CN106892975B - Polypeptide for regulating glycometabolism and application thereof - Google Patents
Polypeptide for regulating glycometabolism and application thereof Download PDFInfo
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Abstract
The present invention relates to polypeptides that modulate carbohydrate metabolism and their use in the manufacture of medicaments for the treatment of disorders associated with carbohydrate metabolism. The polypeptide can reduce blood sugar and promote insulin secretion; can be used for preparing medicaments for treating diabetes and can be applied in combination with the existing treatment scheme. The polypeptide of the invention has the obvious advantages of low effective treatment dose, oral administration and the like.
Description
Technical Field
The invention relates to the field of biological medicine, in particular to a polypeptide for regulating sugar metabolism and application thereof in preparing a medicament for treating diseases related to abnormal sugar metabolism.
Background
Diabetes is a major and growing health problem worldwide. Diabetes mellitus is a chronic disease of disorder of carbohydrate, fat and protein metabolism caused by relative or absolute deficiency of insulin in the body or decreased sensitivity of target cells to insulin, or structural defects of insulin itself. Diabetes is clinically manifested by polydipsia, polyphagia, polyuria and weight loss, accompanied by various complications such as ketoacidosis, gangrene of the extremities, polyneuritis, blindness and renal failure. Diabetes can be classified into type 1 and type 2 according to pathogenesis, with 95% of patients being type 2. Type 2 diabetes, also known as non-insulin dependent diabetes mellitus, is usually developed after age 35-40, and its ability to produce insulin in vivo is not completely lost or is increased in compensatory manner, but insulin resistance is produced in vivo, resulting in relative deficiency.
The existing method for treating the type 2 diabetes mellitus does not have a thorough cure method, and the treatment medicine is necessary to be accompanied for a lifetime and is accompanied with other side effects, so that the existing treatment of the diabetes mellitus has a great defect, and a new medicine with better performance needs to be developed.
Osteocalcin is a vitamin K-dependent calcium-binding protein synthesized and secreted by osteoblasts, a non-collagen acidic glycoprotein, in which the vitamin K-dependent glutamic acid residues in the molecule are osteocalcin and Ca2+Important functional groups for binding[1,2]。
Disclosure of Invention
The present inventors have surprisingly found a polypeptide derived from osteocalcin, which is capable of modulating carbohydrate metabolism; it has the ability to promote insulin secretion, and can maintain blood glucose homeostasis, especially when taken orally, with good results.
Some aspects of the invention relate to polypeptides that modulate carbohydrate metabolism and are represented by formula M1-Za-M2Is shown, in which:
M1、M2each independently is a polypeptide segment having no more than 5, 4, 3, 2, or 1 amino acid residue or is absent;
Zais Tyr-Leu-X1-X2-X3-X4-Gly-Ala-X5-X6-Pro-X7-Pro-Asp-X8-Leu-Glu-Pro, wherein:
X1is Tyr, Asn, Asp or is absent,
X2gln, Asn, His, Pro, Ser or absent,
X3is Trp, Gly or absent,
X4Is Leu or is absent,
X5is a Pro or a Ser,
X6is Ala or Val, or a pharmaceutically acceptable salt thereof,
X7is Tyr or Ser, and the amino acid is,
X8is a Thr or a Pro group,
and said Z isaOptionally having amino acid substitutions, insertions or deletions and said amino acid substitutions, insertionsAnd the total number of deletions is not more than 4, preferably not more than 3, more preferably not more than 2, most preferably not more than 1.
Further, in some embodiments of the invention, Z is in the polypeptideaSelected from one of the following:
SEQ ID NO.1：YLGASVPSPDPLEP，
SEQ ID NO.2：YLYQWLGAPVPYPDPLEP
SEQ ID NO.3：YLYQWLGAPVPYPDPLEPR，
SEQ ID NO.4：YLNNGLGAPAPYPDPLEP，
SEQ ID NO.5：YLYQWLGAPVPYPDTLEP，
SEQ ID NO.6：YLYQWLGAPVPYPDPLEP，
SEQ ID NO.7：YLDHWLGAPAPYPDPLEP，
SEQ ID NO.8：YLDPGLGAPAPYPDPLEP，
SEQ ID NO.9：YLDHGLGAPAPYPDPLEP，
SEQ ID NO.10：YLDQGLGAPAPAPDPLEP，
SEQ ID NO.11：YLDSGLGAPVPYPDPLEP。
further, in other embodiments of the present invention, M is present in the polypeptide1、 M2Are not present; or in yet other embodiments, Z is in the polypeptideaIs YLYQWLGAPVPYPDPLEP, M1Is absent and M2Arg, namely the amino acid sequence of the polypeptide is shown as SEQ ID NO. 3; in still other embodiments, Z is in the polypeptideaIs YLGASVPSPDPLEP, M1Is absent and M2Is Thr, namely the amino acid sequence of the polypeptide is shown as SEQ ID NO. 22.
Further aspects of the invention relate to a polypeptide that regulates sugar metabolism comprising at least 6 consecutive amino acids selected from any one of the following sequences and having a total number of amino acid residues of no more than 18, such as no more than 17, 16, 15, 14:
SEQ ID NO.1：YLGASVPSPDPLEP，
SEQ ID NO.2：YLYQWLGAPVPYPDPLEP
SEQ ID NO.3：YLYQWLGAPVPYPDPLEPR，
SEQ ID NO.4：YLNNGLGAPAPYPDPLEP，
SEQ ID NO.5：YLYQWLGAPVPYPDTLEP，
SEQ ID NO.6：YLYQWLGAPVPYPDPLEP，
SEQ ID NO.7：YLDHWLGAPAPYPDPLEP，
SEQ ID NO.8：YLDPGLGAPAPYPDPLEP，
SEQ ID NO.9：YLDHGLGAPAPYPDPLEP，
SEQ ID NO.10：YLDQGLGAPAPAPDPLEP，
SEQ ID NO.11：YLDSGLGAPVPYPDPLEP。
further, it comprises any of the following:
SEQ ID NO.12：PVPYPDPLEP，
SEQ ID NO.13：PYPDPLEP，
SEQ ID NO.14：PDPLEP，
SEQ ID NO.15：SVPSPDPLEP，
SEQ ID NO.16：PSPDPLEP。
further, it is SEQ ID No. 12: PVPYPDPLEP the flow of the air in the air conditioner,
SEQ ID NO.13：PYPDPLEP，
SEQ ID NO.14：PDPLEP，
SEQ ID NO.15：SVPSPDPLEP，
SEQ ID NO.16：PSPDPLEP。
Some aspects of the invention relate to pharmaceutically acceptable salts of the polypeptides of the invention.
Some aspects of the present invention relate to a polypeptide of the present invention, or a pharmaceutically acceptable salt thereof, having insulin level increasing, blood glucose lowering activity.
Some aspects of the invention relate to polynucleotides encoding the polypeptides of the invention.
Some aspects of the invention relate to a vector comprising the aforementioned polynucleotide.
Some aspects of the invention relate to host cells transfected with the aforementioned vectors and capable of producing the polypeptides of the invention under conditions in which the proteins can be expressed.
Some aspects of the invention relate to pharmaceutical compositions comprising a therapeutically effective amount of a polypeptide of the invention, or a pharmaceutically acceptable salt thereof, as described above.
Some aspects of the invention relate to the use of a polypeptide of the invention or a pharmaceutically acceptable salt or pharmaceutical composition thereof for the manufacture of a medicament for the treatment of a disease associated with abnormal glucose metabolism, which disease may benefit from elevated insulin, reduced blood glucose, preferably type 2 diabetes, insulin resistance, hyperglycemia.
Some aspects of the present invention relate to a method for treating a disease associated with abnormal carbohydrate metabolism, comprising administering to a subject in need thereof a therapeutically effective amount of a polypeptide of the present invention or a pharmaceutically acceptable salt or pharmaceutical composition thereof.
Some aspects of the present invention relate to a method for the treatment of a disease which may benefit from elevated insulin, reduced blood glucose, preferably type 2 diabetes, comprising administering to a subject in need thereof a therapeutically effective amount of a polypeptide of the present invention, or a pharmaceutically acceptable salt or pharmaceutical composition thereof.
In some embodiments, the polypeptide of the invention or a pharmaceutically acceptable salt or pharmaceutical composition thereof may be used for the treatment of diseases associated with abnormal carbohydrate metabolism, preferably diseases benefiting from elevated insulin, reduced blood glucose, most preferably type 2 diabetes.
In some embodiments, the pharmaceutical compositions of the present invention may be administered with any of the pharmaceutical and method compositions known to be useful for treating diseases associated with abnormal carbohydrate and fat metabolism.
In some embodiments, the polypeptide of the present invention or a pharmaceutically acceptable salt or pharmaceutical composition thereof may be administered by various conventional means, preferably orally. The oral administration reduces a plurality of inconveniences of the conventional polypeptide administration, and the polypeptide of the invention has few residues, greatly reduces the cost and has very remarkable potential advantages as a medicament.
Drawings
FIG. 1 shows demonstration of ISAP in vitro experiments1Can promote beta TC-6 cells to express more insulin mRNA, and the expression of the insulin mRNA is analyzed by real-time quantitative PCR. β TC-6 cells were stimulated with different concentrations of the polypeptide for 1 hour after starvation for 4 hours in a sugar-free medium containing 1% FBS prior to stimulation with the polypeptide. All experiments were performed in two parallel groups and repeated 3 times. As a result: mean value of +Standard error of P<0.05,**:P<0.01(t test)
FIG. 2 shows an ISAP1Can promote the islet to express more insulin mRNA and analyze the expression of the insulin mRNA by real-time quantitative PCR. Islets were starved for 4 hours in a sugar-free medium containing 1% FBS prior to polypeptide stimulation and stimulated with different concentrations of polypeptide for 1 hour. All experiments were performed in two parallel groups and repeated 3 times. As a result: mean. + -. standard error of P<0.05, **:P<0.01(t test)
FIG. 3 shows ISAP in a glucose tolerance assay1Results in a decrease in blood glucose levels in the mouse; (A) trend plots for each time point, (B) area under the curve comparison of results in a.
FIG. 4 shows intraperitoneal injection of ISAP1Effect on fasting plasma glucose in obese-T2 DM mice. P<0.05,**P<0.01; compared to the HFD + vehicle group.
FIG. 5 shows intraperitoneal injection of ISAP1Effect on serum insulin levels in obese-T2 DM mice. P<0.05,**P<0.01
FIG. 6 shows via ISAP1OCN (mouse osteocalcin) treated T2DM mouse islets were enlarged. Quantitative statistics of A-E, ISAP are shown in F1Both OCN and OCN promote the increase of islet area. ISAP1Can remarkably recover the size of pancreatic islets in the pancreas of obese-T2 DM mice. (A) Feeding normal feed (ND), high fat feed (HFD) and HFD plus daily intraperitoneal injection of (C) OCN (6pmol/g/D) or (D) ISAP1(20pmol/g/d) or (E) ISAP 1(2pmol/g/d), (F) islet area in serial sections calculated under double-blind conditions. The magnification is 40X. P<0.05,**:P<0.01,***:P<0.001, compared to HFD + vehicle group; each group had 6 mice.
FIG. 7 shows intragastric administration of OCN, ISAP1、ISAP2ISAP3 effect on blood glucose levels in mice fed High Fat (HFD).
FIG. 8 shows an ISAP1And ISAP2Binding to human GPRC6A and internalization thereof, respectively. A) Human GPRC6A was overexpressed in Hela cells; (B) ISAP1OCN and OCN-22; and ISAP2hOCN and hOCN-22; binding to cell membrane of HeLa cell overexpressed by huGPRC6ATesting; (C) OCN labeled with Cy-5 (Cy5-OCN), OCN-22 labeled with Cy-5 (Cy5-Ocn22), ISAP labeled with Cy-52(Cy5-ISAP2) Effect on GPRC6A internalization in Hela cells overexpressing GPRC 6A.
FIG. 9 shows intragastric administration of ISAP1、ISAP4、ISAP5、ISAP6Effect on blood glucose in mice fed high fat diet: (A) effect on blood glucose, (B) effect on GLP 1.
Figure 10 shows a comparison of the sequences of ISAPs from different species.
Detailed Description
Definition of
The "polypeptide capable of regulating sugar metabolism" of the present invention is also referred to as "Insulin Secretion Association Peptide (ISAP)" and refers to a polypeptide derived from osteocalcin or derived therefrom and capable of regulating sugar metabolism.
The term "conservative amino acid substitution" as used herein refers to a substitution of the original amino acid sequence with another amino acid residue having similar properties. For example, lysine residues, arginine residues, and histidine residues are similar in having basic side chains. In addition, aspartic acid residues and glutamic acid residues are similar in having acidic side chains. Further, asparagine residues, glutamine residues, serine residues, threonine residues, tyrosine residues, and cysteine residues are similar in having uncharged polar side chains, and glycine residues, alanine residues, valine residues, leucine residues, isoleucine residues, proline residues, tryptophan residues, phenylalanine residues, and methionine residues are similar in having nonpolar side chains. In addition, tyrosine residues, phenylalanine residues, tryptophan residues, and histidine residues are similar in having aromatic side chains. Thus, it will be apparent to those skilled in the art that amino acid substitutions made in a group of amino acids having similar properties as described above do not result in any change in the properties.
The amino acid abbreviations and Chinese names used herein are as follows
| Name of Chinese | Three letters shorthand | Single letter shorthand |
| Serine | Ser | S |
| Threonine | Thr | T |
| Asparagine | Asn | N |
| Glutamine | Gln | Q |
| Tyrosine | Tyr | Y |
| Cysteine | Cys | C |
| Aspartic acid | Asp | D |
| Glutamic acid | Glu | E |
| Histidine | His | H |
| Lysine | Lys | K |
| Arginine | Arg | R |
| Glycine | Gly | G |
| Alanine | Ala | A |
| Valine | Val | V |
| Leucine | Leu | L |
| Isoleucine | Ile | I |
| Phenylalanine | Phe | F |
| Methionine | Met | M |
| Proline | Pro | P |
| Tryptophan | Trp | W |
The term "disease associated with abnormal glucose metabolism" as used herein refers to a disease characterized by disturbance of glucose metabolism or its complications, such as but not limited to type 2 diabetes, hyperglycemia, insulin resistance, etc., which are genetically or environmentally or both.
Detailed Description
DMEM medium was purchased from Sigma supplemented with 10% FBS (fetal bovine serum), 1% non-essential amino acids, 1g glucose, 0.75g sodium bicarbonate, 0.1g bovine serum albumin and 1.5ml HEPES (4-hydroxyethylpiperazineethanesulfonic acid) per 500ml of medium.
ISAP1The sequence is Tyr-Leu-Gly-Ala-Ser-Val-Pro-Ser-Pro-Asp-Pro-Leu-Glu-Pro-Thr (SEQ ID NO. 22). ISAP2The sequence of (a) is Tyr-Leu-Tyr-Gln-Trp-Leu-Gly-Ala-Ser-Val-Pro-Ser-Pro-Asp-Pro-Leu-Glu-Pro (SEQ ID NO. 2). ISAP3The sequence of (a) is Tyr-Leu-Tyr-Gln-Trp-Leu-Gly-Ala-Ser-Val-Pro-Ser-Pro-Asp-Pro-Leu-Glu-Pro-Arg (SEQ ID NO. 3). ISAP 4The sequence is Ser-Val-Pro-Ser-Pro-Asp-Pro-Leu-Glu-Pro (SEQ ID NO. 15). ISAP5The sequence is Pro-Ser-Pro-Asp-Pro-Leu-Glu-Pro (SEQ ID NO. 16). ISAP6The sequence is Pro-Asp-Pro-Leu-Glu-Pro (SEQ ID NO. 14).
Beta TC-6 was purchased from American Type Culture Collection (ATCC), and this cell line was derived from mouse pancreatic cancer, and has a function of secreting insulin by introducing the large T antigen of SV40 (monkey vacuolating virus 40) into its genome.
All animal experiments were approved by the animal ethics committee of the advanced technology research institute of the chinese academy of sciences, and were performed according to the requirements of the ethics committee.
Example 1
ISAP1Function of (2)
1、ISAP1Effects on β TC-6 cells.
The experimental procedure was as follows:
1. cell plating: suspending beta TC-6 cells at 1.6X 105Per mL density was plated in 24 well plates at 0.5mL per well, 5% CO at 37 ℃2And culturing for 24 hours.
2. ISAP with sugar-free DMEM1The resulting solution was diluted to 2nmol/L, 200pmol/L, 20pmol/L and 2pmol/L, respectively, and stored in an ice bath.
3. Starvation pretreatment of beta TC-6 cells with KRBB buffer (129g NaCl,5g NaHCO)3,4.8g KCl,1.2g KH2PO4,1.2g MgSO4,2.5g CaCl210mM HEPES and 0.1% BSA at pH 7.4) for 4 hours.
4. Discarding the cell culture medium, adding ISAP containing different concentrations into the experimental group and the positive control group respectively 1And glucose, negative control group with medium (blank) added only, 5% CO at 37 deg.C2The reaction lasts for 1 hour.
5. The plate bottom cells were washed twice with sterile PBS, 300. mu.L of Trizol solution was added, cellular RNA was extracted according to the instructions of RNAioso Plus (TaKaRa), treated with DNase, and then SuperScript was addedT(Invitrogen, Canada) kit was reverse transcribed into cDNA by DNAEngine. The fluorescent PCR products at each time point were subjected to real-time monitoring analysis (Light Cycler Roche, Germany) using cDNA as a template by the SYBRGreen method to detect the expression level of the insulin gene.
Primers used for real-time quantitative PCR, murine Ins-1:
in the forward direction 5'-CCAGCTATAATCAGAGACCA-3' of the direction,
and reverse direction 5'-CCAGGTGGGGACCACAAAGA-3'.
GAPDH as internal reference
Positive direction 5'-AGCAGTCCCGTACACTGGCAAAC-3'
Reverse 5 '-TCTGTGGTGATGTAAATGTCCTCT-3'.
The results are shown in FIG. 1, different concentrations of ISAP1Can promote the beta TC-6 cells to express more insulin mRNA.
2、ISAP1The effect on the islets of Langerhans.
Primary islets were isolated from mice, starved for pretreatment, using KRBB buffer (129g NaCl,5g NaHCO) according to the method described in Gregory L.Szot et al, Murine Pancratic Islet Isolation, Journal of Visualized Experiments, 2007 3,4.8g KCl,1.2g KH2PO4,1.2g MgSO4,2.5g CaCl210mM HEPES and 0.1% BSA at pH 7.4) for 4 hours. KRBB solution was discarded, DMEM medium containing 16.8mM glucose was added to the positive control group, and ISAP was added to the experimental group at different concentrations1Sugar-free DMEM culture solution, negative control group only added sugar-free DMEM culture medium (blank), at 37 deg.C, 5% CO2The reaction lasts for 1 hour. The experimental results are shown in FIG. 2, ISAP of different concentrations1All promote the islets to express more insulin mRNA.
3. ISAP in vivo experiments1Influence on sugar metabolism.
3.1 establishment of type 2 diabetes model by high fat feeding
Healthy 6-week-old male C57BL/6 SPF-rated mice were purchased from the center of laboratory animals, Guangdong province, with a body mass of 18-22 g. Divided into two groups, and the two groups are bred in SPF animal houses of the research institute. Control group: mice were given Normal Diet (ND) (normal diet: fat, 5%; carbohydrate, 53%; protein, 23%; total calories 25J/g), ad libitum and water, and fed for 12 weeks. Experimental groups: mice were given High Fat Diet (HFD) (high fat diet D12451, Research Diets, Inc.) and were fed free to eat and drink water for 12 weeks. Detecting the physiological index. Mice fed with high-fat diet weighed more than 40g, and the type 2 diabetes model (T2DM) was considered to be successfully constructed.
3.2 Metabolic assay detection
Glucose Tolerance Tests (GTT) T2DM mice were fasted for 16 hours and injected with saline, ISAP, and 20% Glucose solution intraperitoneally immediately 1 minute after the injection1Or OCN, for 10 min, 30 min, 60 min, 90 min. ISAP1And OCN has a short-term effect in T2DM mice. The results of the experiment are shown in FIG. 3, OCN and ISAP1Blood glucose levels were reduced at each time point compared to the control group (saline injection). Proves the ISAP1Reducing blood sugar.
3.3 Long term administration of ISAP1Influence on sugar metabolism
T2DM mice were used, divided into 5 groups of 6 mice each. Adopting corresponding feed and application mode; first, normal feed (ND) + vehicle; second group, High Fat Diet (HFD) + vehicle; third, HFD + OCN6pmol/g/d + vehicle; fourth group, HFD + ISAP120pmol/g/d + vehicle; fifth group, HFD + ISAP12pmol/g/d + vehicle; d represents daily intraperitoneal injections. Blood glucose levels were measured at weeks 0, 2, and 4, respectively, and approximately 10 microliters of blood was collected from the tip of the tail and measured using a roche glucometer according to the manufacturer's instructions. Results referring to fig. 4, blood glucose levels were reduced compared to the control group (second group). The Ultra Sensitive Mouse Insulin ELISA Kit (cytostalchem, USA) and Multiskan were used at weeks 0 and 6 TMFC microplate reader (Thermo, usa) measures insulin levels in serum. Results see FIG. 5, ISAP1Improves the level of insulin in the serum.
3.4 histomorphometry
After completion of the experiment of step 3.3, mice were euthanized using 90% carbon dioxide, the pancreas was removed, small pieces of tissue were collected, fixed in 4% paraformaldehyde solution for 24 hours, paraffin embedded and sectioned at 5 μm thickness (Leica RM2235, Germany), after which the tissue was stained with hematoxylin and eosin (C0105, Beyotime, china) and observed. Experimental results referring to fig. 6, using 40X magnification, a-E are on the same scale; f is to A toAnd E, quantitative analysis. Compared with the control, the islet area of the experimental group is obviously increased, and the cell volume is not obviously changed, which indicates that the ISAP1Can promote islet proliferation.
4、ISAP1Binding to human GPRC6A
4.1 overexpression of hggprc 6A 1 in Hela cells) cell plating: hela cell suspension at 1.6X 105Per mL density plated in 6-well plates, 2mL DMEM medium per well, 5% CO at 37 ℃2And culturing for 24 hours.
2) The hGPRC6A overexpression vector (pReceiver-M61) was transfected into Hela cells using Lipofectamine2000(Invitrogen) according to the manufacturer's instructions and the normal medium was changed 4 hours after transfection.
3) 48 hours after transfection, the medium was discarded, the plate-bottom cells were washed twice with sterile PBS, 300. mu.L of Trizol solution was added, cellular RNA was extracted according to the instructions of RNAioso Plus (TaKaRa), treated with DNase, and then SuperScript was addedT(Invitrogen, Canada) kit was reverse transcribed into cDNA by DNAEngine. The fluorescent PCR products at each time point were subjected to real-time monitoring analysis (Light Cycler Roche, Germany) using cDNA as a template to detect the expression level of hGPRC 6A. If necessary, puromycin is used for screening to obtain a stable expression cell strain, and then qPCR is used for detecting the gene expression of the stable expression cell strain, the experimental result is shown in figure 6A, the experimental result is shown in figure 8A for stable large-scale expression of human GPRC6A in Hela, and the experimental result is shown in figure 6A for stable large-scale expression of human GPRC6A in Hela.
4.2 ISAP1Binding experiments with cell membranes of HeLa cells overexpressing hGPRC6A,
the results are shown in FIG. 8B, ISAP compared to OCN1Has almost uniform membrane binding capacity, while OCN-22 does not have this capacity, demonstrating ISAP1Is the core domain of OCN, which interacts with the receptor hGPRC 6A.
5. Intragastric administration of ISAP1Effect on blood glucose levels in high fat fed (HFD) mice
Healthy 6-week-old male C57BL/6 mice, with a body mass of 18-22g, divided into 6 groups of 5 mice each, the first group, Normal Diet (ND); second group, high fat diet (H) FD); third, HFD + OCN 2 pmol/g; fourth group, HFD + ISAP 12 pmol/g; fifth group, HFD + ISAP 22 pmol/g; sixth group, HFD + ISAP 32 pmol/g; wherein OCN and ISAP1、ISAP2、 ISAP3The polypeptides were dissolved in physiological saline using a 1ml syringe and a 12-gauge needle, administered daily by a gavage method well known to those skilled in the art, with a gavage volume of 10 μ L/g.
After four weeks, approximately 10 microliters of blood was collected from the tip of the tail and measured using a roche glucometer according to the manufacturer's instructions. Results see FIG. 7, ISAP compared to control1Has blood sugar lowering effect.
Example 2
ISAP2Function ISAP of2Binding to human GPRC6A
2.1 overexpression of hggprc 6A 1 in Hela cells) cell plating: hela cell suspension at 1.6X 105Per mL density plated in 6-well plates, 2mL DMEM medium per well, 5% CO at 37 ℃2And culturing for 24 hours.
2) The hGPRC6 overexpression vector (pReceiver-M61) was transfected into Hela cells using Lipofectamine2000(Invitrogen) according to the manufacturer's instructions and the normal medium was changed 4 hours after transfection.
3) 48 hours after transfection, the medium was discarded, the plate-bottom cells were washed twice with sterile PBS, 300. mu.L of Trizol solution was added, cellular RNA was extracted according to the instructions of RNAioso Plus (TaKaRa), treated with DNase, and then SuperScript was added T(Invitrogen, Canada) kit was reverse transcribed into cDNA by DNAEngine. Using cDNA as template and SYBRGreeThe fluorescent PCR products at each time point were analyzed by real-time monitoring (Light Cycler Roche, Germany) by the n method to detect the expression level of hGPRC 6A. If necessary, puromycin is used for screening to obtain a stable expression cell strain, and then qPCR is used for detecting the gene expression of the stable expression cell strain, the experimental result is shown in figure 6A, the experimental result is shown in figure 8A for stable large-scale expression of human GPRC6A in Hela, and the experimental result is shown in figure 6A for stable large-scale expression of human GPRC6A in Hela.
2.2ISAP2Binding to cell membrane of Hela cell overexpressing hGPRC6ATest (experiment)
The results are shown in FIG. 8B, ISAP compared to hOCN2Having almost uniform membrane binding capacity, while hOCN-22 did not, combined with the results of item 4.2 in example 1, demonstrate ISAP1And ISAP2The core domains of OCN and hOCN, respectively, interact with the receptor hGPRC6A, thus presuming ISAP1And ISAP2With the same function, subsequent signaling and biological events are caused by hgrc 6A.
2.3 OCN, hOCN-22, ISAP labeled with Cy52Internalization of GPRC6A was promoted in Hela cells overexpressing GPRC 6A.
The hGPRC6A-Hela expression cell suspension is added in a proportion of 1.6X 10 5Per mL density was plated in 24-well plates pre-loaded with gelatin-coated coverslips, 0.5mL DMEM per well, 5% CO at 37 ℃2And culturing for 24 hours. Before treatment, cells were starved for 4 hours in serum-free medium, and 100nM of Cy5-OCN, Cy5-hOCN22, Cy5-ISAP was added to each well2Incubation at 37 ℃ for 30 min, fixation of cells using poly-methanol for 30 min, and incubation with Triton X-100 (sigma) for 10 min, DAPI (Sigma) for 10 sec according to the manufacturer's instructions, cells and staining, visualization using a fluorescence confocal microscope and photography, see FIG. 6C for experimental results. As can be seen, Cy5-OCN and Cy5-ISAP2The distribution inside the cells was whereas Cy5-hOCN22 was distributed extracellularly, again illustrating Cy5-OCN and Cy5-ISAP2Can bind to the receptor and enter into the cell through internalization, thereby playing a role in regulating energy metabolism.
2.4 intragastric administration of ISAP2Effect on blood glucose levels in high fat fed (HFD) mice
[01]Healthy 6-week-old male C57BL/6 mice, with a body mass of 18-22g, divided into 6 groups of 5 mice each, the first group, Normal Diet (ND); second group, High Fat Diet (HFD); third, HFD + OCN2 pmol/g; fourth group, HFD + ISAP 12 pmol/g; fifth group, HFD + ISAP 22 pmol/g; sixth group, HFD + ISAP 32 pmol/g; wherein OCN and ISAP1、ISAP2、 ISAP3Administered daily by a gavage method well known to those skilled in the art such thatThe polypeptide was dissolved in physiological saline using a 1ml syringe and a 12-gauge needle, and the gavage volume was 10. mu.L/g.
[02]After four weeks, approximately 10 microliters of blood was collected from the tip of the tail and measured using a roche glucometer according to the manufacturer's instructions. Results see FIG. 7, ISAP compared to control2Has blood sugar lowering effect.
Example 3
ISAP3Functional intragastric administration of ISAP3Effect on blood glucose levels in high fat fed (HFD) mice
Healthy 6-week-old male C57BL/6 mice, with a body mass of 18-22g, divided into 6 groups of 5 mice each, the first group, Normal Diet (ND); second group, High Fat Diet (HFD); third, HFD + OCN 2 pmol/g; fourth group, HFD + ISAP 12 pmol/g; fifth group, HFD + ISAP 22 pmol/g; sixth group, HFD + ISAP 32 pmol/g; wherein OCN and ISAP1、ISAP2、 ISAP3The polypeptides were dissolved in physiological saline using a 1ml syringe and a 12-gauge needle, administered daily by a gavage method well known to those skilled in the art, with a gavage volume of 10 μ L/g.
After four weeks, approximately 10 microliters of blood was collected from the tip of the tail and measured using a roche glucometer according to the manufacturer's instructions. Results see FIG. 7, ISAP compared to control 3Has blood sugar lowering effect.
Combining the results of the experiments of examples 1 and 2, ISAP was compared1、ISAP2、ISAP3The sequence of (A) can be known: with respect to ISAP1,ISAP2Has 4 insertions, 2 substitutions, and 1 deletion; with respect to ISAP1，ISAP3With 4 insertions, 3 substitutions; with respect to ISAP2，ISAP1Has 4 deletions, 2 substitutions, and 1 insertion; in summary, it is assumed that the three polypeptides are variants of each other, and that conservative amino acid substitutions, insertions and deletions, which are well known to those skilled in the art, can be made on the basis of these three sequences, provided that their ability to regulate sugar metabolism is not significantly reduced, for example by not more than 40%, 30%, 20%, 10%. See FIG. 10 for various biological sourcesWherein the sequence of SEQ ID NO. 2: YLYQWLGAPVPYPDPLEP, SEQ ID NO. 4: YLNNGLGAPAPYPDPLEP, SEQ ID NO. 5: YLYQWLGAPVPYPDTLEP, SEQ ID NO. 6: YLYQWLGAPVPYPDPLEP, SEQ ID NO. 7: YLDHWLGAPAPYPDPLEP, SEQ ID NO. 8: YLDPGLGAPAPYPDPLEP, SEQ ID NO. 9: YLDHGLGAPAPYPDPLEP, SEQ ID NO. 10: YLDQGLGAPAPAPDPLEP, SEQ ID NO. 11: YLDSGLGAPVPYPDPLEP, which in most cases do not differ by more than four amino acid substitutions when compared in pairs, and thus it is assumed that these sequences have similar biological functions and are also encompassed by the present invention, preferably wherein the total number of deletions, substitutions and insertions does not exceed 4, e.g., 3, 2, 1. In addition, SEQ ID NO.1 has only one amino acid residue deleted compared to SEQ ID NO.22, and thus it is inferred that SEQ ID NO.1 also has a function similar to SEQ ID NO. 22.
Meanwhile, combining the experimental results of examples 2 and 3, with respect to ISAP2，ISAP1And ISAP3Equivalent to in ISAP2The end has 1 insertion; it is shown that several amino acid residues can be added at the end of a polypeptide, provided that its ability to regulate sugar metabolism is not significantly reduced, e.g. by not more than 40%, 30%, 20%, 10%, preferably by not more than 5, e.g. 4, 3, 2, 1 or 0 amino acid residues at the end.
Example 4
Similar to the method described in example 1, 18 healthy 6 week old male C57BL/6SPF grade mice were selected and divided into 6 groups of 3 mice each. One group was a control group, and mice were given Normal Diet (ND); the remaining 5 groups were given High Fat Diet (HFD), free to ingest and drink, and the following experiments were performed after the high fat diet-fed mice exceeded 40g in weight:
groups 3-6 mice (HFD) were separately gavaged with ISAP daily at 2pmol/g body weight1、ISAP4、ISAP5、ISAP6(ii) a Group 1 mice (ND) and group 2 mice (HFD) were separately gavaged with equal volumes of saline. The procedure was continued for 7 weeks, about 10 microliters of blood was collected from the tip of the tail weekly, blood glucose levels were monitored once using a Roche glucometer, and the results of the experiment, referring to FIG. 9A, were administered ISAP by gavage1、ISAP4、ISAP5、ISAP6All resulted in a significant reduction in blood glucose levels.
Meanwhile, the level of GLP1 in blood was measured using an antibody against GLP1 after the experiment was completed, and the experimental results are shown in fig. 9B. ISAP compared to two control groups1、 ISAP4、ISAP5、ISAP6All resulted in a substantial increase in GLP1 levels. Prove them and ISAP1、ISAP2、ISAP3Biologically functional equivalence, and, with reference to the sequence alignment of FIG. 10, it can also be seen that the sequence of SEQ ID NO. 12: PVPYPDPLEP and SEQ ID NO. 15: SVPSPDPLEP, SEQ ID NO. 13: PYPDPLEP to SEQ ID NO. 16: the PSPDPLEP has a very high similarity in sequence and is also a very conserved sequence between species, and thus should have similar biological activity, and should be included in the scope of the present invention.
It is well known to those skilled in the art that the length of the polypeptide is closely related to the length of the synthesis, and that the weight is 4mg, 6 amino acid residue polypeptide (e.g., ISAP) for the commercial value of this example6) The composite price is 225 yuan (RMB); 8 amino acid residue polypeptides (e.g., ISAP5) The composite price is 300 yuan (RMB); 10 amino acid residue polypeptides (e.g., ISAP4) The composite price is 375 yuan (RMB); 15 amino acid residue polypeptide (e.g., ISAP)1) Composite price 562 yuan (rmb); polypeptide of 46 amino acid residues (e.g. mouse OCN) synthesis price 1675 yuan (rmb); equimolar ISAP taking into account the difference in molecular weight 6The price of the mouse OCN is about one sixtieth of that of the mouse OCN, the cost is greatly reduced, and the mouse OCN is very suitable for large-scale manufacturing and use.
Meanwhile, the polypeptide of the invention can play a role after being taken orally, and compared with the majority of polypeptides which are applied by injection in the prior art, the polypeptide is greatly convenient for patients, and the compliance of the patients is improved, so the polypeptide has huge application prospect in the field of medicine.
Is incorporated by reference
All publications and patents mentioned herein are incorporated by reference in their entirety to the same extent as if each individual publication or patent was specifically and individually indicated to be incorporated by reference. In case of conflict, the present application, including any definitions herein, will control.
Equivalent scheme
While specific embodiments of the invention have been specifically disclosed herein, the foregoing description is intended to be illustrative and not limiting. Many variations of the invention will become apparent to those skilled in the art upon review of this specification and the appended claims. The full scope of the invention should be determined by reference to the claims, their full scope of equivalents, and this specification and such variations.
Sequence listing
<110> Shenzhen advanced technology research institute
<120> polypeptide for regulating sugar metabolism and use thereof
<160> 22
<170> PatentIn version 3.3
<210> 1
<211> 15
<212> PRT
<213> mouse (Mus musculus)
<400> 1
Tyr Leu Gly Ala Ser Val Pro Ser Pro Asp Pro Leu Glu Pro
1 5 10
<210> 2
<211> 18
<212> PRT
<213> human (Homo sapiens)
<400> 2
Tyr Leu Tyr Gln Trp Leu Gly Ala Pro Val Pro Tyr Pro Asp Pro Leu
1 5 10 15
Glu Pro
<210> 3
<211> 19
<212> PRT
<213> Artificial sequence
<220>
<223> ISAP3
<400> 3
Tyr Leu Tyr Gln Trp Leu Gly Ala Pro Val Pro Tyr Pro Asp Pro Leu
1 5 10 15
Glu Pro Arg
<210> 4
<211> 18
<212> PRT
<213> rat (Rattus norvegicus)
<400> 4
Tyr Leu Asn Asn Gly Leu Gly Ala Pro Ala Pro Tyr Pro Asp Pro Leu
1 5 10 15
Glu Pro
<210> 5
<211> 18
<212> PRT
<213> bonobo (Pan trogloytes)
<400> 5
Tyr Leu Tyr Gln Trp Leu Gly Ala Pro Val Pro Tyr Pro Asp Thr Leu
1 5 10 15
Glu Pro
<210> 6
<211> 18
<212> PRT
<213> Kiwi berry (Macaca mulatta)
<400> 6
Tyr Leu Tyr Gln Trp Leu Gly Ala Pro Val Pro Tyr Pro Asp Pro Leu
1 5 10 15
Glu Pro
<210> 7
<211> 18
<212> PRT
<213> cattle (Bos taurus)
<400> 7
Tyr Leu Asp His Trp Leu Gly Ala Pro Ala Pro Tyr Pro Asp Pro Leu
1 5 10 15
Glu Pro
<210> 8
<211> 18
<212> PRT
<213> sheep (Ovis aries)
<400> 8
Tyr Leu Asp Pro Gly Leu Gly Ala Pro Ala Pro Tyr Pro Asp Pro Leu
1 5 10 15
Glu Pro Arg
<210> 9
<211> 18
<212> PRT
<213> wild boar (Sus scrofa)
<400> 9
Tyr Leu Asp His Gly Leu Gly Ala Pro Ala Pro Tyr Pro Asp Pro Leu
1 5 10 15
Glu Pro
<210> 10
<211> 18
<212> PRT
<213> nudease zokor (heterocarphalus glaber)
<400> 10
Tyr Leu Asp Gln Gly Leu Gly Ala Pro Ala Pro Ala Pro Asp Pro Leu
1 5 10 15
Glu Pro
<210> 11
<211> 18
<212> PRT
<213> dog (Canis lupus)
<400> 11
Tyr Leu Asp Ser Gly Leu Gly Ala Pro Val Pro Tyr Pro Asp Pro Leu
1 5 10 15
Glu Pro
<210> 12
<211> 10
<212> PRT
<213> human
<400> 12
Pro Val Pro Tyr Pro Asp Pro Leu Glu Pro
1 5 10
<210> 13
<211> 8
<212> PRT
<213> human
<400> 13
Pro Tyr Pro Asp Pro Leu Glu Pro
1 5
<210> 14
<211> 6
<212> PRT
<213> Artificial sequence
<220>
<223> IASP6
<400> 14
Pro Asp Pro Leu Glu Pro
1 5
<210> 15
<211> 10
<212> PRT
<213> mice
<400> 15
Ser Val Pro Ser Pro Asp Pro Leu Glu Pro
1 5 10
<210> 16
<211> 8
<212> PRT
<213> mice
<400> 16
Pro Ser Pro Asp Pro Leu Glu Pro
1 5
<210> 17
<211> 20
<212> DNA
<213> Artificial sequence
<220>
<223> insulin PCR Forward primer
<400> 17
<210> 18
<211> 20
<212> DNA
<213> Artificial sequence
<220>
<223> insulin PCR reverse primer
<400> 18
<210> 19
<211> 23
<212> DNA
<213> Artificial sequence
<220>
<223> GAPDH forward primer
<400> 19
agcagtcccg tacactggca aac 23
<210> 20
<211> 24
<212> DNA
<213> Artificial sequence
<220>
<223> GAPDH reverse primer
<400> 20
tctgtggtga tgtaaatgtc ctct 24
<210> 21
<211> 18
<212> PRT
<213> Artificial sequence
<220>
<223> a general formula of ISAP
<220>
<221> misc_feature
<222> (3)..(3)
<223> Xaa is Tyr, Asn, Asp or absent
<220>
<221> misc_feature
<222> (4)..(4)
<223> Xaa is Gln, Asn, His, Pro, Ser or absent
<220>
<221> misc_feature
<222> (5)..(5)
<223> Xaa is Trp, Gly or absent
<220>
<221> misc_feature
<222> (6)..(6)
<223> Xaa is Leu or absent
<220>
<221> misc_feature
<222> (9)..(9)
<223> Xaa is Pro or Ser
<220>
<221> misc_feature
<222> (10)..(10)
<223> Xaa is Ala or Val
<220>
<221> misc_feature
<222> (10)..(10)
<223> Xaa is Tyr or Ser
<220>
<221> misc_feature
<222> (10)..(10)
<223> is Thr or Pro
<400> 21
Tyr Leu Xaa Xaa Xaa Xaa Gly Ala Xaa Xaa Pro Xaa Pro Asp Xaa Leu
1 5 10 15
Glu Pro
<210> 22
<211> 15
<212> PRT
<213> mouse (Mus musculus)
<400> 22
Tyr Leu Gly Ala Ser Val Pro Ser Pro Asp Pro Leu Glu Pro Thr
1 5 10 15
Claims (9)
1. A polypeptide that modulates sugar metabolism, the polypeptide being represented by formula Za,
Za is selected from one of the following:
SEQ ID NO.1：YLGASVPSPDPLEP，
SEQ ID NO.4：YLNNGLGAPAPYPDPLEP，
SEQ ID NO.5：YLYQWLGAPVPYPDTLEP，
SEQ ID NO.7：YLDHWLGAPAPYPDPLEP，
SEQ ID NO.8：YLDPGLGAPAPYPDPLEP，
SEQ ID NO.9：YLDHGLGAPAPYPDPLEP，
SEQ ID NO.10：YLDQGLGAPAPAPDPLEP，
SEQ ID NO.11：YLDSGLGAPVPYPDPLEP。
2. a pharmaceutically acceptable salt of the polypeptide of claim 1.
3. A polynucleotide encoding the polypeptide of claim 1.
4. A vector comprising the polynucleotide of claim 3.
5. A host cell transfected with the vector of claim 4 and capable of producing the polypeptide of claim 1 under conditions in which the protein is expressible.
6. A pharmaceutical composition comprising a therapeutically effective amount of the polypeptide of claim 1, or a pharmaceutically acceptable salt of claim 2.
7. Use of the polypeptide of claim 1 or the pharmaceutically acceptable salt of claim 2 or the pharmaceutical composition of claim 6 in the manufacture of a medicament for the treatment of a disease associated with abnormal glucose metabolism, wherein the disease is a disease that would benefit from elevated insulin and reduced blood glucose.
8. The use according to claim 7, wherein the disease may be type 2 diabetes, insulin resistance, hyperglycemia.
9. The polypeptide of claim 1 or the pharmaceutically acceptable salt of claim 2 or the pharmaceutical composition of claim 6, characterized by being orally administrable.
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| WO2018165933A1 (en) * | 2017-03-16 | 2018-09-20 | 深圳先进技术研究院 | Polypeptide for regulating saccharometabolism and uses thereof |
| KR102432962B1 (en) | 2017-03-16 | 2022-08-12 | 쉔첸 인스티튜트스 오브 어드밴스드 테크놀로지 | Polypeptides modulating energy metabolism and uses thereof |
| CN109957006B (en) * | 2017-12-22 | 2021-04-20 | 深圳先进技术研究院 | Metabolin polypeptide artificial antigen, antibody and application |
| CN109957005B (en) * | 2017-12-22 | 2021-04-20 | 深圳先进技术研究院 | Metabolin polypeptide artificial antigen, preparation method thereof, antibody and application |
| CN114288414A (en) * | 2021-12-07 | 2022-04-08 | 深圳先进技术研究院 | Polypeptide crossing blood brain barrier, derivative and application thereof |
| CN117050142A (en) * | 2022-05-05 | 2023-11-14 | 中国科学院深圳先进技术研究院 | Ovarian targeting polypeptide, derivative and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1324831A (en) * | 2000-05-19 | 2001-12-05 | 上海博德基因开发有限公司 | New polypeptide human non-insulin dependent diabetes related protein 25 and polynucleotides for encoding same |
| CN102647995A (en) * | 2009-06-10 | 2012-08-22 | 奥胡斯大学 | SorCS1-like agent for use in the treatment of insulin resistance and diseases related thereto |
| CN103554249A (en) * | 2013-10-25 | 2014-02-05 | 中国科学院深圳先进技术研究院 | Complete antigen of AG15 polypeptide as well as preparation method and antibody thereof |
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| CN101610780A (en) * | 2006-09-13 | 2009-12-23 | 纽约市哥伦比亚大学托管会 | The carboxylation deficiency/the osteocalcin of carboxylation does not improve beta-cell proliferation, insulin secretion, insulin sensitivity, glucose tolerance and reduces fat mass |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1324831A (en) * | 2000-05-19 | 2001-12-05 | 上海博德基因开发有限公司 | New polypeptide human non-insulin dependent diabetes related protein 25 and polynucleotides for encoding same |
| CN102647995A (en) * | 2009-06-10 | 2012-08-22 | 奥胡斯大学 | SorCS1-like agent for use in the treatment of insulin resistance and diseases related thereto |
| CN103554249A (en) * | 2013-10-25 | 2014-02-05 | 中国科学院深圳先进技术研究院 | Complete antigen of AG15 polypeptide as well as preparation method and antibody thereof |
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