Conus Textile Research Articles

Characterization of a Novel α-Conotoxin from Conus textile That Selectively Targets α6/α3β2β3 Nicotinic Acetylcholine Receptors

α6β2 Nicotinic acetylcholine receptors (nAChRs) expressed by dopaminergic neurons in the CNS are potential therapeutic targets for the treatment of several neuropsychiatric diseases, including nicotine addiction and Parkinson disease. However, recent studies indicate that the α6 subunit can also associate with the β4 subunit to form α6β4 nAChRs that are difficult to pharmacologically distinguish from α6β2, α3β4, and α3β2 subtypes. The current study characterized a novel 16-amino acid α-conotoxin (α-CTx) TxIB from Conus textile whose sequence is GCCSDPPCRNKHPDLC-amide as deduced from gene cloning. The peptide and an analog with an additional C-terminal glycine were chemically synthesized and tested on rat nAChRs heterologously expressed in Xenopus laevis oocytes. α-CTx TxIB blocked α6/α3β2β3 nAChR with an IC(50) of 28 nm. In contrast, the peptide showed little or no block of other tested subtypes at concentrations up to 10 μm. The three-dimensional solution structure of α-CTx TxIB was determined using NMR spectroscopy. α-CTx TxIB represents a uniquely selective ligand for probing the structure and function of α6β2 nAChRs.

The Mitochondrial Genome of the Venomous Cone Snail Conus consors

Cone snails are venomous predatory marine neogastropods that belong to the species-rich superfamily of the Conoidea. So far, the mitochondrial genomes of two cone snail species (Conus textile and Conus borgesi) have been described, and these feed on snails and worms, respectively. Here, we report the mitochondrial genome sequence of the fish-hunting cone snail Conus consors and describe a novel putative control region (CR) which seems to be absent in the mitochondrial DNA (mtDNA) of other cone snail species. This possible CR spans about 700 base pairs (bp) and is located between the genes encoding the transfer RNA for phenylalanine (tRNA-Phe, trnF) and cytochrome c oxidase subunit III (cox3). The novel putative CR contains several sequence motifs that suggest a role in mitochondrial replication and transcription.

Secretion and maturation of conotoxins in the venom ducts of Conus textile

The 700 or more species of cone snail attack prey by employing complex venom which can vary considerably both within species and from one species to another. Cone snail venom is remarkable for the high proportion of conotoxins with varied post-translational modifications (PTMs) and for the production of more diverse toxin scaffolds than any other known venomous animal. The venom gland, which is several times longer than its shell, is also unique in being tubular. These unusual characteristics both raise questions, and provide the opportunity for research, concerning the secretion and maturation of conotoxins along the venom duct, a process which is currently not fully understood. This research uses the two mass spectrometric techniques of isotope Coded Affinity Tagging (ICAT) and label-free quantification to study each of five portions of the venom duct of Conus textile snails from New Caledonia. Fifteen conotoxins, several with different post-translational modifications (PTMs) were identified and quantified. One hundred and forty three non-identified conotoxins were also quantified. Distinctive patterns emerged, with the largest group of conotoxins increasing, then peaking in the central–proximal part, before decreasing; whilst the second largest group peaked in the distal region, generally displaying nothing in the first parts. Conotoxins from different superfamilies were commonly found to have similar distributions. A new conotoxin, PCCSKLHDNSCCGL*, was sequenced.A comparison is made with other studies to see how the process varies in cone snails from different regions.

Constrained De Novo Sequencing of Conotoxins

De novo peptide sequencing by mass spectrometry (MS) can determine the amino acid sequence of an unknown peptide without reference to a protein database. MS-based de novo sequencing assumes special importance in focused studies of families of biologically active peptides and proteins, such as hormones, toxins, and antibodies, for which amino acid sequences may be difficult to obtain through genomic methods. These protein families often exhibit sequence homology or characteristic amino acid content; yet, current de novo sequencing approaches do not take advantage of this prior knowledge and, hence, search an unnecessarily large space of possible sequences. Here, we describe an algorithm for de novo sequencing that incorporates sequence constraints into the core graph algorithm and thereby reduces the search space by many orders of magnitude. We demonstrate our algorithm in a study of cysteine-rich toxins from two cone snail species (Conus textile and Conus stercusmuscarum) and report 13 de novo and about 60 total toxins.

121. Venom Composition Changes during Prey Capture in Conus textile

121. Venom Composition Changes during Prey Capture in Conus textile

Geometric morphometric analysis of shell shape variation in Conus (Gastropoda: Conidae)

Geometric morphometric techniques allow for the direct quantification and analysis of variation in biological shape and have been used in studies in systematic biology. However, these techniques have not been used for species discrimination in the gastropod genus Conus, a major taxon of significant tropical reef predators recognized for their peptide-based toxins. Here, we used landmark digitization and analysis to show that five species commonly studied for their conotoxins –Conus consors, Conus miles, Conus stercusmuscarum, Conus striatus, and Conus textile – can be effectively distinguished from each other by their shape, as manifested in the results of a principal components analysis (PCA) and the generated thin-plate splines. Two piscivorous species, C. stercusmuscarum and C. striatus, show clear overlaps in the PCA plot, although each taxon clusters within itself, as does each of the others. The loadings on the first two principal components show that the forms of the shells' aperture and spire are particularly important for discrimination. Phylogenetic analysis using neighbour-joining methods shows that group separations are comparable with published phylogenetic schemes based on molecular data and feeding mode (i.e. piscivory, vermivory, molluscivory). The results of this study establish the utility of geometric morphometric methods in capturing the interspecific differences in shell form in the genus Conus. This may lead to the utilization of these methods on other gastropod taxa and the creation of species-recognition programs based on shell shape. © 2012 The Linnean Society of London, Zoological Journal of the Linnean Society, 2012, 165, 296–310.

Recombinant conotoxin, TxVIA, produced in yeast has insecticidal activity

Conotoxins are a diverse collection of more than 50,000 peptides produced by predatory marine snails of the genus Conus in order to immobilize their prey. Many conotoxins modulate the activity of ion channels, and show high specificity to their targets; as a result, some have valuable pharmaceutical applications. However, obtaining active peptide is difficult and to date has only been achieved though natural collection, chemical synthesis, or the use of prokaryotic expression systems, which often have the disadvantage of requiring subsequent steps to correctly fold the peptide. This paper reports the production of a conotoxin, TxVIA from Conus textile, as a biologically active recombinant protein, using the yeast Pichia pastoris as expression host. The presence of the pro-peptide was found to be necessary for the expression of biologically active conotoxin. We also show that TxVIA is not, as previously reported, mollusc-specific, but also shows insecticidal activity when injected into lepidopteran (cabbage moth) and dipteran (house fly) larvae. In contrast, recombinant TxVIA was not found to be molluscicidal to the grey field slug Deroceras reticulatum.

Role for protein kinase C in controlling Aplysia bag cell neuron excitability

Targeting signalling molecules to ion channels can expedite regulation and assure the proper transition of changes to excitability. In the bag cell neurons of Aplysia, single-channel studies of excised patches have revealed that protein kinase C (PKC) gates a non-selective cation channel through a close, physical association. This channel drives a prolonged afterdischarge and concomitant neuropeptide secretion to provoke reproductive behaviour. However, it is not clear if PKC alters cation channel function and/or the membrane potential at the whole-cell level. Afterdischarge-like depolarizations can be evoked in cultured bag cell neurons by bath-application of Conus textile venom (CtVm), which triggers the cation channel through an apparent intracellular pathway. The present study shows that the CtVm-induced depolarization was reduced by nearly 50% compared to control following dialysis with the G-protein blocker, guanosine-5′-O-2-thiodiphosphate (GDP-β-S), or treatment with either the phospholipase C inhibitor, 1-[6-[[(17β)-3-Methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-1H-pyrrole-2,5-dione (U-73122), or the PKC inhibitor, sphinganine. Neurons exposed to the PKC activator, phorbol 12-myristate 13-acetate (PMA), displayed depolarization with accompanying spiking, and were found to be far more responsive to depolarizing current injection versus control. Immunocytochemical staining for the two typical Aplysia PKC isoforms, Apl I and Apl II, revealed that both kinases were present in unstimulated cultured bag cell neurons. However, in CtVm-treated neurons, the staining intensity for PKC Apl I increased, peaking at 10 min post-application. Conversely, the intensity of PKC Apl II staining decreased over the duration of CtVm exposure. Our results suggest that the CtVm-induced depolarization involves PKC activation, and is consistent with prior work showing PKC closely-associating with the cation channel to produce the depolarization necessary for the afterdischarge and species propagation.

Rapid sensitive analysis of cysteine rich peptide venom components

Disulfide-rich peptide venoms from animals such as snakes, spiders, scorpions, and certain marine snails represent one of nature's great diversity libraries of bioactive molecules. The various species of marine cone shells have alone been estimated to produce >50,000 distinct peptide venoms. These peptides have stimulated considerable interest because of their ability to potently alter the function of specific ion channels. To date, only a small fraction of this immense resource has been characterized because of the difficulty in elucidating their primary structures, which range in size between 10 and 80 aa, include up to 5 disulfide bonds, and can contain extensive posttranslational modifications. The extraordinary complexity of crude venoms and the lack of DNA databases for many of the organisms of interest present major analytical challenges. Here, we describe a strategy that uses mass spectrometry for the elucidation of the mature peptide toxin components of crude venom samples. Key to this strategy is our use of electron transfer dissociation (ETD), a mass spectrometric fragmentation technique that can produce sequence information across the entire peptide backbone. However, because ETD only yields comprehensive sequence coverage when the charge state of the precursor peptide ion is sufficiently high and the m/z ratio is low, we combined ETD with a targeted chemical derivatization strategy to increase the charge state of cysteine-containing peptide toxins. Using this strategy, we obtained full sequences for 31 peptide toxins, using just 7% of the crude venom from the venom gland of a single cone snail (Conus textile).

Remarkable inter- and intra-species complexity of conotoxins revealed by LC/MS

Cone snails have evolved an assortment of venom peptides as an evolutionary strategy for rapid prey immobilization and defence. Earlier studies estimated ∼100 conopeptides per species. In this study we optimized liquid chromatography and electrospray ionization mass spectrometry for the detection of conopeptides in crude venom to characterize conopeptides present in the venom of individual specimens of Conus textile, C. imperialis and C. marmoreus. Using this approach, we have expanded the predicted number of venom peptides 10-fold to an estimate of 1000–1900 conopeptides per species. Our investigation has also revealed a surprisingly high level of intra-species variation that distinguishes cone snails from other venomous species including spiders and scorpions. Given this inherent diversity and variability, more sensitive bioassays and sequencing techniques will be required to fully explore conotoxin bioactivity.

Cloning and isolation of a conus cysteine-rich protein homologous to Tex31 but without proteolytic activity

We cloned and isolated a cysteine-rich protein, designated Mr30, from Conus marmoreus. Mr30 belongs to the cysteine-rich secretory protein family that is highly homologous to Tex31 previously obtained from Conus textile and reported as a protease responsible for processing of pro-conotoxins. Mr30, purified by a procedure similar to that of Tex31, indeed showed low proteolytic activity. However, further investigations revealed that the detected protease activity actually resulted from a trace amount of protease(s) contamination rather than from Mr30 itself. This finding led us to rethink the role of conus cysteine-rich secretory proteins: they were probably not responsible for the processing of pro-conotoxins as previously deduced, but their real biological functions remained to be clarified.

Three-Dimensional Structure of Conotoxin tx3a: An m-1 Branch Peptide of the M-Superfamily

The M-superfamily, one of eight major conotoxin superfamilies found in the venom of the cone snail, contains a Cys framework with disulfide-linked loops labeled 1, 2, and 3 (-CC (1) C (2) C (3) CC-). M-Superfamily conotoxins can be divided into the m-1, -2, -3, and -4 branches, based upon the number of residues located in the third Cys loop between the fourth and fifth Cys residues. Here we provide a three-dimensional solution structure for the m-1 conotoxin tx3a found in the venom of Conus textile. The 15-amino acid peptide, CCSWDVCDHPSCTCC, has disulfide bonds between Cys (1) and Cys (14), Cys (2) and Cys (12), and Cys (7) and Cys (15) typical of the C1-C5, C2-C4, and C3-C6 connectivity pattern seen in m-1 branch peptides. The tertiary structure of tx3a was determined by two-dimensional (1)H NMR in combination with the combined assignment and dynamics algorithm for nuclear magnetic resonance (NMR) applications CYANA program. Input for structure calculations consisted of 62 inter- and intraproton, five phi angle, and four hydrogen bond constraints. The root-mean-square deviation values for the 20 final structures are 0.32 +/- 0.07 and 0.84 +/- 0.11 A for the backbone and heavy atoms, respectively. Surprisingly, the structure of tx3a has a "triple-turn" motif seen in the m-2 branch conotoxin mr3a, which is absent in mr3e, the only other member of the m-1 branch of the M-superfamily whose structure is known. Interestingly, injection of tx3a into mice elicits an excitatory response similar to that of the m-2 branch peptide mr3a, even though the conotoxins have different disulfide connectivity patterns.

The mitochondrial genome of Conus textile, coxI– coxII intergenic sequences and Conoidean evolution

The cone snails belong to the superfamily Conoidea, comprising ∼10,000 venomous marine gastropods. We determined the complete mitochondrial DNA sequence of Conus textile. The gene order is identical in Conus textile, Lophiotoma cerithiformis (another Conoidean gastropod), and the neogastropod Ilyanassa obsoleta, (not in the superfamily Conoidea). However, the intergenic interval between the coxI and coxII genes was much longer in C. textile (165 bp) than in any other previously analyzed gastropod. We used the intergenic region to evaluate evolutionary patterns. In most neogastropods and three conidean families the intergenic interval is small (<30 nucleotides). Within Conus, the variation is from 130 to 170 bp, and each different clade within Conus has a narrower size distribution. In Conasprella, a subgenus traditionally assigned to Conus, the intergenic regions vary between 200 and 500 bp, suggesting that the species in Conasprella are not congeneric with Conus. The intergenic region was used for phylogenetic analysis of a group of fish-hunting Conus, despite the short length resolution was better than using standard markers. Thus, the coxI–coxII intergenic region can be used both to define evolutionary relationships between species in a clade, and to understand broad evolutionary patterns across the large superfamily Conoidea.

AChBP-targeted α-conotoxin correlates distinct binding orientations with nAChR subtype selectivity

Neuronal nAChRs are a diverse family of pentameric ion channels with wide distribution throughout cells of the nervous and immune systems. However, the role of specific subtypes in normal and pathological states remains poorly understood due to the lack of selective probes. Here, we used a binding assay based on acetylcholine-binding protein (AChBP), a homolog of the nicotinic acetylcholine ligand-binding domain, to discover a novel α-conotoxin (α-TxIA) in the venom of Conus textile. α-TxIA bound with high affinity to AChBPs from different species and selectively targeted the α3β2 nAChR subtype. A co-crystal structure of Ac-AChBP with the enhanced potency analog TxIA(A10L), revealed a 20° backbone tilt compared to other AChBP–conotoxin complexes. This reorientation was coordinated by a key salt bridge formed between Arg5 (TxIA) and Asp195 (Ac-AChBP). Mutagenesis studies, biochemical assays and electrophysiological recordings directly correlated the interactions observed in the co-crystal structure to binding affinity at AChBP and different nAChR subtypes. Together, these results establish a new pharmacophore for the design of novel subtype-selective ligands with therapeutic potential in nAChR-related diseases.

An O-conotoxin from the vermivorous Conus spurius active on mice and mollusks

Here, we report the purification, amino acid sequence and a preliminary biological characterization of a peptide, sr7a, from the venom of Conus spurius, a vermivorous species collected in the Yucatan Channel, Mexico. The peptide consists of 32 amino acid residues (CLQFGSTCFLGDDDICCSGECFYSGGTFGICS&; &, amidated C-terminus) and contains six cysteines arranged in the pattern (C–C–CC–C–C) that characterizes the O-superfamily of conotoxins. This superfamily includes several pharmacological families (ω-, κ-, μO-, δ- and γ-conotoxins) that target Ca 2+, K +, Na + and pacemaker voltage-gated ion channels. Compared with other O-conotoxins that were purified from venoms, this peptide displays sequence similarity with ω-SVIA (from Conus striatus), δ-TxVIA/B (from Conus textile), ω-CVID (from Conus catus) and κ-PVIIA (from Conus purpurascens). At a dose of 250 pmol, peptide sr7a elicited hyperactivity when injected intracranially into mice and produced paralysis when injected into the pedal muscle of freshwater snails, Pomacea paludosa, but it had no apparent effect after intramuscular injection into the limpet Patella opea or the freshwater fish Lebistes reticulatus.

Characterization of novel M‐superfamily conotoxins with new disulfide linkage

The M-superfamily with the typical Cys framework (-CC-C-C-CC-) is one of the seven major superfamilies of conotoxins found in the venom of cone snails. Based on the number of residues in the last Cys loop (between C4 and C5), M-superfamily conotoxins can be provisionally categorized into four branches (M-1, M-2, M-3, M-4) [Corpuz GP, Jacobsen RB, Jimenez EC, Watkins M, Walker C, Colledge C, Garrett JE, McDougal O, Li W, Gray WR, et al. (2005) Biochemistry44, 8176-8186]. Here we report the purification of seven M-superfamily conotoxins from Conus marmoreus (five are novel and two are known as mr3a and mr3b) and one known M-1 toxin tx3a from Conus textile. In addition, six novel cDNA sequences of M-superfamily conotoxins have been identified from C. marmoreus, Conus leopardus and Conus quercinus. Most of the above novel conotoxins belong to M-1 and M-2 and only one to M-3. The disulfide analyses of two M-1 conotoxins, mr3e and tx3a, revealed that they possess a new disulfide bond arrangement (C1-C5, C2-C4, C3-C6) which is different from those of the M-4 branch (C1-C4, C2-C5, C3-C6) and M-2 branch (C1-C6, C2-C4, C3-C5). This newly characterized disulfide connectivity was confirmed by comparing the HPLC profiles of native mr3e and its two regioselectively folded isoforms. This is the first report of three different patterns of disulfide connectivity in conotoxins with the same cysteine framework.

Novel α‐conotoxins identified by gene sequencing from cone snails native to Hainan, and their sequence diversity

Conotoxins (CTX) from the venom of marine cone snails (genus Conus) represent large families of proteins, which show a similar precursor organization with surprisingly conserved signal sequence of the precursor peptides, but highly diverse pharmacological activities. By using the conserved sequences found within the genes that encode the alpha-conotoxin precursors, a technique based on RT-PCR was used to identify, respectively, two novel peptides (LiC22, LeD2) from the two worm-hunting Conus species Conus lividus, and Conus litteratus, and one novel peptide (TeA21) from the snail-hunting Conus species Conus textile, all native to Hainan in China. The three peptides share an alpha4/7 subfamily alpha-conotoxins common cysteine pattern (CCX(4)CX(7)C, two disulfide bonds), which are competitive antagonists of nicotinic acetylcholine receptor (nAChRs). The cDNA of LiC22N encodes a precursor of 40 residues, including a propeptide of 19 residues and a mature peptide of 21 residues. The cDNA of LeD2N encodes a precursor of 41 residues, including a propeptide of 21 residues and a mature peptide of 16 residues with three additional Gly residues. The cDNA of TeA21N encodes a precursor of 38 residues, including a propeptide of 20 residues and a mature peptide of 17 residues with an additional residue Gly. The additional residue Gly of LeD2N and TeA21N is a prerequisite for the amidation of the preceding C-terminal Cys. All three sequences are processed at the common signal site -X-Arg- immediately before the mature peptide sequences. The properties of the alpha4/7 conotoxins known so far were discussed in detail. Phylogenetic analysis of the new conotoxins in the present study and the published homologue of alpha4/7 conotoxins from the other Conus species were performed systematically. Patterns of sequence divergence for the three regions of signal, proregion, and mature peptides, both nucleotide acids and residue substitutions in DNA and peptide levels, as well as Cys codon usage were analyzed, which suggest how these separate branches originated. Percent identities of the DNA and amino acid sequences of the signal region exhibited high conservation, whereas the sequences of the mature peptides ranged from almost identical to highly divergent between inter- and intra-species. Notably, the diversity of the proregion was also high, with an intermediate percentage of divergence between that observed in the signal and in the toxin regions. The data presented are new and are of importance, and should attract the interest of researchers in this field. The elucidated cDNAs of these toxins will facilitate a better understanding of the relationship of their structure and function, as well as the process of their evolutionary relationships.

Errata

The FEBS JournalVolume 273, Issue 13 p. 3118-3118 Free Access Errata This article corrects the following: Novel γ-carboxyglutamic acid-containing peptides from the venom of Conus textile Eva Czerwiec, Dario E. Kalume, Peter Roepstorff, Björn Hambe, Bruce Furie, Barbara C. Furie, Johan Stenflo, Volume 273Issue 12The FEBS Journal pages: 2779-2788 First Published online: May 26, 2006 First published: 15 June 2006 https://doi.org/10.1111/j.1742-4658.2006.05355_1.xCitations: 1AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat The paper by Czerwiec et al.[1] contains an error on page 2780. Table 1 should appear as follows: Table 1. Amino acid sequences of conopeptides Gla(1)–TxVI, Gla(2)–TxVI/A, Gla(2)–TxVI/B and Gla(3)–TxVI a obtained by combined Edman degradation and mass spectrometry analysis. Post-translational modifications are highlighted in bold. W, BrTrp; γ, Gla; O, Hyp; #, amidated C-terminus. Name Sequence a Position + 7 in Gla(3)–TxVI displays a microheterogeneity with Gla, Glu and Asp occurring in a ratio of 1 : 1 : 2, respectively (see also supplementary Table S1). The publisher apologises for this error. References 1 Czerwiec E, Kalume DE, Roepstorff P, Hambe B, Furie B, Furie BC and Stenflo J (2006) Novel γ-carboxyglutamic acid-containing peptides from the venom of Conus textile. FEBS J 273, 2779– 2788. Citing Literature Volume273, Issue13July 2006Pages 3118-3118 ReferencesRelatedInformation

Direct cDNA cloning of novel conotoxins of the T-superfamily from Conus textile

The T-superfamily is a large and diverse group of peptides, widely distributed in venom ducts of all major feeding types of Conus. These peptides are likely to be functionally diverse. A directed PCR-based approach using primers based on the conserved signal sequence was applied to investigate new conotoxins of the T-superfamily from Conus textile native to Hainan. Using RT-PCR and 3′-RACE, four novel cDNA sequences encoding precursor peptides were identified in C. textile. They share a common T-superfamily cysteine pattern (CC–CC, with two disulfide bridges). The predicted peptides are small (9–12 amino acids). TeAr193 composed of nine amino acid residues is one of the shortest T-superfamily conotoxins ever found. Patterns of sequence divergence and Cys codon usage define the major T-superfamily branches and suggest how these separate branches arose. The sequences of the signal regions exhibited highest conservation, whereas the sequences of the mature peptides were either almost identical or highly divergent; and conservation of the pro-region was intermediate between that observed in signal and toxin regions. The elucidated cDNAs of the four toxins will facilitate a better understanding of the relationship between structure and function.

Novel γ‐carboxyglutamic acid‐containing peptides from the venom of Conus textile

The cone snail is the only invertebrate system in which the vitamin K-dependent carboxylase (or gamma-carboxylase) and its product gamma-carboxyglutamic acid (Gla) have been identified. It remains the sole source of structural information of invertebrate gamma-carboxylase substrates. Four novel Gla-containing peptides were purified from the venom of Conus textile and characterized using biochemical methods and mass spectrometry. The peptides Gla(1)-TxVI, Gla(2)-TxVI/A, Gla(2)-TxVI/B and Gla(3)-TxVI each have six Cys residues and belong to the O-superfamily of conotoxins. All four conopeptides contain 4-trans-hydroxyproline and the unusual amino acid 6-l-bromotryptophan. Gla(2)-TxVI/A and Gla(2)-TxVI/B are isoforms with an amidated C-terminus that differ at positions +1 and +13. Three isoforms of Gla(3)-TxVI were observed that differ at position +7: Gla(3)-TxVI, Glu7-Gla(3)-TxVI and Asp7-Gla(3)-TxVI. The cDNAs encoding the precursors of the four peptides were cloned. The predicted signal sequences (amino acids -46 to -27) were nearly identical and highly hydrophobic. The predicted propeptide region (-20 to -1) that contains the gamma-carboxylation recognition site (gamma-CRS) is very similar in Gla(2)-TxVI/A, Gla(2)-TxVI/B and Gla(3)-TxVI, but is more divergent for Gla(1)-TxVI. Kinetic studies utilizing the Conusgamma-carboxylase and synthetic peptide substrates localized the gamma-CRS of Gla(1)-TxVI to the region -14 to -1 of the polypeptide precursor: the Km was reduced from 1.8 mm for Gla (1)-TxVI lacking a propeptide to 24 microm when a 14-residue propeptide was attached to the substrate. Similarly, addition of an 18-residue propeptide to Gla(2)-TxVI/B reduced the Km value tenfold.