-collagen–like proteins were
detected both before and after deglycosylation (P14 and P18)
(Table S1). We have confirmed the transcription of a portion
of P14 by PCR amplification of S. pistillata cDNA (Fig. S2).
P14 shows strong similarities to two predicted sequences from
A. digitifera (e-value 10−16) and Favia sp. (e-value 10−41) (Table 1)
(18, 35). Collagen, a fibrillar protein common in extracellular
matrix, is generally considered as a place for noncollagenous
proteins to bind and nucleate the mineral (reviewed in ref. 10),
but may also form sites of nucleation in “holes” of packed collagen molecules (40). Although collagen has been confirmed in
sea pen axial stalks (11) and gorgonian skeletons (12), little work
has been conducted on its presence in scleractinian skeletons. The
extracellular matrix portion of coral cell cultures has been shown
to positively stain for collagen (41), and so it seems highly likely
that collagen-like molecules are a component of S. pistillata SOM.
Strong similarities were observed for a carbonic anhydrase
(P35), which was found only in deglycosylated samples, and gene
sequences from P. damicornis, Favia sp., A. digitifera, Pinctada
maxima, and Emiliania huxleyi (Table 1 and Table S1) (18, 35, 38,
42). The complete sequence of P35 has previously been determined,
and was named S. pistillata carbonic anhydrase 2 (STPCA2; accession number ACE95141.1) by Bertucci et al. (43); it has been
immunolocalized to the cytosol of endo- and ectodermal cells
of S. pistillata tissue slides and has a very high enzyme efficiency
for interconverting HCO3− and metabolic CO2 (43). Although
SPTCA1 and STPCA2 show 35% sequence identity, peptides we
detected by LC-MS/MS were unique to STPCA2. Whereas only
STPCA1 has previously been localized in S. pistillata skeleton
(44), our results strongly suggest that STPCA2 is also present in
the calicoblastic space and is retained in the skeleton after mineralization. Presence of both STPCA1 and -2 in the calcifying
space likely appears to be an enzymatic “bet-hedging,” a strategy
that permits integral pH and bicarbonate control for both coral
skeleton (45) and mollusk shell (46) formation.
The final structural protein of interest is a 184-aa 21-kDa
protein with a theoretical isoelectric point of 5.06 (P12) (Table 1).
This gene contains a secretory signal at the N terminus, ends
with a stop codon, shows significant sequence identity to a predicted protein from Favia sp. (35), contains no known domains,
and is predicted to have an N-linked glycosylation site at Asn-79
(Fig. 1). Finally, it does not contain disproportionate amounts of
acidic, basic, or sulfur-bearing residues. We used PCR amplification of S. pistillata cDNA to confirm the correct transcription of
ENVIRONMENTAL
SCIENCES
remaining five were observed only after proteinase K digestion
(Table S1).
A major problem in isolating and identifying specific proteins
from corals is their posttranslational modification, primarily by
glycosylation (32). Although we consistently observed five distinct protein bands in size-fractionated SOM proteins, there was
always a strong background smear on silver-stained polyacrylamide
gels (Fig. S1). Periodic acid-Schiff staining confirmed the abundance of glycans in these samples (Fig. S1). Hence, the results
presented here are almost certainly a conservative estimate of
the total number of proteins in the skeletal matrix.
Nearly all proteins detected by LC-MS/MS show similarities
to proteins known to play roles in the structure and adhesion
of cells (Table 1). We detected multiple proteins with hits
(e-value ≤ 10−5) to precursor cadherins (P1, P9, P10, and P23)
and von Willebrand factor (P3, P5, P13, P14, P18, and P32)
domains. Blast hits for other proteins include two collagens (P14
and P18), three actins (P6, P7, and P11), and a carbonic anhydrase (P35), among others. Five candidate proteins exhibit poor
(e-value > 10−5) or no blast hits in National Center for Biotechnology Information (NCBI) (P2, P12, P15, P16, and P22).
Although amino acid analyses of SOM from a variety of corals
suggest that highly acidic proteins are compositionally important (25, 34), we only identified two such predicted proteins by
LC-MS/MS.
an internal portion of P12 that contains one of the LC-MS/MS
sequences (Fig. S2).
Consensus structure predictions of P12 in I-TASSER and
Phyre2 (Fig. 1), suggest that it may be related to the secreted
protein noggin that binds to and inhibits the function of some
bone morphogenic proteins (BMPs), of which one, a BMP2/4
ortholog, is known to be present in coral calicoblastic cells (47).
The predicted structure (I-TASSER: Tm-score of 0.74, rmsd of
3.5 Å, and query sequence coverage of 94.6%) exhibits a β-sheet
portion and cystine-knot cytokine fold found in the noggin
protein family (98.9% confidence by Phyre2). In the complete
structure, disulfide bonds could occur between Cys107 and Cys144,
and Cys-137 and Cys181 (Fig. 1). The P12 sequence contains
CXGC and CXC and the last Cys in the knot is immediately
followed by a stop codon, which is standard for this type of fold
(48). These structure predictions suggest that P12 may have a
role in inhibiting skeleton formation by blocking the coral BMP2/4
ortholog’s receptor binding regions.
CARP Subfamily. Two coral acid-rich proteins (CARPs), CARP4
and CARP5, were sequenced by LC-MS/MS and we propose that
they belong to a highly acidic subfamily of proteins that is well
conserved across Order Scleractinia but appears to be absent
from other known biomineralizers. Six internal peptides of
CARP4 (P2) and four internal peptides of CARP5 (P15) were
sequenced by LC-MS/MS with best sequencing following proteinase K digestion (Fig. S2). CARP4 and CARP5 have predicted
isoelectric points of 4.02 and 4.04, respectively. Two sites
of glycosylation, Asn-98 and Asn-126, are predicted for CARP4,
whereas only one, Asn-133, is predicted for CARP5 (Fig. S3).
Detection of CARP5 and the C-terminal end of CARP4 only in
deglycosylated samples supports this prediction. Glycosylation of
CARP4 is also suggested by the difference between its predicted
size and that of a 55-kDa protein that was partially analyzed
by Puverel et al. (49), and which contains two internal peptides
matching CARP4 (Fig. S3). Additionally, anomalous migration
of highly charged proteins in SDS/PAGE systems has previously
been observed for Aspein, a highly acidic molluscan protein (17).
CARPs 4 and 5 exhibit significant sequence identity (31–85%)
with predicted genes from three other stony corals (Fig. S3).
However, similar sequences are absent outside of Order Scleractinia (Table 1). Multiple sequence alignment of CARP4- and
CARP5-like proteins from P. damicornis, A. digitifera, and Favia sp.
reveals several highly conserved regions of this unique coral protein
subfamily (18, 35, 38). The general pattern appears to be a variable N terminus followed by one highly acidic region plus
a highly conserved nonacidic region; this combination of
acidic-plus-nonacidic regions is then repeated before ending
in a variable C terminus (Fig. 2). The duplication and then
variation of the general gene pattern within each species examined could allow for redundancy in supporting the activity of
the protein subfamily. A similar pattern of redundancy, but not
sequence similarity, in a highly acidic protein subfamily is observed in the Asprich proteins of A. rigida (15).
Like the Asprich protein family described by Gotliv et al. (15)
for the mollusk, A. rigida, we propose that the two acidic regions
of the CARP subfamily in corals are templates on which calcium
carbonate nucleation or growth could occur (7). Unlike the Asprich
subfamily, these CARPs contain two nonacidic, yet highly conserved regions that we propose to represent potential protein–
protein interaction sites based on their degree of conservation
(Fig. 2). Binding to structural proteins described above would
allow these highly acidic proteins to be arrayed in an ordered
fashion in the calcifying space for a tighter control by corals
over biomineralization.
Additional, highly acidic proteins will almost certainly be
found in coral skeleton by other methods. The lack of peptide
sequence variability in these proteins makes them poor candidates for identification by LC-MS/MS. Hence, CARPs 4 and
5 should be considered the first, but likely not the only, acidic
proteins involved in coral biomineralization.
4 of 6 | www.pnas.org/cgi/doi/10.1073/pnas.1301419110
A
B
Fig. 2. CARP subfamily general pattern. (A) Conservation of residues in the
CARP subfamily as predicted by ConSurf, with CARP4 as the query. Warmer
(more red) and cooler (more blue) colors represent conserved and variable
amino acid positions, respectively. Residues are predicted to be exposed (e),
buried (b), functional (i.e., highly conserved and exposed; f), or structural
(i.e., highly conserved and buried, s). Numbers indicate residue number of
CARP4. (B) Schematic of the CARP subfamily of SOM proteins. An N-terminal
variable region is followed by a repeat of a highly acidic region plus a highly
conserved nonacidic region; the C terminus is variable.
In summary, the proteomics approach used here identified 36
proteins in S. pistillata SOM. Our results suggest that the in vivo
coral SOM protein complex is laid out as follows: cadherins,
integrins, contactin, and similar adhesion proteins play a dual
role to (i) constitute an extracellular matrix that adheres to newly
formed skeleton and (ii) attach calicoblastic cells to this skeleton-blanketing matrix. Actins and tubulins allow for flexibility of
the calicoblastic space’s size and shape during aragonite crystal
growth. Collagens provide a structural support within the calicoblastic space to which CARP subfamily and analogous proteins can bind as sites of mineral nucleation and growth, and at
least two carbonic anhydrases mediate the subsequent carbonate
chemistry effects. Mineral reworking proteins, although not found
in this study, are also likely present and P12 may modulate their
activity. We suggest that together, these 36 proteins constitute part
of the biomineralization toolkit of Order Scleractinia; some proteins may be part of the general toolkit of all calcium carbonate
mineralizers (e.g., recent proteomic analyses by refs. 50 and 33),
and others, particularly the CARP subfamily described here, are
clearly limited to the aragonite precipitating corals. Whereas
almost certainly more proteins will be discovered in the SOM,
this initial set provides a basis for understanding the spatial
relationships between the major components within the skeleton
and how their relative expression influences rates of calcification.
Future expression studies of the effects of disturbance on coral
biomineralization will be guided by pinpointing the spatial and
temporal arrangement and function of these 36 SOM proteins
in the calicoblastic space.
Methods
Model Organism. S. pistillata, a common hermatypic coral found throughout
the Pacific and Indian oceans (51), has been well studied both in situ and in
laboratory settings. We grew coral nubbins at 28 °C in an 800-L flow through
system, as previously described (34).
SOM Extraction. S. pistillata skeletons were soaked for 4 h in 3% (wt/vol)
sodium hypochlorite, copiously rinsed in deionized water, and dried
overnight at 60 °C. Dried skeletons were ground to a fine powder with an
agate mortar and pestle and again bleached, rinsed, and dried. The skeletal
Drake et al.
Proteomics. SOM complexes were digested either by trypsin or proteinase K,
and masses and charges of the digested peptides were analyzed on a Thermo
LTQ-Orbitrap-Velos ETD mass spectrometer with Dionex U-3000 Rapid Separation nano LC system. The LC-MS/MS data were searched using predicted
gene models from S. pistillata by X! Tandem using an in-house version of
the Global Proteome Machine (GPM USB; Beavis Informatics) with carbamidoethyl on cysteine as a fixed modification and oxidation of methionine
and tryptophan as a variable modification (53). Spectra were also analyzed
against a suite of potential microbial genomes to exclude possible microbial
contamination of the dry skeleton. Data for LC-MS/MS sequenced proteins
have been deposited in GenBank (Table 1).
Gene Confirmation. Internal sequences of predicted genes were confirmed
in DNA and cDNA by PCR using gene-specific primers (Table S2). Holobiont
DNA and cDNA were prepared as previously described from S. pistillata
colonies maintained in in-house aquaria (34). All PCR tubes contained
0.25-μg template, 0.2 mM dNTPs, 1× High Fidelity reaction buffer, 0.5 μM
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Drake et al.
Bioinformatics. LC-MS/MS results were filtered to remove hits from standard contamination (common Repository of Adventitious Proteins, or cRAP,
database). A nonredundant list of all proteins detected with e-values ≤10−10
was used for blast analysis against NCBI and to query a database we created
that contains translated sequences from Homo sapiens (54), Thalassiosira
pseudonana [diatom (55)], Nematostella vectensis [anemone (56)], Strongylocentrotus purpuratus [urchin (57)], E. huxleyi CCMP1516 (coccolithophore;
draft genome), and A. digitifera [hard coral (18)] genomes; a transcriptome
from P. damicornis [hard coral (38)]; and expressed sequence tag (EST)
libraries from Favia sp. [hard coral (35)], Reticulomyxa filosa [foraminiferan
(58)], and P. maxima [oyster (42)]. N. vectensis and R. filosa do not biomineralize; all other comparison species produce calcium- or silica-based
minerals. Predicted proteins from the comparison species with similarities
greater than 35% and e-values ≤10−10 were retained for further analysis.
For CARP subfamily homologs, predicted proteins in comparison species
were combined if they closely mimicked matched S. pistillata CARPs. These
combinations are noted in protein names when they are presented in the
multiple sequence alignment. Residues whose conservation suggests a functional role were predicted in ConSurf (59) using CARP4 as the query sequence.
Structures of selected proteins were predicted using both I-TASSER (60)
and Phyre2 (61). We used these two programs to obtain a consensus in
structure matching, particularly in the case of one S. pistillata protein that
showed no similarity to proteins in the NCBI and contained no known
domains. Glycosylation sites were predicted using the EnsembleGly server
at the AIRL at Iowa State University. Images of predicted structures were
generated in MacPyMOL v1.3r1 (Schrödinger).
ACKNOWLEDGMENTS. We thank Joseph Yaiullo of the Long Island Aquarium
and Frank Natale of the Institute of Marine and Coastal Sciences for
providing and maintaining corals used in this study; N. Kröger for helpful
comments on an earlier draft of this manuscript; and Rutgers Center for
Advanced Biotechnology and Medicine Biological Mass Spectrometry Facility
for generating and helping analyze LC-MS/MS data. This research was
supported by the National Science Foundation Grant EF1041143.
18. Shinzato C, et al. (2011) Using the Acropora digitifera genome to understand coral
responses to environmental change. Nature 476(7360):320–323.
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pp 273–286.
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PNAS Early Edition | 5 of 6
BIOCHEMISTRY
Protein Separation and Characterization. SOM proteins were separated by
SDS/PAGE and bands were visualized by silver staining (Pierce silver stain for
mass spectrometry) and Periodic acid-Schiff staining (Pierce glycoprotein
staining kit). Smearing of proteins in gels precluded extraction of individual
bands for sequencing.
of each primer, and 0.04 units μL−1 of Phusion polymerase (New England
BioLabs) in a 25-μL reaction volume. Amplifications were performed in a
Veriti Thermal Cycler (Applied Biosystems) at 35 cycles of 98 °C for 10 s,
primer-specific annealing temperature for 30 s, and 72 °C for 30–180 s.
PCR products were sequenced by GENEWIZ.
ENVIRONMENTAL
SCIENCES
powder was decalcified in 1 N HCl at room temperature while shaking. HCl
was added gradually so that the solution reached neutral pH within 30 min
of acid addition; more HCl was only added if skeleton powder remained after
30 min. pH of the decalcification solution was brought to neutral with 1 M
NaOH. Water-soluble and -insoluble organic fractions were separated by
centrifugation and analyzed separately. Trichloroacetic acid (TCA)-acetone
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Supporting Information
Drake et al. 10.1073/pnas.1301419110
Fig. S1. Skeletal organic matrix (SOM) proteins separated by SDS/PAGE. (A) Silver staining and (B) Periodic acid-Schiff (PAS) staining of SOM proteins from
decalcified Stylophora pistillata skeleton. Silver staining (A) was performed on glycosylated soluble SOM (lane 2). PAS staining (B) was performed on glycosylated soluble (lane 2) and insoluble (lane 3) SOM, and deglycosylated soluble (lane 4) and insoluble (lane 5) SOM. Lane 1 of each gel contains molecular
weight standards; numbers indicate kilodaltons. Arrows indicate protein bands.
Fig. S1
Fig. S2. Predicted amino acid sequences of 36 S. pistillata proteins. Peptides detected by LC-MS/MS after tryptic digestion are in bold and after proteinase
K digestion are underlined. Translations of internal sequences confirmed by PCR amplification of S. pistillata cDNA using gene-specific primers are highlighted
in gray. Discrepancies between the predicted sequence and that determined by translation of PCR product are in red. The secretion signal peptide of P12, is
crossed out over the portion that is predicted to be cleaved before secretion.
Fig. S2
Fig. S3. Multiple sequence alignment. Aligned sequences of CARP4 and CARP5, two highly acidic predicted proteins detected by LC-MS analysis of deglycosylated S. pistillata SOM, and similar proteins from an A. digitifera genome, a Favia sp. EST library, and a P. damicornis transcriptome. Identical amino acids
are highlighted in gray. Dashes represent gaps. Yellow highlighted residues in CARP4 were previously determined by N-terminal sequencing by ref. 49. Blue
stars denote predicted glycosylation sites of CARP4.
Fig. S3
Table S1. SOM protein primer sets
Table S1
Gene-specific primers used to confirm the DNA and cDNA sequences of selected SOM proteins.
Table S2. Putative homologous proteins from other mineralizers or related organisms
Table S2
The most similar predicted protein sequence from each comparison organism is given. Lack of a similar protein sequence for a given species is noted as “–”.
Drake et al. www.pnas.org/cgi/content/short/1301419110
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A 150 100 75 50 37 25 20 1 2 B 200 100 75 50 1 2 3 4 5 P1 (g11108)
LDSGDNGKVVYNITGGNEEGAFKIDPDTGVIKVKRSLTTVSASTFALDVEAKDKGNPPRSNIAKVNLNVFLPDGPPKFVV
KPVVEEVTEGIKANNRVMVVKAATSEALTYEVVSGNEDGLFRINPSTGEIMVTRELDYEEARERRLVVRVMDTRDRSDQV
TVILQIKNINDNAPTFPGEVNGYVERKVEDDFQIGDPVARLSAFDGDAGDKITYTLSENARASFTINEEGFLIAKKPRKE
FSSPVTFDLTAKDNGSPPRESKVNVQLVLVSYRPEQQHPVREYVREDKEVGSVVAIVPRYFPGGTLSIVYPRKANFTVDN
SGRVRMTTPFDFEARQLYTLTVREQEPSPLSRTNDVDVEINVIDINDNKPVMKMVEFFGRVNTNSRPGTSAYQLKAEDKD
GGLSGRIGYQMVSRGVPFAINPLTEVVETGGKLEDRGGYNVTVFGFDFGVPRQFGDSVYLDIKTVNFKPQFSESSYNFEV
FENDLPGKVIGVVNATSASGARLGFSIVQGDTDNKFSIESTGELVVNSKLDREKTSMYNLKVRTTEQIPRGYSNDVDVRI
SVKNANEYYPYFPLTVYEKSINEDVGSGASVLSITAYDCDCTGCVCKNAELKYSLEGTSVFKIDEVTGLISVGDTPLDYE
TQRIHLFKVVVEDFGEKKFSSRSYVRVSVQNKNDEEPKFQRGEYSIDIAEDAITNKALAAILARDGDGDSSLYTITSGAS
GIFDINRNTGVLSLVQSVLGQKKQYTLTVRATDTAPNPKFDEVRVVINIEDINDNRPVFTVCPKEVTVAENKPTGERVTQ
VTAKDSDNGRNGEVEYMLVTGGERLFEINNSTGLITTITSLDRETADRHTLIVQAEDGGHGRNEAERLLSYCIIEVKVAD
VNDNYPVFFTRQYYGSVFHKAPVGTTVLTVSATDDDTGNNQKVVYSLVGSNDKFEVDGATGSIRTIADLSNFQQTVLLKI
RASNQATPTATKTSSSETTVEINVVNQQPPKFQPSSVYTAEIQEDVEVGTAVVTVKAVSQVDSKNGIAYSLVKSYPSAEQ
NFKVDPGTGVISTASTLNYEQQSSYTLQIRARENNIDLYATCSVNIRLKDVNDDTPTFKLEEYSARVPEETVGGFNVITV
KADDRDTLLSGEVSYSLGASQKFRIVASNGTIFSQEKFDRENQKTHTVFVTATDKGTPPLQAKVAVSVVVVDQNDEPPLF
PNRKYTANVQEDAPIGTSIDDLTATDADIGDNARLEYFISGGEQSQLFRMETVYSPENKGILYLNGKLDYETKPTYTIKV
TATDRKNSDTVDVVIAVTKSWAH*
P2 (g11187)
MIVRDNDDDDDDDDDDDDDDDDDDDDGVDDDDDDDDDFSDDNEEMLSFEVDELEEKDVDGNDVDTMQRHSVDSFDDVDFT
FTKVDTQAKYDGLPVTNVNLSATLPSSSSLEIMVYLFRRAGKVTFGNETFRVEKGTIKFNIRISNWDFCDGSRFDCDEGK
IGEFLDLKLKIKSKDSPAEISDDDRKAAAKRPICEDEDSDDDGVDDPTDSDNDDDDCPSIYDIGGDSEMLLNKGVVTDDD
KYTALPEGYPKFEVEDGEKKFVFRIPKFKKSVLVDPSVNVGRVNGVNSLAHANTGLAFLLFIAAFIVTNYV
P3 (g12510)
MSDATFILMKNAVTQIMDQYGTDRIHYSVIIFGSIVTTQVTFDRRFPEKNELIRQVARYGKVDGNPNLSAALEEAKRIYE
LQSVRPNARKVLVVILDAPSASNTTAVKKAVTELDRKDVLVIGVGIESSANENDLIGITKDDRHLINATLNKPYQEVARE
IIEAIFYGINTAGFSQWGDWSACSKSCRSGGVAGTQERRRICEIPRRGCVGPTVETRECNIQNCEDCGEIGPLEDGSYEA
SSSAQPPRFAKLNTTNPGVNKQGWCPTAEDPEQYLQINL
P4 (g9861)
MVVSQSITTVQESAPMEEEFFNTQITEYEATLKSSADEKEGLQEEILNLQTKLAKLQGDYDRAILDLAATKKDCDLKVKK
FEVQLQNSQKQRDDLKRQLEKLRDDLKKSREALSSAQTEAVNNKRAADRLRDEAEGKNRQIRNLEQKNGSLEDQIEQLKQ
ELSHSEMSAEVEAKRAKDLENELRSSERRVAEQEAIIESLRSTNEDLQRELAISNSRVSTLESQNGKMSTRIRELENELV
RCHARIHDLENEFESVNTKCVQLQSSYDSSVSEVDGLRSRVGGHQSKVNELESRHSVTSKERDDLKLRISPLEKTLAELR
LTLERSENTCDGFEREIDMLKAKNSNLETQLASANKLKTQLRADLDAEKDKSNQLRREITALQTQLGEAQRNLDKFKNDN
VRKQKVIDDLRNTIRGLEAKIHAQENDVKQLEMQKDMAEEEKEAMHDELLDNNRKLAEQEESLRQAQDKLDNVEKELIDS
QKNNEKLENTLENTEKEKRDFENRVLQLQQNLTRVEAELRDALSKKAVLEEDAKQHSAVVSKLTDEIDRLKGLLAEAQRQ
IDAYRRESDSKDATITALKSQISDLDKENDDLRAELSKVKTQLAAVEEEKRKLQIEHNTKNEEINRLTGEIDITVKRNEE
LQSECIVVADKVQSVESSYQDFMSLAGEQDGLLEGEKEKNAQLKKENNALKLKITSLEKKDRSQEVDQLRADLDAANEEI
PKLQDRIQQLVGENDDLSRDILVVQSNLSQKENDLEAAEKDKEKAEDDVHIAQGKIDSLESQLAALRKQKVQLKDNLKDA
NNKIIKAKEESNDLQAKVSQQEKIIEGLKNGNTKKENIIRELTDKIKKLQNELADAKREVSAAKLTAEGLRSDKKDLERK
LADQIKRIEALASEIKDLKAQRNSMETDLSNKTHKLALIESQLNSIKKERENMKEEVKDLNDRLKNLKEENLSFQTEIRR
LEKELEYKNKDNQDKDAVIERLKATKATVDEELLNLKKELMKTQNLLDVAENKTKALENSQRQTNLSMNKLEVTIQNLNR
EREERDRVGSSHDSQYIEIDTLYKASQDRENRTKRELQAVKARLEKVETDYYKIQQESVDRRAKVNSLEKKVRDLQAAND
RKDKEKQAIRSDLEAASNQVADLEVAYDNEAKEKDSFKKQLDDALEKLAKARKQNLELEQTIVENKIQLEEAEKEMTEKE
NAINNLASSKEFLEGQLDAAKSGLDKAQREIDELKSEDASKQNEILELHNKINELEARIVKLTGERDRALDDTAINADKI
ANLEQQIGELVSERTTLLERIEDLKKEISSLREERMEVNKQLTDLQGNISFLTREVENRDKVITDLRNKEELQEKEIDNL
RKNLRRLESENESALQKISELRKAYESSSQKTIMIESKSDSHRDKAKRLDEENVSLKRRITELEITHNVNEEKCQELQDK
LLRAQETIGNLESAPPMESSIVVETETNVEAAAGDGIEIEFLKGDLENLKDKNAQLEKHLRQTLDSESNLKAQMAKQDQT
LKDNEDTILDLERRIQELTIRLKKTDQTDAPGIVSSLRNQKDSLERDNVRMKKELEAVKASQVDTQKRRADVENQLQQIK
RKLNEADFKLNMANTQNENLHQELLRAEEKNRAIEAERESALQEQANLRGGLNETNERLNRAKVDLANLKTQKRDLTGDL
EAVKRDRDDKESTINWHLGNERQLKDEIASLKRKLDNMRDANEKLSHTVDNLNAQKQEREEKIADLEHHM
TLLRDEGEKLRRDLATANASVADFKKKYFNALKDIERLQTELNTKIQRIASLENRIKSLENAKSLLRNEISVHKNTISDQ
KVKLEASRRSDRIPTEITVTKVVGPSLQSAPDMFQFTDDSVPSKKYKDLETSYKKVLREKEDGEKDAATLKNRLAKLQSE
LDETNRVKNAVEGDSMWKAKRISQLESELNDLQSKDMVDSDEALGWKNKIPHLEQDLHAANNKIVRLEAEIETYEKMMKD
YSDDLIKAKERVAQLESKVEDDQEKIEGQRKELMEAYKKEADLEAATKSCHNSKSELNGVIMTLRDKIGALENELQKEAK
QHAHVRGTLDQMKIREDDLKRKIAKLQKQLREVQQQYDSQVQDWSERKYTIDGLRKENADYIATVEKLRRELGMYKNDLD
NMATEKRDLQNELNELTPKMEQLETRLHKALEDRTRFQIELDDTKRALDEVGRRESETVIRQVSGGGLEAGVDMFPELSN
DLESTKNERDRLASDVSSWQNKHDTLQNNYDNLYADKKRLEEKLDSMQKTMNQLDHNNQTLTSDKNRQTVELESAKRKIA
DLLSELDDAKRDKDALKVEYEIIQKKITKFEAEYEIHIKHRHDYDDSGAYAKLEEAYKRSQERENEVQKELLVCYKTIGE
LKHQLENAHETMERLTVTYNTETIRSATLREEVVSYQRRLSELEEKFEINRGANEDMENELTVLQQRMEELSRENSSIQE
AKALVLVEVTNQRNKIARLEQQLEDCESEKKSLRLQLTAMHSKGDAEAHEISKQISQLSEQSLTFQREKQNLERQLSQKH
AETISLQRENDALKSQEYSSSTEKQIESFFEMGGDADLGPSAESTRFEFDNDDFEGGGTTVKTRTVKVRSSKKILLSK
P5 (g11674)
MQGKPNSNRSVSQYKLSYSDDNRVYTPITDQVKKKNKPNPINGGWTEWTAWSACDPCGGNLRSRTRTCTNPVPVFGGLDC
VGANTITQFCPNNCAVDGQWSNWSAWSPCSKTCGPGSTWRTRDCVNPPPSNGGAFCKGQAFQLGSCNDGACPEATTTTAA
TTTEVTTTAATTTEATTTEATTTEAATTTAATTTEAPTTAATTTEATTTEATTTEAPTTAATTTEAATTEATTTEAPTTA
ATTTEATTTEATTTTEATTTEAPTTTAAPTNATPTGIPGLSDIDITFALSATSANSNATYTFMQNTLKTFIDKYRANQIQ
YSILVFGNRVERVVTFNSSFPPTANQLKAAIDAQPALPGGPVLEDALKETFKIFNESQGRPAAKKALVVITDNNSGTDKK
TLSNAVKTVEEQGVLVISVAVGGV
P6 (g11666) see g4601
MCKAGFAGDDAPRAVFPSIVGRPRHQGVMVGMGQKDSYVGDEAQSKRGILTLKYPIEHGIVTNWDDMEKIWHHTFYNELR
VAPEEHPVLLTEAPLNPKANREKMTQLSRLCCLSTPLVEQLVSSLTAEMVVSHTVPIYEGYALPHAILRLDLAGRDLTDY
LMKILTERGYSFTTTAEREIVRDIKEKLCYVALDFEQEMQTAASSSSLEKSYELPDGQVITIGNERFRAPEAMFQPSFLG
MESAGIHETTYNSIMKCDVDIRKDLYANTVLSGGSTMFPGIADRMQKEITALAPPTMKIKIIAPPERKYSVWIGGSILAS
LSTFQQMWISQQEYDESGPSIVHRKCF
P7 (g4601) see g11166 and g13727
MCKAGFAGDDAPRAVFPSIVGRPRHQGVMVGMGQKDSYVGDEAQSKRGILTLKYPIEHGIVTNWDDMEKIMFETFNSPAM
YVAIQAVLSLYASGRTTGIVFDSGDGVSHTVPIYEGYALPHAILRLDLAGRDLTDYLMKILTERGYSFTTTAEREIVRDI
KEKLCYVALDFEQEMQTAASSSSLEKSYELPDGQVITIGNERFRAPEAMFQPSFLGMESAGIHETTYNSIMKCDVDIRKD
LYANTVLSGGSTMFPGIADRMQKEITALAPPTMKIKIIAPPERKYSVWIGGSILASLSTFQQMWISKQEYDESGPSIVHR
KCF
P8 (g9654)
MDKCSNLITDTYFLTVGKPRIKLPKKDSDQEWKFHDWKVKAAWGQKIKNVDGLMKQGHHDGMPVIRWCVTSPCEMKKCER
MAMEFNYKISPRMQWSCVMASCKESCMEEIQRGNADIMTAEGDEIYTAGKMHYLKPIMAETDDHDHVPNYDTKFKKFQKP
DGTLKHFSVALIKKSTAGIKSFKDLSKKKSCHPGVDSKAGFKSPVCSLIKKNVISSVGNVFESAGEFFKESCLPGVQHER
FNPNMTNPESLCNLCKGQGDKDDEFCQILSDKEPYYGCDGALKCLREGVGDVAFFHTHDILMKYDTFSTEFDIVCEKDKF
PLTWANLMKPECHLTEEYPQVLMVNGEKSKNWIKSCTDALMDASTKFKSPTSGGFDMFDSTQYCAKDDSDDKGSEMKNGM
FKVDTNLMKNKDLIFKDCSSSLMLIPEDKRTYEKFLGVEMLGVWKACEQLEPKPRAFICVVGDDQMTKCQDMITKFQKIT
EVKKVSWGCIEAPSKQECVEMVANGLADLVDLNATEMFVAGMDFNLAPFVAENHKDKDAMTESITIGVMLKDGSDFLGDM
QGFKMCNAGVDKLSGFLHPMGWLLANGTVPRRTTPLESIGKYFGESCVPGIDKLDMTSHPLLSHTLNWRGMLKDKIVRWE
DWYDAPLWRQWQDKEDGKTVDYTAMHKIEDFQSPEFKTMIAKMGSFNPRMRKYHTPDIRQVLEKMPEWKMIGWVWHKPED
WTEEQWALFQMWAERSWDQKEDLVSYVEKWTDLDWETFKRWMNAEFQDYSTKDKSLHQDTFIRLSLMCSRYYWLLNEWKN
LQYDDIDMKQCREKLCNTCAGKGDKKCSMDSNNEDYYGYKGALKCLKQKDGNVAFIDAYSFENIIQEDSYKNNDFVLLCP
DGKTVDYTGADSYEKCNFGRIPSRTLVTCNMHDGIWRWKVTKALLVAQEKLPQQVFQDCIFSKNMESFTSIPFVNQTYQV
RLGPMFLRAMESLMQPPVKEKAVKYHTVDCLGEECDGDVEDRLACDECLEGDDVCLENCWDGEPIVMEEMPSHIIPKIPL
KMPLKMVGGVKKITLWKPKCRHSHQMMGKMHMGAMEFGMKGHMHHHDDMRSMSGLIMKGSSPVMHQRWSMMRGKVFPHPK
MSAKPTKFGMKHMMKFGPMMKSGSDKLHMMSSKVMEMHPFKSMPKPFMDEPIHGGKMANHFMETGPLMHSYKSLRGEFYT
NVLFARDGTCNSIVHDLSCFGERREIRMKAGRVGQTTITVPMCPKPTIFKNTTALFTCDNGASFAKPVSLPLKCAYAPCK
PGFWLPDWSLDQYWQGDNSHIGGDHWGSKAWWKNRQGNDNWFLAARDENRVQKKSSVGKQ
P9 (g10811)
MRDISVIVYSILCYFCLLRTAIGQTFDVRVNLEEGKDAGQPVHSFPLPASNEVYTFFPAQDADSKAALTLFQISEQGVVT
TTKPIDFEIGKKNYYDLVAVRRDRGDKEGGVPLSIRISITDTNNFGPTFPSNLYHGRVKENSPVDTIVLGLENCFAEDRD
TEGSITYSISGGNEKGYFKAEGRTVNNINRVFLVLKTTNIKIVRDVNTPKIILTVRAFDGRRFGTTKVSITVLDVNNNDP
KFDKDTYTATINEDTPLMTSVLRVRATDADVGTNGGIYYYISGGSPWFSVDAITGVMKVVSRLPNQAAVDLRVTARDRGR
DGTRSHEAVVKVNINLIADYPPADTASPGANTPPVFPEGSYSANVREDFPVGAAVLVIHAVDRDPPGRNSRIQYRLSGDN
AFRINPDNGVVSLRTNLDYSLQNSYDLNVIAQDQAQNPLSAEASLKITVQQVDKNRFAPEFQAPNKYQRQASIPENRAIN
TKVGSSIIATDSDGSQRPEGQVVYSIASGSGLPYFKIDANTGDLRSVAVLDKEKQPQYNLLIEARDKSLYPLKSNLYMVI
AVTGVEDNFPDFSQPVYSAKVPGNAPRGTFVTAIRAIDQDGDPVTYSIDNAGSAFTIQADSGVITTARPLDPANEERNFV
LSVRATAANKDSRAQVIVNVVSKADSPPTFVNLPYKATVPENLGQIDSLLCLAAKDSQGRFSATGSFDYEAFTGGVYQVQ
ASSGPQQIATESVTIAVTDTKDPPKFSKGSYKFVIDEDTSAGTTITVTNQDGSSGGLVIEDADTTITNFECTIENIKSLE
IENHFTIRLVTSNSNKNAQNSNNVRRRRVEVSVQVYAPGTTFIQFDPASYSKDISENAPTDTNVFKVTAKGPNSIRYSIV
GGNINNAFKITQSGQVQVANSLDRETQATYKLVIRAFKDGTKLASEVTTTINVLDVNDVTPKITFMETQPKELAVEDYSP
KGSFVVKVRSYPLAM
P10 (g11107)
MVLQAVDLDTGINAVIHYELQDNAGGKFKIDQDTGLITTLETLEREGPENEFTVQVKATNRQAGAPLLSGTVTATIKVSD
GNDQSPVFDPLVYKANVPENALPGYLVTKVAAIDKDVGPNAELEYTITAGNDPYEFYIDPRNGRILVSGLLDFDKGKKTY
NLTVTVSDRGNPPKSAPRPALVYINVLDANDNAPIFVPAEYNKKVSESVKPGDTVLLVTAVDKDTGTNAQFQFSITEGDD
ANMFAVRSNANNGSIGEIYTLLQLDRETVPRYNLTVTATDSGGLQGIAVVHLTITDINDNGPWFKPRYYEGTVKAGINPS
QGQPVVTLEAYDPDEAINGPPFTFSLVNTDLSNRFKITKATDKTANMAAFGEFNRDVKQSWEVQVKAEDSGNPTMDNTTF
VYIDVEDDRNRNEPFDGKLTIIVNAYNGRFAGGIIGKAYYRDNDYNGDQDKYSMDVQSFLTLTPETGDITAEANIPVGRY
TFSVKVEETKQRPGNFPKIVTSDVTVIVQSVTNAAIQQSVAVLILEMRKTSFFVADFYTKVQGVLANILTGGDTDQILIF
SIQKAPINRVPLSALFGVEIYLSVKSGGGIMDRLRVLNQLIQNKQRLEELGLKIGSIGIDACAYEQQQVGICKNVVKASS
AFTVVSGDYGQVPATESSLTVVAMNVNLQAEYTSIILPEINCTTGTPCFHGGTCHNAVPKGIICECGREYRGPECQSTTR
TFKGNSYLWLEKLAAYERSTVSLQFMTGTADGLLLYQGPVSEGSNNGLPDVMAVMMVGGYIKLVLSLGPHPNAPLELFMN
RGDRLDDRQWHTVEVIRELKKVTVRIDLCSKAQVVEDDGQVLEKRAACEIEGEVWGSNVYLNGFGPLQIGGVENKLNDAK
ISYAGFQGCIRNIKDNDVMYDLLNPVSEKNTDLGXAFVNSGGNAILRTADGNIISSGIGSGLAGYSSSDAELMGTMITVG
RSGQVHLSSSYMKMYSSFGMRAVVGSSGRVFVGRTATTSTRTIIRSYSYRSYTPIGVKSGGKVNVRVIPTNYDPSGGSIV
ISSTKGGSVVGSSGGKGSAGGGSGASDSGGSASAGGGVVVDGGGAVAGGGGVVVGGSGGVVVGGGTVGHGQYVTGATVLG
GASSADAYGQINAYGTSLGGWTFLGATPNGAKEIEVIGDFGGCTASNSYNGVDLDNDPSIEARRQNVEFGCPCSSGFCLH
GGTCVDAVPPYCMCPPGWTGPRCETVVTSPNP
P11 (g13727) see g11666 and g4601
SLVTQTVKASELDEKGVMVGMGQKDSYVGDEAQSKRGILTLKYPIEHGIVTNWDDMEKIWHHTFYNELRVAPEEHPVLLT
EAPLNPKANREKMTQIMFETFNCPAMYVAIQAVLSLYASGRTTGIVLDSGDGVSHTVPIYEGYALPHAILRLDLAGRDLT
DYLMKILTERGYSFTTTAEREIVRDIKEKLCYVALDFDQELQTAASSSSLEKSYELPDGQVITIGNERFRCPEAMFQPSF
LGMEASGIHETTYNSIMKCDVDIRKDLYANTVLSGGSTMYSGIADRMQKEITALAPSTMKIKIIAPPERKYSVWIGGSIL
ASLSTFQQMWISKQEYDESGPAIVHRKCF
P12 (g2385)
MFIFRHYQKVLLLISQLPLLTLQDETENKCSQTTITEAKDHLRLNGGGPLGTYNENYMAFTLDEVRTNFKNLLTLQPGNF
SALAAWAPLSFPATLESGACVKDSGNCEGCPVVKDLGANYYPRFLNEVNCKSSGQICGQGLGECRTSVKSQRFFRDPSTD
LSTCNKDLEEYYYDMEMCCQCMLL*
P13 (g6918)
MGYNAGDPSNLRFYNIPSSGTVEAHLVDERLGNTNREGVWFFRLDVNPILDLPAKKCYTWIQQQDNIQVPAIRPCPCTLD
QAVADKNFYVDYNQVVAKRSNLTLCAYSVRDLTSRWAQQCCYTEIPGGGRVLSVGPTEGGTPFLIGIPGLPIITDLEAHS
YCCNASLCARYYQLRPSDTCSRYSSRREALIWGDPHFVTLDNKAYTFNGLGEYTMISIDNVGFELQARTKKTSGRGLGTV
FSAAVAKEANTAAVEGRINSQGELEVFVAGIQQNILTVAERGTLISGSNITLIRETAGSISALFPSGISISFSRVSDALA
TTFNGPIEFVNHTKGLLGTWNDDPSDDFTTPDGTVLPADATPQQIHYDFGLKWQIHASQSLFTYQAHENPSTFIDLSYVP
MFIDNITWTNESFRRQAESVCGNNTHCLFDAAVTVDCSFGKSTKELEENNVQVNIKL
P14 (g9951)
IIPDIDVGFAVRDEEVESVGLFTLMRDTISTINHRYGTERVRYTVILYGNINTTVVQFSNRNIDHQELISTVTELGRVPG
AIDLEAKLTAAEALFRSLPSRAGSKKVFIVLTDVTAGGNESTITSAGASLVREGILVFSVNRSEDGTNVVTVTQIDYLNI
PTFTTDRSVVIAETIIRKASS
P15 (g1532)
MVVRNEKDDDDDRDGKDGKDKDDKDDSDSKGDIDDKNDKDSLYDKDKFDDKDDKDDKDNDKDNKDDDDDDDDEDFSDDNE
NLLSFELDEIEEKDADDNEVDDKHSVDSFDDVKFTFGKVNKKSTLDGIHVTTVNLSTYLDDQKASLEIIVYLFHEAGSVR
FISNWNFCDGDESSGECSESKIGEFLDLSLKIKSKGSPKEVDDDDRKKKVCNSKDDDDDDKNEDEDDNDDDDDECPIIYD
VGGDSEMLLNKGVSRAHL
P16 (g11702)
ISYLCMAKFDSDQKTALMIPAKKVFGEVDNWDDKVLAKMCNLLEALPVREMLKLASDVAIKDMLSNLNLSEGQLRVLAVK
ALDLLGSPDKWNKDYIKELGNMVAGLMPSELRQIGAQVVKDSLQFLKGIDFDLDQLSKLDKDDVVNLSKAIDGFLSSDVG
KMTQAVVIAAFPEMKVAKEVAMPVLREFIKKFEEDPSSAGKSIAQLGDFAVALSRGEVDAESVDKVITNLDKLGVLPWDK
TKTLTLIKKIRTKWGDFNGTDADDSDSPNWGFLNMKRLGRLALGIAKEELRDLPIRGIEDVIDVLGKEKDWDRGQ
P17 (g12472)
CTNLPPIFIYKVYMFKFDSTHGRFKGTVEAKDGKLVINGNPISVHAKRDPSEIQWGADGAQYVVESTGVFTTVEKASAHL
KGGAKRVIISAPSADAPMFVMGVNEDKYDPSKMTIVRNM
P18 (g810)
LMEAGVIGSHGQAALRRAEAVHRRASALATIHHHNMAASSVPEAIQIVNLAALKVAQLMEDGLIGHNGLPVTKRVVVATP
IGEGNVQTQYHRTVVKHAQVTKMNIGVAILRDAQSVAVGVRGVSGQAAVRLAMDNEQGTAHALILHHPTMEPLAPGQEYN
LKRAMWGSVQMAAGQPGVNGRPVQNRVVEEHKGGQDLAPTLHPHMAERSVWVAKRSPSSAKNKHVQWMAGGLSGIHGESA
ALHVEVENDQEHEPVLIHHHLVTDQDVLAALWRLNNAKHNRVQWMANFLRGLNGNNAARRVEEELKPGPGGVIVLLLPLV
GRPVLEILSRLEIVAPMSAQLMVYGQTGKVGRSVAGHAVAENNQEYVSVTAHGRPMVGNVVLATIQRQGSAKPRPVQLMA
DGLNGPQGHAMLYVAMENATEQEPNAYTTLAATPTPTATAVPTPTIDPNIPKIDLVFAISATSVSSSRSYELMKNTIKRF
IDRYGVNSIHYSIIVYGDQVVRVINFNRTFPPSANELKTAIDNQLALSGGPVLINALQEAYRVFKESVGRPGAKRVLVVI
ADENSGSSPSFLSRAVRPLEDLGVLVISVGVGDRISRSELNIISPNLLDVISARLNINPSLLAVRIMERILRLNFPDVDV
GFAISAASANSDEIFSLMKQIINTIIDRYGVSKVRFSFIIYGSRVTTRFTFDNAPITQEELIKAVNGTKKVTGDPDLEKA
LEEAEKLFTKSSRPNATKVFVVLTDFVGAGDDNSLIANAVRLRKSGVLILSVGFGQQVNAIGNQMTKVVITQSDYIRVPD
FTTQRPVVIAETIMFKALQGKVHVYECAAYNHA
P19(g20041)
KTENTATIINEDGVIKLGPWEPPSHGIISLLFKTPYPKGTILFNGDTTKDYFHLEIINETSIRLEYNIGNGVSTIDLSLA
NEKELNDRKWHAVTVYRNMLQFGLKLDDQEKHKENPLFKEKELNVGDKLNVGTNPRDVTEGFVGCIRRLVSNHCSSRL
P20 (g6066)
CDFEKKSFCAWLNVPNGNKSLGLDDFDWTLGSGSTPSWQTGPSSDHTTGSAIGTYAFIETSSPRKRGEIARLRSKMFAST
TGKCMSWWFHMYGATVANLNIYMKQVGKLESLVWNLNGSQGNVWKKAEVTITSRTSYQMIFEGVTGRDFQSDIAIDDILF
NEGYCIGLCSSVNSQQRVDCGYVGITKATCVGLRRCCWDDSVPNVPYCFYHPSKIVATLESRQLSVSLGDAAGIILSRTF
LGASTDRAGQHPSPPPQPRLQRGHHPNGTAISSKATVTGTTQKRTISTGGDSLVVHLLLEPVQLRIIQQEVKKFAFEGIR
GQSYQGDIALDDIRVLDGSCPLLRNYMYIETSAPRKPGDYARLESPTHSVTDGNGKCLVFWYHMYGSGIGRLNVYIKRGN
SLGSRVWTASGNQGNRWLRGMITVQSPNTQWKVRNSVCRT
P21 (g18277)
MMAILEHQPNHGNLDMDKITLKDVSCTLNDLGEFNATHLWMKAPLDGCLTNHSTSEDTITYMNSIIAETRASAGSVLISR
EFQAEFPFKCSYPRSAIMSVASFSPREKVIYTRTAEFGNFTFTMDMYETDQYLTPYDSYPVEKKLNEKMHLEVKVKSNDS
KLVLIPERCWATPSQDPKDSKFFAFIDKGCGEDDTLVFNYEENAVQRFTLDAFRFLQESTGSTVYLHCEVEACRKGDNES
RCAKGCIQDNSRKRRSLETDSTIMETVSVGPLTAEDIQPQKQ
P22 (g19762)
MVGAGGANVGVGGGNMGGGLGAGVGGITGGGGGVGAGGIAGGGGTMGGGMVGGVGMTSGGTVGGGGLTGGGMVAGGGIVG
GGVGGNMGGGGGSIGGGIGGVTGAGGNIGGGVGVGGGNVGVGGIGGGVGSIGGNIGGVGAEGGIGGGVGGMGGGMGGIGT
GVGIGGGGVGVGGGIGGANMGGAGAETVGGAKGMEGISVHFCRPCFDGHQELCDYCPSEFQDDDEVEEGYKRHHHHHHKH
DKIHLQLGNDEPRHPERKRPFAKVSGDYEVKQFKQAGPSKGYERHDEHGYRADTFSNRNDDVHPTTNPDTDYYGEYKFDD
SKMTVEDSGKYSEENMKTYQ
P23 (g1057)
MFGLHRCSVSASTTNYNNFNVTEGLPNGTFVGNIGNQSYDIVAITPRDRDDLNLDPISMNITTKIVLDREKLSAYIIDLV
RLNPFKVVTFFINVTDINDNIPTFLVGGYDFEFYEGNPVGARVTAVDDDLGSYAVQNYSIISGNTDNMFALTEYVDAVGR
LGAKIELQPGKQLDREQRDLYSLTVSASDGGTPPQTGFGLVNITVLDVNDNDPVFNPKYYNPNVTENTKQFTPIVEVYAT
DKDIGTYGKVMYSVIHDSTSDPDGHFALRTNEKGIIVNLAKLDREKQDSYSLRVLAHNPGSGGYYKRDLAYVTINILDVN
DNSPEVSYTFYLDDAYQVYEDASVGTKVARVWVTDKDAGKNAEIESVTIQGGNGHLSIAYDAANKVHIISLAKELDREKQ
PTFRVYIYAKDRGDPQQTTQDGFILNVGDINDNPPKFDQDVFIAVVSENATIGTSVVVAHATDKDIGSNAKLVYNISYTS
QATLYKTWFQIDQATGEIRTSARLDREKFARLEFLVTVTDSGVPALSANCTVIVNITDANDNDPIFTKAVYFSTVNENSA
NNTSVLQVLANDIDSGINGIVHYSLATQHRRVPFSIDPNTGLLSTSGQIDFEAQSLYVINVVASDGGGRLDKSILNISVV
DENDNYPVINPIIYDVSVYENLTIDDVVATVMAKDNDSGILSQINFAFSNGNDDGLFFINSSTGVITLIKPLDRETKDFH
QLEVQATDGGGLKSKNSAVIQVTVLDVNDEPPVFEPSSYKFAIDENSDVNTLLGSVYASSKDLGTNAQIYYSISDGNIHN
VFTINSTGTIFTQNNIDHERSPQLLLTVQAKDGGSPPLYGFANVSVTVIDLNDNAPSFESGVIDVNILEDIKIGEVFYNV
TAQDPDSGLFGRVRYTFLSNPNNTFQMNSSTGALTVTHLIDFEGPRQYLIIVLAEDGGSPPLNSTATFRVTVVDVNDHTP
KFSNKSYSTHVDEHTPAGTNILNVSATDADTGNNALISYSFQSGVDTSMFGLRSNGWLFITRQLDREQQEVYRFTVVATD
KGHIPKSSTADVIVYIDDINDHNPKFTQQAYIFSVNENQPNRTFVGQVPASDKDAGANAKISYTFEPPSLEFAIDHNTGI
IRTNQVLNREQRSSYILTVKANDHGTSPRTDHTSVTVTVQDINDNAPTFKKPFYEKAVPEDIPVGSSVLTVEASDPDAGS
NGMITYFFASNPGVFTIDARTGVITTTARLDREKHDRYTLSIGAKDHGNPNQSHVVFVTIHVLDVNDNSPRFVNNSIFVN
IPEKQAIGTVVTTVTAKDEDTGNNGKVRYTIDQGNGGKVFEINETSGVIALRKPLDFEKKSKYQLRIVARDQGQNSQSSF
LYVTVYVLDSNDNRPTFEMNPVTVQLQEGVPLNTNVTTIKAHDYDSGQNSWIRYSIDSQSPGPAKFKVDPSTGVVQTIGS
IDREAVDEYTLNIRATDQAFTESERLSSTLTIFIIVSDVNDNKPIFVSPNETYVMEDEGFDYPVITITAIDNDLGSNAKV
KYRIEHDENRDSFRKFALNAATGLLKLVGNLDHETTPKYVLNISVTDGGTPPQFSFQLLTVFVVDVNDNGPEFNQSLFLG
NVSENQPVGTPVMAVSAYDLDSGENGALTYSIPRGNVNQKFALNGSSGVIYTNSTLDREEKDEYTLTVYAMDNVYPRRVG
TCTVKIKVLDVNDHRPVFIPSRLNLTVLEHRPPFDFHTLRAQDPDIGQNGRIRYIIQHGNEDAKFSVGEFTGVLSTTAEL
DRERKSRYYLVIEARNVVVPFYNASIYVTIFVGDKNDNSPKFVNSSYSVNIRELTKIGTPILRAQANDIDSGKNGEITFS
LSSDALGIFRIDSKTGTIYTLQEFDYYITAAYNFLCRATDKGVAPRETSVGVTVSIIDENNHAPVFDAIPYEATIVSGSS
VPQNELVRVHAEDKDSKSITHMTYRLDNSSQPFFQLESNSGVMRVKQGVSSIPDKVYVLNIIADDGGNLTGRGIVEIIVG
SVSPNRPIFVNITPSFVSIPENSPKSLEVARVLANSSNDVVYSIVDGNSGNAFLVNSQTGIVTVQNPAQLDFERLRDFRL
RIIVSRKSQPSRNDYLTLHVNLTDVNDNKPVFYPANISVQLLEDDGSQSSSFTVRSVANLTTTDKDSGKNQEVTYKIISG
NVDDKFAINAQLGLLTTKGFLDREKHAVYDLVVNATDKGTPSRSSVSHVVVNIVDVNDNIPAFSGPYLVNIKEDLRVGSV
VKRVVATDKDEHPKLMYSFGNDKTIYDVFRIDQLTGDIALLESLDYERNTSYELNVTCSDGRYKSHTTVTVNVKDVNDNA
PEFLESSYQATLSEETDQGTSILKVSAEDRDDGSNRELTYAFVTSLDLFRLNTTSGVIYTAKKIEASDRDSFYFVLVSAV
DRGVPQQHAFVMVQIRINRKPMFKEPAYRARIAEDTLPGTTVLTVSAADDSGMNVAKISYKFKFGNSEGLFRIGRRSGII
KVNKSGLDYESEKNYLMGVEARDESANKSVVVEVNITISDVNDNPPVFDPSEYVKEVYENISIGTTLLRVNATDKDSGAN
GRLVFSILSGNDKQSFRINSRTGEIVTIKRLDHESIKEHTLSIQAIDE
P24 (g15888)
HEVYCFSCEQLPITEHEEEKAKGSFYFKVFGKELFYNYFTYDDVKELIEDGVIRVSNDLKDLLKKGKNWNITRVLRPIEV
KIRQPSCVGLPLDFSVDGVLFLRLNTTVKAEVQPRFFIPLPDQVTLSGKLNI
P25 (g11220)
MPQGRKTITLEVEPSDSIENVKTKIQDKEGIPPDQQRLIFAGKQLEDGRTLSDYNIQKESTLHLVLRLRGGMQIFVKTLT
GKTITLEVEPSDSIENVKTKIQDKEGIPPDQQRLIFAGKQLEDGRTLSDYNIQKESTLHLVLRLRGGMQIFVKTLTGKTI
TLEVEPSDSIENVKTKIQDKEGIPPDQQRLIFAGKQLEDGRTLSDYNIQKESTLHLVLRLRGGMQIFVKTLTGKTITLEV
EPSDSIENVKTKIQDKEGIPPDQQRLIFAGKQLEDGRTLSDYNIQKESTLHLVLRLRGGMQIFVKTLTGKTITLEVEPSD
SIENVKTKIQDKEGIPPDQQRLIFAGKQLEDGRTLSDYNIQKESTLHLVLRLRGGMQIFVKTLTGKTITLEVEPSDSIEN
VKTKIQDKEGIPPDQQRLIFAGKQLEDGRTLSDYNIQKESTLHLVLRLRGGQ
P26 (g1441)
SNTWLMYRKPVQGSNDWSSLYRSSELDGFEIGEVTEVRPNSKGCLETVDNERYPKLFKITFMNDRRHVRLATVVYIHHDA
SKNPVEWTRETVPMNTDQVTLFQRMIEDIEPQKGYEEKWRYISDLPLSLSSDNVPENDGLHPHHMKIVFVTPVAGKLCLR
PKRTKESVFSVGRHHTNLSELCYGKQILGFDVCLSGRYISPHTLHPPAFPFCGPTRLNISVQPSRNPAQKIQFKFGRGDR
FDEYHKTWRAGIYLQGTNPEKKIEADVDYDVQNQRTDVTVDIHNMYPWLRKVCIINQVISDNVVEFDLRFGENCLSDYEV
Q
P27 (g18472)
MLTFQVDSMEEKDSNGNVVGLTSQKKHSWLSSENPNQKFTFSPLNNFTTFQRLPVKRINLSVSLAGPQANLEIQVLLFLK
SGKIEFGNETFNVRSGTFKFNIKVSDWQFCGTNAEVCKNSTTGANEIGQFLDIGMSIGSVAEEPEEVDDDQIPGEDICAP
EEPDEEHECPRIFNMGGNSEMMLNKG
P28 (g11651)
MKLAVAVLLVCVAAPQMILTIVLIILSSTVFGFSLPSFEYGEAFEKAKEIGQEVVREVGRHAEEKKNSLLESGEQAMEKL
RQLKEEANRKAGEIQETLEESGDQAIEKLRELKEQAGQRAEKLKGNAEEILEEQKKSLQETGEQAVEKLTQLKEQASNRA
EEMKNTFHEKRKEAMEKLSNLKEQASQRAEEVKGNVEEVVEETKKTFHEKREEAMEKLRNLKGNAEEMLEEQKKSLQETR
EQAVEKLKQLGEQASNRTEEMKNTFHEKREEAMEKLRNLKEQASQRAEEVKGNIEEMVEETKKKFHEKREEAMEKLRNLK
EEASQRAEQVKGNVEEMYEENRKALQDKREQAMEKLLELKQKALSQKEILKEKKQQLKEKMNQMWDDMKQKAGTNKEQLQ
EAFRQKKQEMKEYWKELNEQAGESKEKFRKIWRENKENWRNKAEEVSVAHSFRYLSCFYASLDKYLPAKDPVYM
P29 (g13377)
MREIVHLQAGQCGNQIGAKFWEVISDEHGIDPTGTYHGDSDLQLERINVYYNEATGGKYVPRAVLVDLEPGTMDSVRSGP
FGQIFRPDNFVFGQSGAGNNWAKGHYTEGAELVDSVLDVVRKEAESCDCLQGFQLTHSLGGGTGSGMGTLLISKIREEYP
DRIMNTFSVVPSPKVSDTVVEPYNATLSVHQLVENTDETYCIDNEALYDICFRTLKLTTPTYGDLNHLVSATMSGVTTCL
RFPGQYRALTVPELTQQMFDAKNMMAACDPRHGRYLTVAAMFRGRMSMKEVDEQMLNVQNKNSSYFVEWIPNNVKTAVCD
IPPRGLKMSATFVGNSTAIQELFKRISEQFTAMFRRKAFLHWYTGEGMDEMEFTE
P30 (g11056)
MGGIKGDVTFSQSQGQSSSEVMVNLTGVSATLKWSIRQLPMIYNGNANLSCKAGAVGDVFDPTMAKASADYETNCQSSSS
SKFQSCAVGDLSTMLGDLDRNQQPTTLSDPKLMIPISGPHSVMGKALVLYDGDMPKACALIGPTQPMKTFVAVFKGPVAG
FVYFRQVDENSDTFVFVDLFFVNGANSKKNFSWQINQGVVSHDASDPSSYCKNVGQTFNPKNMGDVSCNKTIHGKCAIGD
MTSKHGKIEVSLATEMQSSTKAAFIDTNLPLSGDDSVQGETLLLYPSEPTQAIACAKIVRLEPKTVKVSFNGSIQDGVTG
HLMFTQSSPFDATTAKIELTGLNKKAEGYHIHAYPSPPYMQLLPNESCTGLIAGDHWNPFNVDIKTSPPAGTGTDDEYEV
GDLGGKYGTFLNLSSFTKTVMDFNLPMFGRNSIQGRSLVIHKQKIKDNNMRWVCSDLILEVDEGITYFMKATANIRGPAV
KGVVVL
P31 (g20420)
YVVRLGGNDKIKELLLGQDLNDGKPHRVDVVHKDLVINVTLDGDSSQEKSGQITTKYKKLEIDVAIYVGGAPSFQGLLGV
KSNFYFVGCLIDVKFRIVRGTPVREIQLLKKDVVESVKVDMAKDRCSPLAFQPYTFSKPDSEYTFVIDKKTSIAGSFKFR
TYQKDGILLKQGNGQNGFTIKFGGSGVILSLVIKGTPTEVTLPTDVNEPRVDAGNWIAIKFTLSATDIKLEGYNNELPVT
KTPTTAVGNNFFHPDVTAGGFIGCMRDLQVNGKDFKPINGVSKVKGVEFDRCNITDLCVFVPCLNGATCTTDGKGLKCDC
TGTGYKGDVCQLGKYLYTVGCIRN
P32 (g5540)
LPKEINVFDAVGLGKKVFPGVSSTTGQNNRSPAYHFTKQTKDMIASQNAFEEVDKLIHNSSDFIVSAYAKLMANEIYGNP
IVSISSKDGNQLYFLLQISYTPQPYPKDAQLRLGQLFEVYQEDIGEITSRFRGDLQDVKFVKGSPLSDCQRIAKASYCRC
SISCQCTDVVGKKDCVVDGLRYADGKRWTKDKCNICHCKWGQVMCTYTCQVCKDNGQTYMEGETWKSSNDSCHTCKCEDG
KVKCSPPVCAKPDCSGKSGDLVTLKGQCCPICKEDQCKGTGKVFKCGCDVTCSNFHDNDACQSCTEGCYCPEGRVLNDQG
KCMASARCGCLYQGKRYKVRK
P33 (g8985)
MTRLGDTFIRQLHEKGEDFTNLWAFSEFLESVDKSGVPDTKTIPVGLKKMKELGIRNAIIEMDLVYAGINYKKFKVEAIN
ELLTERLRWVHANLAKDSKVVVNFRDLPDGMIKRPKRVFKVIRHLSSLPLDIRPFGIVTEESGKYFPEQLAAWISAVRRE
MDDCGFKDGHLLVHVHEKWGLVDSTQLSCLANGANGIWASMIIQGASMGAASSTVTLMNLVRLGNKKVLKKYNCTALREA
AQEICRATTGQEPYPLQPIYGERALDMVFGMPTKLGINEFDLAKFFGEKPLMRMTTLASAEMIITRLKNIFGEDPQFTIE
RGTRMKEVMLEDLHKNIKEEYMSAAGLAQLFDRSGGQLTGKMADVLAQDEPRKAHAQVLIAEVRAMWDEWDLRDGKRDDQ
LEFDAFYNGFLAPYFGCYRCDETKQALKAMDMDEDGTVDWNEFAVYLKWAMRQYPETKTAEDLLSVAFRKGLIPAMQDE
P34 (g1714)
RYMYIETSSPRVKGETARLVSDRFKIADGHNWCLKFWYHMYGNSVGALKVKLKMYPFNPSKPYYNTLRTTQYNRGDSWYF
DQVRINSADDFEIVFEASVGTSYDADIAIDDIDVTKGYCPSPTPTPTPNACAVKCKSGKCVSSTNMCDFVNDCGVGDNTD
EQNCGDCTFETGTALGHYMYVEASQGDSWDLARFESTWLQQSASTCVVSFWYHMFGSGIGTLYGYVKVGQTYTRLFEEWG
NKGNKWIQGKLYVGRHSGPFKVIFEAERSYNVFGDIAIDDVSFENCTLPPIVSSCKRGQHRCARGSCIDPRRLCDYTDDC
GDNSDEVNCYSYNYRCSFERSLCQWNQLSDDDFDWTRNQGSTASYGTGPMFDHTLGTSAGSYLYAEASWPRKKGDKARLA
SGFLSALPQGDTSCKLRFYFHMFGPDIGSLNVYTRPCNGCAETLVYTRSGNVGNFWDRAEVSLPSTVPFQVVIESVRGSS
YQGDISIDDLSMTQYCRSYNGPIPTAPPPTTAAPTVPACPGFRFKCSNGPCIDWGYVCDYRDDCGDGSDEVNCGPCNFES
GLCNWKDNSVGRYQWSRNKGSTVATGTGPSIDHTCGNAS
P35 (g7349)
LHLVFFNKKYETFQNAVDKPDGLAVLGVLITATCPGNRILGNFAKKLTKVVEEGANTNATATDSIKLNYLMPYNNKQGDE
DDEEEDIASDDPEAVEEEEVDAKKKIKYYTYKGSLTTPPCYESVTWIVFKDKIKISNTQLNKFRKLKAQFGGAP
P36 (g13890)
HYMYIETSSPRKQGDKARLVSEDFSPITIRGRCVKFWYHMYGASIGTLRILEKTGPGNKSESTIWELSGNFGDQWYSGQA
PITSSSPYQVVFEAMRGRSVSGDIAIDDVTFSTSKCSVVPSLAVPPTPPPTTPPPIINNCTFEGGFCSWTNLNGDDFDWT
RSRGSTASWSTGPTVDHTLGTSQGYYVYIETSSPRSPNDKAQLQSGLIQPTTVTSGRCLKFWFHMWGRHVDTLNVYRKDS
SSRV
Formatted Alignments
10
20
S.pistillata_CARP4
S.pistillata_CARP5
Favia_sp._220913.2_220913.6
Favia_sp._48585
Favia_sp._234233
Favia_sp._213894
Favia_sp._38004
P.damicornis_37433_70018
A.digitifera_01440
A.digitifera_06237
A.digitifera_11068
- - - - - - G P
- - - - G P
L
L
I
I
- - - - - - V R
- - - - V R
- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - T V M A F A
- - - - - - M A L I T
- - - - - - - - - M A
T
A
S.pistillata_CARP4
S.pistillata_CARP5
Favia_sp._220913.2_220913.6
Favia_sp._48585
Favia_sp._234233
Favia_sp._213894
Favia_sp._38004
P.damicornis_37433_70018
A.digitifera_01440
A.digitifera_06237
A.digitifera_11068
C
S
A
S
S
S
M
A
T
A
D
Q
R
S
K
D
R
I
L
I
D
K
L
A
F
V
- - - - - - - - - - - - L A D Q V
- - - - - - - - R S G K L
E D D V D
R S G K I
- - - - A R V Q P
S.pistillata_CARP4
S.pistillata_CARP5
Favia_sp._220913.2_220913.6
Favia_sp._48585
Favia_sp._234233
Favia_sp._213894
Favia_sp._38004
P.damicornis_37433_70018
A.digitifera_01440
A.digitifera_06237
A.digitifera_11068
D
D
D
D
N
D
K
D
E
X
D
D
D
E
N
S
D
K
X
X
D
K
V
D
D
D
K
X
X
D D - - D T A
- - T - D D D T V
X - X - D T
- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - A N T V T I R G N D T
- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - D K K N P X X X X X X
- - - - - - - - - - - - - - - - - - - - -
S.pistillata_CARP4
S.pistillata_CARP5
Favia_sp._220913.2_220913.6
Favia_sp._48585
Favia_sp._234233
Favia_sp._213894
Favia_sp._38004
P.damicornis_37433_70018
A.digitifera_01440
A.digitifera_06237
A.digitifera_11068
D
D
D
D
D
D
D
D
X
D
X
D
F
F
F
F
F
F
F
F
X
F
F
F
S
S
G
S
S
S
G
S
X
D
D
S
D
D
D
D
D
E
D
D
X
D
D
D
D
D
D
D
D
S
D
D
X
E
S
D
N
N
N
N
N
N
N
N
X
N
N
N
E
E
E
E
E
E
E
E
X
D
E
E
N
D
D
D
E
D
G
X
D
M
L
L
V
V
L
L
V
L
A
M
S.pistillata_CARP4
S.pistillata_CARP5
Favia_sp._220913.2_220913.6
Favia_sp._48585
Favia_sp._234233
Favia_sp._213894
Favia_sp._38004
P.damicornis_37433_70018
A.digitifera_01440
A.digitifera_06237
A.digitifera_11068
F T K
F G K
F G L
F S A
F S A
F S E
F G L
F T Q
L S H
V S A
L S S
F S
V
V
V
L
L
V
V
L
V
L
V
V
D
N
D
D
D
D
D
D
R
Q
D
D
T
K
R
N
N
S
R
N
T
N
R
Q
K
Q
S
S
Q
Q
S
A
S
N
A
S
A
S
S
S
A
S
S
T
S
S
K
T
T
T
T
S
T
T
R
T
N
T
S.pistillata_CARP4
S.pistillata_CARP5
Favia_sp._220913.2_220913.6
Favia_sp._48585
Favia_sp._234233
Favia_sp._213894
Favia_sp._38004
P.damicornis_37433_70018
A.digitifera_01440
A.digitifera_06237
A.digitifera_11068
E
E
E
E
E
E
N
E
E
E
E
T
T
T
T
T
T
T
T
T
T
T
R
A
R
R
A
A
P
N
K
D
V
V
V
V
V
V
S
V
V
V
V
E
Q
Q
Q
Q
Q
P
K
L
Q
Q
K
S
S
S
S
S
Q
A
S
A
S
G
G
G
G
G
G
G
G
G
G
G
T
T
T
T
T
T
Y
T
T
T
T
S.pistillata_CARP4
S.pistillata_CARP5
Favia_sp._220913.2_220913.6
Favia_sp._48585
Favia_sp._234233
Favia_sp._213894
Favia_sp._38004
P.damicornis_37433_70018
A.digitifera_01440
A.digitifera_06237
A.digitifera_11068
S
S
S
S
S
S
S
S
S
K
K
K
M
M
K
K
K
K
D S P A
G S P K
G S P E
E E P E
A E P E
G S P A
G S P E
- - - - - - - - - D T P I
S P
E
E
E
E
E
E
E
E
E
I
V
V
V
V
V
V
V
V
S
D
D
E
E
D
D
T
S.pistillata_CARP4
S.pistillata_CARP5
Favia_sp._220913.2_220913.6
Favia_sp._48585
Favia_sp._234233
Favia_sp._213894
Favia_sp._38004
P.damicornis_37433_70018
A.digitifera_01440
A.digitifera_06237
A.digitifera_11068
- - - - - - - - - - D N
M E E E E D D
- - - - - - - - - - - - - - - - - - - - N D D
- - - - - - - - - - - - - - - - - - - - D G D
M E E E
D D
D
D
D
D
D
S
D
D
D
N
D
D
E
E
D
D
D
D
S.pistillata_CARP4
S.pistillata_CARP5
Favia_sp._220913.2_220913.6
Favia_sp._48585
Favia_sp._234233
Favia_sp._213894
Favia_sp._38004
P.damicornis_37433_70018
A.digitifera_01440
A.digitifera_06237
A.digitifera_11068
V E - - D G E K K
- - - - - - - - R D A D D D S T K
S T - - G M M K S
S T - - G M M K S
F E - - D D E K K
R D A D D D S T K
- - - - - - - K K
I E - - D E T R K
- - - - - - - K K
I E - - D G E K K
A D D
K K
S.pistillata_CARP4
S.pistillata_CARP5
Favia_sp._220913.2_220913.6
Favia_sp._48585
Favia_sp._234233
Favia_sp._213894
Favia_sp._38004
P.damicornis_37433_70018
A.digitifera_01440
A.digitifera_06237
A.digitifera_11068
A
A
L
L
A
A
L
L
F
L
- - - - - - R L
- R V
- V S
R
- - - - - - - - - - - - C G G
- - C L S
- - C F H
C
- - - - - - - - - - - - - - - - - - V V L V
- - - L A F F
- - - L R L L
L
L
R
I
A
I
E
G
S
L
V
A
V
K
G
D
M
D
N
T
V
T
I
V
D
S
T
D
I
D
M
V
60
G
A
D
G
S
K
K
K
R
N
R
K
K
K
M
K
F
I
D
V
D
V
M
I
D
P
E
D
T
D
K
I
E
E
E
Q
Q
E
E
Q
E
Q
E
E
V
L
L
V
V
V
L
I
L
V
L
D
D
D
D
D
D
D
D
D
E
D
D
E L E E K
E I E E K
E I E E K
S M Q E V
S L Q E V
K I E E K
E I E E K
S L Q E I
E L K E V
S L Q E V
S I E E K
E E K
Y D G L
L D G I
F Q G I
Y Q G I
Y Q G I
L Q G L
F Q G I
Y Q G I
F K G L
Y Q N L
A L G V
Q G I
P
H
P
P
P
P
P
P
A
T
A
P
V
V
V
V
V
V
V
V
V
A
A
V
T
T
I
K
K
I
I
K
I
K
I
V
V
M
V
V
V
M
V
V
V
V
V
N
N
N
N
N
N
N
N
N
T
N
N
F
F
F
F
F
F
F
F
F
F
F
F
W
W
W
W
W
W
W
R
W
W
L
V
F
F
L
V
V
V
F
A
F
F
S
S
A
F
A
L
T
I
I A A F
- - - F Q I T
F L L L
F L L L
I T A C
F Q I T
V F F P
V Q I A
L A M L
L Q A V
I
A
L
L
L
A
H
V
L
T
D
F
V
D
K
I
D
I
X
D
D
T
D
D
R
D
S
X
D
D
D
T
D
D
A
R
T
X
D
K
F
N
K
V
E
L
X
D
D
A
D
D
H
E
Y
X
D
D
D
D
N
D
H
S
F
X
- - - - - - D D D D D G V
- - - - - - N D K D N K D
- - - - - - - - - - - - D
T L Y F E Q D D D D D D D
- - - - - - D D D D D D D
- - - - - - D Q D D D N D
- - - - - - D N D D K D D
- - - - - - D E D D D G E
- - - - - - X X X X X X X
- - - - - - X X X X X X X
- - - - - - X X X X X X X
T L Y F E Q D
D D
D
D
D
D
D
D
D
D
D
X
X
X
D
D
D
D
D
D
D
D
D
X
E
X
D
D
D
D
D
D
N
D
D
X
S
X
D
D
D
E
D
D
D
D
E
X
E
X
D
D
D
D
D
D
I
D
G
X
D
X
D
D
D
D
E
E
D
D
E
X
E
X
D
D
D
V
V
D
D
M
X
V
X
F T
F T
F T
F N
F N
F T
F T
Y T
F Q
F T
F S
F T
V
A
S
S
S
A
S
S
A
E
S
S
D
D
Q
Q
Q
D
Q
A
D
S
D
G
D
G
G
G
G
G
G
G
G
G
G
N
N
N
T
V
M
N
N
D
K
N
N
D
E
D
P
P
D
D
P
E
P
D
V
V
V
V
V
V
V
V
V
V
V
V
D T M - D - - - D - - - G L S G P
G L S G P
G K L - D - - - G L T G P
D - - - K A S E S
G S T - G P
D
D
R
D
D
D
S
L
L
E
S
D
S
D
K
L
K
D
V
T
V
A
G
L
F
S
R
V
L
S
- - - D D D D
- - - - - - - - - - - - N D D V
F S L L
D D N T
- - - E L N K
D
S
S
S
S
S
S
S
S
S
Q
S
S
A
T
V
V
V
A
V
V
T
A
T
T
Y
Y
Y
F
F
Y
H
H
Q
K
L
L
L
L
L
L
L
L
L
L
L
L
P
D
P
A
A
V
P
T
Q
P
E
S
D
D
G
G
E
D
G
N
N
D
Q
P
P
P
Q
P
P
L
S
P
S
K
D
Q
Q
M
D
N
K
M
N
F
F
F
F
F
F
F
N
F
F
C
C
C
C
C
C
C
R
C
C
D - D G D D - D Q G I
D L G N
D - D - - - - S - - - - - D - D
G
- - - - - D
K D S L Y D
- - - - - - - - - L A
- - - - - - - - - D D
D A G D N D
- - - - G N
D N V D D R
- - - - G D
- - - - X X
D
D
D
D
D
K
E
-
Q
D
D
R
R
E
D
K
D
I
E
R
K
K
R
R
K
K
K
K
Q
G
K
H
H
H
H
H
H
H
H
H
H
H
H
S
S
S
G
G
F
S
G
S
S
S
S
V
V
V
L
L
V
V
V
V
V
V
V
V
V
V
V
V
V
V
V
V
V
L
V
Y
Y
Y
M
M
Y
Y
M
Y
V
Y
Y
L
L
L
V
V
L
L
L
L
L
L
L
F
F
F
F
F
F
F
F
F
F
F
F
R
H
R
L
L
R
R
L
L
L
R
A
K
K
G
G
K
K
S
A
K
E
S
S
A
E
Q
A
D
N
N
A
D
N
K R P I C E
- - - V C N
K E P I C M
K E A I C V
K E A I C V
K P A I C K
K E P I C M
- - - - - - - - - - - - - - - K P A V C K
K E A I C
D
D
D
D
D
D
D
D
D
D
D
D
D
D
D
D
D
D
D
E
D
D
D
D
D
D
D
C
C
C
C
C
C
C
C
C
P
P
P
P
P
P
P
P
P
S
I
K
K
K
V
K
H
I
I
I
I
I
I
I
I
I
Y
Y
Y
Y
Y
Y
Y
Y
Y
D
D
D
N
N
D
D
F
N
D
V
V
S
S
V
V
V
V
T
K
V
F
F
F
F
F
F
F
F
F
F
F
R I
- R I
R I
R I
R I
R I
R I
R I
R L
R V
R I
P
P
P
P
P
P
P
P
P
P
P
K
K
K
K
K
K
K
K
K
K
K
F
F
F
F
F
F
F
F
T
F
F
K
G
N
N
N
S
K
S
P
D
K
K
N
N
K
K
K
K
G
D
K
V
I
S
S
H
I
Q
M
S
L
T
F
M
M
F
F
S
F
V
F
N
I
L
L
M
I
C
V
L
I
V
Q
V
V
T
Q
R
H
A
S
H
H
S
A
H
W
- - - Y Y - - F C
- F - C
200
S
S
S
S
S
S
S
S
S
S
S
S
F
F
F
L
L
L
F
L
F
V
F
F
D
D
D
A
A
D
D
V
D
K
K
D
D
D
D
S
S
D
D
S
D
S
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240
250
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C D E G K I G E F
C S E S K I G E F
C S S G K A G E Y
S N Q N E I G Q Y
S N Q N E I G Q Y
C S E G K T G E F
C S S G K A G E Y
- - - - - - - - - - - - - - - - - - - - - - - - C S E G K A G E Y
C S
G K
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L
L
L
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L
L
D
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D
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S - - - - - - - - - - - D - - - - - - - - - - - D X E E E E E E E E E E E
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320
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330
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370
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420
K
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480
350
390
430
400
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K
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440
V
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N
N
G
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G
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490
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300
340
380
G
G
G
G
G
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G
G
G
G
R
E
G
G
G
R
G
R
E
E
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D
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E
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X
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X
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L
L
L
L
L
L
L
V
V
L
V
L
280
K
K
E
Q
Q
K
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D
D
K
D
D
D
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N
D
S
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D
D
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D
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150
190
S
S
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230
S S
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A T
A T
A T
A T
A T
A S
A N
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A D
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100
D
K
T
D
K
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140
180
R
R
R
R
R
R
R
R
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470
D
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L
D
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N
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X
D
D
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D
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V
L
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130
D
D
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460
L
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F
L
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L
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K I S K
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N - - K I S N
K I S
S
N
T
T
N
N
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N
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90
I
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M
M
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M
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N
N
N
N
N
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N
N
N
410
F
F
F
F
F
F
F
F
L
I
F
D
G
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K
T
K
T
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270
D
N
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Q
D
D
S
D
D
N
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50
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- - - - - - - - - - - - - - - - K T I R G
F V C F A
K T I R G
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K
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- - - - - C - - - - - - - - - - - - - - D S E K D D
E - - - - R V S D K K
E - - - - - - - - - K
D
F
F
F
F
F
F
F
F
F
F
F
360
D
D
D
P
P
N
D
D
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L
L
Y
L
L
M
Y
L
L
L
L
L
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D
D
V
D
K
I
D
V
X
K
K
K
K
K
K
K
K
K
K
K
310
D
D
D
D
D
D
D
D
D
I
I
S
N
P
D
D
T
N
D
220
N
T
K
N
N
N
K
N
S
T
N
N
260
I
V
L
L
V
V
P
V
M
V
N
D
S
N
D
N
D
S
N
170
S
S
S
T
T
S
S
T
S
S
S
S
40
V
C
L
A
80
D
D
L
D
D
G
D
G
D
D
120
210
F
V
H
H
L
V
L
V
C
F
R
K
R
K
D
R
E
R
R
R
- - - - - - S V R
- - - - - - - - - - - - - - S V R
160
F
F
F
F
F
F
L
F
F
F
F
- - - - - - - - - - - - - - M
- - - - - - - - - - - - - - - - - - - - - - - - - - - - C F A M F L V A
- - - - - - - C L A L F L V C
- - - - - - - F L C L A L F M
C L A L F L V
70
I
L
M
M
M
N
110
L
L
L
L
L
L
L
L
L
L
L
L
30
Q
Q
F
Q
E
E
V
V
E
E
E
E
E
450
N
N
A
A
A
N
A
S
E
S
S
T
G
G
S
T
N
A
S
P
L
A
G
G
C
A
G
A
L
L
S
S
L
L
L
F
S
I
500