THE INDO-PACIFIC GEMMULA SPECIES IN THE SUBFAMILY TURRINAE: ASPECTS OF FIELD DISTRIBUTION, MOLECULAR PHYLOGENY, RADULAR ANATOMY AND FEEDING ECOLOGY.

The biology, feeding ecology and phylogenetic relationships of marine snails in the family Turridae remain poorly understood. Here we report our study on four deep-water species in the genus Gemmula, a major group in this family. The four species G. speciosa (Reeve 1843), G. sogodensis (Olivera 2005), G. kieneri (Doumet 1940) and G. diomedea (Powell 1964) were collected at five different sites in the Philippines, and their pattern of distribution in the sites, their feeding behaviour as well as their phylogenetic relationships with each other and with other members of the subfamily Turrinae were investigated. The radular morphology (of two Gemmula species) and potential prey (for one Gemmula species) were also examined. Actual feeding observations were also conducted for Gemmula speciosa and compared with two turrids from other genera.All four Gemmula species showed strikingly different patterns of distribution; each species was found to be relatively much more abundant at one site but not at the other sites. Molecular phylogenetic analysis based on 16S sequences correlated with previously reported 12S sequences and revealed that the four species all belong to a well-supported Gemmula clade within the subfamily Turrinae; and that this clade appeared more closely related to the clades Xenuroturris, Turris and Lophiotoma than to the other clades in the subfamily (i.e., Turridrupa, Unedogemmula and Polystira). Morphological analysis of the radula of both G. speciosa and G. sogodensis revealed that the radulae of the two species were similar but differed from the other turrids, Lophiotoma acuta and Unedogemmula bisaya, by the absence of central teeth, consistent with the separation of the Gemmula clade from the Lophiotoma and Unedogemmula clade.To identify the polychaete group that is targeted as prey by species of Gemmula, analysis of regurgitated food fragments was made; phylogenetic analysis of an mtCOI gene fragment that was PCR-amplified from the regurgitated tissue of one specimen (G. diomedea) indicated close affinity of the prey to the terebellid polychaete Amphitritides. Specimens of Gemmula speciosa, when challenged with the terebellid polychaete Loimia sp., were observed to attack the worm suggesting that Gemmula species feed on terebellid polychaetes. Lophiotoma acuta were also observed to feed on the same species of terebellid but were usually group-feeding in contrast to the solitary feeding of G. speciosa. Unedogemmula bisaya did not feed on the terebellid which also supports the separation of the Gemmula and Unedogemmula clade.Two lines of proof (i.e. the molecular phylogenetic analysis and the feeding challenge) supporting the toxin homology findings previously reported, provide consistent evidence that Gemmula is a distinct clade of worm-hunting Turrinae that feeds on Terebellidae.

A rapidly diverging superfamily of peptide toxins in venomous Gemmula species.

The gem turrids (genus Gemmula Weinkauff, 1875) are venomous snails in the family Turridae. A gene superfamily of disulfide-rich peptides expressed in Gemmula venom ducts was characterized. Gemmula speciosa (Reeve, 1843) venom duct cDNA clones revealed two different conotoxin-like prepropeptide precursors, with identical signal sequences, a largely conserved pro region, and a cysteine-rich C-terminal mature peptide region. The conserved signal sequence was used to successfully amplify homologous genes from three other Gemmula species; all had the same pattern of Cys residues in the predicted mature venom peptide. Although the signal sequence and propeptide regions were highly conserved, the mature toxin regions diverged greatly in sequence, except that the Cys residues were conserved. We designate this as the Pg-gene superfamily (Pg-superfamily) of Gemmula venom peptides. Purification of two members of the family directly from G. speciosa venom was achieved; amino acid sequence analysis revealed that these peptides are highly posttranslationally modified. With at least 10-fold as many species of turrids as cone snails, identification of rapidly diversifying gene superfamilies such as the Pg-superfamily of Gemmula is essential before the facile and systematic discovery and characterization of peptide toxins from turrid venoms can be achieved.

The A-superfamily of conotoxins: structural and functional divergence.

The generation of functional novelty in proteins encoded by a gene superfamily is seldom well documented. In this report, we define the A-conotoxin superfamily, which is widely expressed in venoms of the predatory cone snails (Conus), and show how gene products that diverge considerably in structure and function have arisen within the same superfamily. A cDNA clone encoding alpha-conotoxin GI, the first conotoxin characterized, provided initial data that identified the A-superfamily. Conotoxin precursors in the A-superfamily were identified from six Conus species: most (11/16) encoded alpha-conotoxins, but some (5/16) belong to a family of excitatory peptides, the kappaA-conotoxins that target voltage-gated ion channels. alpha-Conotoxins are two-disulfide-bridged nicotinic antagonists, 13-19 amino acids in length; kappaA-conotoxins are larger (31-36 amino acids) with three disulfide bridges. Purification and biochemical characterization of one peptide, kappaA-conotoxin MIVA is reported; five of the other predicted conotoxins were previously venom-purified. A comparative analysis of conotoxins purified from venom, and their precursors reveal novel post-translational processing, as well as mutational events leading to polymorphism. Patterns of sequence divergence and Cys codon usage define the major superfamily branches and suggest how these separate branches arose.

Grafting of "abbreviated" complementarity-determining regions containing specificity-determining residues essential for ligand contact to engineer a less immunogenic humanized monoclonal antibody.

Murine mAb COL-1 reacts with carcinoembryonic Ag (CEA), expressed on a wide range of human carcinomas. In preclinical studies in animals and clinical trials in patients, murine COL-1 showed excellent tumor localization. To circumvent the problem of immunogenicity of the murine Ab in patients, a humanized COL-1 (HuCOL-1) was generated by grafting the complementarity-determining regions (CDRs) of COL-1 onto the frameworks of the variable light and variable heavy regions of human mAbs. To minimize anti-V region responses, a variant of HuCOL-1 was generated by grafting onto the human frameworks only the "abbreviated" CDRs, the stretches of CDR residues that contain the specificity-determining residues that are essential for the surface complementarity of the Ab and its ligand. In competition RIAs, the recombinant variant completely inhibited the binding of radiolabeled murine and humanized COL-1 to CEA. The HuCOL-1 and its variant showed no difference in their binding ability to the CEA expressed on the surface of a CEA-transduced tumor cell line. Compared with HuCOL-1, the HuCOL-1 variant showed lower reactivity to patients' sera carrying anti-V region Abs to COL-1. The final variant of the HuCOL-1, which retains its Ag-binding reactivity and shows significantly lower serum reactivity than that of the parental Ab, can serve as a prototype for the development of a potentially useful clinical reagent.