EP0931262A1 - Biochemical markers of the human endometrium - Google Patents

Biochemical markers of the human endometrium

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Publication number
EP0931262A1
EP0931262A1 EP97939048A EP97939048A EP0931262A1 EP 0931262 A1 EP0931262 A1 EP 0931262A1 EP 97939048 A EP97939048 A EP 97939048A EP 97939048 A EP97939048 A EP 97939048A EP 0931262 A1 EP0931262 A1 EP 0931262A1
Authority
EP
European Patent Office
Prior art keywords
leu
ala
glu
gly
lys
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP97939048A
Other languages
German (de)
French (fr)
Inventor
Inger Byrjalsen
Peter Mose Larsen
Stephen John Fey
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Immunodiagnostic Systems Nordic AS
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Osteometer Biotech AS
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Filing date
Publication date
Priority claimed from GBGB9618600.2A external-priority patent/GB9618600D0/en
Priority claimed from GBGB9707132.8A external-priority patent/GB9707132D0/en
Application filed by Osteometer Biotech AS filed Critical Osteometer Biotech AS
Publication of EP0931262A1 publication Critical patent/EP0931262A1/en
Withdrawn legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57442—Specifically defined cancers of the uterus and endometrial
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/689—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to pregnancy or the gonads
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/36—Gynecology or obstetrics

Definitions

  • the endometrium is the mucous lining of the uterine cavity.
  • the endometrium is the organ in the body that shows the greatest changes under the influence of the sex hormones, oestradiol and progesterone.
  • the endometrium proliferates until progesterone from the corpus luteum transforms the oestrogen-primed proliferative endometrium to a secretory phase endometrium. In due course this is followed by shedding of the fully transformed endometrium during the menstruation, and a new cycle will begin.
  • endometrial status is assessed by histological and biochemical analysis of endometrial biopsies. This is time-consuming, expensive and causes discomfort for the woman. It would be highly desirable to identify biochemical markers which could be measured in body fluids reflecting the endometrial status, obviating the need for endometrial biopsies . The detection of such markers in histological samples would also however be advantageous as an additional method of recognising the histological status of such samples .
  • references to the proteins herein include references to modified forms of the proteins and derivatives of the proteins, including but not restricted to glycosylated, phosphorylated, acetylated, methylated or lipidated forms thereof.
  • the invention provides a method of characterising a biological sample comprising detecting or quantitating therein one or more proteins produced by the endometrium in increased amounts in hyperplasia or in adenocarcinoma as shown by 2D gel electrophoresis comparison of cell lysates of endometrial biopsies from normal endometrium and endometrium showing hyperplasia or adenocarcinoma, excluding variations due to the menstrual cycle, or detecting or quantitating a fragment or breakdown product thereof, or a nucleic acid coding therefor, or an antibody thereto.
  • Said protein, fragment, breakdown product, antibody or nucleic acid may preferably be detected in a body fluid sample but may also be detailed in other forms of sample such as histological samples or cytological samples .
  • the invention includes an immunological binding partner specifically reactive with a protein as defined above with a fragment or breakdown product thereof or with a nucelic acid coding therefor.
  • the invention includes also an assay kit for use m such an analysis method comprising an immunological binding partner as described.
  • This aspect of the invention has resulted from studies aiming to detect endometrial proteins with increased synthesis in endometrial adenocarcinoma as compared to the synthesis during the normal menstrual cycle; to detect endometrial proteins with increased synthesis in endometrial hyperplasia as compared to the synthesis during the normal menstrual cycle; and to detect proteins showing a cycle-related expression during the normal menstrual cycle.
  • the invention relates to the discovery of markers of the "proliferative" phase of the human endometrium.
  • a protein marker for the "secretory" phase of the endometrium has been previously described, see US-A-4 , 489, 166. No similar marker has been described for the proliferative phase although certain candidate proteins were described in Ref . 1.
  • the human endometrium Under influence of the sex hormones, oestradiol and progesterone, the human endometrium undergoes cyclical variation with an oestrogen-dominated phase, i.e. the proliferative phase, an ovulation phase, i.e. the interval phase, a progesterone-dominated phase, i.e. the secretory phase, and finally the endometrium is shed, i.e. the menstrual phase.
  • an oestrogen-dominated phase i.e. the proliferative phase
  • an ovulation phase i.e. the interval phase
  • a progesterone-dominated phase i.e. the secretory phase
  • the endometrium is shed, i.e. the menstrual phase.
  • the same cyclical variation of the endometrium is seen in postmenopausal women receiving sequentially combined hormone replacement therapy.
  • a method of characterising a biological sample comprising detecting or quantitating therein one or more proteins produced by the endometrium in increased amounts during the proliferative phase of the endometrium as shown in 2D gel elctrophoresis comparison of cell lysates of endometrial biopsies from normal endometrium in its proliferative and secretory phases and characterised by one of the following combinations of molecular weight and pi values : -
  • Such a method may preferably be for detecting the phase of the endometrium.
  • This aspect of the invention includes a method of determining the proliferative/secretory phase status of the endometrium comprising the quantitative or qualitative measurement in a sample of any one or more of the proteins defined above or a breakdown product or fragment thereof . It also includes an immunological binding partner for any of the said proteins, breakdown products or fragments or a cell line producing such a binding partner.
  • the sequences and properties of proteins discussed above relate to human proteins, the assay procedures of the invention may be practised on samples arising from other species.
  • references to proteins herein should be understood to include proteins having a degree of homology of at least 60% with the given amino acid sequences irrespective of any modifications of said amino acids.
  • modified amino acids such as phosphorylated, acetylated, amidated, methylated, glycosylated or lipidated derivatives of an amino acid should thus be considered to be the same as the amino acid without any such modification.
  • modified amino acids such as phosphorylated, acetylated, amidated, methylated, glycosylated or lipidated derivatives of an amino acid should thus be considered to be the same as the amino acid without any such modification.
  • Such peptides may be derived from similar proteins from other species, e.g. other mammals such as mouse, rabbit, guinea pig, pig, or cow or may be entirely or predominantly of synthetic origin.
  • the degree of homology may be advantageously be at least 65%, or at least 70%. Under certain circumstances, it is advantageous that the degree of homology is even higher such as at least 80% or at least 90%.
  • Other DNA sequences which encode substantially the same amino acid sequence as a gene encoding a marker protein, i.e. a marker gene may be used in the practice of the present invention. These include, but are not limited to, allelic genes and homologous genes from other species.
  • Nucleic acid fragments comprising a nucleotide sequence which codes for a protein described above or a peptide derived from it as well as nucleic acid fragments which hybridise with these nucleic acid fragments or a part thereof under stringent hybridisation conditions, e.g. 5 mM monovalent ions (O.lxSSC), neutral pH and 65°C are important aspects of the invention.
  • stringent hybridisation conditions e.g. 5 mM monovalent ions (O.lxSSC), neutral pH and 65°C are important aspects of the invention.
  • the term "highly stringent”, when used in conjunction with hybrisidation conditions, is as defined in the art, i.e. 5-10°C under the melting point T m , cf, Sambrook et al, 1989, pages 11.45 - 11.49.
  • nucleic acid is meant a polynucleotide of high molecular weight which can occur as either DNA or RNA and may be either single-stranded or double-stranded.
  • the invention relates to a binding means which specifically binds to a relevant protein or peptide or nucleic acid fragment as described above.
  • the invention relates to an antibody which specifically binds to a relevant protein or peptide or an antigen-binding fragment thereof, i.e. a polyclonal antibody, a monoclonal antibody, chimeric antibody, single chain antibody fragment, Fab and Fab' fragments, and an Fab expression library.
  • both monoclonal and polyclonal antibodies will be useful in providing the basis for one or more assays to detect relevant peptides and proteins.
  • Antibodies which are directed against epitopes that are specific for the proteins will be most useful as cross reaction will be minimised therewith.
  • assay methods and kits may be produced according to standard methodology.
  • the proteins may be obtained in purified form, either by extraction from tissues or by synthesis, and antibodies may be raised thereto or to characterising peptide sequences thereof. Standard assay formats employing such antibodies may be utilised according to the invention.
  • immunoassays are contemplated as including various types of enzyme linked immunoassays (ELISA) , immunoblot techniques, and the like, known in the art. However, it is readily appreciated that utility is not limited to such assays, and useful embodiments including RIAs and other non-enzyme linked antibody binding assays or procedures.
  • ELISA enzyme linked immunoassays
  • the proteins themselves or peptides derived from the protein sequences may be used in detecting auto- antibodies to such proteins.
  • Figure 1 Fluorograph of a two-dimensional gel electrophoresis of [ 35 S]meth ⁇ on ⁇ ne labelled endometrial proteins separated in the first dimension by ISO- elect ⁇ c focusing (IEF; pi 3.5-7) and in the second dimension by sodium dodecyl sulphate polyacrylamide gel electrophoresis. The locations of the spots with increased synthesis in hyperplasia are indicated.
  • Figure 2 Fluorograph of a two-dimensional gel electrophoresis of [ 35 S]meth ⁇ omne labelled endometrial proteins separated in the first dimension by non- equilib ⁇ um pH gradient gel electrophoresis (NEPHGE; pi 6.5-11) and in the second dimension by sodium dodecyl sulphate polyacrylamide gel electrophoresis . The locations of the spots with increased synthesis in hyperplasia are indicated.
  • NEPHGE non- equilib ⁇ um pH gradient gel electrophoresis
  • Figure 3 Fluorograph of a two-dimensional gel electrophoresis of [ 35 S] metmonine labelled endometrial proteins separated in the first dimension oy ISO- elect ⁇ c focusing (IEF; pi 3.5-7) and in the second dimension by sodium dodecyl sulphate polyacrylamide gel elctrophoresis . The locations of the spots with increased synthesis in adenocarcinoma are indicated.
  • Figure 4 Fluorograph of a two-dimensional gel electrophoresis of [ 35 S]meth ⁇ on ⁇ ne labelled endometrial proteins separated m the first dimension by non- equilibnum pH gradient gel electrophoresis (NEPHGE; pi 6.5-11) and in the second dimension by sodium dodecyl sulphate polyacrylamide gel electrophoresis. The locations of the spots with increased synthesis in adenocarcinoma are indicated.
  • NEPHGE non- equilibnum pH gradient gel electrophoresis
  • Figure 5 Fluorograph of a two-dimensional gel electrophoresis of [ 35 S3meth ⁇ on ⁇ ne labelled endometrial proteins separated in the first dimension by ISO- elect ⁇ c focusing (IEF; pi 3.5-7) and in the second dimension by sodium dodecyl sulphate polyacrylamide gel elctrophoresis. The locations of the spots with increased synthesis in proliferative phase endometrium are indicated.
  • Figure 6 Fluorograph of a two-dimensional gel electrophoresis of [ 35 S]meth ⁇ on ⁇ ne labelled endometrial proteins separated in the first dimension by non- equilibrium pH gradient gel electrophoresis (NEPHGE; pi 6.5-11) and in the second dimension by sodium dodecyl sulphate polyacrylamide gel electrophoresis. The locations of the spots with increased synthesis in proliferative phase endometrium are indicated.
  • Figure 7 Tryptic digestion mass spectroscopic characteristics of I#350. The peaks marked with a star are not protein identification specific but represents methodologically non-specific peaks.
  • Figure 8 Tryptic digestion mass spectroscopic characteristics of I#687.
  • the peaks marked with a star are not protein identification specific but represents methodologically non-specific peaks.
  • Figure 9 Tryptic digestion mass spectroscopic characteristics of N#414.
  • the peaks marked with a star are not protein identification specific but represents methodologically non-specific peaks.
  • Figure 10 Tryptic digestion mass spectroscopic characteristics of I#1035. The peaks marked with a star are not protein identification specific but represents methodologically non-specific peaks.
  • Figure 11 Tryptic digestion mass spectroscopic characteristics of N#26. The peaks marked with a star are not protein identification specific but represents methodologically non-specific peaks.
  • Figure 12 Tryptic digestion mass spectroscopic charac- teristics of N#31+N#32. The peaks marked with a star are not protein identification specific but represents methodologically non-specific peaks.
  • endometrial samples were obtained as follows.
  • the proteins of the endometrial biopsies were metabolically labelled with "s-methionine for 20 hours, and total cell lysates were processed for 2D gel electrophoresis, a technique in which proteins are separated in the first dimension according to the isoelectric point and in the second dimension according to the molecular weight. It was possible to study proteins with iso-electric points ranging from 3.5 to 11 and relative molecular weights ranging from 10 to 300 kDa. After electrophoresis the gels were fixed and treated for fluorography . The fluorograms of the 2D gel electrophoresis were subjected to quantitative analysis by computer-aided analysis, by which the density of each spot was quantified, the fluorogram patterns were matched i.e. numbers were assigned to each spot and the same spot was given the same number on all the fluorograms. The density
  • menstrual cycle-related proteins so identified have been identified by amino acid sequence analysis (Ref.2). Selected menstrual cycle-related proteins were excised from several 2D gels, concentrated by ID sodium dodecylsulphate polyacrylamide gel electro- horesis, and cleaved in situ by trypsin. The tryptic fragments were extracted and separated by reverse phase high pressure liquid chromatography. Finally, the partial amino- terminal amino acid sequence of selected tryptic fragments were determined for each protein. For identification the amino acid sequences of the tryptic fragments were compared to previously reported sequences by searching in databases . The hyperplasia and adenocarcinoma associated proteins of the present invention may be sequenced and further characterised by similar methods.
  • spots had increased expression in both hyperplasia and adenocarcinoma. Based on subjective evaluation, preferred subgroups of spots were selected with increased synthesis in hyperplasia and in adenocarcinoma, respectively. The preferred subgroup of spots with increased synthesis in hyperplasia were selected as being the spots showing the highest relative increase in expression in hyperplasia as compared to the samples obtained from women during the normal mentrual cycle and women with irregular proliferative phase endometrium.
  • the preferred subgroup of spots with increased synthesis in adenocarcinoma were selected as the spots showing the highest relative increase in expression in adenocarcinoma as compared to the samples obtained from women during the normal menstrual cycle and women with irregular proliferative phase endometrium.
  • the preferred subgroup of 7 spots with increased synthesis in hyperplasia is given in Table 3
  • the preferred subgroup of 12 spots with increased synthesis in adenocarcinoma is given in Table
  • the information obtained from the 2D-gel electrophoresis with respect to the isoelectric point (pi) and the molecular weight (MW) of a preferred subgroup of these spots which show increased synthesis in proliferative phase endometrium are given in Table 5 and their positions are indicated in Figures 5 and 6.
  • Protein ID ' Accession Identification in protein or nucleotide databases (e.g. SwissProt, Protein Identification Resource (PIR) or EMBL)
  • the proteins of interest may be isolated from endometrial tissue or other protein sources by 2D gel electrophoresis or by using chromatographic techniques. Poly- or monoclonal antibodies towards the protein of interest can be raised, and immunoassays can be established based on such antibodies. Synthetic peptides being fragments characteristic of such proteins may be used for the same purposes . Assays may be based on more than one such protein for measurement at one time.
  • MOLECULE TYPE protein
  • HYPOTHETICAL NO
  • MOLECULE TYPE protein
  • HYPOTHETICAL NO
  • MOLECULE TYPE protein
  • HYPOTHETICAL NO

Abstract

Assay methods are provided for detection or quantitation of any of several proteins which are specifically produced in the endometrium in association with hyperplasia, adenocarcinoma or the proliferative phase of the endometrium. The relevant proteins have been identified by 2D gel electrophoresis with subsequent sequence identification by mass spectroscopic finger printing of tryptic digests.

Description

BIOCHEMICAL MARKERS OF THE HUMAN ENDOMETRIUM
The endometrium is the mucous lining of the uterine cavity. During the menstrual cycle, the endometrium is the organ in the body that shows the greatest changes under the influence of the sex hormones, oestradiol and progesterone. In the oestrogen dominated phase the endometrium proliferates until progesterone from the corpus luteum transforms the oestrogen-primed proliferative endometrium to a secretory phase endometrium. In due course this is followed by shedding of the fully transformed endometrium during the menstruation, and a new cycle will begin.
Persistent unbalanced oestrogen stimulation either due to increased endogenous production of oestrogens, or replacement therapy in which oestrogens are given alone, is associated with increased risk of developing endometrial hyperplasia and subsequently endometrial adenocarcinoma . Histologically, these pathological conditions are characterised by increased thickness of the endometrium and irregular pattern of the endometrial glandular cells. Endometrial adenocarcinoma is a life threatening condition.
At present the endometrial status is assessed by histological and biochemical analysis of endometrial biopsies. This is time-consuming, expensive and causes discomfort for the woman. It would be highly desirable to identify biochemical markers which could be measured in body fluids reflecting the endometrial status, obviating the need for endometrial biopsies . The detection of such markers in histological samples would also however be advantageous as an additional method of recognising the histological status of such samples .
We have now discovered that certain proteins are produced in the endometrium in increased amounts associated with hyperplasia and that certain proteins are produced in increased amounts associated with adenocarcinoma. These two groups of proteins overlap somewhat. The present invention relates in a first aspect to such proteins and to their diagnostic uses. Unless otherwise indicated, references to the proteins herein include references to modified forms of the proteins and derivatives of the proteins, including but not restricted to glycosylated, phosphorylated, acetylated, methylated or lipidated forms thereof.
Thus the invention provides a method of characterising a biological sample comprising detecting or quantitating therein one or more proteins produced by the endometrium in increased amounts in hyperplasia or in adenocarcinoma as shown by 2D gel electrophoresis comparison of cell lysates of endometrial biopsies from normal endometrium and endometrium showing hyperplasia or adenocarcinoma, excluding variations due to the menstrual cycle, or detecting or quantitating a fragment or breakdown product thereof, or a nucleic acid coding therefor, or an antibody thereto.
The invention includes a method of characterising a biological sample comprising detecting or quantitating therein one or more proteins produced by the endometrium in increased amounts in hyperplasia or in adenocarcinoma and characterised by one of the following combinations of molecular weight and pi values:
hyperpl.asia pi MW kDa
6.7 91
6.6 90
6.9 64
6.6 67
6.3 66
6.8 46
5.7 41
5.5 35
5.3 13
6.6 101
5.8 14
7.4 51
8.2 44
9.5 48 adenocarcinoma
P MW (k
6.3 32
6.0 109
6.7 91
6.6 90
6.9 64
6.6 67
6.3 66
6.2 62
6.2 45
5.7 45
5.4 33
6.3 27
6.5 103
6.8 90
6.9 78
5.3 13
6.2 130
6.3 66
6.3 73
8.3 32
8.1 55
8.2 44
6.6 111
7.7 43
9.5 48
8.3 32
7.7 39
or a fragment or breakdown product thereof, or a nucleic acid coding therefor, or an antibody thereto.
Said protein, fragment, breakdown product, antibody or nucleic acid may preferably be detected in a body fluid sample but may also be detailed in other forms of sample such as histological samples or cytological samples . The invention includes an immunological binding partner specifically reactive with a protein as defined above with a fragment or breakdown product thereof or with a nucelic acid coding therefor.
It also includes a cell line producing a monoclonal antibody being such an immunological binding partner.
The invention includes also an assay kit for use m such an analysis method comprising an immunological binding partner as described.
This aspect of the invention has resulted from studies aiming to detect endometrial proteins with increased synthesis in endometrial adenocarcinoma as compared to the synthesis during the normal menstrual cycle; to detect endometrial proteins with increased synthesis in endometrial hyperplasia as compared to the synthesis during the normal menstrual cycle; and to detect proteins showing a cycle-related expression during the normal menstrual cycle. In a second aspect the invention relates to the discovery of markers of the "proliferative" phase of the human endometrium. A protein marker for the "secretory" phase of the endometrium has been previously described, see US-A-4 , 489, 166. No similar marker has been described for the proliferative phase although certain candidate proteins were described in Ref . 1.
Under influence of the sex hormones, oestradiol and progesterone, the human endometrium undergoes cyclical variation with an oestrogen-dominated phase, i.e. the proliferative phase, an ovulation phase, i.e. the interval phase, a progesterone-dominated phase, i.e. the secretory phase, and finally the endometrium is shed, i.e. the menstrual phase. The same cyclical variation of the endometrium is seen in postmenopausal women receiving sequentially combined hormone replacement therapy. The demand for endometrial status assessment has highly increased in the latest decade, not only on account of the extensive research into fertility, but also in order to estimate endometrial response to the large number of combined oestrogens/progestogen preparations used in hormone replacement therapy. It would be highly desirable to identify biochemical markers which could be measured in body fluids reflecting the endometrial status, obviating the need for endometrial biopsies. Studies have suggested that serum placental protein 14 (PP14) , which is produced in the glandular cells of the secretory phase endometrium (Ref. 3), is a reliable marker of the secretory phase endometrium. It has been shown that serum PP14 strongly correlates with the secretory activity of the endometrium in postmenopausal women receiving hormone replacement therapy (Ref. 4,5). No similar marker exists for the proliferative phase endometrium. We have now discovered that certain proteins are produced in the endometrium in increased amounts in proliferative phase endometrium as compared to secretory phase endometrium.
According to this aspect of the invention there is now provided a method of characterising a biological sample comprising detecting or quantitating therein one or more proteins produced by the endometrium in increased amounts during the proliferative phase of the endometrium as shown in 2D gel elctrophoresis comparison of cell lysates of endometrial biopsies from normal endometrium in its proliferative and secretory phases and characterised by one of the following combinations of molecular weight and pi values : -
pl MW(kDa)
6.9 86
5.4 34
5.6 67 5.3 23 6.8 52
8.7 47 8.2 138
6.5 124
7.7 119
7.8 119 8.1 66
7.1 59
6.8 66
7.9 48
7.7 31
6.8 29
7.2 70 8.0 119 6.7 62
or a fragment or breakdown product thereof, or a nucleic acid coding therefor or an antibody thereto.
Such a method may preferably be for detecting the phase of the endometrium.
The preferred features of the first aspect of the invention apply also to this second aspect. This aspect of the invention includes a method of determining the proliferative/secretory phase status of the endometrium comprising the quantitative or qualitative measurement in a sample of any one or more of the proteins defined above or a breakdown product or fragment thereof . It also includes an immunological binding partner for any of the said proteins, breakdown products or fragments or a cell line producing such a binding partner. Whilst the sequences and properties of proteins discussed above relate to human proteins, the assay procedures of the invention may be practised on samples arising from other species. Especially in this context, references to proteins herein should be understood to include proteins having a degree of homology of at least 60% with the given amino acid sequences irrespective of any modifications of said amino acids. When determining homology, modified amino acids such as phosphorylated, acetylated, amidated, methylated, glycosylated or lipidated derivatives of an amino acid should thus be considered to be the same as the amino acid without any such modification. Such peptides may be derived from similar proteins from other species, e.g. other mammals such as mouse, rabbit, guinea pig, pig, or cow or may be entirely or predominantly of synthetic origin.
The degree of homology may be advantageously be at least 65%, or at least 70%. Under certain circumstances, it is advantageous that the degree of homology is even higher such as at least 80% or at least 90%. Other DNA sequences which encode substantially the same amino acid sequence as a gene encoding a marker protein, i.e. a marker gene, may be used in the practice of the present invention. These include, but are not limited to, allelic genes and homologous genes from other species.
Nucleic acid fragments comprising a nucleotide sequence which codes for a protein described above or a peptide derived from it as well as nucleic acid fragments which hybridise with these nucleic acid fragments or a part thereof under stringent hybridisation conditions, e.g. 5 mM monovalent ions (O.lxSSC), neutral pH and 65°C are important aspects of the invention. The term "highly stringent", when used in conjunction with hybrisidation conditions, is as defined in the art, i.e. 5-10°C under the melting point Tm, cf, Sambrook et al, 1989, pages 11.45 - 11.49.
By the term "nucleic acid" is meant a polynucleotide of high molecular weight which can occur as either DNA or RNA and may be either single-stranded or double-stranded. Once the amino acid sequences of the proteins of utility in the present invention are known, it is possible to synthesise DNA or RNA probes which may be used for: i) direct detection of DNA and RNA expressing said proteins on a fixed or frozen tissue section using, e.g. chromogenous , chemiluminescent or immunofluorescent techniques; ii) poly erase chain reaction (PCR) or other amplification techniques; and iii) locating the part or all of the gene, isogene, pseudogene or other related genes either in cDNA libraries, genomic libraries or other collections of genetic material from either the host or other animals, including man. In another aspect, the invention relates to a binding means which specifically binds to a relevant protein or peptide or nucleic acid fragment as described above. In particular, the invention relates to an antibody which specifically binds to a relevant protein or peptide or an antigen-binding fragment thereof, i.e. a polyclonal antibody, a monoclonal antibody, chimeric antibody, single chain antibody fragment, Fab and Fab' fragments, and an Fab expression library.
It is contemplated that both monoclonal and polyclonal antibodies will be useful in providing the basis for one or more assays to detect relevant peptides and proteins. Antibodies which are directed against epitopes that are specific for the proteins will be most useful as cross reaction will be minimised therewith. Based upon the identification of relevant proteins described above, assay methods and kits may be produced according to standard methodology. Thus, the proteins may be obtained in purified form, either by extraction from tissues or by synthesis, and antibodies may be raised thereto or to characterising peptide sequences thereof. Standard assay formats employing such antibodies may be utilised according to the invention. Preferred immunoassays are contemplated as including various types of enzyme linked immunoassays (ELISA) , immunoblot techniques, and the like, known in the art. However, it is readily appreciated that utility is not limited to such assays, and useful embodiments including RIAs and other non-enzyme linked antibody binding assays or procedures. The proteins themselves or peptides derived from the protein sequences may be used in detecting auto- antibodies to such proteins.
Samples of the proteins described above have been subjected to trypsm digestion and the molecular weight of the resulting fragments has been determined by mass spectrometry . This provides a "fingerprint" of the protein which can be matched to date in established data bases available to those working in this field. This procedure has enabled us to identify certain of the proteins as being previously known in other contexts. No matches have been found for certain others, indicating that they have not previously been known.
The invention will be illustrated and explained further by the following description in which the Figures as follows : -
Figure 1 : Fluorograph of a two-dimensional gel electrophoresis of [35S]methιonιne labelled endometrial proteins separated in the first dimension by ISO- electπc focusing (IEF; pi 3.5-7) and in the second dimension by sodium dodecyl sulphate polyacrylamide gel electrophoresis. The locations of the spots with increased synthesis in hyperplasia are indicated. Figure 2: Fluorograph of a two-dimensional gel electrophoresis of [35S]methιomne labelled endometrial proteins separated in the first dimension by non- equilibπum pH gradient gel electrophoresis (NEPHGE; pi 6.5-11) and in the second dimension by sodium dodecyl sulphate polyacrylamide gel electrophoresis . The locations of the spots with increased synthesis in hyperplasia are indicated. Figure 3: Fluorograph of a two-dimensional gel electrophoresis of [35S] metmonine labelled endometrial proteins separated in the first dimension oy ISO- electπc focusing (IEF; pi 3.5-7) and in the second dimension by sodium dodecyl sulphate polyacrylamide gel elctrophoresis . The locations of the spots with increased synthesis in adenocarcinoma are indicated.
Figure 4 : Fluorograph of a two-dimensional gel electrophoresis of [35S]methιonιne labelled endometrial proteins separated m the first dimension by non- equilibnum pH gradient gel electrophoresis (NEPHGE; pi 6.5-11) and in the second dimension by sodium dodecyl sulphate polyacrylamide gel electrophoresis. The locations of the spots with increased synthesis in adenocarcinoma are indicated. Figure 5: Fluorograph of a two-dimensional gel electrophoresis of [35S3methιonιne labelled endometrial proteins separated in the first dimension by ISO- electπc focusing (IEF; pi 3.5-7) and in the second dimension by sodium dodecyl sulphate polyacrylamide gel elctrophoresis. The locations of the spots with increased synthesis in proliferative phase endometrium are indicated. Figure 6: Fluorograph of a two-dimensional gel electrophoresis of [35S]methιonιne labelled endometrial proteins separated in the first dimension by non- equilibrium pH gradient gel electrophoresis (NEPHGE; pi 6.5-11) and in the second dimension by sodium dodecyl sulphate polyacrylamide gel electrophoresis. The locations of the spots with increased synthesis in proliferative phase endometrium are indicated. Figure 7: Tryptic digestion mass spectroscopic characteristics of I#350. The peaks marked with a star are not protein identification specific but represents methodologically non-specific peaks. Figure 8 : Tryptic digestion mass spectroscopic characteristics of I#687. The peaks marked with a star are not protein identification specific but represents methodologically non-specific peaks. Figure 9: Tryptic digestion mass spectroscopic characteristics of N#414. The peaks marked with a star are not protein identification specific but represents methodologically non-specific peaks.
Figure 10: Tryptic digestion mass spectroscopic characteristics of I#1035. The peaks marked with a star are not protein identification specific but represents methodologically non-specific peaks. Figure 11: Tryptic digestion mass spectroscopic characteristics of N#26. The peaks marked with a star are not protein identification specific but represents methodologically non-specific peaks. Figure 12:Tryptic digestion mass spectroscopic charac- teristics of N#31+N#32. The peaks marked with a star are not protein identification specific but represents methodologically non-specific peaks.
To identify proteins expressed at an increased level in differing endometrial conditions, endometrial samples were obtained as follows.
Normal menstrual cycle samples were obtained as described in Ref. 1. Endometrial biopsies were collected from 13 pre-menopausal, regular cycling women (35-50 years old) undergoing endometrial curettage (n=l) or hysterectomy
(removal of the uterus) (n=12) for a variety of medical reasons not related to abnormality or malignancy of the endometrium. None used hormone contraception. For pathological condition samples, endometrial biopsies were collected from 16 patients (41 to 79 years old) undergoing endometrial curettage (n=9) or hysterectomy (n=7) for medical reasons related to abnormality or malignancy of the endometrium. The samples were treated as described in Ref . 1. The proteins of the endometrial biopsies were metabolically labelled with "s-methionine for 20 hours, and total cell lysates were processed for 2D gel electrophoresis, a technique in which proteins are separated in the first dimension according to the isoelectric point and in the second dimension according to the molecular weight. It was possible to study proteins with iso-electric points ranging from 3.5 to 11 and relative molecular weights ranging from 10 to 300 kDa. After electrophoresis the gels were fixed and treated for fluorography . The fluorograms of the 2D gel electrophoresis were subjected to quantitative analysis by computer-aided analysis, by which the density of each spot was quantified, the fluorogram patterns were matched i.e. numbers were assigned to each spot and the same spot was given the same number on all the fluorograms. The density
(quantity synthesis) of each spot was assessed to find proteins with increased synthesis in endometrial adenocarcinoma or hyperplasia and assessed for periodic characteristics during the normal menstrual cycle to find proteins with the menstrual cycle-related synthesis.
Some of the menstrual cycle-related proteins so identified have been identified by amino acid sequence analysis (Ref.2). Selected menstrual cycle-related proteins were excised from several 2D gels, concentrated by ID sodium dodecylsulphate polyacrylamide gel electro- horesis, and cleaved in situ by trypsin. The tryptic fragments were extracted and separated by reverse phase high pressure liquid chromatography. Finally, the partial amino- terminal amino acid sequence of selected tryptic fragments were determined for each protein. For identification the amino acid sequences of the tryptic fragments were compared to previously reported sequences by searching in databases . The hyperplasia and adenocarcinoma associated proteins of the present invention may be sequenced and further characterised by similar methods. Out of a total number of approximately 1,700 spots, 14 spots were found to have increased synthesis in hyperplasia. The locations of these are shown in Figures 1 and 2. Some 27 spots had increased synthesis in adenocarcinoma. The locations of these are shown in Figures 3 and 4. The information obtained from the 2D-gel electrophoresis with respect to the isoelectric point (pi) and the molecular weight (MW) of the spots with increased synthesis in hyperplasia is given in Table 1, and the spots with increased synthesis in adenocarcinoma are listed in Table 2.
Eight spots had increased expression in both hyperplasia and adenocarcinoma. Based on subjective evaluation, preferred subgroups of spots were selected with increased synthesis in hyperplasia and in adenocarcinoma, respectively. The preferred subgroup of spots with increased synthesis in hyperplasia were selected as being the spots showing the highest relative increase in expression in hyperplasia as compared to the samples obtained from women during the normal mentrual cycle and women with irregular proliferative phase endometrium. Similarly, the preferred subgroup of spots with increased synthesis in adenocarcinoma were selected as the spots showing the highest relative increase in expression in adenocarcinoma as compared to the samples obtained from women during the normal menstrual cycle and women with irregular proliferative phase endometrium. The preferred subgroup of 7 spots with increased synthesis in hyperplasia is given in Table 3, and the preferred subgroup of 12 spots with increased synthesis in adenocarcinoma is given in Table
4. TABLE 1
Endometrial proteins with increased synthesis in hyperplasia
Match # Pi MW(kDa)
I#lll 6.7 91
I#121 6.6 90
I#158 6.9 64
I#177 6.6 67
I#191 6.3 66
I#307 6.8 46
I#350 5.7 41
I#405 5.5 35
I#653 5.3 13
I#892 6.6 101
I#1183 5.8 14
N#126 7.4 51
N#148 8.2 44
N#414 9.5 48
Tablβ 2
Endometrial proteins with increased synthesis in adenocarcinoma
Match # p MW(kDa)
I#16 6.3 32
I#53 6.0 109
I#lll 6.7 91
I#121 6.6 90
I#158 6.9 64
I#177 6 . 6 67
I#191 6.3 66
I#194 6.2 62
I#337 6.2 45
I#346 5.7 45
I#436 5.4 33
I#452 6.3 27
I#542 6.5 103
I#558 6.8 90
I#627 6.9 78
I#653 5.3 13
I#788 6.2 130
I#1137 6.3 66
I#1271 6.3 73
N#15 8.3 32
N#91 8.1 55
N#148 8.2 44
N#251 6.6 111
N#354 7.7 43
N#414 9.5 48
N#549 8.3 32
N#551 7.7 39 TABLE 3
Preferred endometrial proteins with increased synthesis in hyperplasia
Match # Pi MW(kDa)
I#lll 6.7 91
I#158 6.9 64
I#191 6.3 66
I#350 5.7 41
I#405 5.5 35
I#653 5.3 13
I#892 6.6 101
TABLE 4
Preferred endometrial proteins with increased synthesis in adenocarcinoma
Match # pi MW(kDa)
I#lll 6.7 91
I#158 6.9 64
I#191 6.3 66
I#194 6.2 62
I#337 6.2 45
I#346 5.7 45
I#452 6.3 27
I#627 6 . 9 78
I#653 5.3 13
N#91 8.1 55
N#354 7.7 43
N#551 7.7 39 Out of the total number of approximately 1,700 spots, 135 had a menstrual cycle-related expression. These 135 spots had maximal expression as follows: 61 spots in proliferative endometrium, 29 spots in interval phase endometrium, 41 in secretory phase endometrium and 4 in late secretory/menstrual phase endometrium. The information obtained from the 2D-gel electrophoresis with respect to the isoelectric point (pi) and the molecular weight (MW) of a preferred subgroup of these spots which show increased synthesis in proliferative phase endometrium are given in Table 5 and their positions are indicated in Figures 5 and 6.
TABLE 5
Endometrial proteins with menstrual cycle-related expression Maximal expression in proliferative phase endometrium
Match # Pi MW(kDa)
I#103 6.9 86
I#590 5.4 34
I#687 5.6 67
I#960 5.3 23
I#1035 6.8 52
N#8 8.7 47
N#21 8.2 138
N#26 6.5 124
N#31 7.7 119
N#32 7.8 119
N#64 8.1 66
N#71 7.1 59
N#74 6.8 66
N#124 7.9 48
N#192 7.7 31
N#207 6.8 29
N#265 7.2 70
N#332 8.0 119
N#342 6.7 62
Fluorographs of gels exemplifying those upon which the identifications given in Tables 1 to 5 above are based appear in Figures 1 to 6. The proteins described above may be further characterised by partial amino acid sequence analysis as described in Ref. 2, or by the more sensitive technique of mass spectrometric peptide mapping. By way of example, we have identified the proteins for which previously given names, data-base accession numbers and amino acid sequences are given in Table 6. Mass spectroscopic characteristics of tryptic digests of further proteins are shown in Figures 7 to 13 which have not matches to any known protein. These proteins can be sequenced by known techniques and are included per se within the scope of the invention.
TABLE 6
Match # Name Amino Acid Sequence ID*
I#191 Human heat MAKAAAIGID LGTTYSCVGV FQHGKVEIIA shock 70 kD NDQGNRTTPS YVAFTDTERL IGDAAKNQVA
And protein 1 LNPQNTVFDA KRLIGRKFGD PWQSDMKHW
PFQVINDGDK PKVQVSYKGE TKAFYPEEIS
I#1137 P08107 SMVLTKMKEI AEAYLGYPVT NAVITVPAYF
NDSQRQATKD AGVIAGLNVL RIINEPTAAA
IAYGLDRTGK GERNVLIFDL GGGTFDVSIL
SEQ ID- TIDDGIFEVK ATAGDTH GG EDFDNRLVNH
No.l FVEEFKRKHK KDISQNKRAV RRLRTACERA
KRTLSSSTQA SLEIDSLFEG IDFYTSITRA
RFEELCSDLF RSTLEPVEKA LRDAKLDKAQ
IHDLVLVGGS TRIPKVQKLL QDFFNGRDLN
KSINPDEAVA YGAAVQAAIL MGDKSENVQD
LLLLDVAPLS LGLETAGGVM TALIKRNSTI
PTKQTQIFTT YSDNQPGVLI QVYEGERAMT
KDNNLLGRFE LSGIPPAPRG VPQIEVTFDI
DANGILNVTA TDKSTGKANK ITITNDKGRL
SKEEIERMVQ EAEKYKAEDE VQRERVSAKN
ALESYAFNMK SAVEDEG KG KISEADKKKV
LDKCQEVISW LDANTLAEKD EFEHKRKELE
QVCNPIISGL YQGAGGPGPG GFGAQGPKGG
SGSGPTIEEV D
I#337 CAMP- ASPPACPSEE DESLKGCELY VQLHGIQQVL dependent KDCIVHLCIS KPERPMKFLR EHFEKLEKEE protein NRQILARQKS NSQSDSHDEE VSPTPPNPW
SEQ ID kinase type KARRRRGGVS AEVYTEEDAV SYVRKVIPKD
I-beta YKTMTALAKA ISKNVLFAHL DDNERSDIFD
No.2 regulatory AMFPVTHIAG ETVIQQGNEG DNFYWDQGE chain VDVYVNGEWV TNISEGGSFG ELALIYGTPR
AATVKAKTD KLWGIDRDSY RRILMGSTLR P31321 KRKMYEEFLS KVSILESLEK WERLTVADRL
EPVQFEDGEK IVVQGEPGDD F IITEGTAS
VLQRRSPNEE YVEVGRLGPS DYFGEIALLL
NRPRAATWA RGPLKCVKLD RPRFERVLGP
CSEILKRNIQ RYNSFISLTV
I#346 Vimentm STRSVSSSSY RRMFGGPGTA SRPSSSRSYV
TTSTRTYSLG SALRPSTSRS LYASSPGGVY
And P08670 ATRSSAVRLR SSVPGVRLLQ DSVDFSLADA
INTEFKNTRT NEKVELQELN DRFANYIDKV
I#405 RFLEQQNKIL LAELEQLKGQ GKSRLGDLYE
EEMRELRRQV DQLTNDKARV EVERDNLAED
IMRLREKLQE EMLQREEAEN
SEQ ID TLQSFRQDVD
NASLARLDLE RKVESLQEEI AFLKKLHEEE
No.3 IQELQAQIQE QHVQIDVDVS KPDLTAALRD
VRQQYESVAA KNLQEAEEWY KSKFADLSEA
ANRNNDALRQ AKQESTEYRR QVQSLTCEVD
ALKGTNESLE RQMREMEENF AVEAANYQDT
IGRLQDEIQN MKEEMARHLR EYQDLLNVKM
ALDIEIATYR KLLEGEESRI SLPLPNFΞSL
NLRETNLDSL PLVDTHSKRT FLIKTVETRD
GQVINETSQH HDDLE
I#452 Heat Shock 27 MTERRVPFSL LRGPSWDPFR DWYPHSRLFD
KD Protein QAFGLPRLPE EWSQWLGGSS WPGYVRPLPP
AAIESPAVAA PAYSRALSRQ LSSGVSEIRH
SEQ ID P04792 TADR RVSLD VNHFAPDELT VKTKDGWEI
TGKHEERQDE HGYISRCFTR KYTLPPGVDP
No.4 TQVSSSLSPE GTLTVEAPMP KLATQSNEIT
And IPVTFESRAQ LGGRSCKIR
Prohibitin MAAKVFESIG KFGLALAVAG GWNSALYNV
DAGHRAVIFD RFRGVQDIW GEGTHFLIPW
P35232 VQKPIIFDCR SRPRNVPVIT GSKDLQNV I
(in TLRILFRPVA SQLPRIFTSI GEDYDERVLP admixture) SITTEILKSV VARFDAGELI TQRELVSRQV
SDDLTERAAT FGLILDDVSL THLTFGKEFT
EAVEAKQVAQ QEAERARFW EKAEQQKKAA
IISAEGDSKA AELIANSLAT AGDGLIELRK
LEAAEDIAYQ LSRSRNITYL PAGQSVLLQL PQ
I#436 Tropomyosin MDAIKKKMQM LKLDKENALD RAEQAEADKK fibroblast AAEDRSKQLE DELVSLQKKL KGTEDELDKY
And isoform TM3 SEALKDAQEK LELAEKKATD AEADVASLNR
RIQLVEEELD RAQERLATAL QKLEEAEKAA
I#590 P09494 DESERGMKVI ESRAQKDEEK MEIQEIQLKE
AKHIAEDADR KYEEVARKLV IIESDLERAE
ERAELSEGQV RQLEEQLRIM DQTLKALMAA
SEQ ID EDKYSQKEDR YEEEIKVLSD KLKEAETRAE
No.5 FAERSVTKLE KSIDDLEEKV AHAKEENLSM HQMLDQTLLE LNNM
I#627 Serotrans- MRLAVGALLV CAVLGLCLAV PDKTVRWCAV ferrin SEHEATKCQS FRDHMKSVIP SDGPSVACVK precursor KASYLDCIRA IAANEADAVT LDAGLVYDAY
SEQ ID LAPNNLKPW AEFYGSKEDP QTFYYAVAW
P02787 KKDSGFQMNQ LRGKKSCHTG LGRSAGWNIP
No.6 IGLLYCDLPE PRKPLEKAVA NFFΞGSCAPC
ADGTDFPQLC QLCPGCGCST LNQYFGYSGA
FKCLKDGAGD VAFVKHSTIF ENLANKADRD
QYELLCLDNT RKPVDEYKDC HLAQVPSHTV
VARSMGGKED LIWELLNQAQ EHFGKDKSKE FQLFSSPHGK DLLFKDSAHG FLKVPPRMDA
KMYLGYEYVT AIRNLREGTC PEAPTDECKP
VKWCALSHHE RLKCDEWSVN SVGKIECVSA
ETTEDCIAKI MNGEADAMSL DGGFVYIAGK
CGLVPVLAEN YNKSDNCEDT PEAGYFAVAV
VKKSASDLTW DNLKGKKSCH TAVGRTAGWN
IPMGLLYNKI NHCRFDEFFS EGCAPGSKKD
SSLCKLCMGS GLNLCEPNNK EGYYGYTGAF
RCLVEKGDVA FVKHQTVPQN TGGKNPDPWA
KNLNEKDYEL LCLDGTRKPV EEYANCHLAR
APNHAWTRK DKEACVHKIL RQQQHLFGSN
VTDCSGNFCL FRSETKDLLF RDDTVCLAKL
HDRNTYEKYL GEEYVKAVGN LRKCSTSSLL EACTFRRP
N#8 47 KD Heat MRSLLLGTLC LLAVALAAEV KKPVEAAAPG
Shock Protein TAEKLSSKAT TLAEPSTGLA FSLYQAMAKD
Precursor QAVENILVSP VWASSLGLV SLGGKATTAS
SEQ ID QAKAVLSAEQ LRDEEVHAGL GELLRSLSNS
P29043 TARNVTWKLG SRLYGPSSVS FADDFVRSSK
No.7 QHYNCEHSKI NFPDKRSALQ SINEWAAQTT DGKLPEVTKD VERTDGALLV NAMFFKPHWD EKFHHKMVDN RGFMVTRSYT VGVTMMHRTG LYNYYDDEKE KLQLVEMPLA HKLSSLIILM PHHVEPLERL EKLLTKEQLK IWMGKMQKKA VAISLPKGW EVTHDLQKHL AGLGLTEAID KNKADLSRMS GKKDLYLASV FHATAFELDT DGNPFDQDIY GREELRSPKL FYADHPFIFL VRDTQSGSLL FIGRLVRLKG DKMRDEL
N#124 Ubiquinol- MKLLTRAGSF SRFYSLKVAP KVKATAAPAG cytochrom C APPQPQDLEF TKLPNGLVIA SLENYSPVSR reductase IGLFIKAGSR YEDFSNLGTT HLLRLTSSLT
SEQ ID complex core TKGASSFKIT RGIEAVGGKL SVTATRENMA protein 2 YTVECLRGDV DILMEFLLNV TTAPEFRRWE
No.8 precursor VADLQPQLKI DKAVAFQNPQ THVIENLHAA AYQNALANPL YCPDYRIGKV TSEELHYFVQ
P22695 NHFTSARMAL IGLGVSHPVL KQVAEQFLNM RGGLGLSGAK ANYRGGEIRE QNGDSLVHAA FVAESAVAGS AEANAFSVLQ HVLGAGPHVK RGSNTTSHLH QAVAKATQQP FDVSAFNASY SDSGLFGIYT ISQATAAGDV IKAAYNQVKR IAQGNLSNTD VQAAKNKLKA GYLMSVESSE CFLEEVGSQA LVAGΞYMPPS TVLQQIDSVA NADIINAAKK FVSGQKSMAA SGNLGHTPFV DEL
N#126 Alpha Enolase SILKIHAREI FDSRGNPTVE VDLFTSKGLF RAAVPSGAST GIYEALELRD NDKTRYMGKG
P06733 VSKAVEHINK TIAPALVSKK LNVTEQEKID
SEQ ID KLMIEMDGTE NKSKFGANAI LGVSLAVCKA
GAVEKGVPLY RHIADLAGNS EVILPVPAFN
No.9 VINGGSHAGN KLAMQEFMIL PVGAANFREA MRIGAEVYHN LKNVIKEKYG KDATNVGDEG
GFAPNILENK EGLELLKTAI GKAGYTDKW
IGMDVAASEF FRSGKYDLDF KSPDDPSRYI
SPDQLADLYK SFIKDYPWS IEDPFDQDDW
GAWQKFTASA GIQWGDDLT VTNPKRIAKA
VNEKSCNCLL LKVNQIGSVT ESLQACKLAQ
ANGWGVMVSH RSGETEDTFI ADLVVGLCTG
QIKTGAPCRS ERLAKYNQLL RIEEELGSKA
KFAGRNFRNP LAK N#148 Phospho- SLSNKLTLDK LDVKGKRVVM glycerate RVDFNVPMKNNQITNNQRIK AAVPSIKFCL kinase 1 DNGAKSWLM
SEQ ID
P00558 SHLGRPDGVP MPDKYSLEPV AVELKSLLGK No.10 DVLFLKDCVG PEVEKACAMP AAGSVILLEN LRFHVEEEGK GKDASGNKVK AEPAKIEAFR ASLSKLGDVY VNDAFGTAHR AHSSMVGVNL PQKAGGFLMK KELNYFAKAL ESPERPFLAI LGGAKVADKI QLINNMLDKV NEMIIGGGMA FTFLKVLNNM EIGTSLFDEE GAKIVKDLMS KAEKNGVKIT LPVDFVTADK FDENAKTGQA TVASGIPAGW MGLDCGPESS KKYAEAVTRA KQIVWNGPVG VFEWEAFARG TKALMDEWK ATSRGCITII GGGDTATCCA KWNTEDKVSH VSTGGGASLE LLEGKVLPGV DALSNIL
N#207 Triose- MAPSRKFFVG GNWKMNGRKQ SLGELIGTLN phosphat AAKVPADTEV VCAPPTAYID FARQKLDPKI isomerase AVAAQNCYKV TNGAFTGEIS PGMIKDCGAT
SEQ ID WWLGHSERR HVFGESDELI GQKVAHALAE
ISHUT GLGVIACIGE KLDEREAGIT EKWFEQTKV No.11 S29743 IADNVKDWSK WLAYEPVWA IGTGKTATPQ QAQEVHEKLR GWLKSNVSDA VAQSTRIIYG GSVTGATCKE LASQPDVDGF LVGGASLKPE FVDIINAKQ
N#332 Hypo-thetical PVPLSFLSTV CDPRVQDGAA ERTGAADGEE
Protein FLGGGGLPAE LFQKKWASF PRTVLSTGMD
KIAA0083 NRYLVLAVNT VQNKEGNCEK RLVITASQSL
SEQ ID ENKELCILRN DWCSVPVEPG DIIHLEGDCT
P51530 SDTWIIDKDF GYLILYPDML ISGTSIASSI No.12 RCMRRAVLSE TFRSSDPATR QMLIGTVLHE VFQKAINNSF APEKLQELAF QTIQEIRHLK EMYRLNLSQD EIKQEVEDYL PSFCKWAGDF MHKNTSTDFP QMQLSLPSDN SKDNSTCNIE WKPMDIEES IWSPRFGLKG KIDVTVGVKI HRGYKTKYKI MPLELKTGKE SNSIEHRSQV VLYTLLSQER RADPEAGLLL YLKTGQMYPV PANHLDKREL LKLRNQMAFS LFHRISKSAT RQKTQLASLP QIIEEEKTCK YCSQIGNCAL YSRAVEQQMD CSSVPIVMLP KIEEETQHLK QTHLEYFSLW CLMLTLESQΞ KDNKKNHQNI WLMPASEMEK SGSCIGNLIR MEHVKIVCDG QYLHNFQCKH GAIPVTNLMA GDRVIVSGEE RSLFALSRGY VKEINMTTVT CLLDRNLSVL PESTLFRLDQ EEKNCDIDTP LGNLSKLMEN TFVSKKLRDL IIDFREPQFI SYLSSVLPHD AKDTVACILK GLNKPQRQAM KKVLLSKDYT LIVGMPGTGK TTTICTLVRI LYACGFSVLL TSYTHSAVDN ILLKLAKFKI GFLRLGQIQK VHPAIQQFTE QEICRSKSIK SLALLEELYN SQLIVATTCM GINHPIFSRK IFDFCIVDEA SQISQPICLG PLFFSRRFVL VGDHQQLPPL VLNREARALG MSESLFKRLE QNKSAWQLT VQYRMNSKIM SLSNKLTYEG KLECGSDKVA NAVINLRHFK DVKLELEFYA DYSDNPWLMG VFEPNNPVCF LNTDKVPAPE QVEKGGVSNV TEAKLIVFLT SIFVKAGCSP SDIGIIAPYR QQLKIINDLL ARSIGMVEVN TVDKYQGRDK SIVLVSFVRS NKDGTVGELL KDWRRLNVAI TRAKHKLILL GCVPSLNCYP PLEKLLNHLN SEKLIIDLPS REHESLCHIL GDFQRE
N#342 Catalase MADSRDPASD QMQHWKEQRA AQKADVLTTG AGNPVGDKLN VITVGPRGPL LVQDWFTDE
P04040 MAHFDRERIP ERWHAKGAG AFGYFEVTHD
SEQ ID ITKYSKAKVF EHIGKKTPIA VRFSTVAGES
GSADTVRDPR GFAVKFYTED GNWDLVGNNT
No.13 PIFFIRDPIL FPSFIHSQKR NPQTHLKDPD MVWDFWSLRP ESLHQVSFLF SDRGIPDGHR HMNGYGSHTF KLVNANGEAV YCKFHYKTDQ GIKNLSVEDA ARLSQEDPDY GIRDLFNAIA TGKYPSWTFY IQVMTFNQAE TFPFNPFDLT KVWPHKDYPL IPVGKLVLNR NPVNYFAEVE QIAFDPSNMP PGIEASPDKM LQGRLFAYPD THRHRLGPNY LHIPVNCPYR ARVANYQRDG PMCMQDNQGG APNYYPNSFG APEQQPSALE HSIQYSGEVR RFNTANDDNV TQVRAFYVNV LNEEQRKRLC ENIAGHLKDA QIFIQKKAVK NFTEVHPDYG SHIQALLDKY NAEKPKNAIH TFVQSGSHLA AREKANL
N#551 HeteroMEKTLETVPL ERKKREKEQF RKLFIGGLSF geneous ETTEESLRNY YEQWGKLTDC WMRDPASKR nuclear SRGFGFVTFS SMAEVDAAMA ARPHSIDGRV
SEQ ID πbonucleo- VEPKRAVARE ESGKPGAHVT VKKLFVGGIK proteins EDTEEHHLRD YFEEYGKIDT IEIITDRQSG
No.14 A2/B1 KKRGFGFVTF DDHDPVDKIV LQKYHTINGH NAEVRKALSR QEMQEVQSSR SGRGGNFGFG
P22626 DSRGGGGNFG PGPGSNFRGG SDGYGSGRGF GDGYNGYGGG PGGGNFGGSP GYGGGRGGYG GGGPGYGNQG GGYGGGYDNY GGGNYGSGNY NDFGNYNQQP SNYGPMKSGN FGGSRNMGGP YGGGNYGPGG SGGSGGYGGR SRY
I#960 Steroid MAAEDVAATG ADPSELEGGG LLHEIFTSPL NLLLLGLCIF
(Prolifer membrane LLYKIVRGDQ PAASDSDDDE PPPLPRLKRR DFTPAELRRF ative binding DGVQDPRILM AINGKVFDVT KGRKFYGPEG PYGVFAGRDA phase protein SRGLATFCLD KEALKDEYDD LSDLTPAQQE TLNDWDSQFT marker) FKYHHVGKLL KEGEEPTVYS DEEEPKDESA RKND
SEQ ID X99714
No.15
I#177 MSKGPAVGID LGTTYSCVGV FQHGKVEIIA NDQGNRTTPS
(Hyperpla Heat shock YVAFTDTERL IGDAAKNQVA MNPTNTVFDA KRLIGRRFDD sia & cognate 71 KD AVVQSDMKHW PFMWNDAGR PKVQVEYKGE TKSFYPEEVS
Cancer protein SMVLTKMKEI AEAYLGKTVT NAWTVPAYF NDSQRQATKD
Marker) AGTIAGLNVL RIINEPTAAA IAYGLDKKVG AERNVLIFDL
P11142 GGGTFDVSIL TIEDGIFEVK STAGDTHLGG EDFDNRMVNH
SEQ ID FIAEFKRKHK KDISENKRAV RRLRTACERA KRTLSSSTQA
No.16 SIEIDSLYEG IDFYTSITRA RFEELNADLF RGTLDPVEKA LRDAKLDKSQ IHDIVLVGGS TRIPKIQKLL QDFFNGKELN KSINPDEAVA YGAAVQAAIL SGDKSENVQD LLLLDVTPLS LGIETAGGVM TVLIKRNTTI PTKQTQTFTT
YSDNQPGVLI QVYEGERAMT KDNNLLGKFE LTGIPPAPRG VPQIEVTFDI DANGILNVSA VDKSTGKENK ITITNDKGRL SKEDIERMVQ EAEKYKAEDE KQRDKVSSKN SLESYAFNMK ATVEDEKLQG KINDEDKQKI LDKCNEIINW LDKNQTAEKE EFEHQQKELE KVCNPIITKL YQSAGGMPGG MPGGFPGGGA
PPSGGASSGP TIEEVD
ID': Accession Identification in protein or nucleotide databases (e.g. SwissProt, Protein Identification Resource (PIR) or EMBL) The proteins of interest may be isolated from endometrial tissue or other protein sources by 2D gel electrophoresis or by using chromatographic techniques. Poly- or monoclonal antibodies towards the protein of interest can be raised, and immunoassays can be established based on such antibodies. Synthetic peptides being fragments characteristic of such proteins may be used for the same purposes . Assays may be based on more than one such protein for measurement at one time.
Ref.1 Byrjalsen et al. Hum Reprod 1995;10:13-18. Ref .2 Byrjalsen et al . , Hum Reprod 1995;10:2760-2766. Ref .3 Jul unen et al . , Endocrinology 1986;118:1782-1786. Ref.4 Byr-jalsen et al., Obstet Gynecol 1992;79:523-528. Ref .5 Byrjalsen et al., Hum Reprod 1992;7:1042-1047.
3EQUENCE ISTING
GENERAL INFORMATION:
(l) APPLICANT:
(A) NAME: Center for Clinical and Basic Research (B) STREET: Ballerup Byve} 222,
(C) CITY: Ballerup
(E) COUNTRY: Denmark
(F) POSTAL CODE (ZIP) : DK-2750 (ii) TITLE OF INVENTION: Biochemical Markers for the Human Endometrium
(in) NUMBER OF SEQUENCES: 16 (lv) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk
(B) COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: Patentln Release #1.0, Version #1.30 (EPO)
(vi) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: GB 9618600.2
(B) TILING DATE: 06-SEP-1996 (vi) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: GB 9707132.8
(B) FILING DATE: 08-APR-1997
(2) INFORMATION FOR SEQ ID NO: 1:
(l) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 641 ammo acids
(B) TYPE: amino acid (C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(n) MOLECULE TYPE: protein (in) HYPOTHETICAL: NO
(lv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE: (A) ORGANISM: homo sapiens
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:
Met Ala Lys Ala Ala Ala He Gly He Asp Leu Gly Thr Thr Tyr Ser 1 5 10 15
Cys Val Gly Val Phe Gin His Gly Lys Val Glu He He Ala Asn Asp 20 25 30
Gin Gly Asn Arg Thr Thr Pro Ser Tyr Val Ala Phe Thr Asp Thr Glu 35 40 45 Arg Leu He Gly ASD Ala Ala Lys Asn Gin Val Ala Leu As.- Pro Gin 50 55 60
Asn Thr Val Phe Asp Ala Lvs Arg Leu He Glv Arg Lys P e Gly Asp 65 70 75^ 80
Pre Val Val Gin Ser Asp Met Lys His Trp Pro Phe Gin Val He Asn 85 90 95
Asp Gly Asp Lys Pro Lys Val Gin Val Ser Tyr Lvs Gly Glu Thr Lys 100 105 ' 110
Ala Phe Tyr Pro Glu Glu He Ser Ser Met Val Leu Thr Lys Met Lys 115 120 125
Glu He Ala Glu Ala Tyr Leu Gly Tyr Pro Val Thr Asn Ala Val He 130 135 140
Thr Val Pro Ala Tyr Phe Asn Asp Ser Gin Arg Gin Ala Thr Lys Asp 145 150 155 160
Ala Gly Val He Ala Gly Leu Asn Val Leu Arg He He Asn Glu Pro 165 170 175
Thr Ala Ala Ala He Ala Tyr Gly Leu Asp Arg Thr Gly Lys Gly Glu 180 185 190
Arg Asn Val Leu He Phe Asp Leu Gly 'Gly Gly Thr Phe Asp Val Ser
195 200 205
He Leu Thr He Asp Asp Gly He Phe Glu Val Lys Ala Thr Ala Gly
210 215 220
ASD Thr His Leu Gly Gly Glu ASD Phe Asp Asn Arg Leu Val Asn His 225 230 ' 235 240
Phe /'al Glu Glu Phe Lys Arg Lys His Lys Lys Asp He Ser Gin Asn 245 250 255 Lys Arg Ala Val Arg Arg Leu Arg Thr Ala Cys Glu Arg Ala Lys Arg
260 265 270
Thr Leu Ser Ser Ser Thr Gin Ala Ser Leu Glu He Asp Ser Leu Phe 275 280 285
Glu Gly He Asp Phe Tyr Thr Ser He Thr Arg Ala Arg Phe Glu Glu 290 295 300
Leu Cys Ser Asp Leu Phe Arg Ser Thr Leu Glu Pro Val Glu Lys Ala 305 310 315 320
Leu Arg Asp Ala Lys Leu Asp Lys Ala Gin He His Asp Leu Val Leu 325 330 335 Val Gly Gly Ser Thr Arg He Pro Lys Val Gin Lys Leu Leu Gin Asp
340 345 350
Phe Phe Asn Gly Arg Asp Leu Asn Lys Ser He Asn Pro Asp Glu Ala
355 360 365
Val Ala Tyr Gly Ala Ala Val Gin Ala Ala He Leu Met Gly Asp Lys
370 375 380 Ser Glu Asn Val Gin Asp Leu Leu Leu Leu Asp Val Ala Pro Leu Ser 385 390" 395 400
Leu Gly Leu Glu Thr Ala Gly Gly Val Met Thr Ala Leu He Lys Arg 405 410 415
Asn Ser Thr He Pro Thr Lys Gin Thr Gin He Phe Thr Thr Tyr Ser 420 425 430
Asp Asn Gin Pro Gly Val Leu He Gin Val Tyr Glu Gly Glu Arg Ala
435 440 445
Met Thr Lys Asp Asn Asn Leu Leu Gly Arg Phe Glu Leu Ser Gly He
450 455 460
Pro Pro Ala Pro Arg Gly Val Pro Gin He Glu Val Thr Phe Asp He 465 470 475 480
Asp Ala Asn Gly He Leu Asn Val Thr Ala Thr Asp Lys Ser Thr Gly 485 490 495
Lys Ala Asn Lys He Thr He Thr Asn Asp Lys Gly Arg Leu Ser Lys 500 505 510
Glu Glu He Glu Arg Met Val Gin Glu Ala Glu Lys Tyr Lys Ala Glu 515 520 525
Asp Glu Val Gin Arg Glu Arg Val Ser Ala Lys Asn Ala Leu Glu Ser 530 535 540
Tyr Ala Phe Asn Met Lys Ser Ala Val Glu Asp Glu Gly Leu Lys Gly 545 550 555 560
Lys He Ser Glu Ala Asp Lys Lys Lys Val Leu Asp Lys Cys Gin Glu 565 570 575
Val He Ser Trp Leu Asp Ala Asn Thr Leu Ala Glu Lys ASD Glu Phe 580 585 590 Glu His Lys Arg Lys Glu Leu Glu Gin Val Cys Asn Pro He He Ser 595 600 605
Gly Leu Tyr Gin Gly Ala Gly Gly Pro Gly Pro Gly Gly Phe Gly Ala 610 615 620
Gin Gly Pro Lys Gly Gly Ser Gly Ser Gly Pro Thr He Glu Glu Val 625 630 635 640
Asp
(2) INFORMATION FOR SEQ ID NO: 2:
(l) SEQUENCE CHARACTERISTICS: (A) LENGTH: 380 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (in) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: homo sapiens
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2: Ala Ser Pro Pro Ala Cys Pro Ser Glu Glu Asp Glu Ser Leu Lys Gly 1 5 10 15
Cys Glu Leu Tyr Val Gin Leu His Gly He Gin Gin Val Leu Lys Asp 20 25 30
Cys He Val His Leu Cys He Ser Lys Pro Glu Arg Pro Met Lys Phe 35 40 45
Leu Arg Glu His Phe Glu Lys Leu Glu Lys Glu Glu Asn Arg Gin He 50 55 60
Leu Ala Arg Gin Lys Ser Asn Ser Gin Ser Asp Ser His Asp Glu Glu 65 70 75 80
Val Ser Pro Thr Pro Pro Asn Pro Val Val Lys Ala Arg Arg Arg Arg
85 90 95 Gly Gly Val Ser Ala Glu Val Tyr Thr Glu Glu Asp Ala Val Ser Tyr
100 105 110
Val Arg Lys Val He Pro Lys Asp Tyr Lys Thr Met Thr Ala Leu Ala
115 120 125
Lys Ala He Ser Lys Asn Val Leu Phe Ala His Leu Asp Asp Asn Glu
130 135 140
Arg Ser Asp He Phe Asp Ala Met Phe Pro Val Thr His He Ala Gly 145 150 155 160
Glu Thr Val He Gin Gin Gly Asn Glu Gly Asp Asn Phe Tyr Val Val
165 170 175 ASD Gin Gly Glu Val Asp Val Tyr Val Asn Gly Glu Trp Val Thr Asn
180 185 190
He Ser Glu Gly Gly Ser Phe Gly Glu Leu Ala Leu He Tyr Gly Thr
195 200 205
Pro Arg Ala Ala Thr Val Lys Ala Lys Thr Asp Leu Lys Leu Trp Gly
210 215 220
He ASD Arg Asp Ser Tyr Arg Arg He Leu Met Gly Ser Thr Leu Arg 225 ' 230 235 240
Lys Arg Lys Met Tyr Glu Glu Phe Leu Ser Lys Val Ser He Leu Glu
245 250 255 Ser Leu Glu Lys Trp Glu Arg Leu Thr Val Ala Asp Arg Leu Glu Pro
260 265 270
Val Gin Phe Glu Asp Gly Glu Lys He Val Val Gin Gly Glu Pro Gly 275 280 285
Asp ASD Phe Tyr He He Thr Glu Gly Thr Ala Ser Val Leu Gin Arg 29θ' 295 300 Arg Ser Pro Asn Glu Glu Tyr Val Glu Val Gly Arg Leu Gly Pro Ser 305 310 315 320
Asp Tvr Phe Gly Glu He Ala Leu Leu Leu Asn Arg Pro Arg Ala Ala 325 330 335
Thr Val Val Ala Arg Gly Pro Leu Lys Cys Val Lys Leu ASD Arg Pro 340 345 350
Arg Phe Glu Arg Val Leu Gly Pro Cys Ser Glu He Leu Lys Arg Asn 355 360 365
He Gin Arg Tyr Asn Ser Phe He Ser Leu Thr Val 370 375 380
(2) INFORMATION FOR SEQ ID NO: 3:
(l) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 465 ammo acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single (D) TOPOLOGY: linear
(i ) MOLECULE TYPE: protein
(in) HYPOTHETICAL: NO
(lv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: homo sapiens
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:
Ser Thr Arg Ser Val Ser Ser Ser Ser Tyr Arg Arg Met Phe Gly Gly 1 5 10 15
Pro Gly Thr Ala Ser Arg Pro Ser Ser Ser Arg Ser Tyr Val Thr Thr 20 25 30
Ser Thr Arg Thr Tyr Ser Leu Gly Ser Ala Leu Arg Pro Ser Thr Ser 35 40 45
Arg Ser Leu Tyr Ala Ser Ser Pro Gly Gly Val Tyr Ala Thr Arg Ser 50 55 60
Ser Ala Val Arg Leu Arg Ser Ser Val Pro Gly Val Arg Leu Leu Gin 65 70 75 80
Asp Ser Val Asp Phe Ser Leu Ala Asp Ala He Asn Thr Glu Phe Lys 85 90 95
Asn Thr Arg Thr Asn Glu Lys Val Glu Leu Gin Glu Leu Asn Asp Arg 100 105 110
Phe Ala Asn Tyr He Asp Lys Val Arg Phe Leu Glu Gin Gin Asn Lys 115 120 125
He Leu Leu Ala Glu Leu Glu Gin Leu Lys Gly Gin Gly Lys Ser Arg 130 135 140 Leu Gly Asp Lou Tyr Glu Glu Glu Met Arg Glu Leu Arg Arg Gin Val 145 150 155 160
Asp C n Leu Thr Asn Asp Lys Ala Arg Val Glu Val CVJ. Arg Asp Asn 165 170 175
Leu Ala Glu Asp He Met Arg Leu Arg Glu Lys Leu Gin Glu Glu Met 180 185 190
Leu Gin Arg Glu Glu Ala Glu Asn Thr Leu Gin Ser Phe Arg Gin Asp 195 200 205
Val ASD Asn Ala Ser Leu Ala Arg Leu Asp Leu Glu Arg Lys Val Glu 210 215 220
Ser Leu Gin Glu Glu He Ala Phe Leu Lys Lys Leu His Glu Glu Glu 225 230 235 240
He Gin Glu Leu Gin Ala Gin He Gin Glu Gin His Val Gin He Asp 245 250 255
Val Asp Val Ser Lys Pro Asp Leu Thr Ala Ala Leu Arg Asp Val Arg 260 265 270
Gin Gin Tyr Glu Ser Val Ala Ala Lys Asn Leu Gin Glu Ala Glu Glu 275 280 285
Trp Tyr Lys Ser Lys Phe Ala Asp Leu Ser Glu Ala Ala Asn Arg Asn 290 295 300
Asn Asp Ala Leu Arg Gin Ala Lys Gin Glu Ser Thr Glu Tyr Arg Arg 305 310 315 320
Gin Val Gin Ser Leu Thr Cys Glu Val Asp Ala Leu Lys Gly Thr Asn 325 330 335 Glu Ser Leu Glu Arg Gin Met Arg Glu Met Glu Glu Asn Phe Ala Val
340 345 350
Glu Ala Ala Asn Tyr Gin Asp Thr He Gly Arg Leu Gin Asp Glu He 355 360 365
Gin Asn Met Lys Glu Glu Met Ala Arg His Leu Arg Glu Tyr Gin Asp 370 375 380
Leu Leu Asn Val Lys Met Ala Leu Asp He Glu He Ala Thr Tyr Arg 385 390 395 400
Lys Leu Leu Glu Gly Glu Glu Ser Arg He Ser Leu Pro Leu Pro Asn 405 410 415 Phe Ser Ser Leu Asn Leu Arg Glu Thr Asn Leu Asp Ser Leu Pro Leu
420 425 430
Val Asp Thr His Ser Lys Arg Thr Phe Leu He Lys Thr Val Glu Thr 435 440 445
Arg Asp Gly Gin Val He Asn Glu Thr Ser Gin His His Asp Asp Leu 450 455 460
Glu 465 (2) INFORMATION FOR SEQ ID NO: 4.
(i) SEQUENCE CHARACTERISTICS: Α) LENGTH: 471 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear (u) MOLECULE TYPE: protein (m) HYPOTHETICAL: NO (IV) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: homo sapiens
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:
Met Thr Glu Arg Arg Val Pro Phe Ser Leu Leu Arg Gly Pro Ser Trp 1 5 10 15
Asp Pro Phe Arg Asp Trp Tyr Pro His Ser Arg Leu Phe Asp Gin Ala
20 25 30 Phe Gly Leu Pro Arg Leu Pro Glu Glu Trp Ser Gin Trp Leu Gly Gly 35 40 45
Ser Ser Trp Pro Gly Tyr Val Arg Pro Leu Pro Pro Ala Ala He Glu 50 55 60
Ser Pro Ala Val Ala Ala Pro Ala Tyr Ser Arg Ala Leu Ser Arg Gin 65 70 75 80
Leu Ser Ser Gly Val Ser Glu He Arg His Thr Ala Asp Arg Trp Arg 85 90 95
Val Ser Leu Asp Val Asn His Phe Ala Pro Asp Glu Leu Thr Val Lys
100 105 110
Thr Lys Asp Gly Val Val Glu He Thr Gly Lys His Glu Glu Arg Gin 115 120 125
Asp Glu His Gly Tyr He Ser Arg Cys Phe Thr Arg Lys Tyr Thr Leu 130 135 140
Pro Pro Gly Val Asp Pro Thr Gin Val Ser Ser Ser Leu Ser Pro Glu 145 150 155 160
Gly Thr Leu Thr Val Glu Ala Pro Met Pro Lys Leu Ala Thr Gin Ser 165 170 175
Asn Glu He Thr He Pro Val Thr Phe Glu Ser Arg Ala Gin Leu Gly
180 185 190 Gly Arg Ser Cys Lys He Arg Met Ala Ala Lys Val Phe Glu Ser He
195 200 205
Gly Lys Phe Gly Leu Ala Leu Ala Val Ala Gly Gly Val Val Asn Ser 210 215 220 Ala Leu Tyr Asn Val Asp Ala Gly His Arg Ala Val He Phe ASD Arg 225 230 235 ' 240
Phe Ar-J. Gly Val Gin ASD He Val Val Gly Glu Gly Thr His Phe Leu 245 250 255
He Pro Trp Val Gin Lys Pro He He Phe Asp Cys Arg Ser Arg Pro 260 265 270
Arg Asn Val Pro Val He Thr Gly Ser Lys Asp Leu Gin Asn Val Asn
275 280 285
He Thr Leu Arg He Leu Phe Arg Pro Val Ala Ser Gin Leu Pro Arg 290 295 300
He Phe Thr Ser He Gly Glu Asp Tyr Asp Glu Arg Val Leu Pro Ser 305 310 315 320
He Thr Thr Glu He Leu Lys Ser Val Val Ala Arg Phe Asp Ala Gly 325 330 335
Glu Leu He Thr Gin Arg Glu Leu Val Ser Arg Gin Val Ser Asp Asp 340 345 350
Leu Thr Glu Arg Ala Ala Thr Phe Gly Leu He Leu ASD Asp Val Ser 355 360 365
Leu Thr His Leu Thr Phe Gly Lys Glu Phe Thr Glu Ala Val Glu Ala 370 375 380
Lys Gin Val Ala Gin Gin Glu Ala Glu Arg Ala Arg Phe Val Val Glu 385 390 395 400
Lys Ala Glu Gin Gin Lys Lys Ala Ala He He Ser Ala Glu Gly Asp 405 410 415
Ser Lys Ala Ala Glu Leu He Ala Asn Ser Leu Ala Thr Ala Gly Asp 420 425 430
Gly Leu He Glu Leu Arg Lys Leu Glu Ala Ala Glu Asp He Ala Tyr 435 440 445
Gin Leu Ser Arg Ser Arg Asn He Thr Tyr Leu Pro Ala Gly Gin Ser 450 455 460
Val Leu Leu Gin Leu Pro Gin 465 470
(2) INFORMATION FOR SEQ ID NO: 5:
(l) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 284 amino acids (B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(in) HYPOTHETICAL: NO
(lv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE:
(A) ORGANISM: homo sapiens (xι) SEQUENCE DESCRIPTION: SEQ ID NO: 5:
Met Asp Ala He Lys Lys Lys Met Gin Met Leu Lys Leu ASD Lys Glu 1 5 10 15
Asn Ala Leu Asp Arg Ala Glu Gin Ala Glu Ala Asp Lys Lys Ala Ala 20 25 30 Glu Asp Arg Ser Lys Gin Leu Glu Asp Glu Leu Val Ser Leu Gin Lys 35 40 45
Lys Leu Lys Gly Thr Glu Asp Glu Leu Asp Lys Tyr Ser Glu Ala Leu 50 55 60
Lys Asp Ala Gin Glu Lys Leu Glu Leu Ala Glu Lys Lys Ala Thr Asp 65 70 75 80
Ala Glu Ala Asp Val Ala Ser Leu Asn Arg Arg He Gin Leu Val Glu 85 90 95
Glu Glu Leu Asp Arg Ala Gin Glu Arg Leu Ala Thr Ala Leu Gin Lys 100 105 110
Leu Glu Glu Ala Glu Lys Ala Ala Asp Glu Ser Glu Arg Gly Met Lys 115 120 125
Val He Glu Ser Arg Ala Gin Lys Asp Glu Glu Lys Met Glu He Gin 130 135 140
Glu He Gin Leu Lys Glu Ala Lys His He Ala Glu Asp Ala Asp Arg 145 150 155 160
Lys Tyr Glu Glu Val Ala Arg Lys Leu Val He He Glu Ser Asp Leu 165 170 175
Glu Arg Ala Glu Glu Arg Ala Glu Leu Ser Glu Gly Gin Val Arg Gin 180 185 190
Leu Glu Glu Gin Leu Arg He Met Asp Gin Thr Leu Lys Ala Leu Met
195 200 205 Ala Ala Glu Asp Lys Tyr Ser Gin Lys Glu Asp Arg Tyr Glu Glu Glu
210 215 220
He Lys Val Leu Ser Asp Lys Leu Lys Glu Ala Glu Thr Arg Ala Glu
225 230 235 240
Phe Ala Glu Arg Ser Val Thr Lys Leu Glu Lys Ser He Asp Asp Leu
245 250 255
Glu Glu Lys Val Ala His Ala Lys Glu Glu Asn Leu Ser Met His Gin 260 265 270
Met Leu Asp Gin Thr Leu Leu Glu Leu Asn Asn Met 275 280 (2) INFORMATION FOR SEQ ID NO: 6:
(i) SEQUENCE CHARACTERISTICS: (A; LENGTH: 698 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (lii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: homo sapiens
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:
Met Arg Leu Ala Val Gly Ala Leu Leu Val Cys Ala Val Leu Gly Leu 1 5 10 15 Cys Leu Ala Val Pro Asp Lys Thr Val Arg Trp Cys Ala Val Ser Glu
20 25 30
His Glu Ala Thr Lys Cys Gin Ser Phe Arg Asp His Met Lys Ser Val 35 40 45
He Pro Ser Asp Gly Pro Ser Val Ala Cys Val Lys Lys Ala Ser Tyr 50 55 60
Leu Asp Cys He Arg Ala He Ala Ala Asn Glu Ala Asp Ala Val Thr 65 70 75 80
Leu Asp Ala Gly Leu Val Tyr Asp Ala Tyr Leu Ala Pro Asn Asn Leu 85 90 95
Lys Pro Val Val Ala Glu Phe Tyr Gly Ser Lys Glu Asp Pro Gin Thr 100 105 110
Phe Tyr Tyr Ala Val Ala Val Val Lys Lys Asp Ser Gly Phe Gin Met 115 120 125
Asn Gin Leu Arg Gly Lys Lys Ser Cys His Thr Gly Leu Gly Arg Ser
130 135 140 Ala Gly Trp Asn He Pro He Gly Leu Leu Tyr Cys Asp Leu Pro Glu 145 150 155 160
Pro Arg Lys Pro Leu Glu Lys Ala Val Ala Asn Phe Phe Ser Gly Ser 165 170 175
Cys Ala Pro Cys Ala Asp Gly Thr Asp Phe Pro Gin Leu Cys Gin Leu 180 185 190
Cys Pro Gly Cys Gly Cys Ser Thr Leu Asn Gin Tyr Phe Gly Tyr Ser 195 200 205
Gly Ala Phe Lys Cys Leu Lys Asp Gly Ala Gly Asp Val Ala Phe Val
210 215 220
Lys His Ser Thr He Phe Glu Asn Leu Ala Asn Lys Ala Asp Arg Asp
225 230 235 240 Gln Tyr Glu Leu Leu Cys Leu Asp Asn Thr Arg Lys Pro Val Asp Glu 245 250 255
T/r Lvs Asp Cys HJ.S Leu Ala Gin Val Pro Ser His Thr Val Val Ala 260 265 270
Arg Ser Met Gly Gly Lys Glu Asp Leu He Trp Glu Leu Leu Asn Gin 275 280 285
Ala Gin Glu His Phe Gly Lys Asp Lys Ser Lys Glu Phe Gin Leu Phe 290 295 300
Ser Ser Pro His Gly Lys Asp Leu Leu Phe Lys Asp Ser Ala His Gly 305 310 315 320
Phe Leu Lys Val Pro Pro Arg Met Asp Ala Lys Met Tyr Leu Gly Tyr 325 330 335
Glu Tyr Val Thr Ala He Arg Asn Leu Arg Glu Gly Thr Cys Pro Glu 340 345 350
Ala Pro Thr Asp Glu Cys Lys Pro Val Lys Trp Cys Ala Leu Ser His 355 360 365
His Glu Arg Leu Lys Cys Asp Glu Trp Ser Val Asn Ser Val Gly Lys 370 375 380
He Glu Cys Val Ser Ala Glu Thr Thr Glu Asp Cys He Ala Lys He 385 390 395 400
Met Asn Gly Glu Ala Asp Ala Met Ser Leu Asp Gly Gly Phe Val Tyr 405 410 415
He Ala Gly Lys Cys Gly Leu Val Pro Val Leu Ala Glu Asn Tyr Asn 420 425 430
Lys Ser Asp Asn Cys Glu Asp Thr Pro Glu Ala Gly Tyr Phe Ala Val 435 440 445
Ala Val Val Lys Lys Ser Ala Ser Asp Leu Thr Trp Asp Asn Leu Lys
450 455 460 Gly Lys Lys Ser Cys His Thr Ala Val Gly Arg Thr Ala Gly Trp Asn
465 470 475 480
He Pro Met Gly Leu Leu Tyr Asn Lys He Asn His Cys Arg Phe Asp 485 490 495
Glu Phe Phe Ser Glu Gly Cys Ala Pro Gly Ser Lys Lys Asp Ser Ser 500 505 510
Leu Cys Lys Leu Cys Met Gly Ser Gly Leu Asn Leu Cys Glu Pro Asn 515 520 525
Asn Lys Glu Gly Tyr Tyr Gly Tyr Thr Gly Ala Phe Arg Cys Leu Val
530 535 540
Glu Lys Gly Asp Val Ala Phe Val Lys His Gin Thr Val Pro Gin Asn 545 550 555 560
Thr Gly Gly Lys Asn Pro Asp Pro Trp Ala Lys Asn Leu Asn Glu Lys 565 570 575
Asp Tyr Glu Leu Leu Cys Leu Asp Gly Thr Arg Lys Pro Val Glu Glu 580 585 590 Tyr Ala Asn Cys His Leu Ala Arg Ala Pro Asn His Ala Val Val Thr 595 600 605
Arg L.ό ASD Lys Glu Ala Cys Val His Lys He Leu Arg Gin Gin Gin 63.0 615 620
His Leu Phe Gly Ser Asn Val Thr Asp Cys Ser Gly Asn Phe Cys Leu 625 630 635 640
Phe Arg Ser Glu Thr Lys Asp Leu Leu Phe Arg Asp Asp Thr Val Cys 645 650 655
Leu Ala Lys Leu His Asp Arg Asn Thr Tyr Glu Lys Tyr Leu Gly Glu 660 665 670
Glu Tyr Val Lys Ala Val Gly Asn Leu Arg Lys Cys Ser Thr Ser Ser 675 680 685
Leu Leu Glu Ala Cys Thr Phe Arg Arg Pro 690 695
(2) INFORMATION FOR SEQ ID NO: 7:
(l) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 417 amino acids
(B) TYPE: amino ac d
(C) STRANDEDNESS: single (D) TOPOLOGY: linear
(n) MOLECULE TYPE: protein
(in) HYPOTHETICAL: NO
(lv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: homo sapiens
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7:
Met Arg Ser Leu Leu Leu Gly Thr Leu Cys Leu Leu Ala Val Ala Leu 1 5 10 15
Ala Ala Glu Val Lys Lys Pro Val Glu Ala Ala Ala Pro Gly Thr Ala 20 25 30 Glu Lys Leu Ser Ser Lys Ala Thr Thr Leu Ala Glu Pro Ser Thr Gly 35 40 45
Leu Ala Phe Ser Leu Tyr Gin Ala Met Ala Lys Asp Gin Ala Val Glu 50 55 60
Asn He Leu Val Ser Pro Val Val Val Ala Ser Ser Leu Gly Leu Val 65 70 75 80
Ser Leu Gly Gly Lys Ala Thr Thr Ala Ser Gin Ala Lys Ala Val Leu 85 90 95
Ser Ala Glu Gin Leu Arg Asp Glu Glu Val His Ala Gly Leu Gly Glu 100 105 110
Leu Leu Arg Ser Leu Ser Asn Ser Thr Ala Arg Asn Val Thr Trp Lys
115 120 125 Leu Gly Ser Arg Leu Tyr Gly Pro Ser Ser Val Ser Phe Ala Asp Asp 130 135 140
Phe Val Arg Ser Ser Lys Gin His Tyr Asn Cys Glu His Ser Lys He 145 150 155 160
Asn Phe Pro Asp Lys Arg Ser Ala Leu Gin Ser He Asn Glu Trp Ala 165 170 175
Ala Gin Thr Thr Asp Gly Lys Leu Pro Glu Val Thr Lys Asp Val Glu 180 185 190
Arg Thr Asp Gly Ala Leu Leu Val Asn Ala Met Phe Phe Lys Pro His 195 200 205
Trp Asp Glu Lys Phe His His Lys Met Val Asp Asn Arg Gly Phe Met 210 215 220
Val Thr Arg Ser Tyr Thr Val Gly Val Thr Met Met His Arg Thr Gly 225 230 235 240
Leu Tyr Asn Tyr Tyr Asp Asp Glu Lys Glu Lys Leu Gin Leu Val Glu
245 250 255
Met Pro Leu Ala His Lys Leu Ser Ser Leu He He Leu Met Pro His
260 265 270
His Val Glu Pro Leu Glu Arg Leu Glu Lys Leu Leu Thr Lys Glu Gin 275 280 285
Leu Lys He Trp Met Gly Lys Met Gin Lys Lys Ala Val Ala He Ser 290 295 300 Leu Pro Lys Gly Val Val Glu Val Thr His Asp Leu Gin Lys His Leu 305 310 315 320
Ala Gly Leu Gly Leu Thr Glu Ala He Asp Lys Asn Lys Ala Asp Leu 325 330 335
Ser Arg Met Ser Gly Lys Lys Asp Leu Tyr Leu Ala Ser Val Phe His
340 345 350
Ala Thr Ala Phe Glu Leu Asp Thr Asp Gly Asn Pro Phe Asp Gin Asp 355 360 365
He Tyr Gly Arg Glu Glu Leu Arg Ser Pro Lys Leu Phe Tyr Ala Asp 370 375 380 His Pro Phe He Phe Leu Val Arg Asp Thr Gin Ser Gly Ser Leu Leu 385 390 395 400
Phe He Gly Arg Leu Val Arg Leu Lys Gly Asp Lys Met Arg Asp Glu 405 410 415
Leu
(2) INFORMATION FOR SEQ ID NO: 8:
(l) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 453 amino acids
(B) TYPE: amino acid (C) STRANDEDNESS : single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein (lii) HYPOTHETICAL: NO
(lv) ANTI-SENSE: MO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: homo sapiens
(xι) SEQUENCE DESCRIPTION: SEQ ID NO: 8:
Met Lys Leu Leu Thr Arg Ala Gly Ser Phe Ser Arg Phe Tyr Ser Leu 1 5 10 15
Lys Val Ala Pro Lys Val Lys Ala Thr Ala Ala Pro Ala Gly Ala Pro 20 25 30
Pro Gin Pro Gin Asp Leu Glu Phe Thr Lys Leu Pro Asn Gly Leu Val 35 40 45
He Ala Ser Leu Glu Asn Tyr Ser Pro Val Ser Arg He Gly Leu Phe 50 55 60 He Lys Ala Gly Ser Arg Tyr Glu Asp Phe Ser Asn Leu Gly Thr Thr 65 70 75 80
His Leu Leu Arg Leu Thr Ser Ser Leu Thr Thr Lys Gly Ala Ser Ser 85 90 95
Phe Lys He Thr Arg Gly He Glu Ala Val Gly Gly Lys Leu Ser Val 100 105 110
Thr Ala Thr Arg Glu Asn Met Ala Tyr Thr Val Glu Cys Leu Arg Gly 115 120 125
Asp Val Asp He Leu Met Glu Phe Leu Leu Asn Val Thr Thr Ala Pro 130 135 140
Glu Phe Arg Arg Trp Glu Val Ala Asp Leu Gin Pro Gin Leu Lys He 145 150 155 160
Asp Lys Ala Val Ala Phe Gin Asn Pro Gin Thr His Val He Glu Asn 165 170 175
Leu His Ala Ala Ala Tyr Gin Asn Ala Leu Ala Asn Pro Leu Tyr Cys 180 185 190
Pro Asp Tyr Arg He Gly Lys Val Thr Ser Glu Glu Leu His Tyr Phe 195 200 205
Val Gin Asn His Phe Thr Ser Ala Arg Met Ala Leu He Gly Leu Gly 210 215 220
Val Ser His Pro Val Leu Lys Gin Val Ala Glu Gin Phe Leu Asn Met
225 230 235 240
Arg Gly Gly Leu Gly Leu Ser Gly Ala Lys Ala Asn Tyr Arg Gly Gly 245 250 255
Glu He Arg Glu Gin Asn Gly Asp Ser Leu Val His Ala Ala Phe Val 260 265 270
Ala Glu Ser Ala Val Ala Gly Ser Ala Glu Ala Asn Ala Phe Ser Val 275 280 285 Leu Gin His Val Leu Gly Ala Gly Pro His Val Lys Arg Gly Ser Asn 290 295 300
Thr Thr Ser His Leu His Gin Ala Val Ala Lys Ala Thr Gin Gin Pro 305 310 315 320
Phe Asp Val Ser Ala Phe Asn Ala Ser Tyr Ser Asp Ser Gly Leu Phe 325 330 335
Gly He Tyr Thr He Ser Gin Ala Thr Ala Ala Gly Asp Val He Lys 340 345 350
Ala Ala Tyr Asn Gin Val Lys Arg He Ala Gin Gly Asn Leu Ser Asn 355 360 365
Thr Asp Val Gin Ala Ala Lys Asn Lys Leu Lys Ala Gly Tyr Leu Met 370 375 380
Ser Val Glu Ser Ser Glu Cys Phe Leu Glu Glu Val Gly Ser Gin Ala 385 390 395 400
Leu Val Ala Gly Ser Tyr Met Pro Pro Ser Thr Val Leu Gin Gin He 405 410 415
Asp Ser Val Ala Asn Ala Asp He He Asn Ala Ala Lys Lys Phe Val 420 425 430
Ser Gly Gin Lys Ser Met Ala Ala Ser Gly Asn Leu Gly His Thr Pro 435 440 445
Phe Val Asp Glu Leu 450 (2) INFORMATION FOR SEQ ID NO: 9:
(l) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 433 ammo acids (B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear (n) MOLECULE TYPE: protein (m) HYPOTHETICAL: NO (lv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: homo sapiens
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9:
Ser He Leu Lys He His Ala Arg Glu He Phe Asp Ser Arg Gly Asn 1 5 10 15
Pro Thr Val Glu Val Asp Leu Phe Thr Ser Lys Gly Leu Phe Arg Ala 20 25 30
Ala Val Pro Ser Gly Ala Ser Thr Gly He Tyr Glu Ala Leu Glu Leu 35 40 45 Arg ASD Asn Asp Lys Thr Arg Tyr Met Gly Lys Gly Val Ser Lys Ala 50 55 60 Val Glu His He Asn Lys Thr He Ala Pro Ala Leu Val Ser Lvs Lys 65 70 75 ' 80
Leu Asn Val Thr Glu Gin Glu Lys He ASD Lys Leu Met He Glu Met 85 90 95
Asp Gly Thr Glu Asn Lys Ser Lys Phe Gly Ala Asn Ala He Leu Gly
100 105 110
Val Ser Leu Ala Val Cys Lys Ala Gly Ala Val Glu Lys Gly Val Pro 115 120 125
Leu Tyr Arg His He Ala Asp Leu Ala Gly Asn Ser Glu Val He Leu 130 135 140
Pro Val Pro Ala Phe Asn Val He Asn Gly Gly Ser His Ala Gly Asn 145 150 155 160
Lys Leu Ala Met Gin Glu Phe Met He Leu Pro Val Gly Ala Ala Asn 165 170 175
Phe Arg Glu Ala Met Arg He Gly Ala Glu Val Tyr His Asn Leu Lys 180 185 190
Asn Val He Lys Glu Lys Tyr Gly Lys ASD Ala Thr Asn Val Gly Asp 195 200 205
Glu Gly Gly Phe Ala Pro Asn He Leu Glu Asn Lys Glu Gly Leu Glu 210 215 220
Leu Leu Lys Thr Ala He Gly Lys Ala Gly Tyr Thr Asp Lys Val Val 225 230 235 240
He Gly Met Asp Val Ala Ala Ser Glu Phe Phe Arg Ser Gly Lys Tyr 245 250 255
Asp Leu Asp Phe Lys Ser Pro Asp Asp Pro Ser Arg Tyr He Ser Pro 260 265 270
Asp Gin Leu Ala Asp Leu Tyr Lys Ser Phe He Lys Asp Tyr Pro Val 275 280 285 Val Ser He Glu Asp Pro Phe Asp Gin Asp Asp Trp Gly Ala Trp Gin 290 295 300
Lys Phe Thr Ala Ser Ala Gly He Gin Val Val Gly Asp Asp Leu Thr
305 310 315 320
Val Thr Asn Pro Lys Arg He Ala Lys Ala Val Asn Glu Lys Ser Cys
325 330 335
Asn Cys Leu Leu Leu Lys Val Asn Gin He Gly Ser Val Thr Glu Ser
340 345 350
Leu Gin Ala Cys Lys Leu Ala Gin Ala Asn Gly Trp Gly Val Met Val 355 360 365
Ser His Arg Ser Gly Glu Thr Glu Asp Thr Phe He Ala Asp Leu Val
370 375 380
Val Gly Leu Cys Thr Gly Gin He Lys Thr Gly Ala Pro Cys Arg Ser 385 390 395 400
Glu Arg Leu Ala Lys Tyr Asn Gin Leu Leu Arg He Glu Glu Glu Leu 405 410 415 Zly Ser Lys Ala Lys Phe Ala Gly Arg Asn Phe Arα Asn Pro Leu Ala 420 425 430
Lys
(2) INFORMATION FOR SEQ ID NO: 10:
(l) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 417 ammo acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single (D) TOPOLOGY: linear
(n) MOLECULE TYPE: protein
(in) HYPOTHETICAL: NO
(lv) ANTI-SENSE: NO
!vι) ORIGINAL SOURCE:
(A) ORGANISM: homo sapiens
[XI) SEQUENCE DESCRIPTION: SEQ ID NO: 10:
Ser Leu Ser Asn Lys Leu Thr Leu Asp Lys Leu Asp Val Lys Gly Lys 1 5 10 15
Arg Val Val Met Arg Val Asp Phe Asn Val Pro Met Lys Asn Asn Gin 20 25 30
He Thr Asn Asn Gin Arg He Lys Ala Ala Val Pro Ser He Lys Phe 35 40 45
Cys Leu Asp Asn Gly Ala Lys Ser Val Val Leu Met Ser His Leu Gly 50 55 60
Arg Pro Asp Gly Val Pro Met Pro ASD Lys Tyr Ser Leu Glu Pro Val 65 70 75 80
Ala Val Glu Leu Lys Ser Leu Leu Gly Lys Asp Val Leu Phe Leu Lys 85 90 95
Asp Cys Val Gly Pro Glu Val Glu Lys Ala Cys Ala Asn Pro Ala Ala 100 105 110
Gly Ser Val He Leu Leu Glu Asn Leu Arg Phe His Val Glu Glu Glu
115 120 125
Gly Lys Gly Lys Asp Ala Ser Gly Asn Lys Val Lys Ala Glu Pro Ala 130 135 140
Lys He Glu Ala Phe Arg Ala Ser Leu Ser Lys Leu Gly Asp Val Tyr 145 150 155 160
Val Asn Asp Ala Phe Gly Thr Ala His Arg Ala His Ser Ser Met Val
165 170 175 Gly Val Asn Leu Pro Gin Lys Ala Gly Gly Phe Leu Met Lys Lys Glu
180 185 190 Leu Asn Tyr Phe Ala Lys Ala Leu Glu Ser Pro Glu Arg Pre Phe Leu 195 200 205
Ala He Leu Gly Gly Ala Lys Val Ala Asp Lys He Gin L<- - He Asn 210 215 220
Asn Met Leu Asp Lys Val Asn Glu Met He He Gly Gly Gly Met Ala 225 230 235 240
Phe Thr Phe Leu Lys Val Leu Asn Asn Met Glu He Gly Thr Ser Leu 245 250 255
Phe Asp Glu Glu Gly Ala Lys He Val Lys Asp Leu Met Ser Lys Ala 260 265 270
Glu Lys Asn Gly Val Lys He Thr Leu Pro Val Asp Phe Val Thr Ala 275 280 285
Asp Lys Phe Asp Glu Asn Ala Lys Thr Gly Gin Ala Thr Val Ala Ser 290 295 300
Gly He Pro Ala Gly Trp Met Gly Leu Asp Cys Gly Pro Glu Ser Ser
305 310 315 320
Lys Lys Tyr Ala Glu Ala Val Thr Arg Ala Lys Gin He Val Trp Asn
325 330 335
Gly Pro Val Gly Val Phe Glu Trp Glu Ala Phe Ala Arg Gly Thr Lys 340 345 350
Ala Leu Met Asp Glu Val Val Lys Ala Thr Ser Arg Gly Cys He Thr 355 360 365 He He Gly Gly Gly Asp Thr Ala Thr Cys Cys Ala Lys Trp Asn Thr
370 375 380
Glu Asp Lys Val Ser His Val Ser Thr Gly Gly Gly Ala Ser Leu Glu 385 390 395 400
Leu Leu Glu Gly Lys Val Leu Pro Gly Val Asp Ala Leu Ser Asn He 405 410 415
Leu
(2) INFORMATION FOR SEQ ID NO: 11:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 249 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein
(iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: nomo sapiens (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11:
Met Ala Pro Ser Arg Lys Phe Phe Val Gly Gly Asn Trp Lys Met Asn 1 5 10 15 Gly Arg Lys Gin Ser Leu Gly Glu Leu He Gly Thr Leu Asn Ala Ala 20 25 30 ;s -al Pro Ala Asp Thr Glu Val Val Cys Ala Pro Pro Thr Ala T /r 35 40 45
He Asp Phe Ala Arg Gin Lys Leu Asp Pro Lys He Ala Val Ala Ala 50 55 60
Gin Asn Cys Tyr Lys Val Thr Asn Gly Ala Phe Thr Gly Glu He Ser 65 70 75 80
Pro Gly Met He Lys Asp Cys Gly Ala Thr Trp Val Val Leu Gly His 85 90 95
Ser Glu Arg Arg His Val Phe Gly Glu Ser Asp Glu Leu He Gly Gin 100 105 110
Lys Val Ala His Ala Leu Ala Glu Gly Leu Gly Val He Ala Cys He 115 120 125
Gly Glu Lys Leu Asp Glu Arg Glu Ala Gly He Thr Glu Lys Val Val 130 135 140
Phe Glu Gin Thr Lys Val He Ala Asp Asn Val Lys Asp Trp Ser Lys 145 150 155 160
Val Val Leu Ala Tyr Glu Pro Val Trp Ala He Gly Thr Gly Lys Thr 165 170 175
Ala Thr Pro Gin Gin Ala Gin Glu Val His Glu Lys Leu Arg Gly Trp
180 185 190 Leu Lys Ser Asn Val Ser Asp Ala Val Ala Gin Ser Thr Arg He He
195 200 205
Tyr Gly Gly Ser Val Thr Gly Ala Thr Cys Lys Glu Leu Ala Ser Gin
210 215 220
Pro Asp Val Asp Gly Phe Leu Val Gly Gly Ala Ser Leu Lys Pro Glu
225 230 235 240
Phe Val Asp He He Asn Ala Lys Gin
245
(2) INFORMATION FOR SEQ ID NO: 12: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1076 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(in) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: homo sapiens (κi) SEQUENCE DESCRIPTION: SEQ ID NO: 12:
Pro Val Pro Leu Ser Phe Leu Ser Thr Val Cys Asp Pro Arα Val Gin 1 5 10 15
Asp Gly Ala Ala Glu Arg Thr Gly Ala Ala Asp Gly Glu Glu Phe Leu 20 25 30 Gly Gly Gly Gly Leu Pro Ala Glu Leu Phe Gin Lys Lys Val Val Ala 35 40 45
Ser Phe Pro Arg Thr Val Leu Ser Thr Gly Met Asp Asn Arg Tyr Leu 50 55 60
Val Leu Ala Val Asn Thr Val Gin Asn Lys Glu Gly Asn Cys Glu Lys 65 70 75 80
Arg Leu Val He Thr Ala Ser Gin Ser Leu Glu Asn Lys Glu Leu Cys 85 90 95
He Leu Arg Asn Asp Trp Cys Ser Val Pro Val Glu Pro Gly Asp He 100 105 110
He His Leu Glu Gly Asp Cys Thr Ser Asp Thr Trp He He Asp Lys 115 120 125
Asp Phe Gly Tyr Leu He Leu Tyr Pro Asp Met Leu He Ser Gly Thr
130 135 140
Ser He Ala Ser Ser He Arg Cys Met Arg Arg Ala Val Leu Ser Glu
145 150 155 160
Thr Phe Arg Ser Ser Asp Pro Ala Thr Arg Gin Met Leu He Gly Thr 165 170 175
Val Leu His Glu Val Phe Gin Lys Ala He Asn Asn Ser Phe Ala Pro 180 185 190 Glu Lys Leu Gin Glu Leu Ala Phe Gin Thr He Gin Glu He Arg His 195 200 205
Leu Lys Glu Met Tyr Arg Leu Asn Leu Ser Gin Asp Glu He Lys Gin 210 215 220
Glu Val Glu Asp Tyr Leu Pro Ser Phe Cys Lys Trp Ala Gly Asp Phe 225 230 235 240
Met His Lvs Asn Thr Ser Thr Asp Phe Pro Gin Met Gin Leu Ser Leu
245 250 255 Pro Ser ASD Asn Ser Lys Asp Asn Ser Thr Cys Asn He Glu Val Val
260 265 270
Lys Pro Met Asp He Glu Glu Ser He Trp Ser Pro Arg Phe Gly Leu 275 280 285
Lys Gly Lvs He Asp Val Thr Val Gly Val Lys He His Arg Gly Tyr
290 295 300 Lys Thr Lvs Tyr Lys He Met Pro Leu Glu Leu Lys Thr Gly Lys Glu
305 " 310 315 320 Ser As n Ser He Glu His Arg Ser Gin Val Val Leu Tyr Thr Leu Leu 325 330 335
Ser Gin Glu Arg Arg Ala Asp Pro Glu Ala Glv Le_ Leu Lo j Tyr Leu 340 345 350
Lys Thr Gly Gin Met Tyr Pro Val Pro Ala Asn His Leu ASD Lvs Arg 355 360 365
Glu Leu Leu Lys Leu Arg Asn Gin Met Ala Phe Ser Leu Phe His Arg 370 375 380
He Ser Lys Ser Ala Thr Arg Gin Lys Thr Gin Leu Ala Ser Leu Pro 385 390 395 400
Gin He He Glu Glu Glu Lys Thr Cys Lys Tyr Cys Ser Gin He Gly 405 410 415
Asn Cys Ala Leu Tyr Ser Arg Ala Val Glu Gin Gin Met Asp Cys Ser 420 425 430
Ser Val Pro He Val Met Leu Pro Lys He Glu Glu Glu Thr Gin His 435 440 445
Leu Lys Gin Thr His Leu Glu Tyr Phe Ser Leu Trp Cys Leu Met Leu 450 455 460
Thr Leu Glu Ser Gin Ser Lys Asp Asn Lys Lys Asn His Gin Asn He 465 470 475 480
Trp Leu Met Pro Ala Ser Glu Met Glu Lys Ser Glv Ser Cys He Gly 485 490 495
Asn Leu He Arg Met Glu His Val Lys He Val Cys Asp Gly Gin Tyr 500 505 510
Leu His Asn Phe Gin Cys Lys His Gly Ala He Pro Val Thr Asn Leu 515 520 525
Met Ala Gly Asp Arg Val He Val Ser Gly Glu Glu Arg Ser Leu Phe 530 535 540
Ala Leu Ser Arg Gly Tyr Val Lys Glu He Asn Met Thr Thr Val Thr 545 550 555 560
Cys Leu Leu Asp Arg Asn Leu Ser Val Leu Pro Glu Ser Thr Leu Phe
565 570 575
Arσ Leu Asp Gin Glu Glu Lys Asn Cvs Asp He ASD Thr Pro Leu Gly 580 585 ' 590
Asn Leu Ser Lys Leu Met Glu Asn Thr Phe Val Ser Lys Lys Leu Arg 595 600 605
Asp Leu He He Asp Phe Arg Glu Pro Gin Phe He Ser Tyr Leu Ser
610 615 620
Ser Val Leu Pro His Asp Ala Lys Asp Thr Val Ala Cys He Leu Lys
625 630 635 640
Gly Leu Asn Lys Pro Gin Arg Gin Ala Met Lys Lys Val Leu Leu Ser 645 650 655
Lvs ASD Tyr Thr Leu He Val Gly Met Pro Gly Thr Gly Lys Thr Thr
660 665 670 Thr He Cys Thr Leu Val Arg He Leu Tyr Ala Cys Gly Phe Ser Val 675 680 635
Leu Leu Thr Ser Tyr Thr His Ser Ala Val ASD Asn He Leu Leu Lys 690 695 700
Leu Ala Lys Phe Lys He Gly Phe Leu Arg Leu Gly Gin He Gin Lys 705 710 715 720
Val His Pro Ala He Gin Gin Phe Thr Glu Gin Glu He Cys Arg Ser 725 730 735
Lys Ser He Lys Ser Leu Ala Leu Leu Glu Glu Leu Tyr Asn Ser Gin 740 745 750
Leu He Val Ala Thr Thr Cys Met Gly He Asn His Pro He Phe Ser 755 760 765
Arg Lys He Phe Asp Phe Cys He Val Asp Glu Ala Ser Gin He Ser 770 775 780
Gin Pro He Cys Leu Gly Pro Leu Phe Phe Ser Arg Arg Phe Val Leu 785 790 795 800
Val Gly Asp His Gin Gin Leu Pro Pro Leu Val Leu Asn Arg Glu Ala 805 810 815
Arg Ala Leu Gly Met Ser Glu Ser Leu Phe Lys Arg Leu Glu Gin Asn 820 825 830
Lys Ser Ala Val Val Gin Leu Thr Val Gin Tyr Arg Met Asn Ser Lys
835 840 845 He Met Ser Leu Ser Asn Lys Leu Thr Tyr Glu Gly Lys Leu Glu Cys
850 855 860
Gly Ser Asp Lys Val Ala Asn Ala Val He Asn Leu Arg His Phe Lys 865 870 875 880
Asp Val Lys Leu Glu Leu Glu Phe Tyr Ala Asp Tyr Ser Asp Asn Pro 885 890 895
Trp Leu Met Gly Val Phe Glu Pro Asn Asn Pro Val Cys Phe Leu Asn 900 905 910
Thr Asp Lys Val Pro Ala Pro Glu Gin Val Glu Lys Gly Gly Val Ser 915 920 925
Asn Val Thr Glu Ala Lys Leu He Val Phe Leu Thr Ser He Phe Val 930 935 940 Lys Ala Gly Cys Ser Pro Ser Asp He Gly He He Ala Pro Tyr Arg 945 950 955 960
Gin Gin Leu Lys He He Asn Asp Leu Leu Ala Arg Ser He Gly Met 965 970 975
Val Glu Val Asn Thr Val Asp Lys Tyr Gin Gly Arg Asp Lys Ser He 980 985 990 Val Leu Val Ser Phe Val Arg Ser Asn Lys Asp Gly Thr Val Gly Glu 995 1000 1005 Leu Leu Lys Asp Trp Arg Arα Leu Asn Val Ala He Thr Arg Ala Lys
1010 1015 1020
His Lvs Leu He Leu Leu Gly Cys Val Pro Ser Leu Asn Cys Tvr Pro 1025 " 1030 1035 ' ' 1040
Pro Leu Glu Lys Leu Leu Asn His Leu Asn Ser Glu Lys Leu He He 1045 1050 1055
Asp Leu Pro Ser Arg Glu His Glu Ser Leu Cys His He Leu Gly Asp 1060 1065 1070
Phe Gin Arg Glu 1075
(2) INFORMATION FOR SEQ ID NO: 13:
(l) SEQUENCE CHARACTERISTICS: (A) LENGTH: 527 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (ill) HYPOTHETICAL: NO (lv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: homo sapiens
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13:
Met Ala Asp Ser Arg Asp Pro Ala Ser Asp Gin Met Gin His Trp Lys 1 5 10 15 Glu Gin Arg Ala Ala Gin Lys Ala Asp Val Leu Thr Thr Gly Ala Gly
20 25 30
Asr. Pro Val Glv Asp Lys Leu Asn Val He Thr Val Gly Pro Arg Gly 35 ' 40 45
Pro Leu Leu Val Gin Asp Val Val Phe Thr Asp Glu Met Ala His Phe 50 55 60
Asp Arg Glu Arg He Pro Glu Arg Val Val His Ala Lys Gly Ala Gly 65 70 75 80
Ala Phe Gly Tyr Phe Glu Val Thr His Asp He Thr Lys Tvr Ser Lys
85 90 95
Ala Lys Val Phe Glu His He Gly Lys Lys Thr Pro He Ala Val Arg 100 105 110 Phe Ser Thr Val Ala Gly Glu Ser Gly Ser Ala Asp Thr Val Arg Asp
115 120 125
Pro Arg Gly Phe Ala Val Lys Phe Tyr Thr Glu Asp Gly Asn Trp Asp 130 135 140
Leu Val Gly Asn Asn Thr Pro He Phe Phe He Arg Asp Pro He Leu 145 150 155 160 Phe Pre Ser Phe He His Ser Gin Lys Arg Asn Pro Gin Thr His Leu 165 170 175
Lys ASD Pro ASD Met Val Trp ASD Phe Tro Ser Leu Am Pro Glu Ser 180 185 " 190
Leu His Gin Val Ser Phe Leu Phe Ser ASD Arg Gly He Pro Asp Gly 195 200 205
His Arg His Met Asn Gly Tyr Gly Ser His Thr Phe Lys Leu Val Asn 210 215 220
Ala Asn Gly Glu Ala Val Tyr Cys Lys Phe His Tyr Lys Thr Asp Gin 225 230 235 240
Gly He Lys Asn Leu Ser Val Glu Asp Ala Ala Arg Leu Ser Gin Glu 245 250 255
Asp Pro Asp Tyr Gly He Arg Asp Leu Phe Asn Ala He Ala Thr Gly 260 265 270
Lys Tyr Pro Ser Trp Thr Phe Tyr He Gin Val Met Thr Phe Asn Gin 275 280 285
Ala Glu Thr Phe Pro Phe Asn Pro Phe Asp Leu Thr Lys Val Trp Pro 290 295 300
His Lys Asp Tyr Pro Leu He Pro Val Gly Lys Leu Val Leu Asn Arg 305 310 315 320
Asn Pro Val Asn Tyr Phe Ala Glu Val Glu Gin He Ala Phe Asp Pro
325 330 335 Ser Asn Met Pro Pro Gly He Glu Ala Ser Pro Asp Lys Met Leu Gin
340 345 350
Gly Arg Leu Phe Ala Tyr Pro Asp Thr His Arg His Arg Leu Gly Pro
355 360 365
Asn Tyr Leu His He Pro Val Asn Cys Pro Tyr Arg Ala Arg Val Ala
370 375 380
Asn Tyr Gin Arg Asp Gly Pro Met Cys Met Gin Asp Asn Gin Gly Gly 385 390 395 400
Ala Pro Asn Tyr Tyr Pro Asn Ser Phe Gly Ala Pro Glu Gin Gin Pro 405 410 415 Ser Ala Leu Glu His Ser He Gin Tyr Ser Gly Glu Val Arg Arg Phe
420 425 430
Asn Thr Ala Asn Asp Asp Asn Val Thr Gin Val Arg Ala Phe Tyr Val 435 440 445
Asn Val Leu Asn Glu Glu Gin Arg Lys Arg Leu Cys Glu Asn He Ala 450 455 460
Gly H s Leu Lys Asp Ala Gin He Phe He Gin Lys Lys Ala Val Lys 465 470 475 480
Asn Phe Thr Glu Val His Pro Asp Tyr Gly Ser His He Gin Ala Leu 485 490 495
Leu ASD Lys Tyr Asn Ala Glu Lys Pro Lys Asn Ala He His Thr Phe 500 505 510 Val Gin Ser Gly Ser His Leu Ala Ala Arg Glu Lys Ala Asn Leu 515 520 525 INFORMATION FOR SEQ ID NO: 14:
(l) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 353 amino acids (C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein (in) HYPOTHETICAL: NO
(lv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE: (A) ORGANISM: homo sapiens
( xi ) SEQUENCE DESCRIPTION : SEQ ID NO : 14 :
Met Gl u Lys Thr Leu Glu Thr Val Pro Leu Glu Arg Lys Lys Arq Glu 1 5 10 15 "
Lys Glu Gin Phe Arg Lys Leu Phe He Gly Gly Leu Ser Phe Glu Thr 20 25 30
Thr Glu Glu Ser Leu Arg Asn Tyr Tyr Glu Gin Trp Gly Lys Leu Thr 35 40 45
Asp Cys Val Val Met Arg Asp Pro Ala Ser Lys Arg Ser Arg Gly Phe 50 55 60
Gly Phe Val Thr Phe Ser Ser Met Ala Glu Val Asp Ala Ala Met Ala
65 70 75 80
Ala Arg Pro His Ser He Asp Gly Arg Val Val Glu Pro Lys Arg Ala
85 90 95
Val Ala Arg Glu Glu Ser Gly Lys Pro Gly Ala His Val Thr Val Lys 100 105 110
Lys Leu Phe Val Gly Gly He Lys Glu Asp Thr Glu Glu His His Leu 115 120 125 Arg Asp Tyr Phe Glu Glu Tyr Gly Lys He Asp Thr He Glu He He 130 135 140
Thr Asp Arg Gin Ser Gly Lys Lys Arg Gly Phe Gly Pne Val Thr Phe 145 150 155 160
Asp Asp His Asp Pro Val Asp Lys He Val Leu Gin Lys Tyr His Thr 165 170 175
He Asn Gly His Asn Ala Glu Val Arg Lys Ala Leu Ser Arg Gin Glu 180 185 190
Met Gin Glu Val Gin Ser Ser Arg Ser Gly Arg Gly Gly Asn Phe Gly 195 200 205
Phe Gly ASD Ser Arg Gly Gly Gly Gly Asn Phe Gly Pro Gly Pro Gly 210 " 215 220 3er Asn Phe Arg Gly Gly Ser Asp Gly Tyr Gly Ser Gly Arα Gly Phe 225 230 235 240
3iy ASD Gly Tyr Asn Gly Tyr Glv Gly Gly Pro Gly Glv C/ Asn Phe 245 250 255
Gly Gly Ser Pro Gly Tyr Gly Gly Gly Arg Gly Gly Tyr Gly Gly Gly 260 265 270
Gly Pro Gly Tyr Gly Asn Gin Gly Gly Gly Tyr Gly Gly Gly Tyr Asp 275 280 285
Asn Tyr Gly Gly Gly Asn Tyr Gly Ser Gly Asn Tyr Asn Asp Phe Gly 290 295 300
Asn Tyr Asn Gin Gin Pro Ser Asn Tyr Gly Pro Met Lys Ser Gly Asn 305 310 315 320
Phe Gly Gly Ser Arg Asn Met Gly Gly Pro Tyr Gly Gly Gly Asn Tyr 325 330 335
Gly Pro Gly Gly Ser Gly Gly Ser Gly Gly Tyr Gly Gly Arg Ser Arg 340 345 350
Tyr
(2) INFORMATION FOR SEQ ID NO: 15:
(l) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 194 amino acids
(B) TYPE: ammo acid
(C) STRANDEDNESS: single (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(ill) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: homo sapiens
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15: Met Ala Ala Glu Asp Val Ala Ala Thr Gly Ala Asp Pro Ser Glu Leu
1 5 10 15
Glu Gly Gly Gly Leu Leu His Glu He Phe Thr Ser Pro Leu Asn Leu 20 25 30
Leu Leu Leu Gly Leu Cys He Phe Leu Leu Tyr Lys He Val Arg Gly
35 40 45 Asp Gin Pro Ala Ala Ser Asp Ser Asp Asp Asp Glu Pro Pro Pro Leu
50 55 60
Pro Arg Leu Lys Arg Arg Asp Phe Thr Pro Ala Glu Leu Arg Arg Phe 65 70 75 80
Asp Gly Val Gin Asp Pro Arg He Leu Met Ala He Asn Gly Lys Val 85 90 95 Phe ASD Val Thr Lvs Gly Arg Lys Phe Tyr Gly Pro Glu Glv Pro Tyr 100 * 105 llC
Glv Val Phe Ala Gly Arg ASD Ala Ser Arg Gly Leu Ala Thr Phe Cys 115 ' 120 125
Leu ASD Lys Glu Ala Leu Lys Asp Glu Tyr Asp Asp Leu Ser Asp Leu 130 135 140
Thr Pro Ala Gin Gin Glu Thr Leu Asn Asp Trp Asp Ser Gin Phe Thr
145 150 155 160
Phe Lys Tyr His His Val Gly Lys Leu Leu Lys Glu Gly Glu Glu Pro 165 170 175
Thr Val Tyr Ser Asp Glu Glu Glu Pro Lys Asp Glu Ser Ala Arg Lys 180 185 190 Asn Asp
(2) INFORMATION FOR SEQ ID NO: 16: (l) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 646 ammo acids
(B) TYPE: ammo acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(in) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: homo sapiens
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16:
Met Ser Lys Gly Pro Ala Val Gly He ASD Leu Gly Thr Thr Tyr Ser 1 5 10 15
Cys Val Gly Val Phe Gin His Gly Lys Val Glu He He Ala Asn Asp 20 25 30 Gin Gly Asn Arg Thr Thr Pro Ser Tyr Val Ala Phe Thr Asp Thr Glu 35 40 45
Arg Leu He Gly Asp Ala Ala Lys Asn Gin Val Ala Met Asn Pro Thr 50 55 60
Asn Thr Val Phe Asp Ala Lys Arg Leu He Gly Arg Arg Phe Asp Asp 65 70 75 80
Ala Val Val Gin Ser Asp Met Lys His Trp Pro Phe Met Val Val Asn 85 90 95
Asp Ala Gly Arg Pro Lys Val Gin Val Glu Tyr Lys Gly Glu Thr Lys 100 105 110
Ser Phe Tyr Pro Glu Glu Val Ser Ser Met Val Leu Thr Lys Met Lys 115 120 125 Glu He Ala Glu Ala Tyr Leu Gly Lvs Thr Val Thr Asn Ala Val Val 130 135 " 140
Thr 7ai Pro Ala Tyr Phe Asn Asp Ser Gin Arg G n Ala Thr Lys ASD 145 150 155 160
Ala Gly Thr He Ala Gly Leu Asn Val Leu Arα He He Asn Glu Pro 165 170 ' 175
Thr Ala Ala Ala He Ala Tyr Gly Leu Asp Lys Lys Val Gly Ala Glu 180 185 190
Arg Asn Val Leu He Phe Asp Leu Glv Gly Gly Thr Phe Asp Val Ser 195 200 ' 205
He Leu Thr He Glu Asp Gly He Pr.e Glu Val Lys Ser Thr Ala Gly 210 215 220
Asp Thr His Leu Gly Gly Glu Asp Phe Asp Asn Arg Met Val Asn His 225 230 235 240
Phe He Ala Glu Phe Lys Arg Lys His Lys Lys Asp He Ser Glu Asn 245 250 255
Lys Arg Ala Val Arg Arg Leu Arg Thr Ala Cys Glu Arg Ala Lys Arg 260 265 270
Thr Leu Ser Ser Ser Thr Gin Ala Ser He Glu He Asp Ser Leu Tyr 275 280 285
Glu Gly He Asp Phe Tyr Thr Ser He Thr Arg Ala Arg Phe Glu Glu
290 295 300 Leu Asn Ala Asp Leu Phe Arg Gly Thr Leu Asp Pro Val Glu Lys Ala
305 310 315 320
Leu Arg Asp Ala Lys Leu Asp Lys Ser Gin He His Asp He Val Leu
325 330 335
Val Gly Gly Ser Thr Arg He Pro Lys He Gin Lys Leu Leu Gin Asp
340 345 350
Phe Phe Asn Gly Lys Glu Leu Asn L s Ser He Asn Pro Asp Glu Ala 355 360 365
Val Ala Tyr Gly Ala Ala Val Gin Ala Ala He Leu Ser Gly Asp Lys 370 375 380 Ser Glu Asn Val Gin Asp Leu Leu Leu Leu ASD Val Thr Pro Leu Ser
385 390 395 400
Leu Gly He Glu Thr Ala Gly Gly Val Met Thr Val Leu He Lys Arg 405 410 415
Asn Thr Thr He Pro Thr Lys Gin Thr Gin Thr Phe Thr Thr Tyr Ser 420 425 430 Asp Asn Gin Pro Gly Val Leu He Gin Val Tyr Glu Gly Glu Arg Ala 435 440 445
Met Thr Lys Asp Asn Asn Leu Leu Gly Lys Phe Glu Leu Thr Gly He
450 455 460
Pro Pro Ala Pro Arg Gly Val Pro Gin He Glu Val Thr Phe Asp He
465 470 475 480 Asp Ala Asn Gly He Leu Asn Val Ser Ala Val Asp Lys Ser Thr Gly 485 490 495
Lvs Glu Asn Lys He Thr He Thr Asn Asp Lys Glv Arg Leu Ser Lys 500 505 510
Glu ASD He Glu Arg Met Val Gin Glu Ala Glu Lys Tyr Lys Ala Glu 515 520 525
Asp Glu Lys Gin Arg Asp Lys Val Ser Ser Lys Asn Ser Leu Glu Ser 530 535 540
Tyr Ala Phe Asn Met Lys Ala Thr Val Glu Asp Glu Lys Leu Gin Gly 545 550 555 560
Lys He Asn Asp Glu Asp Lys Gin Lys He Leu Asp Lys Cys Asn Glu 565 570 575
He He Asn Trp Leu Asp Lys Asn Gin Thr Ala Glu Lys Glu Glu Phe 580 585 590
Glu His Gin Gin Lys Glu Leu Glu Lys Val Cys Asn Pro He He Thr
595 600 605
Lys Leu Tyr Gin Ser Ala Gly Gly Met Pro Gly Gly Met Pro Gly Gly 610 615 620
Phe Pro Gly Gly Gly Ala Pro Pro Ser Gly Gly Ala Ser Ser Gly Pro 625 630 635 640
Thr He Glu Glu Val Asp 645

Claims

1. A method of characterising a biological sample comprising detecting or quantitating therein one or more proteins produced by the endometrium in increased amounts in hyperplasia or in adenocarcinoma as shown by 2D gel electrophoresis comparison of cell lysates of endometrial biopsies from normal endometrium and endometrium showing hyperplasia or adenocarcinoma, excluding variations due to the menstrual cycle, or detecting or quantitating a fragment or breakdown product thereof, or a nucleic acid coding therefor or antibodies thereto.
2. A method of characterising a biological sample comprising detecting or quantitating therein one or more proteins produced by the endometrium in increased amounts in hyperplasia or in adenocarcinoma and characterised by one of the following combinations of molecular weight and pi values :
hyperpl.asia
Pi MW kDa
6.7 91
6.6 90
6.9 64
6.6 67
6.3 66
6.8 46
5.7 41
5.5 35
5.3 13
6.6 101
5.8 14
7.4 51
8.2 44
9.5 48 adenoc,arcinoma pi MW (kDa)
6.3 32
6.0 109
6.7 91
6.6 90
6.9 64
6.6 67
6.3 66
6.2 62
6.2 45
5.7 45
5.4 33
6.3 27
6.5 103
6.8 90
6.9 78
5.3 13
6.2 130
6.3 66
6.3 73
8.3 32
8.1 55
8.2 44
6.6 111
7.7 43
9.5 48
8.3 32
7.7 39
or a fragment or breakdown product thereof, or a nucleic acid coding therefor or antibodies thereto.
3. A method as claimed in Claim 1 or Claim 2, wherein said protein, fragment, breakdown product, antibodies, or nucleic acid is detected in a body fluid sample.
4. An immunological binding partner specifically reactive with a protein as defined in Claim 1 or Claim 2 or with a fragment or breakdown product thereof or with a nucleic acid coding therefor.
5. A cell line producing a monoclonal antibody being an immunological binding partner as claimed in Claim 4.
6. An assay kit for use in a method as claimed in Claim 1 or Claim 2, comprising an immunological binding partner as claimed in Claim 4.
7. A method of characterising a biological sample comprising detecting or quantitating therein one or more proteins produced by the endometrium in increased amounts during the proliferative phase of the endometrium as shown in 2D gel electrophoresis comparison of cell lysates of endometrial biopsies from normal endometrium in its proliferative and secretory phases and characterised by one of the following combinations of molecular weight and pi values : -
pl MW(kDa)
6.9 86
5.4 34
5.6 67 5.3 23 6.8 52
8.7 47 8.2 138
6.5 124
7.7 119
7.8 119 8.1 66
7.1 58
6.8 66
7.9 48
7.7 31
6.8 29
7.2 70 8.0 119 6.7 62
or a fragment or breakdown product thereof, or a nucleic acid coding therefor, or an antibody thereto.
8. A method as claimed in Claim 7, for detecting the phase of the endometrium.
9. A method as claimed in Claim 7 or Claim 8, wherein said protein, fragment, or breakdown product is detected in a body fluid sample .
10. An immunological binding partner specifically reactive with a protein as defined in Claim 7 or with a fragment or breakdown product thereof or with a nucleic acid coding therefor .
11. A cell line producing a monoclonal antibody being an immunological binding partner as claimed in Claim 10.
125 12. An assay kit for use in a method as claimed in Claim 7 or Claim 8, comprising an immunological binding partner as claimed in Claim 10.
13. A protein produced by the endometrium in increased 10 amounts in hyperplasia or in adenocarcinoma as shown by 2D gel electrophoresis comparison of cell lysates of endo-metrial biopsies from normal endometrium and endometrium showing hyperplasia or adenocarcinoma, excluding variations due to the menstrual cycle, and 15 characterised by one of the following combinations of molecular weight and pi values :
hyperpl,asia pi MW kDa
20 6.7 91
6 . 6 90
6.9 64
6.8 46
5.7 41
25 5.3 13
6.6 101
5.8 14
9.5 48
30 adenocarcinoma pi MW (kDa)
6.3 32
6.0 109
6.7 91
6.6 90
6.9 64
6.2 62
6.5 103
6.8 90
5.3 13
6.2 130
6.3 66
6.3 73
8.3 32
8.1 55
6.6 111
7.7 43
9.5 48
8.3 32
14. A protein produced by the endometrium in increased amounts during the proliferative phase of the endometrium as shown in 2D gel electrophoresis comparison of cell lysates of endometrial biopsies from normal endometrium in its proliferative and secretory phases and characterised by one of the following combinations of molecular weight and pi values : -
pl MW(kDa)
6.9 86
5.6 67 6.8 52 8.2 138 6.5 124
7.7 119
7.8 119 8.1 66 7.1 58 6.8 66 7.7 31
15. A protein as claimed in Claim 13 or Claim 14, characterised by the properties : -
PI MW(kDa)
5.7 41
5.6 67 9.5 48
6.8 52 6.5 124
7.7 119
7.8 119 and by the respective tryptic digestion MS spectra shown in Figures 7 to 12.
16. The use of a protein as defined in any one of Claims 1,
2 or 7 or a fragment thereof, for detecting autoantibodies to a said protein.
EP97939048A 1996-09-06 1997-09-05 Biochemical markers of the human endometrium Withdrawn EP0931262A1 (en)

Applications Claiming Priority (5)

Application Number Priority Date Filing Date Title
GBGB9618600.2A GB9618600D0 (en) 1996-09-06 1996-09-06 Biochemical markers of the human endometrium
GB9618600 1996-09-06
GBGB9707132.8A GB9707132D0 (en) 1997-04-08 1997-04-08 Biochemical markers of the human endometrium
GB9707132 1997-04-08
PCT/GB1997/002394 WO1998010291A1 (en) 1996-09-06 1997-09-05 Biochemical markers of the human endometrium

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US6743595B1 (en) 1999-01-25 2004-06-01 Metriogene Biosciences Inc. Method and diagnostic kit for diagnosis of endometriosis
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US7504222B2 (en) 2001-10-31 2009-03-17 Millennium Pharmaceuticals, Inc. Compositions, kits, and methods for identification, assessment, prevention, and therapy of breast cancer
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WO2005021579A2 (en) * 2003-08-28 2005-03-10 Biorexis Pharmaceutical Corporation Epo mimetic peptides and fusion proteins
EP2251695B1 (en) * 2003-12-23 2013-07-17 Mount Sinai Hospital Corporation Markers associated with endometrial disease
EP1767944A1 (en) * 2005-09-26 2007-03-28 ProteoSys AG Phosphorylated or non-phosphorylated mPR as diagnostic marker or therapeutic target
EP2015785A4 (en) * 2006-04-24 2009-07-15 Shantha West Inc Agrm2 antigen
WO2008037449A2 (en) * 2006-09-26 2008-04-03 Proteosys Ag Use of at least one isoform of progesterone receptor membrane component 1 (pgrmc1)
EP1906185A1 (en) * 2006-09-26 2008-04-02 ProteoSys AG Use of at least one isoform of progesterone receptor membrane component 1 (PGRMC1)

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AU6960694A (en) * 1993-05-28 1994-12-20 Medical University Of South Carolina Endometrial proteins, antigenic compositions and methods for detecting endometriosis

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