FEBS Letters 580 (2006) 1587–1591
Mammalian L -to-D -amino-acid-residue isomerase from platypus venom Allan M. Torresa, Maria Tsampazia, Chryssanthi Tsampazia, Eleanor C. Kennetta, Katherine Belovb, Dominic P. Geraghtyc, Paramjit S. Bansald, Paul F. Alewoodd, Philip W. Kuchela,* a
School of Molecular and Microbial Biosciences, University of Sydney, Building G08, Sydney, NSW 2006, Australia b Faculty of Veterinary Science, University of Sydney, Camden, NSW 2570, Australia c School of Human Life Sciences, University of Tasmania, Launceston, Tasmania 7250, Australia d Institute for Molecular Bioscience, University of Queensland, Brisbane, Qld 4072, Australia Received 7 December 2005; revised 20 January 2006; accepted 24 January 2006 Available online 3 February 2006 Edited by Maurice Montal
Abstract The presence of D -amino-acid-containing polypeptides, defensin-like peptide (DLP)-2 and Ornithorhyncus venom C-type natriuretic peptide (OvCNP)b, in platypus venom suggested the existence of a mammalian D -amino-acid-residue isomerase(s) responsible for the modification of the all-L -amino acid precursors. We show here that this enzyme(s) is present in the venom gland extract and is responsible for the creation of DLP-2 from DLP-4 and OvCNPb from OvCNPa. The isomerisation reaction is freely reversible and under well defined laboratory conditions catalyses the interconversion of the DLPs to full equilibration. The isomerase is 50–60 kDa and is inhibited by methanol and the peptidase inhibitor amastatin. This is the first known L -to-D -amino-acid-residue isomerase in a mammal. 2006 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. Keywords: DLP; Peptide isomerase; Platypus venom peptide
1. Introduction The Australian duck-bill platypus, Ornithorhynchus anatinus, is unique among mammals as the adult male has pelvic venom (crural) glands, and a venomous spur on each hind limb [1]. The venomous spurs are used as offensive and defensive weapons and the toxins cause excruciating pain to victims [2]. Platypus venom contains many non-protein and protein components (see Fig. 1) whose roles are yet to be established. Among these is a group of four polypeptides of 5 kDa referred to as defensin-like peptides (DLPs) [3–5], whose tertiary structures resemble that of b-defensins, but they have not thus far shown anti-microbial properties. Natriuretic peptides, Ornithorhyncus venom C-type natriuretic peptide (OvCNPa and OvCNPb), are better understood, as in rats they cause smooth muscle relaxation and mast cell histamine release. The natriuretic peptides are the most physiologically active components discovered in the venom to date [6–10]. We previously discovered that OvCNPb and DLP-2 each contain a D -amino acid residue at position 2, while their coun* Corresponding author. Fax: +61 2 951 4726. E-mail address:
[email protected] (P.W. Kuchel).
Abbreviations: DLP, defensin-like peptide; MW, molecular weight; OvCNP, Ornithorhyncus venom C-type natriuretic peptide; RP-HPLC, reverse-phase HPLC
terparts OvCNPa and DLP-4 have all L -forms [11,12]. The occurrence of biologically active D -amino-acid-containingpeptides in higher organisms is rare but has been reported in frogs [13–15], molluscs [16–19], spiders [20,21], and crustaceans [22–24]. D -amino-acid-containing peptides are more stable in tissues as they are less susceptible to protease degradation. Some of these unusual peptides are more potent than the regular all L -forms of the peptides [15,16,20]. They most certainly are created post-translationally from regular peptides via a special enzyme referred to as peptidylaminoacyl-L /-D -isomerase [25,26] or simply, here, isomerase. In our recent paper on DLP structure, we showed that the platypus venom gland extract has isomerase activity that converts DLP-4 to DLP-2 [12]. Therefore, we set out to characterize the enzyme responsible for this activity and to ultimately determine whether orthologous isomerases and hence D -amino-acid-containing peptides are found in other mammals, including humans. In this study, we introduce the isomerase in platypus venom and describe some of its biochemical and biophysical properties. To investigate the specificity of this enzyme, two 12-residue peptide analogues that incorporate the first seven residues of DLP-2 and DLP-4 were chemically synthesized and tested for interconversion. We also show that the platypus venom gland extract contains isomerase activity that interconverts OvCNPa and OvCNPb, suggesting a possible broad-range substrate-specificity isomerase that acts on both DLPs and OvCNPs.
2. Materials and methods 2.1. Preparation of venom-gland extracts, DLP, OvCNP, and DLP analogue samples The venom glands used were obtained from adult male platypuses in Tasmania, that had been killed accidentally on roads. Details of the preparation of the venom-gland extract are described in Torres et al. [12]. Native DLP-2, DLP-4, OvCNPa and OvCNPb were isolated from the venom gland extracts by reverse-phase HPLC (RP-HPLC). Synthetic versions of these peptides, including the two DLP analogues, were produced manually using 2-(1-H-benzotriazol-1-yl-)1,1,3,3 tetramethyluronium hexafluorophosphate activation of Boc-amino acids with in situ neutralization chemistry, as previously described [11,27]. 2.2. Assay of L -to-D -amino-acid-residue isomerase An Amicon Ultrafree-MC with 30 kDa nominal molecular weight (MW) cut-off was used to fractionate the venom-gland extract. The isomerase-active retentate that contained components with nominal MW > 30 k was washed twice by reconstituting it with two volumes
0014-5793/$32.00 2006 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.febslet.2006.01.089
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of conversion) and found that the relative peak intensities of the two DLPs became equalized after 1 h of incubation and then remained constant (see Fig. 2A), suggesting that the conversion was reversible and went to full equilibration. The reversibility of the conversion was tested by using DLP-2 as a substrate instead of DLP-4. As shown in Fig. 2B, the peak corresponding to DLP-4 appeared after 1 h and remained relatively constant after 4 h of incubation. This confirmed that the isomerization process was indeed reversible and that the enzyme interconverts DLP-2 and DLP-4.
A
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LLHDHPNPRK YPKANKKGLS KGCFGLKLDR IGSTSGLGC
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IMFFEMQSRS RS
Fig. 1. RP-HPLC of platypus venom gland extract and primary structures of peptides acted upon by the isomerase. (A) RP-HPLC of 50 lL platypus venom gland extract. The components were eluted with a gradient of 20–45% acetonitrile containing 0.1% (v/v) TFA at 1.0 mL min 1, for 40 min. (B) Primary structures of DLP-2/DLP-4, OvCNPa/ OvCNPb, and synthetic DLP-2A/DLP-4A. The second amino-acid residues shown in bold are in the L -form in DLP-4, OvCNPa, and DLP-4A but are in the D -form in DLP-2, OvCNPb, DLP-2A. The disulfide-bonding patterns are shown by lines below the sequences.
of 50 mM phosphate containing 0.9% (w/v) NaCl (phosphate buffered saline, PBS) at pH 7.4 and repeating the filtration. Finally, the retentate was reconstituted to the original volume with 50 mM PBS. A typical incubation mixture consisted of 20 lL of fractionated venom gland extract, 20 lL of 50 mM PBS, 10 lL of peptide (0.3 mM) and 6 lL of 40 mM EDTA. This mixture was incubated either at 32 C (the average body temperature of the platypus) or 37 C in a water bath, for 24 h and sampled at specified times to monitor the conversion to the other form by RP-HPLC. 2.3. Chromatography A Phenomenex Synergi 4 l Hydro-RP analytical column (250 mm · 4.60 mm) was used in RP-HPLC. The solvent system consisted of 0.1% (v/v) trifluoroacetic acid in water (Buffer A) and 0.1% (v/v) trifluoroacetic acid in acetonitrile (Buffer B). Samples with volumes ranging from 5 to 200 lL were applied at a flow rate of 1 mL min 1, and separated components were detected at light wavelengths of 215 or 280 nm. The elution gradients for DLP, OvCNP, and DLP analogues were 28–35% B for 11 min, 25–31% B for 10 min, and 5– 60% B for 15 min, respectively. Size-exclusion chromatography was performed using a Pharmacia Superdex 75 HR 10/30 column equilibrated with buffer containing 50 mM PBS at pH 7.4. Gland extracts with typical volumes ranging from 50 to 200 lL were eluted at 500 lL min 1 flow rate and were monitored at light wavelengths of both 215 and 280 nm.
3. Results 3.1. Interconversion of DLP-4 and DLP-2 In our previous study [12], the isomerase activity was confirmed when DLP-2 was observed after a 4 h incubation at 33 C of a mixture containing DLP-4 and fractionated gland extract containing proteins with MW > 30 k. In this study, we performed the experiment at 37 C (to increase the rate
3.2. Interconversion of DLP-2 and DLP-4 analogues Two 12-residue DLP analogues, DLP-4A and DLP-2A, were assayed to determine if smaller peptides are acted upon by enzyme in the fractionated gland extract. These two peptides are identical in their first seven residues with DLP-2 and DLP-4, and are followed by Ser-Arg-Ser-Arg-Ser to make them soluble in aqueous solution. As shown in Figs. 2C and D, these analogues were converted to the other form in 1 h (at the same enzyme concentration as used previously) and conversion reached full equilibration in 3–4 h. 3.3. Interconversion of OvCNPa and OvCNPb An RP-HPLC peak corresponding to OvCNPb was clearly seen after 2 h of incubation of fractionated venom gland extract (MW > 30 k) and OvCNPa (see Fig. 2E), and its intensity increased up to 24 h. Thus, an active isomerase with an MW > 30 k converted OvCNPa to OvCNPb in the venom gland extract. The dramatic decrease in OvCNPa between 2 and 24 h may be attributed to degradation possibly due to protease activity and/or by other means, such as aggregation, as we found that OvCNPa by itself was not very stable in solution. The conversion of OvCNPa to OvCNPb was reversible, as OvCNPa was formed when OvCNPb was incubated using a similar >30 kDa gland extract (see Fig. 2F). The conversion of OvCNPs was much slower than that of the DLPs for a given amount of extract. 3.4. Inhibition of DLP isomerase with amastatin and methanol Amastatin has limited solubility in water so it was dissolved in methanol prior to addition to an incubation mixture containing DLP-2. This meant that methanol would always be present in samples containing amastatin hence two control experiments, one with and one without methanol, were required. Fig. 3 shows RP-HPLC chromatograms of three samples after 3 h of incubation at 37 C. The sample with 0.23 mM amastatin and methanol showed complete inhibition, while partial inhibition was observed for the sample containing methanol alone. 3.5. Size-exclusion chromatography Figs. 4A and B show the size-exclusion chromatogram and SDS–PAGE gel of the venom gland extract. Five obvious bands are evident in the gel (Fig. 4B); a major protein with molecular size 57 kDa exists with four minor proteins of 4, 67, 80, and 110 kDa. Size-exclusion chromatography using Superdex 75 detected at a light wavelength of 215 nm (Fig. 4A) clearly showed a large major peak corresponding to a molecular size of 57 kDa, a smaller one corresponding to 110 kDa, while the 80 and 67 kDa peaks appeared as a shoulder between the other two peaks.
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A
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Fig. 2. Isomerization of DLPs and OvCNPs. RP-HPLC traces of incubation mixtures containing the specified peptides sampled at various incubation times at 37 C. The peptides used were: (A) DLP-4; (B) DLP-2; (C) DLP-4A; (D) DLP-2A; (E) OvCNPa and; (F) OvCNPb. To decrease protease degradation of OvCNPs during long incubation times, the sample in (E) was incubated at 37 C for the first 5 h and at 32 C thereafter.
DLP-4
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isomerase activity was measured in fractions 2 and 4, which all contained proteins at or near 57 kDa, respectively; while fractions 1, and 5 onwards were devoid of any activity. These results imply that the active component has a size between 50– 80 kDa. The size was narrowed down even further by noting that all active fractions contained the 57 kDa protein, and that the 66 and 80 kDa proteins were most abundant in fraction 2 and were less abundant (if even present) in fraction 4. Therefore, the DLP isomerase was concluded to have a size closer to 57 than to 66 kDa.
Methanol
4. Discussion
Methanol + amastatin 8.0
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Elution Time (min) Fig. 3. Amastatin and methanol inhibition of the DLP isomerase. RPHPLC traces of an incubation mixture after 3 h at 37 C. All samples contained the same amounts of DLP-2, >30 kDa fraction of venom gland extract, and 40 mM EDTA.
Figs. 4C–E present the size-exclusion profiles and relative isomerase activities of the different 1.5 mL fractions of a venom gland extract. As shown, no isomerase activity was observed for fraction 1 which contained most of the 110-kDa fraction. Maximum isomerase activity was located in fraction 3 (f3) from the Superdex 75 column. The fact that this fraction contained most of the abundant 57 kDa component suggested that the DLP isomerase has a size at or near 57 kDa. Lower
We have shown here that platypus venom gland extracts contain an isomerase(s) that interconverts the second residue in DLP-4/-2 and OvCNPa/b between the L -form and D -form. This platypus isomerase is the first such enzyme to be discovered in a mammal. Its presence opens up the possibility that other D -amino-acid-containing peptides and their respective isomerase(s) may be present in higher organisms, including humans. The functions of these D -amino-acid-containing peptides in platypus venom are not clear since the true roles of the DLPs and OvCNPs in venom action are yet to be established. However, these unusual peptides are likely to be physiologically important in the venom as the DLPs are the most abundant polypeptides in the venom [3], while OvCNPs had been shown to possess potent antihypertensive activity [7,8]. The venom isomerase from the funnel-web spider, Agelenopsis aperta, that acts on the third amino-acid residue from the C-terminus was characterized approximately a decade ago and was found to be similar in structure to serine proteases [20,21]. More recently, Jilek and co-workers discovered an isomerase of 52 kDa from frog skin that converts the L -amino acid at position 2 of its substrate to the D -form [26]. It is tempting to speculate that the isomerase in the platypus is
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Acknowledgments: We thank Dr Jamie I. Vandenberg for valuable discussions, Mr. Nicholas Mok for conducting initial experiments in this work, Dr. Bill Bubb for help with the NMR experiments, and Mr. Bill Lowe for expert technical assistance. This work was supported by an Australian Research Council Discovery Grant to P.W.K and Dr. J.I. Vandenberg.
References DLP-2
DLP-2
f3
the platypus isomerase bears similarity of its active site with some aminopeptidases since they both act on or near the Nterminus of a peptide. This was investigated by studying the effects of a competitive inhibitor of aminopeptidases, amastatin, on the DLP-2/DLP-4 conversion by a fractionated venom gland extract. The results showed that amastatin was inhibitory, although the presence of methanol reduced the rate of conversion. Moreover, the experiments using the 12-residue DLP analogues convincingly demonstrated that residues 8– 42 of DLP-2 or DLP-4 are not critical for the isomerase activity. Efforts are underway to purify the isomerase using complementary chromatographic techniques such as ion-exchange, and RP-HPLC. Our results show that there are many proteinaceous components in the extract with MW between 50 and 60 k and that the enzyme is only present in minute quantities; this makes further characterization complicated. We are currently sequencing bands isolated from active fractions on the SDS–PAGE gel and are constructing a cDNA library of the platypus venom gland so that we can isolate and express putative isomerase genes and test their activities. Once isomerase activity is demonstrated in these expressed proteins, we will search for homologues in therian (marsupial and eutherian) mammals. The platypus last shared a common ancestor with humans over 210 million years ago, yet it is possible that this enigmatic creature may reveal new insights into human biology and medicine.
f5 f4
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Fig. 4. Size exclusion chromatography of the whole gland extract and isomerase activities of fractionated samples. (A) Size exclusion chromatogram of 50 lL of gland extract using a Superdex 75 HR column detected at a light wavelength of 215 nm. The largest peak which corresponded to a protein with size 57 kDa by column calibration was collected (fraction M) and was analyzed further by gel electrophoresis. (B) SDS–PAGE gel of MW standards, the whole extract, and fraction M that shows protein bands with sizes of 57, 67, 80, and 110 kDa. (C) Size-exclusion chromatogram profiles of 50 lL fraction, f3 ( ), f4 (- - -), and f5 detected ated samples, f1 (——), f2 at a light wavelength of 215 nm. The samples were obtained by collecting 1.5 mL fractions during size-exclusion chromatography of 100 lL gland extract. The inset shows a histogram of isomerase activity. (D,E) RP-HPLC traces of the incubation mixture containing DLP-4 for each fraction after 1 h (D) and 26 h (E) of incubation at 37 C.
related to the frog isomerase, but it is also possible that it could be different since many proteins and peptides discovered in the platypus venom are unique [4,10]. We considered it likely that
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