3.2 Mambaquaretins form a distinct group of Kunitz-type snake
toxins
Amino acid sequences of Kunitz peptides were aligned according to MQ1
with representative sequences of dendrotoxins and three BPTI sequences
(P00974, P00975 and P04815, Fig. 3A). This alignment was used to
reconstruct the phylogenetic relationships of Kunitz-type snake toxins.
The phylogram showed that all nine MQs constitute a well-supported
monophyletic group, sister to ion-channel modulators isolated from
mambas (so-called dendrotoxins). MQs are organized into two sub-groups:
MQ1 to MQ6 are very closely-related to each other, whereas MQ7 to MQ9
are more divergent (Fig. 3B, red background). MQ8 is a natural variant
of the dendro-B (P00983, named here MQ7), a weak trypsin inhibitor
(Strydom and Joubert, 1981). Both diverge from MQ1 by 19 mutations. MQ9
is the most original sequence with 21 mutations compared to MQ1 and a
one residue shorter loop 1 (Fig. 3A). All MQs are a sister group to a
branch composed of seven dendrotoxins (Fig. 3B, blue background). The
peptides DTX-R55 (Q7LZS8) and DTX-E (P00984) have never been
characterized and are probably active on serine proteases as they
possess the dyad Lys15-Ala16. The five other dendrotoxins are potassium
and calcium channel inhibitors, which are not active on V2R up to 10 µM
(Fig. 2D).
The next group is composed of two cobra toxins (Fig. 3B, green
background). P19859 is an uncharacterized toxin from the Indian cobra
Naja naja (Shafqat et al., 1990). Q5ZPJ7 is a weak chymotrypsin
inhibitor from the Chinese cobra Naja atra (Zhou et al., 2004). The
synthetic versions of P19859 and Q5ZPJ7 (Supplementary Fig. 4) bind V2R
(Fig. 2D) and antagonize cAMP production (Fig. 2E, Table 1) with
Ki and Kinact equivalent to those of
other MQs. Injected in rats, both cobra toxins raise diuresis (2-fold
increase) with a concomitant decrease of urine osmolality, demonstrating
an aquaretic effect (Table 2). The other sequences used to build the
phylogram belong for the most part to viper or Australian snakes. Among
them, we checked the activity of five selected toxins (E7FL11, B4ESA3,
D5J9Q8, F8J2F6 and C1IC51, Supplementary Fig. 4) distributed all along
the dendrogram and having a dyad composition compatible with V2R
activity (Asn15-Gly16, Asn15-Ala16 or His15-Gly16). None of them were
active on V2R up to 10 µM (Fig. 1D).
The nine MQs constitute a new phylogenic Kunitz group associated with a
V2R activity. The comparison between the 11 V2R-active toxin sequences
and the non-active V2R Kunitz peptides highlighted 5 positions that
might play positive roles in binding to the V2R (Fig. 3A, supplementary
Fig. 5). The positions 17, 28, and 44 are strictly conserved in
V2R-active Kunitz and highly variable in non-active V2R Kunitz. The
position 34 is occupied by a threonine residue for 10 toxins and by an
asparagine for one. It is highly variable in non-active V2R Kunitz.
Finally, the position 10 is basic (Lys) or neutral (Ala, Ser) in
V2R-active Kunitz whereas it is mostly acidic in other Kunitz groups
(Fig. 3A, supplementary Fig. 5).