Original Article
A new recombinant pituitary adenylate cycla-activating peptide-derived peptide efficiently promotes gluco uptake and gluco-dependent insulin cretion
Yi Ma,Tianjie Luo,Wenna Xu,Zulu Ye,and An Hong*
Department of Cell Biology,Institute of Biological Medicine,Jinan University,Guangzhou510632,China
*Correspondence address.Tel:þ86-20-85223266;Fax:þ86-20-85221983;E-mail:
economist>狗屎的英文The recombinant peptide,DBAYL,a promising thera-peutic peptide for type2diabetes,is a new,potent,and highly lective agonist for VPAC2generated through site-directed mutagenesis bad on quence alignments of pi-tuitary adenylate cycla-activating peptide(PACAP), vasoactive intestinal peptide(VIP),and related analogs. The recombinant DBAYL was ud to evaluate its effect and mechanism in blood gluco metabolism and utiliza-tion.As much as28.9mg recombinant DBAYL peptide with purity over98%can be obtained from1l of Luria-Bertani medium culture by the method established in this study and the prepared DBAYL with four mutations (N10Q,
V18L,N29Q,and M added to the N-terminal) were much more stable than BAY55-9837.The half-life of recombinant DBAYL was about25folds compared with that of BAY55-9837in vitro.The bioactivity assay of DBAYL showed that it displaced[125I]PACAP38and [125I]VIP from VPAC2with a half-maximal inhibitory concentration of48.4+6.9and47.1+4.9nM,respective-ly,which were significantly lower than that of BAY55-9837,one established VPAC2agonists.DBAYL enhances the cAMP accumulation in CHO cells expressing human VPAC2with a half-maximal stimulatory concentration (EC50)of0.68nM,whereas the receptor potency of DBAYL at human VPAC1(EC50of737nM)was only1/1083 of that at human VPAC2,and DBAYL had no activity toward human PAC1receptor.Western blot analysis of the key proteins of insulin receptor signaling pathway:insulin re-ceptor substrate1(IRS-1)and gluco transporter4 (GLUT4)indicated that the DBAYL could significantly induce the insulin-stimulated IRS-1and GLUT4expression more efficiently than BAY55-9837and VIP in adipocytes. Compared with BAY55-9837and PACAP38,the recombinant peptide DBAYL can more efficiently promote insulin relea and decrea plasma gluco level in Institute of Cancer Rearch(ICR)mice.The results suggested that DBAYL could efficiently improve gluco uptake and gluco-depend-ent insulin cretion by VPAC2-mediated effect.Keywords pituitary adenylate cycla-activating peptide; type2diabetes;insulin;VPAC2-mediated effect;recombinant peptide
Received:June29,2012Accepted:August6,2012 Introduction
Pituitary adenylate cycla-activating polypeptide(PACAP) is a member of the superfamily of metabolic,neuroendo-crine,and neurotransmitter peptide hormones and belongs to the cretin,glucagons,and vasoactive intestinal peptide (VIP)family[1,2].PACAP exists as either a38-amino acid (PACAP38)or27-amino acid(PACAP27)peptide. PACAP27corresponds to the N-terminal27-amino acid portion of PACAP38and exhibits the same biological activ-ity as PACAP38[3,4].The action of PACAP is mediated through three G protein-coupled receptors,PAC1,VPAC1, and VPAC2.PAC1receptor exhibits high affinity for PACAP38and PACAP27,but much lower affinity for VIP. VPAC1and VPAC2receptors exhibit similar high affinity for PACAP38,PACAP27,and VIP[5].PACAP is widely distributed in the brain and peripheral organs,notably in the endocrine pancreas,gonads,respiratory,and urogenital tracts,which has been shown to have effects on many pathological states including Parkinson’s dia[6],dia-betes[7,8],ischemia[9],traumatic injury[10],immuno-logical disorders[11,12],myeloma kidney injury,and so on [13].Most of the neuroprotective actions of PACAP are mediated through the lective PAC1receptor whereas the effects on peripheral organs often involve VPAC1or VPAC2receptor.PACAP has been shown to increa insulin cretion from the pancreas through VP
华盛顿大学圣路易斯分校
AC2recep-tor[14,15].But the role of PACAP in the control of gluco homeostasis is complex,becau it also plays a role in increasing glucagon and catecholamine cretion,which increas gluco output from the liver through VPAC1-mediated effect[16].Therefore,PACAP derivative as
Acta Biochim Biophys Sin2012,44:948–956|ªThe Author2012.Published by ABBS Editorial Office in association with Oxford University Press on behalf of the Institute of Biochemistry and Cell Biology,Shanghai Institutes for Biological Sciences,Chine Academy of Sciences.DOI:10.1093/abbs/gms078.
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VPAC2-specific agonist,which would stimulate gluco-dependent insulin cretion from pancreatic
b-cell without leading to incread gluco production by the liver could be ud for clinical treatment of type2diabetes. Development of BAY55-9837,an established highly lect-ive VPAC2agonist,as a potential peptide therapeutic for the treatment of type2diabetes was limited by its poor peptide stability[14].To overcome the limitation,the re-combinant peptide DBAYL with32amino acids was designed and generated through site-directed mutagenesis by gene-recombination technology.The recombinant DBAYL (N10Q,V18L,N29Q,and M added to the N-terminal)were much more stable than BAY55-9837.DBAYL enhances the cAMP accumulation in VPAC2-CHO cells with higher bio-activity than BAY55-9837.DBAYL could more efficiently induce the expression of the key proteins of insulin receptor signaling pathway including insulin receptor substrate1 (IRS1)and gluco transporter4(GLUT4)than BAY55-9837in adipocytes[17,18].In addition,DBAYL treatment incread the insulin-stimulated GLUT4translocation to the plasma membrane.Corresponding to the results,gluco uptake activity of differentiated3T3-L1adipocytes treated with DBAYL were significantly improved,which was better than BAY55-9837.
Thus,insulin signal transduction was more efficiently improved by DBAYL through VPAC2-mediated effect. DBAYL,a novel recombinant PACAP-derived peptide,as highly lective agonist for VPAC2,can hopefully be a peptide therapeutic for type2diabetes through efficiently promoting gluco uptake and gluco-dependent insulin cretion.
Materials and Methods
Materials
Chitin beads and the plasmid pKYB-MCS were purchad from New England Biolabs(NEB,Ipswich,USA). Escherichia coli strain ER2566was kept in our laboratory. All the restriction enzymes were purchad from New England Biolabs.T4DNA liga was obtained from TaKaRa (Dalian,China).Synthetic peptides were purchad from Sinoasis Pharmaceuticals(Guangzhou,China).Primer syn-thesis and DNA quencing were performed by Invitrogen Company,Guangzhou Branch(Guangzhou,China).VPAC2-CHO cell line was constructed in our laboratory.3T3-L1 adipocytes were provided by Dr Zhang WJ(College of Life Sciences,Wuhan University,Wuhan,China). Construction and identification of the expression plasmid pKY-DBAYL
The DBAYL gene was designed according to the bias li for the codons to ensure its high expression.The gene was synthesized and amplified in two steps as
described previously[7]using three oligonucleotides primers:F1:50-GGTGGTCATATGCATAGCGATGCGGT GTTTACCGATCAGTATACCCGTCTGCGTAAA-30,con-taining an Nde I site(underlined);F2:50-CAGATATT TTTTCGCCGCCAGCTGTTTACGCAGACGGGT-
30;F3:
50-CCACCATGCTCTTCCGCAATAACGTTTCTGTTTAA TGCTCTGCAGATATTTTTT-30,containing an Sap I site (underlined);GGTGGT at the50end of F1and CCACCA
at the50end of F3are the protecting bas.After polymer-
a chain reaction(PCR)products were purified by the
PCR clean-up kit(Qiagen,Hilden,Germany)and digested
with Nde I and Sap I,the DNA fragment was directly ligated
to a gel-purified Nde I/Sap I digested pKYB-MCS vector (NEB)to yield the expression plasmid pKY-DBAYL.
pKY-DBAYL containing DBAYL gene was confirmed by
DNA quencing using the T7promoter as the quencing
primer(Fig.1).
Expression of fusion protein
The recombinant expression vector pKY-DBAYL was transformed into li strain ER2566with the opti-
mized procedure[19].Briefly,the cells were grown at378C
to a density of OD600¼0.8and induced by adding isopro-
pyl b-D-thiogalactoside to a final concentration of0.5
mM.
Figure1The constructed recombinant expression vector
pKY-DBAYL(A)The amino acid quence of RBAYL and rBAY.(B)
longerThe construction map of the expression plasmid pKY-DBAYL.
PACAP-derived peptide promotes gluco uptake and gluco-dependent insulin cretion
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The induced cells were incubated for6h at358C and col-lected by centrifugation at10,621g for20min.Sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE)was ud to identify the expression of the fusion protein.The cell pellet was resuspended in buffer A con-taining2
0mM Tris-HCl(pH8.0),500mM NaCl, and1mM EDTA by gentle shaking for20min,and then disrupted with JN-3000PLUS low-temperature ultra-high-pressure continuous flow cell crusher(JNBIO, Guangzhou,China)at the following conditions:diluted bac-teria concentration of18%by buffer A,crushing pressure of 1700bar and cooling temperature of38C.The lysate was then centrifuged at10,621g for30min at48C and the supernatant was subjected to purification and preparation of target peptide by chitin beads affinity chromatography. Preparation and identification of the recombinant peptide DBAYL
The supernatant(1.5l)was pasd through a column (4.5cmÂ20cm)packed with25ml chitin beads at a flow rate of0.5ml/min.After the supernatant was loaded on the column,the flow rate was raid to2ml/min and the column was thoroughly washed with more than10bed volume of buffer A.Then80ml of buffer B containing 20mM Tris-HCl(pH8.0),500mM NaCl,1mM EDTA, and100mM b-mercaptoethanol was then quickly pasd through the column to distribute b-mercaptoethanol evenly throughout the resin and the column flow was stopped.The column was incubated at258C for24h.Fractions contain-ing DBAYL were obtained by eluting the column with buffer A.Then the recombinant peptide DBAYL was puri-fied and prepared by rever-pha high-performance liquid chromatography(HPLC)system using4.6mmÂ150mm 300SB-C18Sep-Pak column(Agilent Technolog
ies, Beijing,China)through gradient elution with increasing concentration of acetonitrile from2%to55%for45min at 1ml/min.The eluate containing DBAYL was dried by ly-ophilization.Prepared DBAYL at a final concentration of 1mg/ml in45%acetonitrile containing0.1%trifluoroacetic acid was analyzed by4000Q TRAP electrospray ionization-mass spectrometry(ESI-MS;Applied Biosystems,Foster City,USA).Peptide concentrations were determined by com-paring the OD280values of peptide stock solutions in the assay buffer with the predicted extinction coefficient[20]. Stability assay
工作室英文
DBAYL,BAY55-9837,PACAP38,or VIP at a final concen-tration of1mg/ml in20mM sodium phosphate buffer(pH 8.0)containing150mM sodium chloride were incubated at 378C.At different time points,samples were collected and analyzed by liquid chromatography mass spectrometry,a rapid and nsitive method to detect degradation of polypep-tide in the formulations.A2-ml sample was injected into HPLC-ESI-MS system containing 1.0mmÂ150mm300 SB-C18Sep-Pak(Agilent Technologies,Santa Clara,USA) column and analyzed under the condition of increasing con-centration of acetonitrile from2%to55%for55min at 0.05ml/min by HPLC-ESI-MS system.
Competition receptor binding assay
The potential of DBAYL to displace[125I]PACAP38 and[125I]VIP by competitively binding to the human VPAC2receptor was examined in VPAC2-CHO cell mem-brane prepared previously[7].Briefly,10mg of membrane was incubated with0.1nM[125I]PACAP38(Phoenix Pharmaceuticals,Mountain View,USA)or[125I]VIP (PerkinElmer Life and Analytical Sciences,Boston,USA) in the prence of increasing concentrations of DBAYL peptide,in a total volume of100ml of20mM HEPES (pH7.4)containing150mM NaCl,0.5%BSA,2mM MgCl2,and0.1mg/ml bacitracin at378C.After being incu-bated for20min,the membrane was collected on GF/C filters pretreated with0.1%polyethylenimine.The filters were washed with25mM cold Na3PO4containing1% BSA and counted on a gamma counter.Non-specific binding was defined as the residual binding in the prence of1mM recombinant PACAP38)or VIP and was always,20%of the total binding.The assay of PACAP38,VIP,and BAY55-9837were taken as the positive controls.[K15,R16,L27]VIP(1–7)/GRF(8–27),a VPAC1-specific agonist,was ud as the negative control in the receptor binding assay[21].Each assay was per-formed at least three times.
Assay of cAMP accumulation induced by DBAYL Human PACAP receptor-transfected cells,VPAC1-CHO, VPAC2-CHO,and PAC1-CHO cells,cultured in the Dulbecco’s modified Eagle’s medium at378C were scraped off with rubber policeman and washed with PBS twice. The density of the cells
was adjusted to2Â106cells/ml. DBAYL or rPACAP38was added to the500-ml cell sus-pension,and the concentrations of the peptide were ranged from1Â10212to1Â1025M.The mixtures were incu-bated at378C for5min,then two volumes of0.2M HCl was added,and the mixtures were incubated at room tem-perature for another20min.Cells were lyd by pipetting up and down until the suspension was homogeneous.The precipitate was removed by centrifugation at225g for 10min,and the supernatant was transferred into test tube and cAMP concentrations were measured by using the cyclic AMP enzyme immunoassay kit(Cayman Chemical Company,Ann Arbor,USA).
点钞方法Western blot analysis of IRS-1and GLUT4induced by DBAYL
Cell culture and induction of3T3-L1adipocytes were carried out as described previously[22].Differentiated
PACAP-derived peptide promotes gluco uptake and gluco-dependent insulin cretion
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3T3-L1adipocytes were incubated with100nM insulin for 20min[23].After being washed twice with PB
the logicS buffer, differentiated3T3-L1adipocytes were cultured for48h in medium,respectively,containing0and1m M of DBAYL, BAY55-9837or VIP.Then the total protein was extracted. After the total protein was parated by12%SDS-PAGE and transferred onto poly(vinylidene difluoride)membranes (Immobilon P;Millipore,Billerica,USA),the membranes were incubated with the IRS-1rabbit mAb(Cell Signaling Technology,Boston,USA)or anti-GLUT4antibody(Santa Cruz Biotechnology,Santa Cruz,USA)for2h at room temperature.The horradish peroxide(HRP)-conjugated goat-anti-rabbit IgG(Immunology Consultants Laboratory, Portland,USA)or sheep-anti-mou HRP-IgG(BioFX Laboratories,Owings Mills,USA)was ud as the cond antibody.Protein bands were visualized by using an ECL kit(Santa Cruz Biotechnology)and densitometric analysis of the results of western blot was performed with image analysis software[24].
To evaluate the effect of DBAYL on GLUT4translocat-ing to the plasma membrane,differentiated3T3-L1adipo-cytes were incubated with100nM insulin for20min.After being washed twice with PBS buffer,differentiated3T3-L1 adipocytes were cultured for48h in medium containing 1m M of DBAYL.Another experiment group that differen-tiated3T3-L1adipocytes were cultured for48h in medium containing1m M of DBAYL without insulin treatment to determine whether the manner of the
DBAYL effect on GLUT4translocating to the plasma membrane is insulin-dependent or non-insulin-dependent.
Then plasma membrane lawns were prepared by sonic-ation as described previously[25].GLUT4contents of the plasma membrane lawns were determined by immunoblot-ting using anti-GLUT4antibody performed with an ECL kit. Effect of DBAYL on gluco uptake activity Differentiated3T3-L1adipocytes were incubated with 100nM insulin for20min[23].After being washed twice with PBS buffer,the3T3-L1adipocytes were cultured for 48h in medium,respectively,containing0,1,and5m M of DBAYL or BAY55-9837.The gluco level of the cell culture supernatants was determined with Gluco assay kit-gluco oxida method(Applygen Technologies Inc., Beijing,China).
Effect of DBAYL on insulin relea and gluco disposal in ICR mice
Eighteen male ICR mice weighing25–30g were houd at room temperature on a12/12h light/dark cycle.ICR mice fasted over-night(12h)and were randomly divided into three groups according to their weight(six per group).The prepared DBAYL(0.5m g/kg)that is dissolved in the normal saline was intraperitoneally injected into the ICR
mice and10min later,gluco dissolved in distilled water
(2g/kg)was given to ICR mice by gavage.The experimen-
tal groups with the same do or volume of BAY55-9837
and rPACAP38were as positive controls and the groups
with normal saline as a negative control.At15min after gavage,blood samples were collected from the tail vein and
the plasma gluco levels were determined using OneTouch
Ultra Meter(Johnson&Johnson,Johnson,USA)and the plasma insulin was measured using RIA kit(Linco Rearch,Charles,USA)in the First Affiliated Hospital of
Jinan University(Guangzhou,China).
Results
Expression and preparation for DBAYL
The fusion proteins consisting of target peptide-,intein-
and chitin-binding DBAYL-intein-CBD)were expresd through a recombinant peptide expression vector,
pKY-DBAYL,li strain ER2566.The fusion pro-
teins were purified using chitin affinity column.The cleav-
age of intein was induced by b-mercaptoethanol and the
target peptide,DBAYL was relead.Then the recombin-
ant peptide DBAYL was further purified and prepared by rever pha HPLC system.About28.9mg of recombin-
ant DBAYL peptide over98%of purity can be obtained
from1l of Luria-Bertani medium.The prepared DBAYL
钟彬娴
was analyzed and identified by ESI-MS.Figure2 showed that the molecular weight of DBAYL from
ESI-MS was3916.6Da,which was consistent with the theoretical value(3916.5Da).The purity of prepared DBAYL was over98%by the analytical HPLC determin-
ation method.
Peptide stability improved by site-directed mutagenesis
The recombinant DBAYL was tested together with
BAY55-9837,PACAP38,and VIP for stability at378C in
20mM sodium phosphate buffer(pH8.0)containing
150mM sodium chloride.After4weeks at378C,the main peptide peaks for BAY55-9837,PACAP38,and VIP were remarkably diminished and the slower migrating peak emerged,probably as a result of peptide degradation.On
the other hand,DBAYL exhibited dramatic improvement
in stability,losing only7.7%of the main peak.The sta-
bility data in4weeks showed that the half-life of recom-
binant DBAYL was about25folds compared with that of
BAY55-9837in vitro,and the half-life of wild-type PACAP38and VIP is slightly shorter than the BAY55-
9837in vitro(Fig.3).
DBAY L lectively binding to VPAC2receptor
Human VPAC2receptor-transfected cells,and VPAC2-
CHO cells,were ud for competition receptor binding assay.Competition binding of[125I]PACAP38or[125I]VIP
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on membranes purified from CHO cells identified DBAYL as a VPAC2-lective peptide (Fig.4).DBAYL competi-tively displaced [125I]PACAP38from VPAC2,with a half-maximal inhibitory concentration (IC50)of 48.4+6.9nM,and the IC50of the recombinant PACAP38,VIP,and BAY55-9837were 18.1+5.3,21.2+4.0,and 68.3+8.1nM,respectively [Fig.4(A)].DBAYL competitively displaced [125I]VIP from VPAC2with an IC50of 47.1+4.9nM,and the IC50for rPACAP38,VIP,and BAY55-9837at human VPAC2were 19.7+4.9,18.0+2.6,and 70.3+3.7nM,respectively [Fig.4(B)].Whereas the IC50for VIP(1–7)/GRF(8–27),an established human VPAC1-specific agonist,at human VPAC2was over 20m M.The results showed that DBAYL could competi-tively displace [125I]PACAP38and [125I]VIP by binding to human VPAC2receptor in VPAC2-CHO cells.In two com-petition receptor-binding experiments,the IC50of DBAYL was significantly lower than that of BAY55-9837,the established VPAC2-specific agonist.
Receptor potency of DBAYL at PACAP receptors subtypes
The accumulation of cAMP in human PACAP receptor-transfected cells (VPAC1-CHO,VPAC2-CHO,and
PAC1-
break a legFigure 2The ESI-MS of the prepared recombinant DBAYL Prepared DBAYL at 1mg/ml in 45%acetonitrile containing 0.1%TFA was analyzed
by electrospray ionization time-of-flight mass序列号英语
spectrometry.
Figure 3Stability analysis of peptides at 1mg/ml in aqueous solution during incubation at 378C Sample (2-ml)was injected into HPLC-ESI-MS system and analyzed under the condition of increasing concentration of acetonitrile from 2%to 55%for 55min at 0.05ml/min.
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