CN106892975B

CN106892975B - Polypeptide for regulating glycometabolism and application thereof

Info

Publication number: CN106892975B
Application number: CN201710156024.XA
Authority: CN
Inventors: 任培根; 张键; 滕斌; 李健; 姚振宇
Original assignee: Shenzhen Institute of Advanced Technology of CAS
Current assignee: Shenzhen Zhongke Xinjin Biotechnology Co Ltd
Priority date: 2017-03-16
Filing date: 2017-03-16
Publication date: 2021-12-21
Anticipated expiration: 2037-03-16
Also published as: CN106892975A

Abstract

The present invention relates to a polypeptide for regulating sugar metabolism and its use in preparing a medicament for treating diseases related to sugar metabolism. The polypeptide of the present invention can lower blood sugar and promote insulin secretion; it can be used to prepare a medicament for treating diabetes, and can be administered in combination with existing treatment regimens. The polypeptide of the present invention has significant advantages such as low therapeutically effective dose and oral administration.

Description

Polypeptide for regulating glycometabolism and application thereof

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-Z_a-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;

Z_ais 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 is_aOptionally 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 polypeptide_aSelected 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 polypeptide_aIs 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 polypeptide_aIs 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 added^T(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 added^T(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 SYBRGr_eeThe 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

ccagctataa tcagagacca 20

<210> 18

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> insulin PCR reverse primer

<400> 18

ccaggtgggg accacaaaga 20

<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

1. A polypeptide that regulates sugar metabolism, the polypeptide is represented by formula Za,

Za is selected from one of the following:

SEQ ID NO. 1: YLGASVPPSPDPLEP,

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 can be expressed.

6. A pharmaceutical composition comprising a therapeutically effective amount of the polypeptide of claim 1, or the 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 preparation of a medicament for the treatment of a disease related to abnormal glucose metabolism, wherein the The disease is a disease that can benefit from an increase in insulin and a decrease in blood sugar.

8. The use of 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, which is orally administrable.