CN1796410A - Function and application of MAAT1p15 antigen correlated to melanoma - Google Patents

Abstract

This invention publishes the function of melanoma-associated antigen MAAT1p15 (melanoma-associated antigen recogmzed by cytotoxic T lymphocytes p15). It derivatively comprises an amino acid residue sequence of SEQ ID No.1 within which one or several amino acid residues is substituted, deleted or added and performs the same activities as those of SEQ ID No.1. Yeast two-hybrid identification implemented with the intracellular domain of LRP6 as bait protein, the interaction between MAAT1p15 and LRP6 is identified and further confirmed in mammalian cells. Results from luciferase report system also reveals that MAAT1p15 significantly enhances the transcription activity of Wnt1 and LRP6-responsive downstream genes, and such a synergetic activation is promoted with an increasing transfection amount of MAAT1p15. Therefore, it is quite probable that MAAT1p15 participates in the signal pathway of Wnt through interaction with LRP6 and thus helps receptor LRP6 with signal conduction. The identification of the function of MAAT1p15 provides new clues for investigating the signal conduction mechanism of Wnt and establishes a new base for further elucidating the molecular mechanisms of the emergence and development of melanoma.

Description

Function and the application of melanic related antigen MAAT1 p15

Technical field

The present invention relates to function and the application of melanic related antigen MAAT1 p15 (melanoma-associated antigen recognized bycytotoxic Tlymphocytes p15).

Background technology

Wnt is a kind of glycoprotein factor, by the form performance function of paracrine.Normal Wnt signal path is of crucial importance in embryo development procedure, has not only participated in the formation of embryo's dorso ventral axis, and relevant with a plurality of growth incidents such as cell polarity foundation, cell fate decisions; The tumour that then often causes the abnormal activation of Wnt signal path takes place as colorectal carcinoma, liver cancer, ovarian cancer, prostate cancer, skin carcinoma etc.Therefore understanding the transmission mechanism of Wnt signal in depth, is from the prerequisite of the generation evolution of molecular level understanding relative disease, also can have very important significance for prevention and treatment of diseases provides necessary theoretical basis.

Seven transmembrane proteins of Frizzled gene family coding are found the earliest Wnt acceptors.Proteic seven of Frizzled strides the film district and has G protein coupling receptor characteristic, but to so far, still without any studies confirm that Frizzled albumen can be by providing signal in conjunction with G albumen.

(low density-lipoprotein receptor related protein LRP6) is the single-transmembrane receptor of being cloned out in 1998 to LDH receptor related protein.Result of study subsequently shows that LRP6 participates in the Wnt signal transduction pathway fruit bat in three kinds of model animals of Xenopus laevis and mouse, and therefore is considered to the accessory receptor of seven-transmembrane acceptor Frizzled.The classical path of Wnt signal conduction has tentatively been determined in present research, after being the coreceptor LRP6/Frizzled interaction of Wnt part and cell surface, activate Dishevelled albumen, and then make in the tenuigenin beta-catenin white (β-catenin) assemble to nucleus, thus activate the expression of downstream gene.Though there has been a large amount of reports that each link of Wnt signal transduction pathway is explained, LRP6 is as the acceptor of the Wnt signal pathway of latest find, and it activates the proteic molecular mechanism in downstream and still imperfectly understands.Utilize yeast two-hybrid system from mouse 11.5d embryo cDNA library, to screen the interaction protein of LRP6 intracellular region, and utilize the luciferase reporting system to detect the regulating effect of positive colony to the Wnt signal path, for the transmission mechanism of studying the Wnt signal provides new clue and enlightenment.

Summary of the invention

The function that the purpose of this invention is to provide melanic related antigen MAAT1 p15 (melanoma-associated antigenrecognized by cytotoxic Tlymphocytes p15).

Melanic related antigen MAAT1 p15 be in the sequence table in the sequence table amino acid residue sequence of SEQ ID No.1 or with the amino acid residue sequence of SEQ ID No.1 through replacement, disappearance or the interpolation of one or several amino-acid residue and have identical with SEQ ID No.1 active by SEQ ID No.1 deutero-albumen.

257 amino acid of albumen total length of SEQ ID No.1 in the sequence table, it can be discerned by cytotoxic T lymphocyte, and the extensive distribution or the integral part of mitochondrial ribosomal protein are also arranged in other healthy tissuess.Retrieval finds that except mouse, the people, also there is MAAT1 p15 homologous gene in fruit bat in the species such as nematode among the GenBank, and the high conservative of numerous protein is similar in this and the Wnt signal path.As shown in Figure 1, the homology between these homologous genes is very high, and mouse and people's gene order homology is 85%, is respectively 40% and 32% with the gene order homology of nematode and fruit bat.

We are the yeast two-hybrid screening that bait protein carries out with the intracellular region of LRP6, have found the interaction between melanic related antigen MAAT1 p15 and the LRP6, and have verified this interactional verity in mammalian cell.Luciferase reporting system detected result also shows, MAAT1 p15 can obviously strengthen the downstream gene transcriptional activity of Wnt1 and LRP6 response, and the effect of this synergistic activation increasing and strengthen with MAAT1 p15 transfection amount.Therefore, MAAT1 p15 probably be by and LRP6 between interaction participated in the path of Wnt signal, assist acceptor LRP6 to carry out the signal conduction.

Have report to find that MAAT1 p15 has unusual high expression level and can be discerned by cytotoxic T lymphocyte in melanoma, it may and eliminate the crucial effect of bringing into play in the melanoma cell in immune system recognition.Its sequence is very conservative on evolving, nematode, and fruit bat all exists height homologous gene in the species such as mouse and people, and this prompting MAAT1 p15 may have crucial function in vivo.The concrete function of MAAT1 p15 it be unclear that, analyzing it according to its unusual high expression level in melanoma may be relevant with melanomatous generation, and data presentation of the present invention, it can participate in the conductive process of Wnt signal, assists to activate the expression of Wnt downstream gene.Also there is report to show that Wnt signal transduction pathway and melanomatous generation and transfer process also have confidential relation.Wnt downstream responses factor-beta-catenin and LEF-1 all have unusual high expression level in K-1735, and cause the overactivity of Wnt signal, and the stimulation of the Wnt5a factor can strengthen the transfer ability of melanoma cell.The present invention shows that MAAT1 p15 can interact with LRP6 and conduction has synergistic activation to the Wnt signal path, and this is consistent with MAAT1 p15 and Wnt signal path are high expression level in melanoma report.This consistence prompting, MAAT1 p15 may participate in the promotion adjusting of Wnt signal path to melanomatous generation and transfer process.

In a word, interactional discovery not only provides new clue for studying Wnt signal conduction mechanism between MAAT1 p15 and the LRP6, and is that the molecular mechanism of further illustrating melanomatous generation evolution has been established new basis.

Description of drawings

Fig. 1 is that the aminoacid sequence of MAAT1 p15 in people, mouse, nematode and fruit bat compares.The wire frame district represents that sequence homology between the species is 100% conserved regions among the figure.

Fig. 2 is MAAT1 p15 albumen and bait protein LRP6 betagalactosidase activity filter paper analysis result in yeast.

Fig. 3 is the interaction situation of MAAT1 p15 and LRP6 in the 293T cell.Last figure is with anti-HA antibody mediated immunity post precipitation, the Western detected result of sediment composite.Middle figure and figure below are then represented LRP6 and the proteic expression of MAAT1 p15 in the cell pyrolysis liquid respectively.1 is with the cell pyrolysis liquid behind HA-pCS2/LRP6C and the Flag-pcDNA6/MAAT1 p15 cotransfection; 2 is with the cell pyrolysis liquid after the independent transfection of Flag-pcDNA6/MAAT1 p15.

Fig. 4 is the influence that MAAT1 p15 (A) and MAAT1 p15C (B) transcribe the downstream gene of Wnt response.

Embodiment

The structure of embodiment 1, LRP6C and MAAT1 p15 albumen correlated expression carrier.

Reading frame design Auele Specific Primer according to gene order among the GenBank and expression vector carries out the PCR reaction, the clone has obtained Yeast expression carrier pGBKT7/LRP6C respectively, pACT2/MAAT1 p15, carrier for expression of eukaryon HA-pcDNA6/LRP6C (intracellular region), Flag-pcDNA6/MAAT1 p15C (lacking 15 amino acid whose fragments of aminoterminal), Flag-pcDNA6/MAAT1 p15 (total length).Clone LRP6 expression vector is a template with plasmid pCS2/LRP6, and clone MAAT1 p15C is a template with the library plasmid that contains MAAT1 p15C, and clone MAAT1 p15 full-length gene is a template with mouse embryo cDNA library.Reaction conditions is: 95 ℃ of sex change 5min at first; 94 ℃ of sex change 30s of PCR circulation then, 55 ℃ (LRP6C) or 60 ℃ (MAAT1 p15) 30s that anneal, 72 ℃ of extension 60s carry out 30 circulations; Last 72 ℃ are extended 10min.Primer is given birth to worker biotech company by Shanghai and is synthesized, and checking order after the clone finishes, it is correct to identify that fusion rotein is read frame.

The primer of PCR reaction is as follows:

F/LRP6C/pGBKT7：5’-TATGAATTCAGGATGTTGTGTCCACGTATG-3’

F/LRP6C/pcDNA6：5’-TATGAATTCTAGGATGTTGTGTCCACGTATG-3’

R/LRP6C：5’-TATAGTCGACTCAGGAGGAGTCTGTACAGG-3’

F/MAAT1?p15/pACT2：5’-TATGAATTCGAATGCCGCTGCACAGGTAT-3’

F/MAAT1?p15/pcDNA6：5’-TATGAATTCTATGCCGCTGCACAGGTAT-3’

F/MAAT1?p15C/pcDNA6：5’-TATGAATTCTCTGCGGCAGGGCATCTG-3’

R/MAAT1?p15：5’-TATTCTAGATCAGGCGTGGTCACCGGC-3’

The screening of embodiment 2, LRP6 interaction protein and evaluation.

In order to further investigate the transmission mechanism of Wnt signal path, utilize yeast two-hybrid system to screen, in the hope of finding interactional albumen with acceptor LRP6.Member in the Wnt signal path is all very conservative on evolving, sequence homology in each species is all very high, the protein sequence total length homology of the people of acceptor LRP6 and mouse is 93%, long 216 amino acid of intracellular region, and homology reaches 98%, so the intracellular region of personnel selection LRP6 is as bait protein screening mice embryonic cDNA library.At first bait protein plasmid pGBKT7/LRP6C is converted among the yeast strain AH109, detectionof, the result is negative, shows that LRP6C itself does not have the self activation ability, can be used as bait protein and carries out yeast two-hybrid screening.Through library screening and evaluation, obtain 18 positive colonies, No. 13 clones wherein are melanic related antigen (melanoma-associated antigen recognized by cytotoxic Tlymphocytes p15, MAAT1 p15) fragment (16-257aa), 15 amino acid of aminoterminal disappearance, back called after MAAT1 p15C.

In order to verify the yeast two-hybrid screening result, cloned MAAT1 p15 full-length gene with PCR method, made up and caught protein expression vector, with the common again transformed yeast of bait protein LRP6C, and carry out β-gal color reaction and detect.The result shows, LRP6C and one are contained irrelevant proteic AD plasmid and are transformed jointly, perhaps MAAT1 p15 transforms jointly with the pGBKT7 plasmid that does not contain LRP6C, its yeast cell all can not be grown on the flat board of three scarce (Leu-/Trp-/His-) substratum, and LRP6C and the common transformed yeast cells of MAAT1 p15 lack well-grown on the culture medium flat plates three.Select well-grown bacterium colony from two flat boards that lack (Leu-/Trp-) substratum and do β-gal color reaction, have only LRP6C and the common transformed yeast cells of MAAT1 p15 to show blue (Fig. 2), illustrate that there are interaction really in the intracellular region of LRP6 in yeast cell and MAAT1 p15.

Embodiment 3, LRP6 and the interaction of MAAT1 p15 in mammalian cell

With expression vector Flag-pcDNA6/MAAT1 p15 and the common transfection 293T of the HA-pcDNA6/LRP6C cell that builds, can detect overexpression band HA LRP6C fusion rotein (among Fig. 3) and the band Flag MAAT1 p15 fusion rotein (under Fig. 3).In order to confirm that LRP6C and MAAT1 p15 can interact in Mammals, carried out the co-immunoprecipitation experiment, cell pyrolysis liquid is done the Western detection with HA antibody precipitation, Flag antibody.By Fig. 3 result as seen, do not have positive signal to occur separately behind the cell pyrolysis liquid co-immunoprecipitation of transfection Flag-pcDNA6/MAAT1 p15, then have positive signal to occur behind HA-pcDNA6/LRP6C and the common cells transfected lysate of the Flag-pcDNA6/MAAT1 p15 co-immunoprecipitation.Show that LRP6C and MAAT1 p15 can interact in mammalian cell.

Embodiment 4, DDIP are to the checking of Wnt signal path regulating effect.

The promoter sequence of LEF-1 luciferase reporting plasmid has 6 multiple LEF-1 binding sites.Under the hormesis of the Wnt factor, LEF-1 albumen combines and activates transcribing of downstream luciferase reporter gene with these sites.The luciferase catalytic substrate of expressing discharges fluorescence, and (Top Count) can detect intensity of fluorescence by the luciferase reporting analyser, thereby can reflect the level that reporter gene is transcribed.In order whether to influence the conduction of Wnt signal path after verifying MAAT1 p15 and LRP6 combining, utilize this luciferase reporting system, observe the adjusting of this gene pairs Wnt signal path by overexpression MAAT1 p15 gene in cell.

The result is shown in Fig. 4 A, and transfection Wnt1 can activate the luciferase reporting system, and the reporter gene uciferase activity is compared with control group has increased about 3 times, and when single expression MAAT1 p15, reporter gene is not activated.Uciferase activity obviously strengthens after MAAT1 p15 and the Wnt1 coexpression, and prompting MAAT1 p15 can work in coordination with Wnt1 and promote reporter gene expression.And along with the increase of MAAT1 p15 transfection amount, the effect of this assistance activated also strengthens (Fig. 4 A, 3-5 row) gradually, and cotransfection MAAT p15 is during to 600ng, more than the twice when uciferase activity reaches independent transfection Wnt1.

Above results suggest, MAAT1 p15 may with cell endogenous LRP6 combination, thereby the signal path under cooperative response Wnt1 token stimulus conduction.Therefore and then observe the influence of MAAT1 p15 because overexpression LRP6 also can activate the Wnt signal path, to LRP6.When single expression LRP6, the reporter gene uciferase activity has improved about 3 times, and when expressing MAAT1 p15 gradually, LRP6 also strengthens (Fig. 4 A, 6-8 row) gradually to the activation of reporter gene.This shows that MAAT1 p15 may be the accessory protein of LRP6, with the transduction that can strengthen the Wnt signal after LRP6 combines.

Owing to obtained the fragment MAAT1 p15C of MAAT1 p15 during yeast two-hybrid screening, so further studied the activation whether MAAT1 p15C also can influence LRP6.We use MAAT1 p15C and repeat the system experimentation of LEF-1 luciferase reporting.The result shows that MAAT1 p15C is the same with total length, and all can work in coordination with increases the reporter gene transcriptional level (Fig. 4 B) that the Wnt signal is mediated.

Sequence table

<160>1

<210>1

<211>257

<212>PRT

＜213〉mouse (Mus musculus)

<400>1

1?maftasqrwe?egqarqigfr?frwalnperl?amplhkypvw?lwkrlqlreg?icsrlpghyl

61?rsleeertpt?pvhyrphgak?fkinpkngqr?ervedvpipi?yfppesqrgl?wggegwilgq

121?iyanndklsk?rlkkvwkpql?ferefyseil?dkkftvtvtm?rtldlideay?gldfyilktp

181?kedlcskfgm?dlkrgmllrl?arqdpqlhpe?dperraaiyd?kykefaipee?eaewvgltle

241?eaiekqrlle?ekdpvplfki?yvaeliqqlq?qqalsepavv?qkrasgq

Claims (5)

1, the function of melanic related antigen MAAT1 p15 (melanoma-associated antigen recognized by cytotoxic Tlymphocytes p15).MAAT1 p15 be the amino acid residue sequence of SEQ ID No.1 in the sequence table or with the amino acid residue sequence of SEQID No.1 through replacement, disappearance or the interpolation of one or several amino-acid residue and have identical with SEQ IDNo.1 active by SEQ ID No.1 deutero-albumen.

2, the expression vector that contains the described protein coding gene of claim 1.

3, the clone that contains the described protein coding gene of claim 1.

4, described albumen of claim 1 and encoding gene thereof the application in making up the screening anti-tumor medicine model.

5, described albumen of claim 1 and encoding gene thereof the application in the medicine of preparation treatment cancer.

Images