Báo cáo khoa học: Activity of the plant peptide aglycin in mammalian systems potx

Activity of the plant peptide aglycin in mammalian systems Xin-Peng Dun 1 , Jian-He Wang 1 , Lei Chen 1 , Jie Lu 1 , Fa-Fang Li 1 , Yan-Ying Zhao 1 , Ella Cederlund 2 , Galina Bryzgalova 3 , Suad Efendic 3 , Hans Jo ¨ rnvall 2 , Zheng-Wang Chen 1,2 and Tomas Bergman 2 1 School of Life Science and Technology, Huazhong University of Science and Technology, Wuhan, China 2 Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden 3 Department of Molecular Medicine and Surgery, Karolinska University Hospital, Stockholm, Sweden Polypeptide hormones have long been recognized as important regulatory molecules in animals and the human. Since the discovery of secretin in 1902 [1], and insulin in 1921 [2,3], polypeptides have been assigned signaling functions in the regulation of physiological processes, and several peptides have been used as drugs in specific diseases. Discovery of polypeptide sig- nals in plant defense, growth, and development shows the presence of peptide signaling also in plants [4,5]. It has been reported that plant peptides may be found in animals through alimentary absorption or through coexistence as homologous counterparts in animals, sharing common structures [6,7]. In the present study, we have isolated a bioactive pep- tide, aglycin, from porcine intestine and found it to be identical to a segment of the hormone-like plant poly- peptide albumin 1 B precursor (PA1B, chain b) from pea seeds (Pisum sativum) [8]. PA1B, chain b, is involved in plant signal transduction to regulate growth and dif- ferentiation and is increasingly expressed during seed development (SwissProt entry P62927). In total, six iso- forms of this polypeptide have been described, revealing sequence homology (PA1A–F, SwissProt entries P62926–62931, respectively). In relation to the PA1B chain b sequence, the other five isoforms are 75–94% identical (BLAST search at http://www.pir.uniprot.org). Keywords aglycin; albumin 1 B precursor; blood glucose; mice; voltage-dependent anion- selective channel protein 1 Correspondence T. Bergman, Medical Biochemistry and Biophysics, Karolinska Institutet, SE-171 77 Stockholm, Sweden Fax: +46 8 337 462 Tel: +46 8 524 87780 E-mail: Tomas.Bergman@ki.se Z W. Chen, School of Life Science and Technology, Huazhong University of Science and Technology, Wuhan 430074, China Fax ⁄ Tel: +86 27 8779 2027 E-mail: zwchen21@hotmail.com (Received 3 October 2006, revised 23 November 2006, accepted 29 November 2006) doi:10.1111/j.1742-4658.2006.05619.x A 37 residue peptide, aglycin, has been purified from porcine intestine. The sequence is identical to that of residues 27–63 of plant albumin 1 B precur- sor (PA1B, chain b) from pea seeds. Aglycin resists in vitro proteolysis by pepsin, trypsin and Glu-C protease, compatible with its intestinal occur- rence and an exogenous origin from plant food. When subcutaneously injected into mice (at 10 lgÆg )1 body weight), aglycin has a hyperglycemic effect resulting in a doubling of the blood glucose level within 60 min. Using surface plasmon resonance biosensor technology, an aglycin binding protein with an apparent molecular mass of 34 kDa was detected in mem- brane protein extracts from porcine and mice pancreas. The polypeptide was purified by affinity chromatography and identified through peptide mass fingerprinting as the voltage-dependent anion-selective channel pro- tein 1. The results indicate that aglycin has the potential to interfere with mammalian physiology. Abbreviations CTIP, concentrate of thermostable intestinal polypeptides; HRP, horseradish peroxidase; PA1B, albumin 1 B precursor; VDAC-1, voltage- dependent anion-selective channel protein 1. FEBS Journal 274 (2007) 751–759 ª 2007 The Authors Journal compilation ª 2007 FEBS 751 We now show that aglycin interferes with mamma- lian physiology as revealed by an increase of blood glucose concentration in mice upon subcutaneous injection. Furthermore, a protein purified by binding to aglycin in porcine and mice pancreas membrane protein extracts is identified as the voltage-dependent anion-selective channel protein 1 (VDAC-1) [9]. Hence aglycin apparently has several effects in mammalian systems. Results Aglycin was purified as outlined in Fig. 1, monitoring glucose-induced insulin release from pancreatic b-cells [10]. Fraction 4 from Sephadex G-25 fine chromato- graphy was active. Ion-exchange chromatography of this fraction revealed an active component eluting at 0.1 m ammonium bicarbonate. This fraction was lyophilized and further purified by reversed phase high performance liquid chromatography (RP-HPLC) (Fig. 1). An average yield of 6 lg aglycin was obtained with 2.0 mg lyophilized peptides from 0.1 m ammo- nium bicarbonate fraction, corresponding to about 3 lg pure peptide from 1 kg tissue (wet). The molecu- lar mass determined by matrix-assisted laser desorp- tion ⁄ ionization time-of-flight (MALDI-TOF) mass spectrometry was 3742.3 Da. Edman degradation revealed the amino acid sequence ASCNGVCSPFEM PPCGSSACRCIPVGLVVGYCRHPSG (37 residues). Database searches with this sequence revealed that it is identical to residues 27–63 of the polypeptide PA1B from pea seeds [8]. After exposure of aglycin to common proteases, samples were analyzed by RP-HPLC. The results show that pepsin, trypsin and Glu-C protease do not affect aglycin to any appreciable extent because essentially no proteolytic products were observed after 12 h of incubation (Fig. 2). Mass measurements revealed only the intact material, both before and after the proteo- lytic treatment (data not shown). Aglycin subcutaneously injected into mice at a dose of 10 lgÆg )1 (n ¼ 20) was found to enhance the blood glucose concentration with a peak value at 60 min cor- responding to a doubling of the glucose concentration in relation to the saline group (Fig. 3). When the pea albumin isoform PA1F, chain b (purified from pea seeds, 91% identical sequence to that of aglycin) was tested for the effect on blood glucose, a similar hyper- glycemic pattern was observed (data not shown). Ana- lysis by electrospray mass spectrometry of the PA1F isoform directly before testing its influence on blood glucose concentration revealed a mass value within Fig. 1. Purification of aglycin from pig intestine. Details of the purifi- cation scheme and chromatographic steps are given in the text. Fig. 2. Aglycin stability against proteolytic cleavage. After treatment with pepsin, trypsin or Glu-C protease, the reaction mixtures were analyzed by RP-HPLC (chromatogram after pepsin treatment shown). No significant hydrolysis products were observed for any of these enzymes after 12 h of incubation. Aglycin activity in mammals X P. Dun et al. 752 FEBS Journal 274 (2007) 751–759 ª 2007 The Authors Journal compilation ª 2007 FEBS 0.02 Da of that for the fully oxidized peptide, showing disulfide bridges to be intact in the biologically active peptide. Aglycin immobilized onto an surface plasmon reson- ance biosensor chip (coupling efficiency checked by atomic force microscopy) was tested for interaction with protein components in the microsomal membrane fraction of homogenized porcine tissues. Significant change of the refractive index (increase by 7 · 10 )4 ) was detected with the pancreatic extract only, showing that one or several binding proteins are abundantly present in this tissue preparation (Fig. 4). Using affinity chromatography with immobilized aglycin, a binding protein was recovered from porcine pancreas membrane extract. ELISA at various condi- tions confirmed the interaction between aglycin and the purified protein (Fig. 5). The binding protein was also purified from mice pancreas extract using the same protocol and tested in ELISA with the same monoclonal antibody (data not shown). SDS ⁄ PAGE of the fraction from affinity chromatography revealed a single band at 34 kDa for both the porcine and mice preparations (Fig. 6). The aglycin binding protein was identified by peptide mass fingerprinting using MALDI-TOF mass spectrometry after tryptic in-gel Fig. 3. The effect of aglycin on blood glucose concentration in nor- mal mice. Means ± SEM are shown. The probability of random dif- ference between saline and aglycin groups is < 0.001 (n ¼ 20). Fig. 4. Surface plasmon resonance measurements reveal the exist- ence of a binding protein in membrane extracts tested. P, pan- creas; L, liver; K, kidney; M, muscle. Upward arrows indicate beginning of injection of the extract, downward arrows indicate beginning of washing. An aglycin interacting protein is present in the pancreatic extract. Fig. 5. ELISA of the purified protein from porcine pancreas mem- brane extract reveals that it binds to aglycin. (A) Wells coated with the purified protein followed by addition of aglycin, and then mono- clonal antibody against aglycin. (B) Wells coated with the purified protein followed by monoclonal antibody but without previous addi- tion of aglycin. (C) After blocking with gelatin, aglycin was added, followed by monoclonal antibody. (D) Wells coated with aglycin fol- lowed by the monoclonal antibody. Fig. 6. SDS ⁄ PAGE of the pancreatic aglycin binding protein purified by affinity chromatography. Lane A, protein molecular mass mark- ers; lane B, the aglycin binding protein isolated from mice; lane C, the aglycin binding protein isolated from pig. Staining was with Coomassie blue and numbers indicate molecular masses in kDa. X P. Dun et al. Aglycin activity in mammals FEBS Journal 274 (2007) 751–759 ª 2007 The Authors Journal compilation ª 2007 FEBS 753 digestion of the material from mice pancreas as the 282 residue voltage-dependent anion-selective channel protein 1 (VDAC-1, Sus scrofa SwissProt Q9MZ16, theoretical mass 30.6 kDa [9,11]) at a sequence cover- age of 60%. Discussion We have isolated the thermostable plant peptide agly- cin from pig intestine and investigated its possible interactions and activities in mammalian systems. The results reveal that it has a clear physiological effect in raising the blood glucose concentration in mice about two-fold upon subcutaneous injection. Furthermore, specific binding to the ion channel protein VDAC-1 was detected in the membrane protein extracts from porcine and mice pancreas. Aglycin is a single-chain, 37 residue polypeptide, containing six half-cystine residues at positions 3, 7, 15, 20, 22 and 32, an N-terminal alanine and a C-ter- minal glycine. A search for the aglycin amino acid sequence in the SwissProt database revealed that it is identical to residues 27–63 of the plant polypeptide PA1B from pea seeds, first reported in 1986 [8]. PA1B was later characterized as the pea counterpart of a 4 kDa hormone-like peptide in soybean [12] associated with plant cell proliferation and differentiation [13]. Interestingly, insulin and insulin-like growth factor I and II from mammals are able to compete with the 4 kDa peptide in binding to the receptor-like protein basic 7S globulin isolated from soybean [12,14,15]. Due to the similarity with animal insulin in binding the basic 7S globulin and stimulating its protein kinase activity [16], the 4 kDa soybean peptide was initially designated leginsulin [12] but this name was later aban- doned to avoid any confusion with insulin [13]. We have now purified PA1B, chain b, from a porcine intestinal extract of thermostable polypeptides and detected novel activities ⁄ interactions. Therefore, we believe that PA1B, chain b, deserves a descriptive name and suggest aglycin to emphasize the first and the last residue in the amino acid sequence. Considering the sequences of all the six known isoforms of the peptide, all but one (PA1C, chain b, N-terminal residue Ile), starts with an Ala residue and ends with a Gly residue which makes the name even more appropriate. Structurally aglycin belongs to the cystine-knot pep- tide family that has been found in several sources (plants, fungi, animal venoms, insects) [17]. The mem- bers reveal diverse biological activities and are com- monly ion channel blockers and toxins as well as enzyme inhibitors [17]. The cystine-knot structural motif consists of a ring-like structure formed by two disulfide bonds and their connection held together by a third disulfide bond. This motif is invariably associated with a nearby b-sheet structure and the overall design appears highly efficient for structure stabilization. Aglycin has furthermore been described as an entomo- toxin because of its highly toxic activity against cereal weevils (Sitophilus spp.) [18]. A high-affinity binding site in the insect gut has been detected and character- ized, but the identity of the corresponding target pro- tein and the mechanisms involved are unknown [18]. There are reports describing homologous counter- parts to plant peptides in the animal kingdom with identical or very similar sequences [6,7]. We now find that aglycin significantly resists hydrolysis by trypsin, pepsin and Glu-C protease in vitro, and it is conceiv- able that aglycin isolated in this study is of exogenous origin from plant food sources. This conclusion is sup- ported by results from studies on soluble proteins pre- sent in ileal digests from pigs on pea diets where albumin PA1B was found totally resistant to gastric and small intestine digestion [19]. However, it cannot yet be excluded that aglycin, or a structural homolog of aglycin, exists in animals and thus represent a cross-kingdom bioactive peptide family. Interestingly, similarity searches using the Swiss Institute of Bio- informatics (SIB) BLAST network service (http:// www.expasy.org/cgi-bin/blast.pl) identified a mouse protein segment with 60% identity (15 ⁄ 25, 72% posi- tives) to the aglycin sequence (TrEMBL Q9D7N2), and a human protein segment with 58% identity (10 ⁄ 17, 58% positives) to the aglycin sequence (TrEM- BL Q76B61). In these alignments, the four residue sequence PCGS (aglycin residues 14–17) was common to both protein segments. The presence of aglycin-like sequences in proteins from mouse and man indicates that the plant peptide aglycin may have structurally related counterparts in mammals generated by frag- mentation of larger precursor proteins. Subcutaneous injection of aglycin at a dose of 10 lgÆg )1 body weight increases the blood glucose con- centration in normal mice (Kunming type) about two- fold. Another aglycin isoform (PA1F, chain b) was also tested employing C57BL ⁄ 6 mice ( n ¼ 6) with similar results. In other words, aglycin is bioactive in a mam- malian system represented by the mice with statistically significant effects on the blood glucose level. However, it should be emphasized that even though aglycin, like insulin, influences the concentration of glucose in blood, the effect is opposite that of insulin, increasing rather than decreasing the blood glucose concentration. VDAC-1 was purified and identified as a specific aglycin binding protein. A high-affinity protein binding site for aglycin in the gut of cereal weevils has been Aglycin activity in mammals X P. Dun et al. 754 FEBS Journal 274 (2007) 751–759 ª 2007 The Authors Journal compilation ª 2007 FEBS described but without identification of the correspond- ing protein target [18]. It is therefore likely that the tar- get for aglycin binding is an ion channel protein of the VDAC type. Gressent et al. [18] points out that the binding activity was found in the microsomal fraction, as we also did in this study, and of the two well-known activities for members of the cystine-knot peptide family, ion channel toxicity and enzyme inhibition [20], the latter has so far not been demonstrated for aglycin [18]. Furthermore, ion channel blockers have with few exceptions been described only for venoms originating from the animal kingdom which makes the present finding that the plant peptide aglycin binds to VDAC-1 in porcine pancreas even more interesting, in particular because all cystine-knot plant peptides for which the target is known are enzyme inhibitors [17]. Aglycin increases blood glucose concentration in mice (see above). It is tempting to suggest that the mechanism of enhancement involves binding to VDAC-1. The VDAC-1 protein is involved in energy metabolism of cells, and is mainly distributed to the outer mitochondrial membrane where it controls energy homeostasis by transport of ATP and ADP [21]. How- ever, VDAC-1 was now purified from pancreatic cell membranes, not from mitochondrial membranes, which implicates a novel function of VDAC-1 (i.e., interaction with aglycin) facilitated by its distribution to the cell membranes of pancreatic b-, a- and pp-cells that pro- duce and secret insulin, glucagon and pancreatic pep- tide, respectively. It has been reported that VDAC-1 has been found also in other types of secretory cell membranes such as those of B-lymphocytes and mem- branes associated with cell secretion [22,23]. Further- more, VDAC-1 was recently identified as a NADH– ferricyanide reductase in the plasma membrane [24]. Members of the VDAC protein family are found in both animals and plants. VDAC-1 (SwissProt entry Q9MZ16), now identified as a binding partner to agly- cin, forms channels through both the outer mitochond- rial membrane and the plasma membrane. This allows diffusion of small hydrophilic molecules. Despite its name, VDAC-1 is permeable to both anions and cati- ons depending on the actual membrane potential. To speculate, the mechanism behind the increase of blood glucose concentration could potentially involve the flux of calcium ions through the plasma membrane of pan- creatic b-cells. Because aglycin, upon binding to VDAC-1, probably blocks the channel function and consequently slows down or stops the transport of pos- itive ions such as calcium into the b-cell, this will lead to low levels of cellular calcium that potentially can affect insulin secretion resulting in lower than normal insulin exocytosis and elevated blood glucose levels. In conclusion, aglycin is a plant albumin fragment which binds to VDAC-1 in membrane protein extracts from porcine and mice pancreas. It can also increase blood glucose concentration when injected subcutane- ously into mice. Therefore, aglycin represents a plant peptide with physiological effects in mammalian sys- tems. Experimental procedures Purification and identification of aglycin The starting material for peptide purification was a concen- trate of thermostable intestinal polypeptides (CTIP) from porcine gut [10,25]. Bioactivity during the purification pro- cess was monitored as the effect on glucose-induced insulin release from isolated pancreatic b-cells (rat) [10]. A 30 g quantity of CTIP was dissolved in 0.24 L water [containing 0.5% (v ⁄ v) thiodiglycol], and 1.08 L isopropanol was added to the clear solution. After vigorous stirring, a precipi- tate (the first fraction) was removed by centrifugation (7000 g, 25 min, Yingtai instrument GL21MC, rotor GL21MC30110, Changsha, China). To the supernatant, additionally 1.32 L isopropanol (precooled to )20 °C) was added. After 24 h at )20 °C, a precipitate was collected by suction filtration. This precipitate of crude peptides (11.5 g, the second fraction) was dissolved in 500 mL 0.2 m acetic acid and chromatographed on a Sephadex G-25 fine (Phar- macia, Uppsala, Sweden) column (10 · 90 cm) in 0.2 m acetic acid. After lyophilization, fraction 4 (1.2 g dry mater- ial) was found to be bioactive and was partly soluble in 120 mL 0.01 m ammonium bicarbonate, pH 8.0. Insoluble material was removed by centrifugation (12 000 g, 25 min, Yingtai instrument GL21MC, rotor GL21MC30107). The supernatant was chromatographed on an Express Ion-Exchange C (Whatman, Maidstone, UK) column (2.5 · 60 cm) by stepwise elution with 0.01, 0.02, 0.05, 0.1 and 0.2 m ammonium bicarbonate, pH 8.0. The polypeptide fractions eluted with 0.1 m ammonium bicarbonate exhib- ited activity and were lyophilized (90.5 mg dry material). An aliquot (2.0 mg) of this material was subjected to RP-HPLC using an Agilent 1100 system (Agilent Technol- ogies, Wilmington, DE, USA) fitted with a Zorbax C 18 col- umn (4.6 · 150 mm, 5 l m particles). Eluent A was 0.1% trifluoroacetic acid in water, and eluent B, 0.1% trifluoro- acetic acid in acetonitrile. A linear gradient of 10–60% eluent B in 50 min (1 mLÆmin )1 ) was employed. Eluted components absorbing at 214 nm were collected, lyophilized and analyzed for bioactivity. After establishing the amino acid sequence and hence identity of aglycin (below), the peptide was purified from pea seeds to acquire sufficient amounts for further charac- terization and analysis. A similar protocol was then employed and the homogeneity of the preparations was X P. Dun et al. Aglycin activity in mammals FEBS Journal 274 (2007) 751–759 ª 2007 The Authors Journal compilation ª 2007 FEBS 755 checked by coelution of plant and animal material in RP-HPLC and by electrophoresis [26]. Structural analysis of aglycin Molecular masses of components recovered from RP-HPLC were determined using MALDI-TOF mass spectrometry in an Applied Biosystems (Foster City, CA, USA) Voyager 4307 instrument, using a-cyano-4-hydroxycinnamic acid at 10 mgÆmL )1 70% acetonitrile, 0.1% trifluoroacetic acid as matrix. The PA1F, chain b, preparation was analyzed by electrospray mass spectrometry directly before biological test- ing using a Waters (Manchester, UK) Q-TOF Ultima instru- ment fitted with a PicoTip nanospray emitter (New Objective, Woburn, MA, USA). Edman degradation was carried out without prior reduction and alkylation of the sample in an Applied Biosystems Procise HT instrument. Cysteine residues were indirectly identified by the presence of gaps in the other- wise clearly interpretable sequence. Computer searches of peptide sequences were performed in the SwissProt and TrEMBL databases. Protein concentrations were determined using the Bio-Rad (Hercules, CA, USA) protein assay. Stability against proteolytic cleavage Stability of aglycin towards pepsin (Calbiochem, San Diego, CA, USA), trypsin (Promega, Madison, WI, USA) and Glu-C protease (Roche Diagnostics, Basel, Switzerland) was tested. For pepsin, 2 lLat1lgÆlL )1 was added to 20 lg aglycin dissolved in 18 lL water (adjusted to pH 2.0 with 1 m HCl); for trypsin, 2 lLat1lgÆlL )1 was added to 20 lg aglycin dissolved in 18 lL 1% ammonium bicarbonate. Both reaction mixtures were incubated at 37 °C for 12 h. For Glu-C protease, 2 lLat1lgÆlL )1 was added to 20 lg aglycin dissolved in 18 l L 1% ammonium acetate, followed by incubation at 25 °C for 12 h. The reaction mixtures were analyzed by RP-HPLC on a Zorbax C 18 column (4.6 · 150 mm, 5 lm particles). HPLC conditions were elu- ent A, 0.1% trifluoroacetic acid in water; eluent B, 0.1% tri- fluoroacetic acid in acetonitrile; flow rate, 1 mLÆmin )1 ; gradient 10–60% B, 0–50 min; and detection at 214 nm. Effect on blood glucose in mice Normal mice (Kunming, 18–20 g, n ¼ 40) were obtained from the standard animal center of China Medical College (Beijing, China). The mice were fasted for 8 h and the ini- tial blood glucose concentration was determined with Accu- Chek Advantage blood glucose monitor (Roche Diagnos- tics). The mice were then divided into an equal number of animals receiving saline and aglycin, respectively. For ani- mals in the saline group, 100 lL 0.9% NaCl was injected subcutaneously, and in the aglycin group, the aglycin pep- tide at 10 lgÆg )1 body weight (in 0.9% NaCl at 2 lgÆlL )1 ). After injection, the blood glucose concentration was meas- ured at time points 20, 40, 60 and 80 min. Blood for deter- mination of glucose concentration was taken from the tail of each animal. Blood glucose values are given as means of data collected from 20 animals ± SEM. A statistical com- parison between the groups was performed with the Stu- dent t-test. P < 0.05 was considered significant. Animal experiments were designed and carried out according to the directive 86 ⁄ 609 ⁄ EEC to minimize pain and discomfort. The effect on blood glucose was further tested using one of the other polypeptide isoforms, PA1F, chain b, for which the sequence is 91% identical to that of PA1B, chain b (three amino acid replacements: Ser17Thr, Val29Ile and His34Asn). The experimental conditions were the same (except that C57BL ⁄ 6 mice were used and aglycin was injected into six animals). Detection of an aglycin binding protein Tissue extracts Fresh porcine pancreas, liver, kidney and muscle (500 g each) were collected from a local slaughter house and immediately washed with 0.25 m sucrose (precooled to 4 °C). After removal of connective tissue and fat, the material was cut into small pieces and washed with buffer A (50 mm Hepes, pH 7.6, containing 1 mm phenyl- methanesulfonyl fluoride, 1 mm dithiothreitol, 1 mm EDTA, 0.2 mgÆmL )1 soybean trypsin inhibitor, 2 lgÆmL )1 aprotinin, 5 lgÆmL )1 leupeptin and 1 mgÆmL )1 bacitracin), disintegrated in a JJ-2 homogenizer (GuoHua Instrument Co., Wuhan, China) with two volumes of buffer A contain- ing 0.25 m sucrose, centrifuged first at 600 g for 10 min at 4 °C, and then the supernatants again at 12 000 g for 15 min at 4 °C (Yingtai instrument GL21MC, rotor GL21MC30107). The second supernatants were finally cen- trifuged at 200 000 g for 60 min at 4 °C (rotor TLA-100, Beckman Coulter, Fullerton, CA, USA) to obtain a micro- somal membrane pellet, washed once with buffer A, dis- solved to a final protein concentration of approximately 10 mgÆmL )1 with buffer A containing 1% Triton X-100, stirred for 45 min at 4 °C, then centrifuged again at 200 000 g for 45 min at 4 °C (rotor TLA-100, Beckman Coulter, Fullerton, CA, USA) [27]. The clear supernatant was used for surface plasmon resonance measurements to detect binding proteins and for subsequent isolation by affinity chromatography (below). Pancreas from 20 mice was similarly processed and the extract also used for isola- tion and characterization of aglycin binding proteins. Biosensor analysis Spreeta biosensor and software (American TI Corp., Attle- boro, MA, USA) was used to detect aglycin binding pro- teins. Aglycin was immobilized onto the surface plasmon Aglycin activity in mammals X P. Dun et al. 756 FEBS Journal 274 (2007) 751–759 ª 2007 The Authors Journal compilation ª 2007 FEBS resonance sensor chip according to the manufacturer’s pro- tocol. The efficiency of immobilization was evaluated by atomic force microscopy using a NanoIIIa instrument (Digital Instrument Company, Santa Barbara, CA, USA). Extracts of membrane proteins prepared from pancreas, liver, kidney and muscle (above) were diluted with Hepes- buffered saline (HBS: 10 mm Hepes, pH 7.4, containing 0.15 m NaCl) to final protein concentration 200 lgÆmL )1 . Aliquots were injected over the sensor chip surface at a flow rate of 20 lLÆmin )1 for 2–3 min at 25 °C. After each injection, the sensor chip was thoroughly washed with HBS containing 0.05% Triton X-100 and equilibrated with HBS. Binding interactions were continuously monitored and plot- ted as refractive index versus time and displayed in a sen- sorgram [28]. Affinity purification Aglycin (5 mg) was coupled to 1 mL CNBr-activated Sepharose 4B (Amersham Pharmacia Biotech, Uppsala, Sweden) according to the manufacturer’s protocol. The affinity resin was transferred to a column and equili- brated with buffer B (50 mm Hepes, pH 7.6, containing 0.1% Triton X-100) at 4 °C. The pancreatic membrane protein extract (porcine or mice) was diluted three-fold with buffer A and applied at 0.5 mLÆmin )1 (4 °C). After adsorption, the column was washed with buffer C (buf- fer B containing 1 mm phenylmethanesulfonyl fluoride and 1 mm dithiothreitol), followed by thorough washing with buffer C containing 1 m NaCl, at 40 mLÆh )1 . For elution, monitoring was at 254 nm, with first buffer D (50 mm acetate, pH 5.0, containing 1 m NaCl and 0.1% Triton X-100, 1 mm phenylmethanesulfonyl fluoride, 1mm dithiothreitol), and then buffer D containing 1.5 m urea at 20 mLÆh )1 . Eluted fractions (2 mL) were collected in tubes containing 1 mL 0.5 m Tris ⁄ HCl, pH 8.25, and pooled according to the peak patterns. After immediate dialysis against 10 mm Hepes buffer, 0.1% Triton X-100, pH 7.6 [27], 2 mL aliquots were taken for interaction studies between pancreatic proteins and aglycin by ELISA (below). Remaining parts of the fractions were lyophilized for protein characterization. ELISA measurements The interaction between porcine pancreas proteins and aglycin was studied in an ELISA array. Briefly, 96 well pol- yvinylchloride plates were coated with 50 lL porcine pan- creas protein fraction from affinity chromatography (10 lgÆmL )1 in 50 mm Na 2 CO 3 ⁄ NaHCO 3 , pH 9.6) and incubated at 4 °C overnight. The wells were washed with 20 mm NaCl ⁄ P i containing 0.1% gelatin, then blocked with 20 mm NaCl ⁄ P i containing 1% gelatin for 1 h at 37 °C. The wells were washed three times with NaCl ⁄ P i -T (20 mm NaCl ⁄ P i , 0.1% Tween-20) containing 0.1% gelatin, before incubation overnight at 4 °C with aglycin, 50 lLofa 50 lgÆmL )1 solution in NaCl ⁄ P i -T. The wells were washed with NaCl ⁄ P i -T, followed by addition of an antiaglycin monoclonal antibody (prepared in our laboratory as des- cribed [29]) at a 1 : 20 000 dilution of 2 mgÆmL )1 in NaCl ⁄ P i -T and incubation 1 h at 37 °C. After removal of nonbinding antibodies with NaCl ⁄ P i -T (five times, 3 min each), horseradish peroxidase (HRP) labeled rabbit anti- mouse IgG secondary antibody was applied and incubated for 1 h at 37 °C. Non-adsorbed IgG-HRP complex was thoroughly removed by washing with NaCl ⁄ P i -T. Bound HRP was monitored by addition of o-phenylenediamine and detection at 492 nm. Identification of an aglycin binding protein Gel electrophoresis SDS ⁄ PAGE of fractions from the affinity purification was carried out in 0.75 mm 12% slab gels (Bio-Rad) [30]. Sam- ples were dissolved in 5% SDS containing 20 mm dithio- threitol and incubated for 12 h at room temperature. The electrophoresis was conducted in the presence of 0.1% SDS and 20 mm dithiothreitol. Rabbit phosphorylase b (97.4 kDa), bovine serum albumin (66.2 kDa), rabbit actin (43.0 kDa), bovine carbonic anhydrase (31.0 kDa) and trypsin inhibitor (20.1 kDa) were used as molecular mass standards. In-gel digestion and peptide mass fingerprinting After separation by SDS ⁄ PAGE, the gel was stained with Coomassie blue and the single band detected from the aglycin binding fraction was excised and cut into small pieces (1 mm 2 ). The pieces were placed in a 0.65 mL silic- onized tube, washed twice with 250 lL 100 mm ammo- nium bicarbonate, vortexed in 250 lL 50% (v ⁄ v) acetonitrile ⁄ 100 mm ammonium bicarbonate for 10 min, and dehydrated in 150 lL neat acetonitrile until the gel turned opaque. It was then dried in a Speed Vac (Globule Medical Instrument, Ramsey, MN, USA) for 20 min and subsequently reswelled in trypsin solution (three-fold the gel volume; 300 lL 100 mm ammonium bicarbonate con- taining 3 lg trypsinÆmL )1 , Calbiochem, San Diego, CA, USA) for 10 min. After addition of 100 lL 100 mm ammonium bicarbonate, digestion was carried out at 37 °C overnight. The solution was then transferred to a 0.65 mL siliconized tube and the gel pieces were extracted twice under sonication (10 min) with 50 lL 50% (v ⁄ v) acetonit- rile containing 5% trifluoroacetic acid. The digest and extracts were combined and concentrated under vacuum. Peptide mass fingerprints were determined by MALDI- TOF mass spectrometry in a Tof Spec instrument (Micro- mass, Manchester, UK) [31,32] and submitted to data- base searches using the mascot software (http://www. matrixscience.com). X P. Dun et al. Aglycin activity in mammals FEBS Journal 274 (2007) 751–759 ª 2007 The Authors Journal compilation ª 2007 FEBS 757 Acknowledgements This work was supported by grants from the National Science Foundation of China (30370647 and 30470823), the Chinese 863 Program (2002AA214061), the Swedish Research Council (03X-3532, 629-2002- 8654 and 621-2003-3616), the Swedish Cancer Society (4159), the Wallenberg Consortium North (WCN), the Juvenile Diabetes Foundation (JDFI-4-99-647), the European Commission (LSHC-CT-2003–503297), and Karolinska Institutet. References 1 Bayliss WM & Starling EH (1902) On the causation of the so-called ‘peripheral reflex secretion’ of the pancreas. Proc Roy Soc Lond 69, 352–353. 2 Banting FG, Best CH, Collip JB, Campbell WR & Fletcher AA (1922) Pancreatic extracts in the treatment of diabetes mellitus. Preliminary report. Can Med Assoc J 12, 141–146. 3 Macleod JJR (1922) The source of insulin. A study of the effect produced on blood sugar by extracts of the pancreas and principal islets of fishes. J Metab Res 2, 149–172. 4 Ryan CA, Pearce G, Scheer J & Moura DS (2002) Poly- peptide Hormones. Plant Cell Suppl. 251–264. 5 Lindsey K (2001) Plant peptide hormones: the long and the short of it. Current Biology 11, R741–R743. 6 Oliveira AEA, Machado OLT, Gomes VM, Neto JX, Pereira AC, Vieira JGH, Fernandez KVS & Xavier- Filho J (1999) Jack bean seed coat contains a protein with complete sequence homology to bovine insulin. Protein Peptide Lett 6, 15–22. 7 Maryani MM, Morse MV, Bradley G, Irving HR, Cahill DM & Gehring CA (2003) In situ localization associates biologically active plant natriuretic peptide immuno-analogues with conductive tissue and stomata. J Exp Bot 54, 1553–1564. 8 Higgins TJ, Chandler PM, Randall PJ, Spencer D, Beach LR, Blagrove RJ, Kortt AA & Inglis AS (1986) Gene structure, protein structure, and regulation of the synthesis of a sulfur-rich protein in pea seeds. J Biol Chem 261, 11124–11130. 9 Strausberg RL, Feingold EA, Grouse LH, Derge JG, Klausner RD, Collins FS, Wagner L, Shenmen CM, Schuler GD, Altschul SF et al. (2002) Generation and initial analysis of more than 15 000 full-length human and mouse cDNA sequences. Proc Natl Acad Sci USA 99, 16899–16903. 10 Chen Z-W, Agerberth B, Gell K, Andersson M, Mutt V, O ¨ stenson CG, Efendic S & Jo ¨ rnvall H (1988) Isola- tion and characterization of porcine diazepam-binding inhibitor, a polypeptide not only of cerebral occurrence but also common in intestinal tissues and with effects on regulation of insulin release. Eur J Biochem 174, 239–245. 11 Yu WH, Wolfgang W & Forte M (1995) Subcellular localization of human voltage-dependent anion channel isoforms. J Biol Chem 270, 13998–14006. 12 Watanabe Y, Barbashov SF, Komatsu S, Hemmings AM, Miyagi M, Tsunasawa S & Hirano H (1994) A peptide that stimulates phosphorylation of the plant insulin-binding protein. Isolation, primary structure and cDNA cloning. Eur J Biochem 224, 167–172. 13 Yamazaki T, Takaoka M, Katoh E, Hanada K, Sakita M, Sakata K, Nishiuchi Y & Hirano H (2003) A possible physiological function and the tertiary structure of a 4-kDa peptide in legumes. Eur J Biochem 270, 1269–1276. 14 Komatsu S & Hirano H (1991) Plant basic 7S globulin- like proteins have insulin and insulin-like growth factor binding activity. FEBS Lett 294, 210–212. 15 Hanada K & Hirano H (2004) Interaction of a 43-kDa receptor-like protein with a 4-kDa hormone-like peptide in soybean. Biochemistry 43, 12105–12112. 16 Hanada K, Nishiuchi Y & Hirano H (2003) Amino acid residues on the surface of soybean 4-kDa peptide involved in the interaction with its binding protein. Eur J Biochem 270, 2583–2592. 17 Norton RS & Pallaghy PK (1998) The cystine knot structure of ion channel toxins and related polypeptides. Toxicon 36, 1573–1583. 18 Gressent F, Rahioui I & Rahbe Y (2003) Characteriza- tion of a high-affinity binding site for the pea albumin 1b entomotoxin in the weevil Sitophilus. Eur J Biochem 270, 2429–2435. 19 Le Gall M, Quillien L, Gue ´ guen J, Rogniaux H & Se ` ve B (2005) Identification of dietary and endogenous ileal protein losses in pigs by immunoblotting and mass spec- trometry. J Nutr 135, 1215–1222. 20 Craik DJ, Daly NL & Waine C (2001) The cystine knot motif in toxins and implications for drug design. Toxi- con 39, 43–60. 21 Blachly-Dyson E, Zambronicz EB, Yu WH, Adams V, McCabe ER, Adelman J, Colombini M & Forte M (1993) Cloning and functional expression in yeast of two human isoforms of the outer mitochondrial mem- brane channel, the voltage-dependent anion channel. J Biol Chem 268, 1835–1841. 22 Thinnes FP, Gotz H, Kayser H, Benz R, Schmidt WE, Kratzin HD & Hilschmann N (1989) Identification of human porins. I. Purification of a porin from human B-lymphocytes (Porin 31HL) and the topochemical proof of its expression on the plasmalemma of the progenitor cell. Biol Chem Hoppe Seyler 370, 1253– 1264. 23 Buettner R, Papoutsoglou G, Scemes E, Spray DC & Dermietzel R (2000) Evidence for secretory pathway localization of a voltage-dependent anion channel iso- form. Proc Natl Acad Sci USA 97, 3201–3206. Aglycin activity in mammals X P. Dun et al. 758 FEBS Journal 274 (2007) 751–759 ª 2007 The Authors Journal compilation ª 2007 FEBS 24 Lawen A, Ly JD, Lane DJ, Zarschler K, Messina A & DePinto VC (2005) Voltage-dependent anion-selective channel 1 (VDAC1): a mitochondrial protein rediscov- ered as a novel enzyme in the plasma membrane. Int J Biochem Cell Biol 37, 277–282. 25 Lee JY, Boman A, Sun CX, Andersson M, Jo ¨ rnvall H, Mutt V & Boman HG (1989) Antibacterial peptides from pig intestine: isolation of a mam- malian cecropin. Proc Natl Acad Sci USA 86, 9159–9162. 26 Schagger H & von-Jagow G (1987) Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Anal Biochem 166, 368–379. 27 Fujita-Yamaguchi Y, Choi S, Sakamoto Y & Itakura K (1983) Purification of insulin receptor with full binding activity. J Biol Chem 258, 5045–5049. 28 Bender V, Ali M, Amon M, Diefenbach E & Manolios N (2004) T cell antigen receptor peptide–lipid mem- brane interactions using surface plasmon resonance. J Biol Chem 279, 54002–54007. 29 Wang J-H, Dun X-P, Qu L-N, Zhao Y-Y, Yang T-B & Chen Z-W (2005) Preparation and identification of monoclonal antibodies against pea albumin 1b (PA1b). Hybridoma 24, 197–200. 30 Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–685. 31 Jeno P, Mini T, Moes S, Hintermann E & Horst M (1995) Internal sequences from proteins digested in polyacrylamide gels. Anal Biochem 224, 75–82. 32 Medzihradszky KF, Leffler H, Baldwin MA & Burlin- game AL (2001) Protein identification by in-gel digestion, high-performance liquid chromatography, and mass spec- trometry: peptide analysis by complementary ionization techniques. J Am Soc Mass Spectrom 12, 215–221. X P. Dun et al. Aglycin activity in mammals FEBS Journal 274 (2007) 751–759 ª 2007 The Authors Journal compilation ª 2007 FEBS 759 . though aglycin, like insulin, in uences the concentration of glucose in blood, the effect is opposite that of insulin, increasing rather than decreasing the. as a specific aglycin binding protein. A high-affinity protein binding site for aglycin in the gut of cereal weevils has been Aglycin activity in mammals X