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 mic
Trang 1Xin-Peng Dun1, Jian-He Wang1, Lei Chen1, Jie Lu1, Fa-Fang Li1, Yan-Ying Zhao1, Ella Cederlund2, Galina Bryzgalova3, Suad Efendic3, Hans Jo¨rnvall2, Zheng-Wang Chen1,2and Tomas Bergman2
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.
Trang 2We 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 chromatochromato-graphy 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 (n¼ 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.
Trang 30.02 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.
Trang 4digestion 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)1body 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
Trang 5described 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 C18 col-umn (4.6· 150 mm, 5 lm 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
Trang 6checked 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 ustest-ing 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 lL at 1 lgÆlL)1 was added to 20 lg
aglycin dissolved in 18 lL water (adjusted to pH 2.0 with
1 m HCl); for trypsin, 2 lL at 1 lgÆlL)1was added to 20 lg
aglycin dissolved in 18 lL 1% ammonium bicarbonate
Both reaction mixtures were incubated at 37C for 12 h
For Glu-C protease, 2 lL at 1 lgÆlL)1was added to 20 lg
aglycin dissolved in 18 lL 1% ammonium acetate, followed
by incubation at 25C for 12 h The reaction mixtures
were analyzed by RP-HPLC on a Zorbax C18 column
(4.6· 150 mm, 5 lm particles) HPLC conditions were
elu-ent A, 0.1% trifluoroacetic acid in water; eluelu-ent 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, n¼ 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)1body 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
4C) 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
4C, and then the supernatants again at 12 000 g for
15 min at 4C (Yingtai instrument GL21MC, rotor GL21MC30107) The second supernatants were finally cen-trifuged at 200 000 g for 60 min at 4C (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 4C, then centrifuged again at
200 000 g for 45 min at 4C (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 characterizaisola-tion 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
Trang 7resonance 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 25C 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 4C The pancreatic membrane
protein extract (porcine or mice) was diluted three-fold
with buffer A and applied at 0.5 mLÆmin)1 (4C) 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,
1 mm 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 Na2CO3⁄ NaHCO3, pH 9.6) and
incubated at 4C overnight The wells were washed with
20 mm NaCl⁄ Picontaining 0.1% gelatin, then blocked with
20 mm NaCl⁄ Pi containing 1% gelatin for 1 h at 37C
The wells were washed three times with NaCl⁄ Pi-T (20 mm
NaCl⁄ Pi, 0.1% Tween-20) containing 0.1% gelatin, before
incubation overnight at 4C with aglycin, 50 lL of a
50 lgÆmL)1 solution in NaCl⁄ Pi-T The wells were washed with NaCl⁄ Pi-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⁄ Pi-T and incubation 1 h at 37C After removal of nonbinding antibodies with NaCl⁄ Pi-T (five times, 3 min each), horseradish peroxidase (HRP) labeled rabbit anti-mouse IgG secondary antibody was applied and incubated for 1 h at 37C Non-adsorbed IgG-HRP complex was thoroughly removed by washing with NaCl⁄ Pi-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 mm2) 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 37C 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)
Trang 8This 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
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