Enzyme Catalyzing Dephosphorylation

1. Field of the invention

This invention relates to a novel enzyme which catalyzes a dephosphorylation and protein. The protein having an enzyme activity of the present invention supplies a phosphoric acid which is particularly necessary for of an oosperm at a growing period thereof and is a substance capable of exterminating injurious insects by inhibiting a growth effect of the oosperm or an activity thereof.

2. Prior art

Biology has made remarkable progress of late. As a result, various molecules explaining morphogenesis have been identified as of today. There have been known growth factors which drastically induce morphogenesis, adhesion factors which mediate between cells, and the like.

However, there are many morphologies of differentiation which cannot be explained by the known growth factors or adhesion factors. Among these, researches on protease which take notice of development have scarcely been carried out so that the present inventor aimed the function of protease at the development process. Protease can make reaction irreversible by decomposing a protein so that it can be considered that it would be inevitable for development of an organism having a property of channeling or direction. Thus, it can be considered that if the function of the protease at the development stage can be clarified, a novel viewpoint can be obtained for understanding the development.

Up to this day, as a protease which participates in the development, Sarcophaga peregrina cathepsin L has been found. The sarcophaga peregrina cathepsin L is shown to be essential for morphogenesis at metamorphosis stage of complete metamorphosis insects. In the complete metamorphosis insects, there exists. The imaginal disk is existing in a larva body and differentiated to an imago tissue by stimulation of ecdysone which is a metamorphosis hormone at the period of chrysalis.

It has been known that an imaginal disk was extracted from a larva body and morphogenesis to an imago tissue can be reproduced in an in vitro culture system in which the imaginal disk cultured in the presence of ecdysone. There has been also reported by using the above culture system that it is essential for differentiate the imaginal disk to an imago leg to secrete cathepsin L out of cells by the induction of ecdysone and to decompose a protein on a basement membrane of the imaginal disk. This demonstrates that intracellular lysosome enzyme cathepsin L has an important function in morphogenesis in addition to the function of decomposing an unnecessary protein in a cell.

The action of cathepsin L at metamorphosis stage has now been elucidated with a certain extent but the action of cathepsin at a development stage other than the metamorphosis stage has not yet been known. It is important to elucidate the function of cathepsin L at the development stage since it makes possible to control development by promoting of inhibiting et al. Thus, the present inventor has analyzed gene expression and protein expression of cathepsin L.

As a result, I have obtained an interesting knowledge particularly at the initial stage of embryogenesis. It has been clarified that cathepsin L exists as maternal mRNA and a precursor protein in a non-fertilized egg and exists as a matured protein from the initial stage of embryogenesis. That is, with regard to fertilized eggs and non-fertilized eggs of sarcophaga peregrina (Non-fertilized eggs are collected by separating imagoes immediately after eclosion to male and female, grown in separate cages and collected from abdomen of female after 5 days from eclosion using a pincette in an insect saline. Fertilized eggs are collected from female which is bred in a cage simultaneously with male. After eclosion, embryo at the fifth day is called as "early", at the 7th days as "middle" and at the 9th days as "late".), presence of cathepsin L is tested. The results are shown in FIG. 1. In FIG. 1, 50 .mu.g of homogenate protein is used in respective lanes, and Lane 1 shows a non-fertilized egg, Lane 2 early egg embryo, Lane 3 middle egg embryo and Lane 4 late egg embryo. An arrow shows the position of a matured type cathepsin L (35 kDa). As can be clearly seen from Lanes 1 and 2 in FIG. 1, the presence of a matured type cathepsin L is confirmed from early embryo.

Early embryogenesis of complete metamorphosis insects may look like different from the other organisms. At the early stage of embryogenesis, however, embryo has not yet differentiated to the respective tissues and many reserved life phenomena which are similar to the other species can be found. For example, determination of dorso-ventical axis is made by a concentration gradient of a growth factor along with the body axis, and it has been reported that it is completely the same mechanism between drosophila (fruit fly) and African clawed toad.

Accordingly, if the function of cathepsin L can be clarified at the early embryogenesis, there is a great possibility of showing the function of protease common in embryogenesis not only in insects, but also in a common organisms.

From the above fact, the present inventor has conducted identification of an intravital substrate at the early embryogenesis was subjected to cloning to clarify an important function of cathepsin L at the early embryogenesis which can be considered to be common to a living body. It has also been desired to prepare an antibody to the protein.

The present inventor tried to identify an intravital substrate at the early embryogenesis to clarify the function of cathepsin L in the early embryogenesis. And analysis of localization of a substrate protein and cloning of cDNA are carried out as mentioned below.

(1) A protein with a molecular weight of 100 kDa on SDS-PAGE which can be considered to be an intravital substrate of cathepsin L at the early embryogenesis in a non-fertilized egg homogenated protein is determined.

(2) From the analysis of localization in the early embryo, it is found that the 100 kDa protein exists at the center portion of embryo in which cells have not yet been formed and cathepsin L is similarly localized.

(3) By cloning of cDNA which encodes the 100 kDa protein, the 100 kDa protein is found to be a novel protein.

(4) An antibody to the 100 kDa protein is prepared.

That is, the present invention relates to a protein with 100 kDa having an action as a substrate protein of cathepsin L and having an amino acid sequence shown by SEQ ID. NO: 1 of the Sequence Listing and also the invention relates to DNA having a base sequence encoding the same.

he present invention also relates to an enzyme which catalyzes a dephosphorylation comprising a protein having an amino acid sequence shown by SEQ ID. NO: 1 of the Sequence Listing which is obtained by analyzing the function possessed by the said protein with 100 kDa.

The present invention further relates to an antibody to the above-mentioned protein with 100 kDa.

FIG. 1 is a drawing showing an immunoblotting of cathepsin L in a fertilized egg and a non-fertilized egg.

FIG. 2 is a drawing showing an immunoblotting of a protein with 100 kDa in a fertilized egg and a non-fertilized egg.

FIG. 3 is a drawing showing inhibition of proteolysis of a protein with 100 kDa in vivo by E64, etc.

FIG. 4 is a graph showing the results of FIG. 3 as a function of

FIG. 5 shown a comparison of amino acid sequences between 100 kDa protein and other protein.

The protein of the present invention is explained below.

An intravital substrate of cathepsin L was identified by homogenizing non-fertilized eggs of sarcophaga peregrina, adding a purified cathepsin L thereto, incubating and detecting a protein which disappears by SDS-PAGE. As the result, the protein with a molecular weight of 100 kDa on SDS-PAGE was disappeared depending on the amount of the purified cathepsin L added. This protein with about 100 kDa was also disappeared in the stage of actual embryogenesis. From the above facts, the protein with a molecular weight of about 100 kDa was made a candidate of an intravital substrate of cathepsin L.

To confirm that the 100 kDa protein disappears in the actual embryogenesis, manifestation of the 100 kDa protein at embryogenesis was analyzed by an immunoblotting method. For this purpose, an antibody to the 100 kDa protein is firstly prepared. That is, non-fertilized egg homogenated protein was subjected to SDS-PAGE, the portion corresponding to 100 kDa protein was cut off from a gel to perform SDS-urea PAGE. Then, a single band was cut from the gel, immuno-sensitized to a rabbit to obtain an antiserum, and the antiserum was subjected to affinity purification to obtain an anti-100 kDa antibody.

By using the thus obtained antibody, an immunoblotting analysis was carried out to embryo homogenates at the respective embryogenesis stage. As the result, it was found that almost all the portions of the 100 kDa protein was decomposed at the early embryogenesis and completely decomposed after the middle stage (see FIG. 2).

In FIG. 2, each 10 .mu.g of embryos of non-fertilized eggs and fertilized eggs at the respective stage was used. Lane 1 shows non-fertilized eggs, Lane 2 the early embryo, Lane 3 the middle embryo, Lane 4 the late embryo, and an arrow shows the position of the 100 kDa protein. As can be clearly seen from Lane 1 and Lane 2, it can be confirmed that the 100 kDa protein existing in the non-fertilized egg almost disappeared at the early stage in the actual embryogenesis.

From the above facts, the protein having a molecular weight of about 100 kDa which is

(a) a protein disappearing on SDS-PAGE by treating a purified cathepsin L, and

(b) a protein disappearing at the early embryogenesis could be identified.

It is the problem whether cathepsin L hydrolyses the 100 kDa protein as an intravital substrate in vivo or not.

Thus, to examine whether the 100 kDa protein was an intravital substrate of cathepsin L or not, a cysteine protease inhibitor E64 was injected to embryo of sarcophaga peregrina at the early stage. As the result, it was found that a rate of hydrolysis of the 100 kDa protein became late in the embryo to which E64 was injected, as compared to that in the embryo to which a serine protease inhibitor, namely benzamizine, and that in the non-treated embryo (see FIG. 3).

That is, in the experiment shown in FIG. 3, a cysteine protease inhibitor E64, insect saline, and a serine protease inhibitor, benzamizine were each injected to embryo of sarcophaga peregrina at the early stage, and the embryos were recovered at 0 hour, after 7 hours and 17 hours. Thereafter, an embryo of each fly was homogenized and whether decomposition of the 100 kDa protein was inhibited or not was examined by the immunoblotting. Three samples were checked with respect to each point of time. Lane 1 is the sample at 0 hour after injection, Lane 2 after 7 hours and Lane 3 after 17 hours. In FIG. 3, left upper drawing is in the case of E64, right upper drawing is in the case of insect saline, left lower drawing is a non-treated case, and right lower drawing is in the case of benzamizine. FIG. 4 shows quantitative analysis values obtained in the experiments shown in FIG. 3 by using an image analyzer. The obtained value of the sample at 0 hour is made 100%. Error bars show the standard deviation of the measured values of three samples.

These results strongly suggests that the 100 kDa protein is an intravital substrate of cathepsin L.

Next, analysis of localization and cloning of cDNA were carried out to know the function of the 100 kDa protein identified by the present inventor on early embryogenesis.

A frozen slice of an early embryo of sarcophaga peregrina was prepared and a fluorescent antibody method was performed by using an anti-100 kDa antibody to know localization of the substrate protein. As the result, fluorescence was detected all over the center portion of the embryo (Figure not shown). At the center portion of the embryo at this stage, cells are not yet formed. On the other hand, when localization of cathepsin L was analyzed in the same fluorescent antibody method, fluorescence was observed in the cells and also fluorescence was observed all over the center portion of the embryo as in the localization of 100 kDa. From the above facts, it can be considered that the 100 kDa protein would be hydrolyzed by cathepsin L existing over the center portion of the embryo prior to forming cells. This results do not contradict to the matter that the 100 kDa protein is an intravital substrate of cathepsin L. Also, this protein can be considered to be a protein contained in granules of a yolk.

On the other hand, lysosome enzyme cathepsin L is an intracellular small organ lysosome and has been considered to have a function of decomposing unnecessary proteins. Also, cathepsin L functions in a lysosome, and cells secrete cathepsin L in the course of morphogenesis of imaginal disk. It is shown to be essential to decompose a protein on a basement membrane. It can be now found that cathepsin L already exists in early embryogenesis before forming cells. From this fact, it can be considered to be important that cathepsin L is prepared at the center portion of embryo in early embryogenesis before forming cells and the protein is decomposed therein.

Moreover, fluorescence caused by the 100 kDa protein and cathepsin L was detected from whole portion of the non-fertilized egg by the fluorescence antibody method, so that it is suggested that they exist in the whole portion of the non-fertilized egg.

Next, cloning of cDNA was carried out to know the true nature of the 100 kDa protein.

To obtain cDNA which encodes the 100 kDa protein, a total RNA was firstly obtained from ovary of sarcophaga peregrina to prepare a cDNA library (ZAP-cDNA synthesis kit of stratagene was used). Then, immunoscreening was performed to the library by using an antibody to the 100 kDa protein. As the result, 22 positive clones were obtained from 36,000 clones. When Southern blotting hybridization was performed to the resulting positive clones with a nucleic acid sequence expected from a partial amino acid sequence already determined as a probe, signals were observed in 13 clones. These clones were analyzed and found that one of which had an open reading frame which encodes 724 amino acids and contained five parts of amino acid sequences previously determined (see an amino acid sequence of SEQ ID NO: 1 of the Sequence Listing). From the above, it is concluded that the clone is cDNA of the 100 kDa protein.

An amino acid sequence of the protein with about 100 kDa of the present invention and a base sequence encoding the same are shown in SEQ ID NO: 1 and NO: 2 of the Sequence Listing, respectively.

Homology search was conducted by using the whole length of the amino acid sequence of this 100 kDa protein to the already existing data base but no homologous showing significance can be found out.

However, the middle portion of the 100 kDa protein showed a high homology with a protein belonging to myotubularin-related family which is considered to be tyrosine-phosphatase as shown in SEQ ID NO8 and SEQ ID NO: 9.

SEQ ID NO: 8 shows an amino acid sequence of human myotubularin in comparison with 100 kDa protein and homologue thereof.

SEQ ID NO: 9 shows an amino acid sequence of C. elegance myotubularin.

Both of lower lines in SEQ ID NO: 8 and SEQ ID NO: 9 show amino acid sequences of the 100 kDa protein. Asterisks mean a coincident amino acid and dots mean an amino acid having similar characteristics. The numeral at the left end shows the position from the first methionine. A sequence preserved in tyrosine phosphatase is underlined. The sequence showed 22% homology with 135 amino acids of myotubularin-like protein of C. elegance and 32% homology with 69 amino acids of human myotubularin. And yet, the sequence contained 13 amino acids which are well preserved in thyrosine-phosphatase therebetween. Cysteine which is considered to be necessary for an activity is also preserved (FIG. 5). In FIG. 5, asterisks mean a coincident amino acid and dots mean an amino acid having similar characteristics. hIA-2 in FIG. 5 shows human receptor-type partial amino acid sequence, hPTN3 human intracellular type partial amino acid sequence, dPTP Drosophila receptor-type partial amino acid sequence, rOSTP rat receptor-type partial amino acid sequence, C.e. C. elegans partial amino acid sequence, and S.c. S. cerevisiae partial amino acid sequence, respectively. From the above facts, the 100 kDa protein can be considered to be thyrosine-phosphatase. An interesting point is that there is no homology in the preserved amino acid sequences at the N-terminal side and the C-terminal side.

In addition, as a motif structure, there exist 11 portions of N-type sugar chain addition site. There is no sequence which is considered to be a signal sequence or a transmembrane domain.

In addition, the 100 kDa protein is a serine-enriched protein (12% of the whole), especially serine exists mainly at the C-terminal side. Thyrosine is also enriched and the total amount of serine and thyrosine is about 20% of the whole amino acids. Many phosphated sites exist over the whole sequence.

According to the above, it can be clarified that the 100 kDa protein is structurally novel protein.

Next, the function of the 100 kDa protein of the present invention is investigated.

If the 100 kDa protein is phosphatase, it would release a phosphoric acid by its dephosphorylation activity to supply a phosphoric acid necessary for early embryogenesis in view of the fact that many phosphated sites exist in the 100 kDa protein. In embryogenesis at the early stage, a large amount of polyphosphoric acid exists. A phosphoric acid at early embryogenesis may be supplied by subjecting the 100 kDa protein to dephosphorylation from the polyphosphoric acid.

The present inventor tried to identify an intravital substrate in early embryogenesis to clarify the function of cathepsin L in early embryogenesis, and the protein with a molecular weight of 100 kDa on SDS-PAGE is identified as an intravital substrate. There is substantially no example which identifies the substrate of protease in early embryogenesis by not using genetic means but biochemical means.

Next, from the analytical result of localization in embryo at the early stage by an indirect fluorescence antibody method, it can be found that the 100 kDa protein is localized as in the embryo in which no cell has been formed. It can be also found that cathepsin L exists at the center portion of embryo at early embryogenesis. From the above facts, it can be considered that lysosome enzyme cathepsin L is prepared at the center portion of embryo at early embryogenesis prior to formation of cells and it may be important to decompose the protein at the portion. Also, this result suggests that cathepsin L exists in the 100 kDa protein.

According to cDNA cloning, the 100 kDa protein is a structurally novel protein and the middle portion showed a high homology as myotubularin which is considered to have thyrosine-phosphatase. From the above fact, there is a possibility that the 100 kDa protein is considered to be thyrosine-phosphatase.

According to the present invention, the function of the protein of the present invention at the early stage of a fertilized egg is clarified so that a substance which is available for controlling growth such as growth or inhibition of a fertilized egg and an antibody thereof are provided by the present invention.

In the following, the present invention is explained in more detail by referring to Examples, but the present invention is not limited by these Examples.

Imaginal of sarcophaga peregrina was bred at 27.degree. C. Larvae were bred in a Tupperware always filling with water and end aged larvae were made chrysalis by drying with a dryer.

Non-fertilized eggs were collected by separating imagoes immediately after eclosion to male and female, grown in separate cages and collected from abdomen of female after 5 days from eclosion by a pincette in an insect saline.

Fertilized eggs are collected from female which is grown in a cage simultaneously with male. After eclosion, embryo at the fifth day is called as "early", at the 7th days as "middle" and at the 9th days as "late".

Non-fertilized eggs of sarcophaga peregrina were homogenized with a 50 mM Na-phosphate buffer (pH 5.8), 500 .mu.g of protein was subjected to SDS-PAGE and after CBB staining, the portion corresponding to 100 kDa protein was cut out from a gel. When the cut out gel was substituted with a SDS sample buffer for 4 hours, then SDS-urea PAGE was performed, a single band and a broad band were detected. A recovered rate of the single band was estimated to be 60% or more from the comparison with a molecular weight marker and CBB staining strength. Also, this band was decreased depending on embryogenesis on SDS-urea PAGE. From the above procedure, the 100 kDa protein was judged to be purified. In the following, the single band on SDS-urea PAGE is treated as the 100 kDa protein.

15 .mu.g of the 100 kDa protein purified by the two-dimensional electrophoresis (the first dimension; SDS-PAGE, the second dimension; SDS-urea PAGE) was homogenized in 0.5 ml of PBS, suspended with an equal amount of an adjuvant and the mixture was made an emulsion by an ultrasonic wave to prepare an immuno antigen for one use. A complete adjuvant was used only for the first sensitization and an incomplete adjuvant was used after the second sensitization.

It was sensitized to a female albino rabbit four times with an interval of every one week. Blood was collected from ear vein and allowed to stand at 37.degree. C. for one hour, and then allowed to stand at 4.degree. C. overnight to obtain serum from supernatant.

The 100 kDa protein (50 .mu.g) subjected to SDS-urea PAGE was transferred to a PVDF membrane to cut out the objective band. This was subjected to blocking by a skim milk sol (20 mM Tris/HCl, pH=7.9, 5% skim milk) and after reacting it with 10 ml of antiserum for 4 hours, 200 mM of glycine/HCl (pH=2.8) was added to extract an antibody. The extract was neutralized with 1M KOH.

According to the guanidine hot phenol method, whole RNA of sarcophaga peregrina (egg maturation late) was obtained. Migrated genome was removed from 300 .mu.g of whole RNA by CsCl ultracentrifugation with 230,000 g at 20.degree. C. for 12 hours, and 10 .mu.g of a poly(A)mRNA was purified by using oligotex-dT30. Purified 5 .mu.g of the poly(A)mRNA was used for preparing a cDNA library. For preparation of cDNA library, ZAP-cDNA synthesis kit (stratagene) was used. As the result, a cDNA library with a library size of 3.9.times.10.sup.5 (390,000) pfu was structured.

Phage cDNA library was infected to an E. coli (XL1-blue MRF') and seeded on a NZY plate. After culturing at 42.degree. C. for 3.5 hours, the protein was induced at 37.degree. C. overnight by 20 mM of IPTG-treated nitrocellulose filter, and the nitrocellulose filter adsorbed a protein was subjected to blocking with a 3% gelatin solution. Next, an anti 100 kDa antibody (4 .mu.g/ml) purified by affinity was reacted at 27.degree. C. for 2 hours, and reacted with an anti-rabbit IgG antibody to which an alkali phosphatase is conjugated at 27.degree. C. for 2 hours. Then, by a coloration reaction of alkali phosphatase, a positive plaque was detected.

A non-fertilized egg homogenate (50 .mu.g) was subjected to SDS-PAGE, and then, the position corresponding to the 100 kDa protein was cut out from the gel and subjected to SDS-urea PAGE. The gel was transferred to a nitrocellulose filter, stained by ponceau S and cut out the 100 kDa corresponding portion by a razor. Then, it is decolorized by 0.2 mM NaOH, treated by 0.5% PVP, added a 1/100 amount of lysyl end peptidase (in case of trypsine, a 1/10 amount was added) to the mixture and subjected to decolorization reaction under the conditions of 100 mM NH.sub.4 HCO.sub.3 (pH=8) at 37.degree. C. for 15 hours. The decomposed product was applied to C18HPLC and eluted by the gradient of 1%/min from H2O to acetonitrile in the presence of 0.05% TFA to fractionate peaks. The respective peaks were dried by a centrifugal evaporator and the sequences were determined by an automatic peptide sequencer.

The determined sequences are as follows. Treated by lysyl end peptidase

SEQ ID NO: 3 Phe Met Ala Val Ser Ser/Met Ser/Ala

SEQ ID NO: 4 Asp Leu His Asp Leu Leu Glu Phe Ile Phe Glu Lys

Treated by trypsine

SEQ ID NO: 5 Ile Phe Ile Gln Tyr Gly Lys

SEQ ID NO: 6 Phe Asn Asp Ala Leu Val Pro

SEQ ID NO: 7 Ala Val Pro Val Thr Met Tyr

From an early embryo collected from abdomen of imaginal female, cut pieces were prepared each 10 .mu.m, fixed by 1.5% HCHO, and blocked by 1% FCS/TBS. They were reacted with a primary antibody diluted to 1/20 to 1/100 for one hour, then reacted with a FITC conjugated secondary antibody and 100 ng of DAPI for 30 minutes, encapsulated by 2.5% DABC 0.50% glycerol and observed by a fluorescent microscope.