# Patent

## Title
Acetylation of 3-hydroxymethyl cephalosporins

## Abstract

This application is a continuation of now abandoned application, Ser. No. 07/134,301 filed on Dec. 15, 1987, abandoned.

## Background
This invention relates to the amino acid sequence of a human .alpha..sub.2 -plasmin inhibitor (or .alpha..sub.2 -antiplasmin), and cDNA and genomic DNA encoding this sequence.

The human .alpha..sub.2 -plasmin inhibitor was first isolated and purified by Aoki and Moroi. It is known to be a strong plasmin inhibitor which instantaneously inhibits the esterase activity of plasmin, a fibrinolytic enzyme, and is a single-chain glycoprotein having a molecular weight of about 67,000 and containing 11.7% of sugar chains [M. Moroi & N. Aoki: The Journal of Biological Chemistry, 251, 5956-5965 (1976)].

On the other hand, it is known that the human .alpha..sub.2 -plasmin inhibitor has three types of active sites. One is a site of inhibiting the protrolytic action of plasmin (to be referred to sometimes as the "reactive site"). A second is a site of binding to plasmin at the carboxyl terminal portion B. Wiman & D. Collen: European Journal of Biochemistry, 84, 573-578 (1978)]. A third is a site of binding to fibrin [Y. Sakata et al.: Thrombosis Research, 16, 279-282 (1979)].

It has already been determined that among the three active sites of the human .alpha..sub.2 -plasmin inhibitor, the fibrin binding site is Gln which is the second amino acid from the amino terminus of the human .alpha..sub.2 -plasmin inhibitor [T. Tamaki & N. Aoki: The Journal of Biological Chemistry, 257, 14767-14772 (1982)]. A peptide fragment composed of 26 amino acids including the plasmin binding site is also known. It has been reported that this peptide fragment exists near the carboxy-terminus of the human .alpha..sub.2 -plasmin inhibitor [see T. Sasaki et al.: The Journal of Biochemistry, 99, 1699-1705 (1986)], but its position is not clear. It has been reported that the reactive site of the human .alpha..sub.2 -plasmin inhibitor is Leu-Met [see B. Wiman Chemistry, 254, 9291-9297 (1979)], or it is described that this reactive site is Arg-Met [see H. R. Lijnen et al.: Thrombosis Research, 39, 625-630 (1985)]. But its position has not yet been determined clearly. If the structures and positions of these plasmin binding sites and the reactive site and their vicinities are determined, it will be very beneficial and interesting to the utilization and development of human .alpha..sub.2 -plasmin inhibitor.

On the other hand, if a DNA of human .alpha..sub.2 -plasmin inhibitor or its fragment is obtained, it will become possible to produce part or the whole of the human .alpha..sub.2 -plasmin inhibitor by a gene manipulation technique. The utilization of such a gene or its fragment will permit diagnosis of human .alpha..sub.2 -plasmin inhibitor deficiency.

As a result of studies on human .alpha..sub.2 -plasmin inhibitor gene, the present inventors isolated complementary DNA (to be referred to as cDNA) of human .alpha..sub.2 -plasmin inhibitor containing 1209 nucleotides coding for the sequence of 403 amino acids from the carboxy-terminus of the human .alpha..sub.2 -plasmin inhibitor, and elucidated the detailed structure of the human .alpha..sub.2 -plasmin inhibitor on the carboxy-terminus side by analyzing the base sequence of the cDNA of human .alpha..sub.2 -plasmin inhibitor. This has led to the present invention.

Specifically, according to one aspect of this invention, there is provided cDNA encoding a human .alpha..sub.2 -plasmin inhibitor precursor represented by the sequence of amino acids from the -39th Met to the 452nd Lys in FIG. 1 of the accompanying drawings, preferably cDNA represented by the base sequence from the 7th A to the 1479th G in FIG. 1. The invention also provides an amino acid sequence of a human .alpha..sub.2 -plasmin inhibitor precursor represented by the sequence of 452 amino acids from the -39th Met to the 452nd Lys in FIG. 1.

In the amino acid sequence composed of 491 amino acids shown in FIG. 1, the amino acid sequence from the 1st Asn to the 452nd Lys shows the protein of the human .alpha..sub.2 -plasmin inhibitor itself, and the amino acid sequence from the -39th Met to the -1st Pro shows the leader sequence.

The present inventors furthered their studies on the elucidation of the structure of a genomic DNA coding for the human .alpha..sub.2 -plasmin inhibitor, and isolated human genomic DNAs coding for an amino acid sequence composed of 39 amino acids and corresponding to the propeptide of human .alpha..sub.2 -plasmin inhibitor and an amino acid sequence composed of 452 amino acids and constituting the human .alpha..sub.2 -plasmin inhibitor. Analysis of the base sequences of these amino acids has led to the discovery that the genomic DNA is a DNA composed of exons II, III, IV, V, VI, VII, VIII, IX and X, as shown in FIG. 4, bonded to each other via introns and encoding human .alpha..sub.2 -plasmin inhibitor protein, and that exon I shown in FIG. 4 is further bonded to the upstream end of exon II via an intron.

Thus, according to another aspect of this invention, there is provided a genomic DNA composed of 9 exons from exon II to X and 8 introns interposed between the adjoining exons, or a genomic gene consisting of the above genomic gene and an exon I bonded to the upstream end of the exon II via an intron.

Part of exon II, exon III and part of exon IV in the genomic DNA of the invention encode a propeptide of 39 amino acids. The remaining part of exon IV, exon V, exon VI, exon VII, exon VII, exon VIII, exon IX and part of exon X encode human .alpha..sub.2 -plasmin inhibitor. The restriction endonuclease map of the genomic DNA of the human .alpha..sub.2 -plasmin inhibitor with regard to six restriction endonucleases is shown in FIG. 3 (2). This genomic gene was obtained as clones of three lambda phages shown in FIG. 3 (3).

The base sequence and amino acid sequence of the genomic DNA of human .alpha..sub.2 -plasmin inhibitor are shown in FIG. 4. In FIG. 4, the exons are the underlined portions of the base sequence. Exon I does not encode the amino acid sequence. Exon II consists of 67 bases in total, four bases (GAAC) in the non-coding region and 63 bases encoding 21 amino acids in part of the propeptide. Exon III consists of 39 bases encoding 13 amino acids in part of the propeptide. Exon IV consists of 63 bases in total, 15 bases encoding 5 amino acids in part of the propeptide and 48 based encoding 16 amino acids in the N-terminus portion of the .alpha..sub.2 -plasmin inhibitor. Exon V consists of 202 bases encoding 67 1/3 amino acids of .alpha..sub.2 -plasmin inhibitor. Exon VI consists of 144 bases encoding 48 amino acids of .alpha..sub.2 -plasmin inhibitor. Exon VII consists of 204 bases encoding 68 amino acids of .alpha..sub.2 -plasmin inhibitor. Exon VIII consists of 143 bases encoding 47 2/3 amino acids of .alpha..sub.2 -plasmin inhibitor. Exon IX consists of 205 bases encoding 68 1/3 amino acids of .alpha..sub.2 -plasmin inhibitor. Exon X consists of 1160 bases in total, 410 bases encoding 136 2/3 amino acids on the C-terminus of .alpha..sub.2 -plasmin inhibitor and 759 bases in the non-coding region. The DNA base sequences of these exons are underlined.

The following Examples illustrate isolation of messenger RNA ("mRNA" for short) from human liver cells, construction of a cDNA library, screening of human .alpha..sub.2 -plasmin inhibitor, recloning of human .alpha..sub.2 -plasmin inhibitor cDNA, preparation of a restriction endonuclease cleavage map of human .alpha..sub.2 -plasmin inhibitor, determination of the base sequence of human .alpha..sub.2 -plasmin inhibitor cDNA, cloning of human .alpha..sub.2 -plasmin inhibitor genomic DNA, preparation of a restriction endonuclease map of the human .alpha..sub.2 -plasmin inhibitor genomic DNA, and determination of the base sequence of the human .alpha..sub.2 -plasmin inhibitor genomic DNA in detail.

It should be understood that in the present specification and the accompanying drawings, amino acids and polypeptides are abbreviated by the method of IUPAC-IUB, Commission on Biological Nomenclature (CBN). For example, the following abbreviations are used.

The DNA sequence will be shown in abbreviations by bases contained in the constituent deoxyribonucleotides. For example, the following abbreviations are used.

A: adenine (representing deoxyadenylic acid)

C: cytosine (representing deoxycytidylic acid)

G: guanine (representing deoxyguanylic acid)

T: thymine (representing deoxythymidylic acid)

FIG. 1 shows the amino acid sequence of human .alpha..sub.2 -plasmin inhibitor in this invention and a cDNA thereof.

FIG. 2 is a restriction endonuclease map of the cDNA of human .alpha..sub.2 -plasmin inhibitor. The white portion is a coding region of the amino acid sequence of the invention and the black portion is a non-coding region.

FIG. 3 (1) shows introns-exons of the genomic DNA of human .alpha..sub.2 -plasmin inhibitor. The ten exons are shown by rectangles. Exon I, part of exon II and part of exon X are non-coding regions which do not encode amino acids. The non-coding region of exon X is shown by hatchings.

FIG. 3 (2) is a restriction endonuclease map of the genomic DNA of human .alpha..sub.2 -plasmin inhibitor in accordance with this invention. The symbols show the following endonucleases.

FIG. 3 (3) shows DNA fragments in phage clones encoding the genomic DNA of human .alpha..sub.2 -plasmin inhibitor in accordance with this invention. This figure also shows the sequence of a pUC19 plasmid, pP162, and charon 4A phage, lambda P12, encoding separate portions of the genomic DNA of the invention. The plasmid and phage were deposited with the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Md. 20852 on Nov. 13, 1991 under the Budapest Treat and bear ATCC Designation Nos. 75142 and 75143, respectively.

FIGS. 3 (1) to (3) correspond to each other vertically.

FIG. 4 shows the DNA base sequence and amino acid sequence of the genomic DNA of human .alpha..sub.2 -plasmin inhibitor in accordance with this invention. The exon portions are underlined, and the corresponding amino acids are indicated beneath. A sequence presumably corresponding to the TATA box upstream of exon I and the transcriptional site are surrounded by lines. The polyadenylation recognition site downstream of 3'-terminus is surrounded by a line.

mRNA was isolated from human liver cells in accordance with the guanidine thiocyanate method [see J. M. Chirgwin et al.: Biochemistry, 18, 5294-5299 (1979)]. To 2.times.10.sup.8 human liver cells was added 5 ml of GTC solution (6M guanidine isothiocyanate, 5mM sodium citrate, 0.1M 20-mercaptoethanol, 0.5% sodium N-lauroylsarcosinate), and the mixture was homogenized. The homogenate was superimposed on 3.8 ml of an aqueous solution containing 5.7M CsCl and 0.1M EDTA. They were ultracentrifuged using an RPS-50T rotor (Hitachi) at 35,000 rpm for 15 hours. After ultracentrifugation, the solution was carefully removed, and the residue was rinsed three times with about 1 ml of ethanol, and dissolved in 1.4 ml of water. The solution was treated with ethanol to form a precipitate. The precipitate was dissolved in 0.5 ml of a washing solution composed of 0.5M NaCl, 18mM of Tris-HCl (pH 7.5), 1mM EDTA and 0.05% of SDS, and the solution was passed through 0.5 ml of Oligo(dT) cellulose column. The column was washed with the above washing solution, and eluted with an eluent composed of 10mM Tris-HCl (pH 7.5), 1mM EDTA and 0.05% SDS to give about 31 micrograms of polyA+mRNA.

cDNA was synthesized from the polyA+mRNA derived from human liver cells by the method of Gubler and Hoffman [see U. Gubler & B. J. Hoffman: Gene, 25, 263-269 (1983)] using a cDNA synthesis kit made by Amersham Co.

A single-stranded cDNA was synthesized in a yield of about 30% by adding 5 micrograms of Oligo(dT) 12-18 to 5 micrograms of polyA+mRNA in the presence of 50 units of RNase inhibiting enzyme (HPRI) derived from human placenta and causing transcriptase to act at 42.degree. C. for 1.5 hours. Four units of E. coli ribonuclease H and 115 units of E. coli DNA polymerase I were added to the resulting reaction solution, and reacted at 12.degree. C. for 1 hour and then at 22.degree. C. for 1 hour. The reaction mixture was left to stand at 70.degree. C. for 10 minutes to deactivate the enzymes. Then, 10 units of T4-DNA polymerase was added and reacted at 47.degree. C. for 10 minutes to obtain a double-stranded cDNA in a yield of about 95%. The double-stranded cDNA was reacted with 20 units of EcoRI methylase at 37.degree. C. for 1 hour. To the reaction product was bonded 16 units of EcoRI (made by Takara Shuzo). Then, 16 units of EcoRI (made by Takara Shuzo) was added and the mixture was reacted at 37.degree. C. for 2 hours. The reaction mixture was passed through a column of Sephasrose L-4B to purify it and 0.34 microgram of cDNA was obtained. This cDNA (0.4 microgram) and 1.0 microgram of .lambda.gt10 arm (made by Vector Cloning Systems) were ligated to obtain a hybrid DNA having the cDNA derived from human liver cells inserted into it. The resulting hybrid DNA was packaged in vitro [see A. Becker & M. Gold: Proc. Natl. Acad. Sci. USA, 72, 581 (1975)] to obtain a cDNA library derived from human liver cells.

The library of cDNA derived from human liver cells obtained in Example 2 was transfected into E. coli C 600 hfl.sup.- strain to form plaques. Clones containing human .alpha..sub.2 -plasmin inhibitor DNA were selected in accordance with the Benton and Davis plaque hybridization method using synthetic DNAs P-1 and P-2 labelled with [.sup.32 P] [see W. D. Benton & R. W. Davis: Science, 196, 180 (1977)]. The synthetic DNAs used as probes were synthesized by using a DNA sequence corresponding to the partial amino acid sequence of human .alpha..sub.2 -plasmin inhibitor reported by Collen et al. [see D. Collen et al.: Thrombosis and Haemostasis, 48, 311-314 (1982)] as a DNA synthesizer (made by Applied Biosystems). ##STR1##

Plasmid pUC8 (2 micrograms) was digested with restriction endonuclease EcoRI in accordance with Example 2, and 1.9 units of alkaline phosphatase (E. coli C75: a product of Takara Shuzo) was added. The mixture was reacted at 58.degree. C. for 2 hours. After the reaction, the reaction solution was extracted with phenol three times to deactivate and remove the alkaline phosphatase in the reaction solution. The EcoRI-digested cDNA fragment of human .alpha..sub.2 -plasmin inhibitor obtained in Example 3 was added to the resulting EcoRI/alkaline phosphatase treated solution of pUC8, and 2 units of T4-DNA ligase was reacted with the mixture at 12.degree. C. for 16 hours to perform ligation.

E. coli LE 392 was transformed with hybrid DNA prepared by ligating the EcoRI-digested cDNA fragment of human .alpha..sub.2 -plasmin inhibitor in accordance with an ordinary CaCl.sub.2 method [see M. V. Norgard et al.: Gene, 3, 297 (1978)]. The transformants were inoculated in L-broth plates containing ampicillin in a concentration of 50 micrograms/ml. The plates were cultivated overnight at 37.degree. C. to grow the transformants. DNAs were prepared from the resulting colonies by a known method, and by agarose gel electrophoresis, the desired hybrid DNAs were determined. They were named pPI 41, pPI 39 and pPI 142. -plasmin inhibitor cDNA

Lambda gt10DNA containing the human .alpha..sub.2 -plasmin inhibitor cDNA obtained in Example 3 was digested with EcoRI to cut out the inserted human .alpha..sub.2 -plasmin inhibitor cDNA and isolated by 0.8% agarose gel electrophoresis. This human .alpha..sub.2 -plasmin inhibitor cDNA fragment (0.1 microgram) was dissolved in 10 microliters of a buffer for restriction endonucleases an aqueous solution containing 100mM NaCl, 50mM Tris-HCl (pH 7.5), 10mM MgCl.sub.2 and 1mM dithiothreitol for digestion with EcoRI; an aqueous solution containing 50mM NaCl, 10mM Tris-HCl (pH 7.5), 10mM MgCl.sub.2 and 1mM dithiothreitol for digestion with BamHI, PstI and HindIII; and an aqueous solution containing 20mM KCl, 10mM Tris-HCl (pH 8.0), 10mM MgCl.sub.2 and 1mM dithiothreitol for digestion with SmaI, respectively], and digested at 37.degree. C. for 1 hour. When the digestion was carried out using two restriction endonucleases, the cDNA fragment was first treated with one restriction endonuclease which acted at a low salt concentration. Then, the salt concentration was raised to a predetermined concentration, and the DNA fragment was treated with the other enzyme which acted at a high salt concentration.

After digestion, 1 microliter of 0.25% bromophenol blue and a 50% aqueous solution of glycerol were added, and the mixture was subjected to electrophoresis using 0.8%-1.2% agarose containing 1 microgram/ml of ethidium bromide. At the time of electrophoresis, a digestion product of the DNA of lambda phage with HindIII was used as a molecular size marker for the DNA fragment. After the electrophoresis, ultraviolet light was irradiated on the gel, and the digestion pattern was observed. The patterns of digestions with various restriction endonucleases alone or combinations of two restriction endonucleases were analyzed. The restriction endonuclease map thus obtained of the human .alpha..sub.2 -plasmin inhibitor cDNA having a molecular size of about 2.2 Kb is shown in FIG. 2. inhibitor cDNA

The base sequence of the human .alpha..sub.2 -plasmin inhibitor cDNA was determined by the Sanger's dideoxy sequence method [see F. Sanger et al.: Proc. Natl. Acad. Sci. USA, 74, 5463-5467 (1977)].

For example, The EcoRI fragment of the human .alpha..sub.2 -plasmin inhibitor cDNA was inserted into the EcoRI site of M13 mp18 or M13 mp19 vector, and E. coli JM105 strain was transformed with the vector. A single-stranded hybrid DNA was prepared by a known method using the transformed JM105. The single-stranded DNA was subjected to sequence reaction using an M13 sequence kit (made by Amersham), and then subjected to electrophoresis using 6% polyacrylamide gel containing 7M urea. The gel was radioautographed overnight at -80.degree. C. The separation pattern was analyzed to obtain data for determination of the base sequence of human .alpha..sub.2 -plasmin inhibitor.

As a result, the base sequence encoding the human .alpha..sub.2 -plasmin inhibitor was determined, and is shown in FIG. 1.

DNA extracted from human placenta was digested with restriction endonucleases AluI and HaeIII, and inserted into Charon 4A bacteriophage vector arm via an EcoRi linker to construct a library. The plaques (1.2.times.10.sup.6) of the library were screened using cDNA fragments corresponding to an amino acid sequence from the 31st to the 130th amino acids from the N-terminus of the amino acid sequence of .alpha..sub.2 -plasmin inhibitor and a cDNA fragment corresponding to the 179th to the 429th amino acids from the N-terminus of the amino acid sequence of .alpha..sub.2 -plasmin inhibitor as probes [see W. D. Benton & R. W. Davis: Science, 196, 180-182 (1977)]. For the screening of clones upstream of 3', a synthetic DNA (5'ACTCCCCTGCCAGCC3') composed of 15 bases was used as a probe. When cDNAs were used as probes, the cDNA fragments were labelled with .sup.32 P by nick translation. The synthetic DNA was labelled on the 5' side with T4 polynucleotidekinase.

As a result of screening, three clones, .lambda.PI1, .lambda.PI2 and .lambda.PI6, encoding the entire region of the genomic DNA of .alpha..sub.2 -plasmin inhibitor were obtained. The correspondence of the clones to the genomic DNA is shown in FIGS. 3 (1), (2) and (3). .alpha..sub.2 -plasmin inhibitor genomic DNA

The phage DNA containing human .alpha..sub.2 -plasmin inhibitor genomic DNA obtained in Example 7 was digested with EcoRI, and the inserted human .alpha..sub.2 -plasmin inhibitor genomic DNA was cut out and isolated by 0.9-1.5% agarose gel electrophoresis. This human .alpha..sub.2 -plasmin inhibitor DNA fragment (0.1 to 0.5 microgram) was dissolved in 10 microliters of a buffer for restriction endonucleases and digested with 2 units of various restriction endonucleases at 37.degree. C. for 1 hour.

When digestion was carried out using two restriction endonucleases, the DNA fragment was first treated with one restriction endonuclease which acted at a low salt concentration. Then, the salt concentration was raised to a predetermined concentration, and the DNA fragment was treated with the other restriction endonuclease which acted at a high salt concentration.

After digestion, 1 microliter of 0.25% bromophenol blue and a 50% aqueous solution of glycerol were added, and the mixture was subjected to electrophoresis using 0.8%-1.2% agarose containing 1 microgram/ml of ethidium bromide. At the time of electrophoresis, a digestion product of the DNA of lambda phage with HindIII was used as a molecular size marker for the DNA fragment. After the electrophoresis, ultraviolet light was irradiated on the gel, and the digestion pattern was observed. The patterns of digestions with various restriction endonucleases alone or combinations of two restriction were analyzed. The resulting restriction endonuclease map of human .alpha..sub.2 -plasmin inhibitor genomic DNA so obtained is shown in FIG. 3 (2). inhibitor genomic DNA

The base sequence of the human .alpha..sub.2 -plasmin inhibitor genomic DNA was determined by the Sanger's dideoxy sequence method [see F. Sanger et al.: Proc. Natl. Acad. Sci. USA, 74, 5463-5467 (1977)].

For example, The EcoRI fragment of the human .alpha..sub.2 -plasmin inhibitor genomic DNA was inserted into the EcoRI site of M13 mp18 or M13 mp19 vector, and E. coli JM105 strain was transformed with the vector. A single-stranded hybrid DNA was prepared by a known method using the transformed JM105. The single-stranded DNA was subjected to sequence reaction using an M13 sequence kit (made by Amersham), and then subjected to electrophoresis using 6% polyacrylamide gel containing 7M urea. The gel was radioautographed overnight at -80.degree. C. The separation pattern was analyzed to obtain data for determination of the base sequence of human .alpha..sub.2 -plasmin inhibitor.

As a result, the base sequence of the genomic DNA encoding the human .alpha..sub.2 -plasmin inhibitor was determined, and is shown in FIG. 4.

## Claims

1. cDNA encoding a human .alpha..sub.2 -plasmin inhibitor precursor protein which consists of the following amino acid sequence: ##STR2##

2. The cDNA of claim 1 which consists of the following base sequence: ##STR3##

3. cDNA encoding a leader amino acid sequence which consists of the following amino acid sequence: ##STR4##

4. cDNA of claim 3 which consists of the following base sequence: ##STR5##

5. Genomic DNA encoding a human .alpha..sub.2 -plasmin inhibitor protein consisting of exons II, III, IV, V, VI, VII, VIII, IX and X having base sequences as shown below, said exons being connected to one another via introns which have base sequences or sizes as shown below, a first portion of said genomic DNA consisting of from exons II to VII and being contained in the base sequence of pUC19 plasmid, pPI62 bearing ATCC Designation No. 75142, and a second portion of said genomic DNA consisting of from exons VII to X and being contained in the base sequence of charon 4A phage, lambda PI2 bearing ATCC Designation No. 75143: ##STR6##

6. The genomic DNA of claim 5 consisting of the following base sequence, wherein exon I having the following base sequence is linked upstream of exon II via intron I having a size of about 8kb, the first portion of said genomic DNA consisting of from exons I to VII and being contained in the base sequence of pUC19 plasmid, pPI62 bearing ATCC Designation No. 75142, and the second portion of said genomic DNA consisting of from exons VII to X and being contained in the base sequence of charon 4A phage, lambda PI2 bearing ATCC Designation No. 75143: ##STR7##

7. Genomic DNA encoding a leader amino acid sequence which consists of exon II, intron 2, exon III, intron 3 and exon IV having the following base sequences, said genomic DNA being contained in the base sequence of pUC19 plasmid, pPI62 bearing ATCC Designation No. 75142: ##STR8##