Supplementary Material
Harnessing disorder: onychophorans use highly unstructured proteins, not silks, for prey capture.
Victoria S. Haritos*, Ajay Niranjane#, Sarah Weisman, Holly E. Trueman, Alagacone Sriskantha and
Tara D. Sutherland
Supplementary Fig 1.
Image of several intertwined Euperipatoides rowelli
Supplementary Fig 2. Thread drawn from a pool of Eu. rowelli slime gland secretion.
Supplementary Figure 3. WAXS pattern of threads drawn from Eu. rowelli slime gland
secretion. The three visible spots are detector artefacts also seen in air scattering patterns. No
spots or rings attributable to scattering from ordered structure are detected. The scale bar
represents 5 nm-1. WAXS measurements were performed at the high-flux ChemMatCARS
beamline at the Advanced Photon Source. A bundle of threads drawn from onychophoran
slime were mounted in air, perpendicular to the beam, with WAXS patterns collected in
transmission. An optical microscope alignment system was used to confirm centred
positioning of samples in the X-ray beam. Wavelength was 0.0496 nm, the nominal sample to
detector distance was 0.58 m, and the beam was placed off-centre to the detector to give
detectable q-range (q = 4πsinθ/λ) of approximately 0.07 to 26 nm-1, as calibrated by a silver
behenate standard.
Carbohydrate-binding proteins
Also abundant with 14 copies in the cDNA library were sequences encoding cysteine-rich proteins
with high homology to several families of lectins and carbohydrate binding proteins. All sequences
obtained in full length had secretion signal peptide motifs (Er_Lec1-5). These sequences were mapped
to three distinct carbohydrate-binding families – (a) galactose lectin (pfam02140, Er_Lec1 & 2) one of
the most widespread carbohydrate binding lectin types; (b) H-type lectin (pfam09458, Er_Lec3& 4)
generally involved with self-non-self recognition of cells through binding of carbohydrate; and (3) the
fibrinogen-related domains (pfam00147, Er_Lec5) which are acetyl group recognition lectins involved
in innate immunity. Er_Lec1 was matched to a dimer at ~25 kDa obtained under non-reducing
conditions on SDS-PAGE but was disassociated when the slime sample was run under full reducing
conditions. BLASTp analysis against the NCBI protein database returned moderately low matches
between Er_Lec1-4 sequences (≤40% identity) and lectins isolated from fish skin, amphioxus and sea
anemone. The closest match was found between Er_Lec2 and a rhamnose-binding lectin from Amur
catfish (Silurus asotus, BAA87860, Hosono et al. 1999, 49% identity). tBLASTn search of Er_Lec
proteins against the translated ESTs from Roeding et al. 2007 found no homologous sequences.
Small secreted peptides
We also identified a high frequency of sequences that encoded small peptides (≤14 kDa) in the cDNA
library and all had N-terminal secretion signal peptides in the predicted peptide sequence (Er_Pep
Er_Prot; Table 1). Of these, eight were most similar to protease inhibitors: seven ESTs contained
either a single or double thyroglobulin type I repeat domain (pfam 00086; Er_ProtA) proposed to be
inhibitors of cysteine proteases, and one (Er_ProtB) had homology with BPTI/Kunitz family (pfam
00014) of serine protease inhibitors. The other 28 ESTs encoded 6 groups of small peptides (<6 kDa
for mature peptide) with no identified conserved domains. The most numerous ESTs arose from the
glycine-rich peptide group (Er_Pep1) with modest sequence identity (41%) to amphibian defensive
peptides such P05226 from Xenopus borealis and to ESTs from the onychophoran Epiperipatus sp.
(e.g. AM499786, AM499778) (Roeding et al. 2007). One EST (Er_Pep6) coding for a leucine-rich
peptide from slime was matched to the anti-microbial peptide gastrin (Equus callabus,
NP_001075979, 41% identity). The second most abundant group of peptides (Er_Pep2) were cysteinerich having six representatives in the mature peptide regularly spaced between intervals of 10 other
amino acid residues. A proline- and cysteine-rich peptide group (Er_Pep3) was also identified but
there were no significant matches under default settings to any proteins or ESTs in the Genbank
database.
proline
hydrophobic
charged/polar
80
70
60
50
Mol% 40
30
20
10
0
Crude slime HMW slime
Supplementary Fig 4.
Er_P1
Globular
Predicted amino acid character for the high molecular weight proline-rich
protein (encoded by Er_P1) compared with the measured amino acid content of crude Eu.
kanangrensis slime and that of the high molecular weight fraction (HMW, >600 kDa)
(Benkendorff et al. 1999). The average amino acid distribution for a folded protein from
the SWISS-PROT data base (data from Tompa 2002) is also given.
Benkendorff, K., Beardmore, K., Gooley, A.A., Packer, N.H. & Tait, N.N. 1999 Characterisation of
the slime gland secretion from the peripatus, Euperipatoides kanangrensis (Onychophora:
Peripatopsidae). Comp. Biochem. Physiol. B 124, 457-465.
Hosono, M., Ishikawa, K., Mineki, R., Murayama, K., Numata, C.,Ogawa, Y., Takayanagi, Y. &
Nitta,K. 1999 Tandem repeat structure of rhamnose-binding lectin from catfish (Silurus asotus)
eggs. Biochim. Biophys. Acta 1472, 668-675.
Roeding, F., Hagner-Holler, S., Ruhberg, H., Ebersberger, I., von Haeseler, A., Kube, M., Reinhardt,
R. & Burmester, T. 2007 EST sequencing of onychophora and phylogenomic analysis of Metazoa.
Mol. Phylogen. Evol. 45, 942-951.
Tompa, P. 2002 Intrinsically unstructured proteins. Trends Biochem. Sci. 275, 27-33.