CN112904014A

CN112904014A - Application of urine TBC1D5a protein and polypeptide fragment thereof in normal pregnancy or gestational diabetes

Info

Publication number: CN112904014A
Application number: CN201911229413.6A
Authority: CN
Inventors: 张曼; 胡智颖
Original assignee: Individual
Current assignee: Individual
Priority date: 2019-12-04
Filing date: 2019-12-04
Publication date: 2021-06-04
Anticipated expiration: 2039-12-04
Also published as: CN112904014B

Abstract

The invention provides application of a human urine TBC1 domain family member 5 subtype a (TBC1 domain family member 5 isoform a, TBC1D5a) and a polypeptide fragment thereof, in particular to application of urine TBC1D5a protein and a polypeptide fragment thereof in preparation of a preparation for monitoring normal pregnancy or gestational diabetes. The research proves that compared with a normal women group (normal control) with normal reproductive age, the TBC1D5a and the polypeptide fragment thereof have higher expression in normal pregnancy and gestational diabetes groups, and can be used for monitoring and auxiliary diagnosis of patients with gestational state or gestational diabetes. The invention exerts the advantages of noninvasive acquisition, large-scale repeated sampling and convenient storage of the urine specimen, and utilizes the urine specimen to detect the urine TBC1D5a protein and the polypeptide fragment thereof.

Description

Application of urine TBC1D5a protein and polypeptide fragment thereof in normal pregnancy or gestational diabetes

Technical Field

The invention relates to a new application of urine TBC1 domain family member 5 subtype a (TBC1D5a) protein and a polypeptide fragment thereof, in particular to an application of urine TBC1D5a protein and a polypeptide fragment thereof in monitoring normal pregnancy or gestational diabetes.

Background

In the process of pregnancy, in order to meet the requirements of fetal growth and development, a series of changes occur in the reproductive, blood circulation, urinary, respiratory, digestive and endocrine systems of pregnant women, especially in the endocrine system, the secretion of gonadotropin is reduced, and the secretion of prolactin, thyroid stimulating hormone, adrenocorticotropic hormone, glucocorticoid, aldosterone, thyroid hormone and the like is increased. In the middle and late gestation period, part of pregnant women have reduced insulin sensitivity, and have decomposition and antagonism on insulin, so that insulin activity is reduced, and the pregnant women can not normally compensate the physiological change to cause Gestational Diabetes (GDM). If the blood sugar level is not well controlled, the incidence rate of adverse pregnancy fatalities of gestational diabetes women in gestational period hypertension diseases, abortion, cesarean section, premature delivery, neonatal hypoglycemia, neonatal hyperbilirubinemia and the like is obviously higher than that of normal pregnant and lying-in women. For the patients with gestational diabetes who are diagnosed, the later blood sugar control standard and control requirement are stricter, and the monitoring is more frequent. The method aims to improve the life quality and medical compliance of the gestational diabetes patients, relieve the pain of blood sampling for multiple times of gestational blood sugar detection, and formulate more refined and personalized monitoring indexes aiming at different gestational periods, and hope to realize painless, convenient, rapid and easily repeated urine detection or monitoring of the in vivo sugar level and sugar metabolism condition through the research of urine protein or polypeptide, and also lay a foundation for the further research of the urine polypeptide detection kit.

Human TBC1 domain family member subtype 5a (TBC1D5a) protein is one of the members of the TBC family protein. Most of TBC proteins are down-regulated molecules of specific Rab proteins, which can selectively promote the hydrolysis and inactivation of specific Rab proteins. The functional RAB-GAP is formed by combining with RAB7, and the functional RAB-GAP plays roles in regulating ATG9 transportation, autophagy and regulating reverse transport complex. Autophagy generally maintains the metabolism of stressed cells by promoting intracellular catabolism and nutrient cycling. For stresses requiring increased glycolytic demand, the core autophagy mechanism also promotes glucose uptake and glycolytic flux by promoting cell surface expression of the glucose transporter GLUT1/SLC2a 1. During metabolic stress, LC3+ autophagy compartment binds and sequesters the rabgap protein TBC1D5 away from its inhibitory interaction with the reverse transcriptase complex, thereby recruiting reverse transcriptase to the endoplasmic membrane and GLUT1 plasma membrane translocation. In contrast, in autophagy deficient cells, TBC1D5 remains unchanged from its inhibitory interaction with reverse transcriptase, resulting in the incorrect entry of glut1 into the lysosomal compartment. In addition, deletion of TBC1D5 in autophagy-deficient cells rescues recruitment of reverse transcriptase to the plasma membrane and circulation on the surface of GLUT 1. Thus, during metabolic stress, TBC1D5 shuttles to autophagosomes contributing to reverse transcription dependent GLUT1 transport. However, the mechanism of physiological changes of TBC1D5 in pregnancy is not reported in the literature.

Compared with the common clinical blood sample, the urine can be collected in a non-invasive, continuous and large amount; without homeostatic regulation, more various and larger changes can be accumulated, and many pathophysiological changes of the body can be reflected in urine. Some protein polypeptides with relatively small molecular weight, such as hormones and cytokines, are excreted into urine quickly after entering blood, and the probability that the proteins and polypeptides are detected in urine is much higher than that in blood; before urine is collected, a possible protein degradation process in urine is completed, so that urine protein can be kept stable for a longer time. In order to relieve the pain of blood glucose detection of GDM patients in blood sampling for multiple times, the experiment hopes to realize monitoring and understanding of the glucose level and the glucose metabolism level in GDM patients through detection of urine protein or polypeptide on the basis of the previous methodology exploration.

Disclosure of Invention

The invention aims to provide application of urine human TBC1 domain family member 5 subtype a (TBC1D5a) protein and polypeptide fragments thereof in preparation of preparations for monitoring normal pregnancy or gestational diabetes.

Preferably, the amino acid sequence of the urine TBC1D5a is shown in SEQ ID NO.1 (MYHSLSETRH PLQPEEQEVG IDPLSSYSNK SGGDSNKNGR RTSSTLDSEG TFNSYRKEWE ELFVNNNYLA TIRQKGINGQ LRSSRFRSIC WKLFLCVLPQ DKSQWISRIE ELRAWYSNIK EIHITNPRKV VGQQDLMINN PLSQDEGSLW NKFFQDKELR SMIEQDVKRT FPEMQFFQQE NVRKILTDVL FCYARENEQL LYKQGMHELL APIVFVLHCD HQAFLHASES AQPSEEMKTV LNPEYLEHDA YAVFSQLMET AEPWFSTFEH DGQKGKETLM TPIPFARPQD LGPTIAIVTK VNQIQDHLLK KHDIELYMHL NRLEIAPQIY GLRWVRLLFG REFPLQDLLV VWDALFADGL SLGLVDYIFV AMLLYIRDAL ISSNYQTCLG LLMHYPFIGD VHSLILKALF LRDPKRNPRP VTYQFHPNLD YYKARGADLM NKSRTNAKGA PLNINKVSNS LINFGRKLIS PAMAPGSAGG PVPGGNSSSS SSVVIPTRTS AEAPSHHLQQ QQQQQRLMKS ESMPVQLNKG DVVTGSDAQV SVPVQTLTDL QGLSSKNISS SPSVESLPGG REFTGSPPSS ATKKDSFFSN ISRSRSHSKT MGRKESEEEL EAQISFLQGQ LNDLDAMCKY CAKVMDTHLV NIQDVILQEN LEKEDQILVS LAGLKQIKDI LKGSLRFNQS QLEAEENEQI TIADNHYCSS GQGQGRGQGQ SVQMSGAIKQ ASSETPGCTD RGNSDDFILI SKDDDGSSAR GSFSGQAQPL RTLRSTSGKS QAPVCSPLVF SDPLMGPASA SSSNPSSSPD DDSSKDSGFT IVSPLDI); or an amino acid sequence which is derived from the amino acid sequence shown in SEQ ID NO.1 and has the same function with the amino acid sequence shown in SEQ ID NO. 1.

Preferably, the TBC1D5a protein and polypeptide fragments thereof are derived from urine.

Preferably, the urine TBC1D5a protein and polypeptide fragments thereof are highly expressed in normal pregnant or gestational diabetic patients.

Preferably, the preparation is a detection kit for the TBC1D5a protein and polypeptide fragments thereof in urine of patients with normal pregnancy or gestational diabetes.

Preferably, the kit comprises one or more of an aptamer antibody or antibody fragment capable of specifically binding to TBC1D5a protein and polypeptide fragments thereof.

Preferably, the kit further comprises a component selected from the group consisting of: the kit comprises a solid phase carrier, a diluent, a reference substance, a standard substance, a quality control substance, a detection antibody, a second antibody diluent, a luminescent reagent, a washing solution, a color development solution and a stop solution, wherein the solid phase carrier is any one or a combination of a plurality of the solid phase carrier, the diluent, the reference substance, the standard substance, the quality control substance, the detection antibody, the second antibody and.

Preferably, the standard comprises TBC1D5a protein standard, humanized tag antibody standard; preferably, the quality control product comprises: TBC1D5a protein control product, humanized label antibody quality control product; preferably, the solid support comprises: microparticles, microspheres, slides, test strips, plastic beads, liquid phase chips, microwell plates, or affinity membranes.

Preferably, the solid phase carrier is made of any one of polyvinyl chloride, polystyrene, polyacrylamide and cellulose.

The inventor firstly collects urine samples of women of normal childbearing age, women of normal pregnancy and patients with gestational diabetes, takes supernatant after centrifugation, and utilizes weak cation exchange magnetic beads to purify and separate the urine samples. Uniformly mixing 1 mu l of sample and 10 mu l of matrix, taking 1 mu l of point on an Anchorchip target plate, ionizing the sample, carrying out mass spectrometry, and collecting data within the range of 1000-10000Da to obtain a mass spectrometry polypeptide diagram consisting of protein peaks with different mass-to-charge ratios. All mass spectrograms of normal fertile age women, normal pregnant women and gestational diabetes groups were analyzed using the BioExplorer analysis software to screen differential polypeptides. Then, the inventor carries out matrix-assisted laser desorption ionization time-of-flight mass spectrometry identification on the differential polypeptide with statistical significance, searches a database to obtain the TBC1D5a protein with differential expression, and compared with a normal fertile age group, the TBC1D5a protein is highly expressed in urine of normal pregnant women and pregnant diabetic groups.

Compared with the normal childbearing group, the TBC1D5a protein and the polypeptide fragment thereof are high in expression in urine of normal pregnant women and pregnant diabetes patients and have better consistency with fasting blood glucose and glycosylated hemoglobin. Therefore, the detection of the urine TBC1D5a protein and the polypeptide fragment thereof can be used for monitoring or auxiliary diagnosis of normal pregnancy or gestational diabetes.

The invention exerts the advantages of non-invasive acquisition, large-scale repeated sampling and convenient preservation of the urine specimen, and utilizes the urine specimen to detect the urine coagulation TBC1D5a protein and the polypeptide fragment thereof.

In order to make the aforementioned and other objects, features and advantages of the present invention comprehensible, preferred embodiments accompanied with figures are described in detail below.

Drawings

FIG. 1 is a graph of the peak area distribution of TBC1D5a protein in urine from women of normal child bearing age, women of normal pregnancy and gestational diabetic patients.

FIG. 2 is a scattergram of TBC1D5a protein expression in urine samples from women of normal child bearing age, women of normal pregnancy and gestational diabetic patients.

FIG. 3 is a scattergram of TBC1D5a protein expression in the GDM-M and GDM-L groups.

FIG. 4 is a comparison of the uniformity of TBC1D5a protein expression in mid-gestation with Fasting Plasma Glucose (FPG) in gestation diabetic patients.

FIG. 5 is a comparison of the uniformity of expression of TBC1D5a protein in the mid-gestation period with glycated hemoglobin (HbA 1C%) in gestational diabetic patients.

Fig. 6 is a ROC curve of urine TBC1D5a protein expressed in urine of gestational diabetes patients.

Detailed Description

Example 1Collection and processing of urine specimens

60 gestational diabetes patients (GDM) are collected in obstetrical outpatient service and inpatient clinical diagnosis of Beijing century bed hospital affiliated to capital medical university, wherein 30 gestational middle (GDM-M, 13-27 weekend) samples and 30 gestational late (GDM-L, 28 weeks and later) samples are collected; 31 normal pregnant women (N) in routine obstetrical examination in the outpatient clinic; 31 pregnant women (T) with normal health examination, namely clean mid-stream urine specimens of a control group, are all 15-49 years old. After urine collection, 400 Xg centrifugation for 5min, supernatant was collected and split charged and frozen at-80 ℃. Inclusion criteria were:

the first pregnancy test and the first establishment of files are carried out in our hospital from the early pregnancy (8-12 weeks) of patients with gestational diabetes and normal pregnant women, and the data are complete;

75g Oral Glucose Tolerance Test (OGTT) is carried out at 24-28 weeks of pregnancy, and the results all accord with the diagnosis standard of gestational diabetes in ninth edition 'gynaecology obstetrics'; exclusion criteria:

acute and chronic infectious, tumor, cardiovascular disease;

previous presence of impaired glucose tolerance;

severe liver and kidney dysfunction;

those with mental and intellectual disabilities. A comparison of the clinical characteristics of these subjects is shown in Table 1-1.

TABLE 1-1 women of Normal gestational age (T), pregnant women of Normal pregnancy (N) and patients with Gestational Diabetes Mellitus (GDM)

Note: Alb/Cr indicates a urine microalbumin to creatinine ratio < 30.

Example 2Magnetic bead purification and urine polypeptide screening

And (3) enriching the polypeptide in the urine by using weak cation exchange magnetic beads, and analyzing the polypeptide spectrum in the urine by using a matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS). After calibration of the instrument with standards, 1. mu.l of the eluate was mixed with 10. mu.l of matrix (0.3% of. alpha. -cyano-4-hydroxycinnamic acid, HCCA) and 1. mu.l of the spot was applied to an Anchorchip (Autoflex MALDI TOF, Bruker-Dalton) target plate and dried at room temperature. And ionizing the sample by irradiating the nitrogen laser, and then carrying out mass spectrometry, and acquiring data in the range of 1000-10000Da to obtain a mass spectrogram consisting of protein peaks with different mass-to-charge ratios. For each MALDI crystallisation spot, a total of 400 laser shots (50 shots at each of the 8 different positions of each crystallisation spot) were made, and the average represented a sample, thereby obtaining a polypeptide profile for all samples. Three biological replicates were performed per sample. The mass spectrograms of normal pregnant women (T), normal pregnant women (N) and Gestational Diabetes Mellitus (GDM) groups were analyzed using BioExplorer analysis software to screen differential polypeptides. The polypeptide peaks of mass to charge ratio (m/z) 1290.6 were statistically different (P < 0.05) between the 3 groups, and the distribution of the mean peak areas is shown in fig. 1.

Example 4Identification and analysis of differential Polypeptides

From the sample tube, 20ul was taken for LC-MS/MS analysis. An EASY-nLC1000 HPLC system was used for the separation. Liquid phase a was 0.1% formic acid in acetonitrile (2% acetonitrile) and liquid phase B was 0.1% formic acid in acetonitrile (98% acetonitrile). The 100% A solution was equilibrated on a 100X 100mm BEHnanoACquisity column at a flow rate of 400 nl/min. The chromatographic spray needle is PN: FS 360-20-10-N-20-C12. Samples were separated by capillary HPLC and analyzed by Q-exact mass spectrometer (Thermo). The ion source voltage is 3.5kv, the analysis time is 120min, and the scanning range of the parent ion is 300-2000 m/z. The mass-to-charge ratios of the polypeptides and polypeptide fragments were collected by collecting 20 fragments (MS 2scan, HCD) after each complete scan. At M/Z200, the MS1 resolution was 70,000, and the MS2 resolution was 17,500. Data analysis software PD (Proteome scanner 1.4, thermo) was used for the search. The search database is Mascot. The parent ion error was set to 20ppm, the fragment ion error was set to 1Da, the digestion mode was non-digestion, and the modification was oxidation (M) of methionine. Data card value, Percolator algorithm, FDR < 1%. The differentially expressed polypeptide peak of mass to charge (m/z) 1290.6 was retrieved in the database and identified as TBC1D5a protein.

Compared with the normal control group, TBC1D5a protein is highly expressed in urine of normal pregnant women (N) and Gestational Diabetes (GDM) patients, as shown in fig. 2, TBC1D5a protein has significant differences in expression in urine of three groups of normal pregnant women, normal pregnant women and gestational diabetes patients, and also in two groups of normal pregnant women and gestational diabetes patients.

Further dividing Gestational Diabetes Mellitus (GDM) into a middle gestational period (GDM-M, 13-27 weekend) and a late gestational period (GDM-L, 28 weeks and later) according to gestational weeks, as shown in figure 3, the peak values of the TBC1D5a protein in the GDM-M polypeptide are higher than the peak value of the GDM-L polypeptide, and the difference is not significant.

The TBC1D5a protein is respectively subjected to trend analysis with Fasting Plasma Glucose (FPG) and glycosylated hemoglobin (HbA 1C%) results detected when OGTT is performed in the middle gestation period of GDM patients, and the trend is more consistent with the variation trend of FPG and HbA 1C%. As shown in fig. 4 and 5.

Further, the inventor detects 1290.6 polypeptide peaks in urine of 60 gestational diabetes patients and 31 normal pregnant women, establishes a ROC curve with an area under the curve being 0.661, and as shown in FIG. 6, the response TBC1D5a protein has good sensitivity and specificity.

Although the present invention has been described with respect to the preferred embodiments, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims.

Sequence listing

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Claims

1. The use of urine human TBC1 domain family member subtype 5a (TBC1D5a) protein and its polypeptide fragment in the preparation of a preparation for monitoring normal pregnancy or gestational diabetes.

2. The use of claim 1, wherein the urine TBC1D5a protein has the amino acid sequence shown in SEQ ID No. 1; or an amino acid sequence which is derived from the amino acid sequence shown in SEQ ID NO.1 and has the same function with the amino acid sequence shown in SEQ ID NO. 1.

3. The use of claim 1, wherein the TBC1D5a protein and polypeptide fragments thereof are derived from urine.

4. The use of claim 1, wherein said urine TBC1D5a protein and polypeptide fragments thereof are highly expressed in a normal pregnant woman or a pregnant diabetic patient.

5. The use of claim 1, wherein the preparation is a TBC1D5a protein and its polypeptide fragment detection kit for urine of a normal pregnant woman or a pregnant diabetic patient.

6. The use of claim 5, wherein the kit comprises one or more of an aptamer antibody or antibody fragment capable of specifically binding to TBC1D5a protein and polypeptide fragments thereof.

7. The use according to claim 5, wherein the kit further comprises a component selected from the group consisting of: the kit comprises a solid phase carrier, a diluent, a reference substance, a standard substance, a quality control substance, a detection antibody, a second antibody diluent, a luminescent reagent, a washing solution, a color development solution and a stop solution, wherein the solid phase carrier is any one or a combination of a plurality of the solid phase carrier, the diluent, the reference substance, the standard substance, the quality control substance, the detection antibody, the second antibody and.

8. The use of claim 7, wherein the standard comprises a TBC1D5a protein standard, a humanized labeled antibody standard; preferably, the quality control product comprises: TBC1D5a protein control product, humanized label antibody quality control product; preferably, the solid support comprises: microparticles, microspheres, slides, test strips, plastic beads, liquid phase chips, microwell plates, or affinity membranes.

9. The use of claim 8, wherein the solid phase carrier is made of any one of polyvinyl chloride, polystyrene, polyacrylamide and cellulose.