Supplementary Text A
H3: The DNA sequence data allowed clarification of some of the Leu/Ile residue assignments
in the peptide sequences (Peptides 2,8, 10,11); these two residues have the same mass, and
thus cannot be resolved by mass spectrometry (Table 1). The DNA data also allowed the
occasional correction of the mass spectrometry data. For peptide 8, residue 4 is a Lys, rather
than a Gln; these residues have nearly identical masses and can difficult to distinguish in
mass spectrometry. For peptide 11, the N-terminal residues VQD, which were the least
confident assignments from mass spectrometry, were LDN in the cDNA-derived sequence.
An alternative explanation for such discrepancies would be the presence of multiple isoforms
for the protein.
H8: The cDNA-predicted amino acid sequence contained both of the H8 peptides identified
by mass spectrometry, and allowed correct assignment of the Leu/Ile residues as Leu. It also
allowed the revision of the sequence for Peptide 1, where an extra Thr is present at residue 3.
An alternative explanation for such discrepancies would be the presence of multiple isoforms
for the protein.
H9: The predicted N-terminal sequence from the cDNA clone differs slightly from Peptide 1
(Table 1) and the Edman data, with the cloned sequence suggesting that residue 5 may be a
Ser and that residue 8 may be a Cys. Peptide 3 could also be identified in the cDNA-derived
sequence, which allowed its Leu and Ile residues to be resolved from one another.
1
Supplementary Text B. Ambiguities in peptide sequencing by mass spectrometry
Phenylalanine (F) may be a methionine (M) adduct, methionine sulphoxide, which has almost
the same mass. Likewise, glutamine (Q) may be lysine (K), as both residues have very
similar masses. Trypsin cleaves C-terminal to arginine (R) and lysine (K) residues, but either
can be present internally in a tryptic peptide if digestion is incomplete, or definitely if one of
these residues is followed immediately by a proline residue.
2
Supplementary Table S1.
Amino acid composition of cDNA-predicted polypeptide segments compared with actual
composition of H. dofleinii bands.
Band
%
lengtha
Asx
Ser
Glx
Gly
His
Arg
Thr
Ala
Pro
Tyr
Val
Met
Lys
Ile
Leu
Phe
H3
H8
H9
cDNAb
40%
AAAc
cDNAb
20%
AAAc
cDNAb,d
100%
AAAc
10.5
7.3
7.7
8.9
3.2
3.2
5.7
10.9
3.6
4.5
6.5
2.8
6.9
2.8
8.9
3.6
8.7
11.6
13.3
16.8
1.0
5.2
5.2
7.0
4.9
2.5
5.5
0.4
4.9
3.8
7.0
2.4
12.2
3.0
16.6
7.6
1.5
3.0
3.0
4.5
3.0
3.0
6.1
1.5
4.5
9.1
13.6
4.5
9.7
10.2
15.0
13.6
0.7
5.5
4.9
6.8
6.3
3.0
5.5
0.4
5.0
3.5
7.5
2.5
17.9
8.3
6.2
6.2
2.8
4.8
1.4
9.0
4.8
6.2
7.6
0
5.5
4.1
4.8
4.1
15.0
8.0
11.6
10.5
1.3
4.4
4.6
8.0
3.1
2.4
5.7
5.0
7.0
4.8
5.8
2.6
Correlation with composition of H. dofleinii band (R)e and statistical significance (P)f
R (cDNA)
0.82
0.62
0.65
P (cDNA)
< 0.0001
0.011
0.006
R (SP ave)
0.89
0.88
0.89
P (SP ave)
< 0.0001
< 0.0001
< 0.0001
a
% length is an estimate of the completeness of the clone, i.e. the number of amino acid residues predicted
from the cDNA segment as a percentage of the total number of residues in the best-matching S. purpuratus
protein (H3, H8) or in the complete H. dofleinii ORF (H9).
b
Amino acid compositions of cDNA-predicted polypeptides were calculated using
http://web.expasy.org/protparam/
c
Amino acid analysis data of Peng et al. (2011).
d
Excluding signal peptide.
e
Spearman correlation coefficients (R-values) for linear regression of amino acid composition of each H.
dofleinii band against that of the cognate cDNA-derived polypeptide segment (“cDNA”) or against the
average composition of the SwissProt database (“SP ave”; Tompa 2002), calculated using GraphPad InStat
v3.01.
f
Significance (two-tailed non-parametric P-values) of the correlation of amino acid composition of each H.
dofleinii band with that of the cognate cDNA-derived polypeptide segment (“cDNA”) or with the average
composition of the SwissProt database (“SP ave”; Tompa 2002), calculated using GraphPad Instat v3.01.
3
Supp. Table S2. Amino acid composition of best-match S. purpuratus proteins compared with actual composition
of H. dofleinii bands.
Band a
Type
Species
Asx
Ser
Glx
Gly
His
Arg
Thr
Ala
Pro
Tyr
Val
Met
Lys
Ile
Leu
Phe
H2
H3
H6
H7
H8
H9
Peptide/nitrate
transporter
Spb
Hd
Transketolase
FBP aldolase
Transaldolase-like
Exportin-6-like
C-type lectin
6.9
8.3
6.5
6.6
1.3
3.4
10.8
9.0
4.4
4.2
7.7
3.1
7.6
9.7
5.1
9.7
9.8
16.2
16.1
1.1
3.6
3.9
7.8
3.2
1.8
4.6
n.d.
8.8
4.1
7.0
2.4
b
b
b
Sp
Hd
Sp
Hd
Sp
Hd
Sp
Hd
Spb
Hd
10.1
6.3
6.9
8.2
2.1
4.3
5.5
13.0
4.5
2.7
8.7
1.6
5.8
5.9
7.6
3.7
8.7
11.6
13.3
16.8
1.0
5.2
5.2
7.0
4.9
2.5
5.5
0.4
4.9
3.8
7.0
2.4
9.9
4.4
11.3
8.5
1.4
3.8
6.8
12.3
4.7
3.3
7.4
0.8
6.0
4.4
9.6
2.7
12.1
7.0
13.2
7.8
1.3
4.9
5.1
9.2
3.9
2.4
6.4
3.2
8.3
5.6
7.3
2.5
10.3
6.7
12.1
4.8
3.3
4.5
10.6
2.4
2.4
7.3
3.0
10.0
6.4
10.3
2.7
13.1
6.5
12.7
8.1
n.d.
4.1
5.0
9.4
3.2
1.9
6.4
3.8
9.4
5.3
8.3
3.0
8.5
8.8
11.8
3.8
2.7
3.8
4.9
4.4
4.4
2.5
5.8
4.4
4.7
8.2
13.4
6.8
9.7
10.2
15.0
13.6
0.7
5.5
4.9
6.8
6.3
3.0
5.5
0.4
5.0
3.5
7.5
2.5
11.3
13.8
8.1
7.5
4.4
1.3
6.9
3.1
3.8
5.0
5.7
1.9
3.8
3.8
5.7
3.8
15.0
8.0
11.6
10.5
1.3
4.4
4.6
8.0
3.1
2.4
5.7
5.0
7.0
4.8
5.8
2.6
Correlation with composition of H. dofleinii band (R) and statistical significance (P)
0.39
0.83
0.89
0.94
R (Echin)
0.15
< 0.0001
< 0.0001
< 0.0001
P (Echin)
0.90
0.89
0.94
0.92
R (SwPrt ave)
< 0.0001
< 0.0001
< 0.0001
< 0.0001
P (SwPrt ave)
a
b
0.45
0.083
0.88
< 0.0001
0.50
0.047
0.89
< 0.0001
H4 is omitted because no amino acid analysis data is available for this band. Amino acid compositions of S. purpuratus proteins (“Sp”)
were calculated from translated ORF sequences using http://web.expasy.org/protparam/, amino acid analysis data for H. dofleinii proteins
(Hd) are from Peng et al. (2011).
4
Supp. Fig. S1. SignalP v4.1 predicition for cDNA-derived protein corresponding to H9.
The server is at http://www.cbs.dtu.dk/services/SignalP/. Parameters are calculated as follows:
C-score (raw cleavage site score)
The output from the CS networks, which are trained to distinguish signal peptide cleavage sites from
everything else. Note the position numbering of the cleavage site: the C-score is trained to be high at the
position immediately after the cleavage site (the first residue in the mature protein).
S-score (signal peptide score)
The output from the SP networks, which are trained to distinguish positions within signal peptides from
positions in the mature part of the proteins and from proteins without signal peptides.
Y-score (combined cleavage site score)
A combination (geometric average) of the C-score and the slope of the S-score, resulting in a better
cleavage site prediction than the raw C-score alone. This is due to the fact that multiple high-peaking Cscores can be found in one sequence, where only one is the true cleavage site. The Y-score distinguishes
between C-score peaks by choosing the one where the slope of the S-score is steep.
The graphical output from SignalP (above) shows the three different scores, C, S and Y, for each position in the
sequence.
Parameter summary:
Measure Position Value
max. C
21
0.548
max. Y
21
0.699
max. S
17
0.935
mean S
1-20
0.891
D
1-20
0.803
Cutoff
0.450
Signal peptide?
YES
5
Cleavage site between pos. 20 and 21: NRA-ED D=0.803 D-cutoff=0.450
Networks=SignalP-noTM
In the parameter summary, the maximal values of the three scores are reported. In addition, the following two
scores are shown:
mean S
The average S-score of the possible signal peptide (from position 1 to the position immediately before
the maximal Y-score).
D-score (discrimination score)
A weighted average of the mean S and the max. Y scores. This is the score that is used to discriminate
signal peptides from non-signal peptides.
For non-secretory proteins all the scores represented in the SignalP output should ideally be very low (close to
the negative target value of 0.1).
6
SpEchL2
TfCL
Hd band 9
Consensus
SpEchL2
TfCL
Hd band 9
Consensus
SpEchL2
TfCL
Hd band 9
Consensus
SpEchL2
TfCL
Hd band 9
Consensus
----MFNKIVTILVIASSAVMLPVLPGCQAGGCGCPPLWTAFQNNCYRYFSVKNITWHGA 56
MLLFLFLFGLALGAVAPSGVDQEHIEALLQ--VNCPLLWISFNNHCYKYVSNR-MTWVDA 57
-----MASKFAVLLLLYTMVVVNRAEDKVS--FGCPKDWYAYNDNCYHYNAERRFTSTSG 53
:
.:: : : *
.** * :::::**:* : : :* ..
CYkff
ktW dA
EMHCSGFSVPCSDVDSTISLGHLTSIHSKEEMTFLSVLYESIRSKVVTSTTYVWIGLHDQ 116
ELHCVSQDA------------NLVSIHSLEEHNFVKALIK----KSDITEERTWIGLSDI 101
ASYCRDHGA------------KLLYISSIEEFKFAGAIASS--RDADVLIPSLHIGLNNN 99
:* . ..
:* * * ** .* .: .
.
*** :
e fC
LVSI S eE Fl
WIGL
TTEDSWEWSDGSSL----DYEIWEPGQPSSHNGNQDCVMFFSSNKYKWNDLACDSDGDTA 172
HKEGTWMWSDGSKV----DFVTWNQGQPDNHLANENCVHTNYYIAKKWNDALCSELYAFV 157
AREGDFVWSDGKKLSQAPNVVVWEPNQPNDLGHNQNCVVYRINN-YNVNDAPCSYVAGVF 158
*. : ****..:
:
*: .**..
*::**
: ** *.
w WsDGs
y nW gePnn
e CV l
g Wnd C
fI
YHGSAYVCKLPQW 185
CQSRTVFS----- 165
CKKPRAN------ 165
:
Ck
Supp. Fig. S2. Multiple sequence alignment of C-lectins with the polypeptide from H.
dofleinii band 9 cDNA. TfCL is a C-type lectin from the fish Trachidermus fasciatus
(AFW17073.1); SpEchL2 is echinoidin-like isoform 2 from the holothurian S. purpuratus
(XP_003726044.1); Consensus shows conserved motifs taken from the sequence logo for Ctype lectin class 2 (http://prosite.expasy.org/cgi-bin/prosite/sequence_logo.cgi?ac=PS50041),
to which these sequences conform better than to class 1; capitals indicate more strongly
conserved residues. The alignment was performed using Clustal-W2 at http://www.ebi.ac.uk;
* identical : highly conserved . conserved. The invariant cysteines are highlighted in cyan
(the pair that form one disulphide bond) and magenta (the pair that form the other). Predicted
ligand-binding residues for the two known proteins (taken from their NCBI RefSeq entries)
are shown in red.
7
Yeast TK
Hd band 3
Yeast TK
Hd band 3
Yeast TK
Hd band 3
Yeast TK
Hd band 3
Yeast TK
Hd band 3
Yeast TK
Hd band 3
1 MTQFTDIDKLAVSTIRILAVDTVSKANSGHPGAPLGMAPAAHVL-WSQMR
:...:.:|..::::.:.|.||||...:..|....|| :..|:
1 --------EDVANKLREDSIESTTAAGSGHPTTCMSAAEVMSVLFFHTMK
49
42
50 MNPTNPDWINRDRFVLSNGHAVALLYSMLHLTGYDLSIEDLKQFRQLGSR
.....|.....|||::|.|||..:||:.....|. ..:|:||..|::.|.
43 YKVDVPKDPANDRFIMSKGHAAPILYAAWAEAGL-FPVENLKNLRKIDSD
99
100 TPGHPEFELPGVEVTTGPLGQGISNAVGMAMAQANLAATYNKPGFTLSDN
..|||...|..|:|.||.||||:|...|||
|.......:|.
92 LEGHPTPRLSFVDVATGSLGQGLSVGAGMA---------YTGKYLDKADY
149
150 YTYVFLGDGCLQEGISSEASSLAGHLKLGNLIAIYDDNKITIDGATSISF
.||..||||...||...||.:.|.:.||.||:||:|.|::.....||:..
133 RTYCLLGDGESAEGSVWEAMAFASYYKLDNLVAIFDVNRLGQSQPTSLQH
199
91
132
182
200 D-EDVAKRYEAYGWEVLYVENGN--EDLAGIAKAIAQAKLSKDKPTLIKM
| |....|.||:|:.. ||.:|: |||
|||:..|...||||:.|..
183 DMETYRLRCEAFGFNT-YVVDGHSVEDL---AKALHDASTVKDKPSCILA
246
247 TTTIGYGSLHAGSHSV---HGAPLKADDVKQLKSKFGFNPDKSFVVPQEV…
.|..|.|: .|...:
||
229 KTYKGKGA--KGIEDLEGWHG-----------------------------
293
228
247
Supp. Fig. 3. Sequence alignment of yeast transketolase (partial) with polypeptide from
H. dofleinii band 3 cDNA. “Yeast TK” is the N-terminal portion of Saccharomyces
cerevisiae transketolase (AAA35168.1) with catalytically essential residues highlighted in red
and residues involved in cofactor or metal binding highlighted in cyan. These are as
identified in http://www.uniprot.org/uniprot/P23254, Singleton et al. [1996, Biochemistry 35,
15865-15869], Wikner et al. [1997 Eur. J. Biochem. 233, 750-755] and Gerhardt et al. [2003,
Plant Physiology 132, 1941–1949]. Sequence alignment is by EMBOSS Needle at
http://www.ebi.ac.uk; key: “|” identical, “:” highly conserved; “.” conserved.
8