CA2411017A1 - Epididymis-specific proteins with fibronectin type ii modules - Google Patents
Epididymis-specific proteins with fibronectin type ii modules Download PDFInfo
- Publication number
- CA2411017A1 CA2411017A1 CA002411017A CA2411017A CA2411017A1 CA 2411017 A1 CA2411017 A1 CA 2411017A1 CA 002411017 A CA002411017 A CA 002411017A CA 2411017 A CA2411017 A CA 2411017A CA 2411017 A1 CA2411017 A1 CA 2411017A1
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- protein
- sequences
- epididymis
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- 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
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- A61P15/00—Drugs for genital or sexual disorders; Contraceptives
- A61P15/08—Drugs for genital or sexual disorders; Contraceptives for gonadal disorders or for enhancing fertility, e.g. inducers of ovulation or of spermatogenesis
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Abstract
The invention relates to novel epididymis-specific proteins with fibronectin type II modules, to derivatives and fragments thereof, to DNA sequences that code for these, and to their utilization for producing pharmaceutical compositions used for diagnosing and treating male infertility as well as for contraception.
Description
IHF Institut fur Hormon-and Fortpflanzungsforschung GmbH
Grandweg 64 22529 Hamburg (P 57007 Me/LA) June 2001 Eiaidid~rmis-specific oroteigs with fibronectin type II
modules The invention relates to novel, epididymis-specific proteins with fibronectin type II modules and also to derivatives and fragments of same, to DNA sequences coding for these and to their use far the preparation of pharmaceutical compositions for the diagnosis and treatment of male infertility and for contraception.
The epididymis (parorchis) plays a key role in the maturation of the spermatozoa. In humans it consists of a single duct roughly 5 m long, which is coiled in a strongly meander-like manner. The functionally still immature spermatozoa formed in the testicle (testis) are transported to the epididymis and subjected for a passage lasting 2 to 3 days to a maturation process in the course of which they acquire amongst other things their directed mobility, their ability to engage in capacitation and in the acrosome reaction.
Grandweg 64 22529 Hamburg (P 57007 Me/LA) June 2001 Eiaidid~rmis-specific oroteigs with fibronectin type II
modules The invention relates to novel, epididymis-specific proteins with fibronectin type II modules and also to derivatives and fragments of same, to DNA sequences coding for these and to their use far the preparation of pharmaceutical compositions for the diagnosis and treatment of male infertility and for contraception.
The epididymis (parorchis) plays a key role in the maturation of the spermatozoa. In humans it consists of a single duct roughly 5 m long, which is coiled in a strongly meander-like manner. The functionally still immature spermatozoa formed in the testicle (testis) are transported to the epididymis and subjected for a passage lasting 2 to 3 days to a maturation process in the course of which they acquire amongst other things their directed mobility, their ability to engage in capacitation and in the acrosome reaction.
In the epididymis the spermatozoa are located in the epididymal fluid, which is also called epididymal plasma. This plasma is a secretory product of the testicle, of the rete testis and of the epididymis itself. The fluid of the ejaculate, the so-called seminal plasma, also contains secretory products of the accessory glands. Both -the epididymal plasma and the seminal plasma are a complex mixture of various molecules such as proteins, salts and water which serves as the medium for the transport and the preservation of the spermatozoa. It is assumed that the individual constituents of the seminal plasma have various functions for the fertility of the ejaculate.
Understanding of the functions of the individual constituents of the epididymal and seminal plasmas is a decisive pre-condition for the ability to intervene in the maturation process of the spermatozoa in the epididymis. Such an inter-vention can for example be necessary to deal with male infertility and for contraception. To date, however, the post-testicular maturation process and the interplay between the individual components of the epididymal/seminal plasma and molecules on the surface of the spermatozoa have been only inadequately researched. In the past, it was possible to ident-ify some proteins which are a constituent of human epididymal fluid and of seminal plasma. These include among others the epididymis-specific proteins HE1 to HE5 (Kirchhoff, C. 1998)).
Most studies in the parorchis have, however, been carried out using animal models. Almost everything that is known comes from work on rats, mice, hamsters, boars, bulls or occasionally apes, the species-specific differences being great. It was possible to identify in the seminal plasma of bovines a family of closely related proteins with two fibronectin type II
domains, which is called the BSP protein family (Desnoyers and Manjunath, 1992; Mizller et al., 1998). BSP proteins display multiple binding properties, among others, they bind to heparin, a glucosaminoglycan which plays an important role in the capacitation of spermatozoa (Parrish et al., 1988, Chandonnet et al., 1990}, and to apolipoprotein-A-I in free form or associated with HDL (Manjunath et al., 1989; Chandonnet et al, 1990). It is_ therefore assumed that BSP proteins accelerate the capacitation of spermatozoa which is induced by heparin and HDL, and that they induce the cholesterol efflux from epididymal spermatozoa (Therien et al., 1998}.
It was not previously possible to also identify in humans similar proteins which are related to the BSP proteins.
Generally, what is learnt from animal tests transfers poorly to humans, because of the high tissue- and species-specificity of the proteins of the epididymal/seminal plasma (T6pfer-Petersen et al. , 1995) . As a result, because deficient knowledge of the factors involved, a targeted influence on the fertilization process is possible only in rare cases today.
The problem underlying the present invention thus resides in providing means by which the epididymal metabolism can be influenced in a targeted manner in humans or in related mammals.
According to one aspect of the invention, this problem is solved by an epididymis-specific protein which is characterized in that it comprises the amino acid sequence shown in the sequence listing under SEQ ID NO: 2.
According to another aspect of the invention, the problem is further solved by proteins which are characterized in that they are derivatives, isoforms or homologues of the aforementioned protein, in which the protein a) has the same epididymis specificity and antigenicity; and b) has a homology of at least 55a compared with the amino acid sequence given in SEQ ID NO: 2.
Knowledge of these novel proteins makes it possible to prepare reversible male contraceptives which have a post-testicular action, i.e. do not act in the testis and do not affect the hormonal balance. Such_ contraceptives are not known in the state of the art.
According to the invention a protein has the same epididymis specificity as the protein with the SEQ ID NO: 2 if it is expressed in the same tissues as this protein. A protein furthermore has the same antigenicity as the protein with the SEQ ID NO: 2 if it is bound by antibodies which also bind to a protein with the SEQ ID NO: 2.
Within the framework of the present invention, it has surprisingly been found that at least in humans, dogs and horses a family of proteins related to HSP proteins exists which are not homologous to the BSP proteins. Therefore it was not possible in the past to discover this -novel protein family according to the invention by means of standard methods. This family is as a rule characterized by four fibronectin type II
modules, although it was possible to also identify by means of RT-PCR individual members having only three or one fibronectin type II (Fn2) modules. Previously, only seminal plasma proteins with two Fn2 modules were known.
According to the invention, the term ~~fibronectin type II
modules~~ refers to collagen-binding sites in proteins which inter olio also occur in fibronectin (Banyai et al., 1985;
Constantine et al., 1990 . According to the invention, all deviations (deletions, insertions, replacements, etc.) in the amino acid sequence are included if the consensus sequence remains.
The consensus sequence of the fibronectin type II module comprises the following sequence (50% consensus):
uss-GssCsFPFpYcG+pYccCTs-GrscshhWCoTTssYDcDc+WGFC.
The capital letters represent amino acids in the single-letter code, the meaning of the remaining characters being represented in Table 1. _ Table 1 C ass Code Amino acs Alcoholic o 5, T
Aliphatic i Z, L, V
Any - A, C, D, E,F, G; ~~ __1'K~~~~' _N'.
P, Q, R, S,T, V, W, Y
Aromatic a F, H, W, Y
Charged c D, E, H, K,R
Hydrophobic h A, C, F, G,H, I, K, L,M, R, T, V, W, Y
Negatively _ D~ E _ charged Polar p C, D, E, H,K, N, Q, R,S, T
Positively + H, K, R
charged Small s A, C, D, G,N, P, S, T,V
Very small a A, G, S
,Freely rotatableT A, C, D, E,G, H, K, N, I I Q. R- s, T-l The Fn2 modules of the proteins according to the invention are similar, but not homologous to known Fn2 modules of previously known seminal plasma proteins of ungulates (see Figure 2b).
The proteins according to the invention are epididymis-specific (see Figure 3), i.e. they are expressed primarily in the epididymis.
All naturally occurring allelic variants and isoforms of the proteins according to the invention are referred to as derivatives within the meaning of this invention, the various forms being able to differ from one another in each case as regards sequence length and also in respect of deletions, substitutions, insertions, replacements or additions of amino acids. In particular, ali naturally occurring variations of the proteins according to the invention are included in which there are differences at the N-terminal.
Within the meaning of the present invention proteins which are derivded from species other than humans are referred to as homologues.
The term ~~proteins or polypeptides having the same antigenicity~~ describes molecules according to the invention which react with the same antibodies.
Compared with the amino acid sequence given in SEQ ID NO: 2, the derivatives according to the invention preferably have a homology of at least 65%, particularly preferably of at least 75o and especially preferred of at least BO%.
According to a preferred embodiment of the invention, the derivatives, isoforms or homologues according to the invention have the same biological activity as the protein with the amino acid sequence given in SEQ ID NO: 2.
According to another preferred embodiment of the invention, the homologues according to the invention are derived from a species selected from humans, primates, dog, horse, bovines, pig.
According to a particularly preferred embodiment, the homologues according to the invention comprise one of the sequences represented in SEQ ID NO: 4, 33 and 34.
The_amino acid sequence shown in SEQ ID NO: 2 represents the amino acid sequence of human HE12, while the amino acid sequences shown in SEQ ID NO: 4 represent the amino acid sequence CEl2a from the dog. The amino acid sequences shown in SEQ ID NO: 33 and 34 are a section cut from the amino acid sequence CEl2b tSEQ ID NO: 33) and CEl2c (SEQ ID NO: ' 3 4 ) f rom the dog .
A subject-matter of the invention relates in particular to derivatives of the proteins according to the invention which comprise at least one fibronectin type II module. The deriv-atives preferably comprise four fibronectin type II modules. No proteins which have four fibronection type II modules have previously been known in the state of the art. An increased number of fibronectin type II modules is expected to lead to a more pronounced binding with the binding partners (see above).
The invention further relates to fragments of the proteins and derivatives according to the invention which have the same biological actuvity and/or antigenicity as the proteins or derivatives according to the invention themselves.
The invention furthermore relates to DNA sequences which code for the proteins, derivatives, isoforms, homologues or fragments according to the invention. These DNA sequences according to the invention are selected in particular from a) the nucleotide sequences given in SEQ ID NO: 1 or SEQ ID NO:
3, 5 and 12, b) sequences homologous to the aforementioned sequences with a degree of homology of at least 55%, preferably 65%, especially preferred 80%, and c) syngenic or complementary sequences of the sequences according to a) and b), or fragments of same, if the fragments code for proteins or polypeptides with the same biological activity and/or antigenicity.
The nucleic acid sequence shown in SEQ ID NO: 1 represents the HE12 sequence from a human, the nucleic acid sequence shown in SEQ ID NO: 3 represents the CEl2a sequence from the dog, _g_ the nucleic acid sequence shown in SEQ ID NO: 5 is the CEl2b sequence from the dog, while the nucleic acid sequence shown in SEQ ID NO: 12 represents a section cut from the coding sequence for the homologue in the horse.
According to the invention the term "syngenic sequence" covers all sequences which were derived from the same or a homologous gene and code for the proteins, derivatives and fragments according to the invention or can be used for the preparation of probes. The term also covers in particular sequences having deviations caused by the degeneration of the genetic code. In particular the term "syngenic sequence" also covers those sequences in which, although the coding region agrees wholly or partly with the coding regions of the sequences represented in SEQ ID NO: 1, 3, 5 and 32, the 5'- and 3'-untranslated regions are different. Further, the term "syngenic sequence" also covers those nucleotide sequences in which the region coding for the mature protein fully or partially complies with the coding regions of the sequences shown in SEQ ID NO: 1, 3, 5 and 12, however in which the regions coding for the signal sequences are different.
According to a preferred embodiment, the nucleic acids according to the invention comprise one of the nucleotide sequences represented in SEQ ID NO: 6, 7, 8, 9, 10 and 11.
The present invention furthermore relates to fragments of the DNA sequences according to the invention which have at least a length of 40 nucleic acids, preferably of 100 nucleic acids, and which in particular are suitable as probes.
The DNA sequences according to the invention can be obtained by searching gene banks comprising epididymis tissue with probes, the probes being fragments of the proteins represented in SEQ
ID NO: 1, 3 and 5. Such procedures are known to a person _g_ skilled in the art {Sambrook et al., 19895. The measurement of the degree of homology is also known to a person skilled in the art (Altschul, et al., 1997).
The present invention also relates to a vector molecule which comprises one or more of the DNA sequences according to the invention. According to a preferred embodiment, the vector molecule according to the invention comprises the DNA sequences according to the invention inserted in a way suitable for expression in suitable host organisms.
Vectors which are suitable within the framework of the present invention to receive the DNA sequences according to the invention include in particular all plasmid and viral vectors that are suitable for the transformation of bacteria, yeasts, insect and mammal cells.
The invention further covers microorganisms which have the registration number DSM 13473 or DSM 13474. These microorganisms have been registered at the German Micro-organisms and Cell Cultures Collection, Mascheroder Weg 1b, D-38124 Brunswick. The microorganism with the registration number DSM 13474 contains a plasmid with a DNA sequence with the nucleotides 156-827 from SEQ ID NO: 1 and thus the complete open reading frame of human HE32-DNA. The microorganism with the registration number DSM 13473 contains a plasmid with a DNA
sequence with the nucleotides 162-917 and thus the complete reading frame for the mature CE12 protein and also for part of the signal sequence.
All prokaryotic or eukaryotic host cells transformed with the vectors according to the invention are a further subject-matter of the invention. Suitable host cells are for example E. coli, Pichia pastoris, Saccharomyces cerevisiae, Sf9, mammal cell lines, primary cell cultures. CHO, BHK or COS cells for example can be used as mammal cell line.
A method of preparing the proteins, derivatives or fragments according to the invention, wherein host cells according to the invention are cultured under conditions which allow the expression of the DNA sequence, and the expression product is obtained from the culture mixture are a further subject of the invention. The conditions to be chosen depend on the vectors and host cells used in each case and are known to a person skilled in the art (Sambrook et al.).
Furthermore, the proteins, derivatives, isafarms, homologues, fragments or DNA sequences according to the invention can also be chemically synthesized by known methods.
The invention also relates to antibodies which are suitable to react in an immunoreaction with the proteins, derivatives, homologues or fragments according to the invention. According to preferred embodiments these antibodies are polyclonal or monoclonal antibodies. The antibodies according to the invention can be prepared with the help of the high-purity proteins according to the invention in a way known to a person skilled in the art. Such antibodies can be prepared on the basis of the complete protein and on the basis of fragments and active derivatives of same if these possess the same antigenicity icf. example 5).
The antibodies can be labelled and serve to detect the proteins, derivatives, homologues and fragments according to the invention in vitro or in viva.
Pharmaceutical compositions which comprise the proteins, derivatives, homologues, fragments, DNA sequences or antibodies according to the invention as active ingredient are a further subject of the present invention. Alongside the active active ingredient, such pharmaceutical compositions regularly further comprise physiologically compatible carriers which are known to a person skilled in the art.
The use of the proteins, derivatives, homologues, fragments, DNA sequences and antibodies according to the invention for the preparation of pharmaceutical compositions for the diagnosis of male reproductive disorders, for the treatment of male reproductive disorders, in particular infertility, and for the preparation of a medicament for contraception is a further subject of the invention.
According to a preferred embodiment, the complementary DNA
sequences according to the invention are used as antisense DNA
molecules for the preparation of the aforementioned pharmaceutical compositions, the antisense molecules being suitable for the suppression of the preparation of the DNA
sequences according to the invention in vivo.
The pharmaceutical compositions according to the invention are preferably used for the modulation of the heparin and apolipoprotein A-I bonds in the epididymis. Their use for the modulation of the capacitation of spermatozoa is especially preferred.
A novel family of epididymis-specific proteins is thus made available with the present invention, which are characterized by the presence of heparin-binding sites. The proteins, DNA
sequences and antibodies according to the invention can be used as a reversible male contraceptive which has a post-testicular action and does not affect the hormonal balance. Such a contraceptive is not known in the state of the art.
The invention is described further in the following by means of figures and examples.
Examples Example 1 Cloning of novel epididymis~specific cDNAs A dog epididymis cDNA library (Ellerbrock et al., 1994) was searched using the subtraction-hybridization strategy described in Kirchhoff et al. (1990). Roughly 10,000 independent cDNA
clones were plated in low density (ca. 500 pfu (plaque forming units) per plate) and searched by means of a mufti-step procedure. 32P-labelled single-strand cDNA pools from dog epididymis were used as probes. 32P-labelled single-strand cDNA
pools from dog liver, testis and lung were used as negative controls. Epididymis-specific plaques were purified by means of gradual dilution, and the incorporated cDNA was obtained by in vivo excision, as recently described (Ellerbrack et al., 1994).
The recombinant plasmid DNAs obtained were amplified and purified, and the cDNA clones were sequenced by means of standard procedures (Sambrook et al., 1989}. This procedure led to the identification of a novel cDNA clone family in the dog, which was named CE12. Two variants with differences in the open reading frame, CEl2a and CEl2b (Figure la to c) were found. Both contained four fibronectin type II (Fn2) modules arranged as a tandem. These modules were similar, but not homologous vis-a-vis known Fn2 modules of other seminal plasma proteins (Figure 2b).
A roughly 400 by cDNA fragment which was common to CEl2a and b was used to search a human epididymis cDNA library (Kirchhoff et al., 1993). The same procedure would be possible with a 750 by PCR product which can be obtained with the following primers: 5'GATCTGTTTACTTCATCTTG3' (SEQ ID NO: 19) and 5'CCTTTATTGGTGGTTATGCC3' (SEQ ID NO: 20). This happened with procedures known to a person skilled in the art (Sambrook et al., 1989). A number of cross-hybridizing clones (0.030 of the library) were isolated, the longest cDNA inserts were sequenced in both directions and the sequences compared. The HE12 cDNA
sequence obtained was almost 80o identical to the CEl2b cDNA.
As neither the CE12 nor the HE12 cDNAs were complete at the 5' end, an inverse PCR technique was carried out in order to obtain the sequence information that was still missing. To this end, a modified technique was used which had recently been described by Gebhardt et al. (1999}. Human and dog epididymis cDNAs which had been prepared from epididymis RNA extracts of other individuals were used. Antisense primers whose sequence was located in the known sequences of the partial cDNA clones were used for the reverse transcription (CE12 cDNA synthesis primer: 5'GTTGTTTTCATCCTCCATGC3' (SEQ ID NO: 13}; HE12 cDNA
synthesis primer: 5' TCCATGCAATCAGAGACCAC3' (SEQ ID NO: 14)).
ug whole epididymis RNA were used. 8uperscript~ Reverse Transcriptase (Gibco-BRL, Karlsruhe, Germany) was used as enzyme. The synthesis of the second strand was carried out with E. coli DNA polymerase after the incorporation of strand breaks with RNase H (both enzymes from Stratagene, Heidelberg, Germany). Smooth ends were prepared with T4 DNA polymerase (Biolabs, Schwalbach/Taunus, Germany} and ligated by means of T4 DNA ligase (Boehringer, Mannheim, Germany), in order to concatemerize and/or circularize the DNA. The inverse PCR was carried out using the following sense primers (CE12:
5'GTGAAACCAATGAGTATGG3' (SEQ ID NO: 15}; HE12: 5'GTGGTGCTCAGT-CACCTCTG3' (SEQ ID NO: 16)) and the corresponding antisense primers: (CE12: 5'GGTAATCGTCTGCCATGC3' (SEQ ID NO: 17); HE12:
5'CGTGGGTAATCTTCACTCTG3' (SEQ ID NO: 18)).
In this way, six independent CE12 and four independent HE12 coding PCR products were subcloned and sequenced. All the HE12 PCR products were essentially co-linear and ended almost at the same 5' site. Combining the sequences of the HE12 cDNA seq-uences obtained from the library and those of the PCR fragments produced an HE12 cDNA clone that was 1025 nucleotides long.
The HE12 clone displays an open reading frame (ORF) of 223 codons with an ATG start codon at nucleotides 156-158 and a TGA
stop codon at nucleotides 825-827 (Figure 2a). The ORF is followed by a 198-nucleotide-long 3' untranslated region which contains an AATAAA polyadenylation signal at nucleotides 1010-1015. The ORF produces a novel human epididymal protein. This comprises an N-terminal signal peptide (amino acid 1-23) which is typical of secretory proteins and which is immediately followed by four Fn2 units arranged in tandem (Figure 2b).
In contrast to this, different CE12 RT PCR fragments formed which resulted from an mRNA length polymorphism. These fragments had different 5' UTRs and extensive deletions or insertions even within their ORFs, which leads to at least two different CE12 proteins (Figure 1b, c). If the obtained sequences are combined, a CEl2a cDNA clone with 1343 nucleotides results iFigure la). The ORF in this long cDNA
variant starts at nucleotide 129 and contains two methionines in the reading frame at positions -39 and -23, the second being homologous to the position of the start methionine of the HE12 cDNA sequence in Figure 2a. Only if this methionine is assumed as start codon can a clear signal peptide sequence be read out from the sequence. In addition, CE12 cDNA clones were obtained which display different 5' UTRs, in which there was only one, namely the second ATG start codon. It is therefore assumed that each of these CE12 sequences have different promoters.
A further polymorphism in the CE12 clones was discovered immed-iately after the assumed signal peptide sequence, which prob-bably led to different N terminals in the CE12 clones. The CEl2a and b clones differ by virtue of 22 codons which are either present in the cDNAs or absent and for which there is no counterpart in the HE12 cDNA sequence (Figure 2). The tv~ro variants in CE12 cDNAs occur with a frequency of 1 _ 3. The N-terminal as it was derived for CEl2a contains a consensus sequence for an N-glycosylation site at asparagine +4 (Asn +4), this site being missing from CEl2b (Figure lc). The amino acids which result from the immediately following codon can be either D or N (Figure lc). The occurrence of smaller, roughly 1 kb CE12 mRNAs in the Northern blot (see below) make it possible that there are additional length polymorphisms also in the 3' untranslated region, although this was not confirmed by the found cDNA clones. However, the 3' UTR of the CE12 mRNA appears to be clearly longer than that of the HE12 mRNA and contains two possible polyadenylation signals, of which only the first is homologous to the human counterpart. On the other hand, the sequences which code for the Fn2 units are identical in all CE12 cDNA variants and also highly homologous to corresponding regions of the HE12 cDNA (roughly 85o sequence identity) (Figure 2b). A comparison of the predicted CE12 and HE12 protein sequences with those of the BSP-A1/PDC-109 and BSP-A3 seminal plasma proteins (accession numbers P02784 and P04557) showed a sequence identity of below 550. This also rules out any cross-hybridization with- the BSPs at nucleic acid level under the stringency conditions used in this study.
Example 2 Analysis of the structure of the epididymal fibronectin type II domain proteins at cDNA level Since proteins which contain four Fn2 units arranged in a tandem were previously unknown, PCR analyses were carried out in order to rule out the possibility that cloning artefacts occurred during the preparation of the libraries.
Oligonucleotide primer pairs were used in the RT-PCR analysis.
The RT-PCR was carried out using standard methods. The following primary pairs were used for the amplification of CE12, HE12 and EQ12 cDNA fragments from 1 ~g of foetal epididymal RNA:
CE12: 750 by product; primers used 5'GATCTGTTTACTTCATCTTG3' (SEQ ID NO: 19} and 5'CCTTTATTGGTGGTTATGCC3' (SEQ ID NO: 20);
HE12: 837 by product; primers used 5'CAACCTTCCTTCTCTATTCC3' (SEQ ID NO: 21} and 5'TCAAGACTCCTTTATTGGTG3' (SEQ ID NO: 22);
EQ12: 752 by product; primers used 5'GGGTCTTCTTACTTCTCTTG3' (SEQ ID NO: 23) and 5'TCCTTTATTGGTGGTTATGCAC3' (SEQ ID NO: 24}.
Each of the products contained those cDNA regions which code for four Fn2 units. As shown, human and dog epididymal cDNAs were used for the PCR amplification. As an additional control, in order to rule out any cDNA probes contamination, a corresponding primer set was also used in the case of the epididymal horse cDNA preparation. In every case an amplified sequence of roughly 800 by was obtained. Subcloning and sequencing confirmed the occurrence of epididymal mRNAs which code for four highly preserved Fn2 units arranged in tandem.
Such proteins occurred not only in humans and dogs but also in horses (see Figure 2c}. During the RT-PCR analyses of the human epididymal cDNA, shorter PCR fragments were also observed, but in much smaller quantities. Subcloning and sequencing of these fragments showed that they originated in variants which contained fewer than four Fn2 motives. These variants most probably contain three or one Fn2 modules, the variant with only one Fn2 module occurring in much smaller quantities.
Corresponding products were not found in dogs and horses.
Example 3 Investigation of the tissue distribution of CE12 and HE12 mRNAs by northern blot analysis Northern blot analyses were carried out with whole RNA extracts of different dog and human tissues in order to investigate the distribution of the CE12 and HE12 mRNAs (Figures 3 and 4). RNA
from different tissues was extracted in 15 to 20 volumes of a chaotropic solution as described in Pera et al. (1996). 5 to ~g of the total RNA/column, depending on the type of experiment, were separated by means of denaturing agarose gel electrophoresis and transferred onto Amersham-Buckler, Brunswick (Germany) nylon membranes. Equal quantities of RNA
were assured by ethidium bromide staining before the transfer.
Digoxigenin (DIG)-labelled cDNA probes were prepared by PCR
amplification of purified fragments according to the instruct-ions provided by the manufacturer (Boehringer, Mannheim, Germany). The sequences of the primers were as follows:
CE12, 404 by product; primers used 5'CTCTCCTTGGTGTGCAACC3' (SEQ
ID NO: 25) and 5'AAGTGGCAGGGAAAGCCAG3' (SEQ ID NO: 26);
HE12, 400 by product; primers used 5'CAACCTTCCTTCTCTATTCC3' (SEQ ID NO: 27) and 5'TCCATGCAATCAGAGACCAC3' (SEQ ID NO: 28);
CE1, 730 by product; primers used 5'AAGCAGCAGAGCTGGAG3' (SEQ ID
NO: 29) and 5'AATGTCACCTTCACCAG3' (SEQ ID NO: 30);
CE4, 370 by product; primers used 5'ACGCAGGAGTGCGTCTCGGA3' (SEQ
ID NO: 31) and 5'AGCGGTCACTTTATTGGTTG3' (SEQ ID NO: 32).
The PCR reactions were carried out as described (Pera. et al.
1996). The hybridization with the non-radioactive probes was carried out using the DTG labelling kit (Boehringer, Mannheim) as recently described (Pera et al., 1996).
It was shown that in both species (human and dog) the mRNAs were present only in the epididymis, but not in the other tissues investigated, _the tissues that were investigated including testis, seminal vesicles and prostate (Figures 3 and 4). A single hybridizing band of roughly 1.1 kb was found in human epididymal extracts, whereas at least two or more hybrid-izing bands were present in dog epididymal RNA preparations.
The bands in the dog mRNA occurred in a wide range from roughly 1.0 to 1.5 kb, which supports the assumption that in the dog a length polymorphism occurs in the case of CE12 mRNA.
The expression pattern of the human and dog mRNAs was investigated along the length of the epididymis. HE12 mRNA was found in all regions of the human epididymis, although a higher content was found in the more proximal parts of the organ (Figure 4). In the dog epididymis CE4 mRNA, which is known to be present in all regions of the organ (Beiglbock et al., 1998;
Gebhardt et al., 1999), served as internal control (Figure 3).
The regionalization of the CE12 expression was clearer in dogs than in humans, and all the CE12-hybridizing mRNAs showed the same pattern with a maximum expression in the corpus region.
Under unilateral-cryptorchidic conditions, in which one testis plus epididymis remains in the abdomen, normal quantities and expression patterns of the mRNA were observed in the scrotal epididymis, which served as control. A reduction in the quantities of CE12 mRNA was found in the abdominal organs. If the different regions of the dog epididymis are considered, the reduction was at its clearest in the caput region, while the quantities of CE12 mRNA in the corpus appeared not to be affected (Figure 3}.
A hybridization under low stringency conditions using hetero-logous DIG-labelled CE12 cDNA as probe showed that cross-hybridizing mRNAs are present in the epididymis of other kinds of mammal including humans, bovines, horses, pigs and apes (Macaca mullata) (Figure 5a, b). Although not investigated in detail, the largest quantities in the pig parorchis were found in the caput region, which is an exception compared with the other mammals. No cross-hybridization with either CE12 or HE12 cDNA probes was found with RNA extracts from rodents, rabbits and guinea pig epididymes.
Example 4 Localization of C812 mRNAs by in situ hybridization The epididymes of two dogs of average size were incubated for 6 hours in Bouin's solution, washed in 70a ethanol and embedded in paraffin wax. 5- to 7-~m sections were prepared and subjected to a non-radioactive hybridization procedure as described (Beiglbock et al., 1998; Gebhardt et al., 1999), using in vitro labelled sense and antisense cRNA probes of sub-cloned Accl/XbaI-digested CE12 cDNA fragments which comprised 404 by of the open reading frame (nucleotides 295-&69, cf.
Figure 1). The labelling was carried out in the presence of dioxigenin UTP (Boehringer-Mannheim). The detection was carried out as suggested by the manufacturer. Alternative methods are known to a person skilled in the art (Kirchhoff et aI. 1991;
Pera et al. 1994; otherwise: see above). Negative control checks were carried out in_ parallel at adjacent sections, using the corresponding dioxigenin-labelled sense strand cRNAs. The sections were analysed by means of light microscopy.
CE12 transcripts were localized in tissue sections of the dog epididymis (see Figure 6a and b). The in situ hybridization showed a strong and highly specific marking of the epithelium of the epididymal duct, while the lumen of the duct which contains the spermatozoa and the tissue between the duct showed no signal. Within the epithelium, the signal was at its strongest in the basal region. In good agreement with the Northern blot results, the maximum coloration was found in the distal caput and in the corpus, whereas the proximal caput appeared to be unmarked and the distal parts of the organ were only weakly marked (Figures 7a-d).
Example 5 Preparation of antibodies A 20-acid oligopeptide (NFi3-SSFDENQQWKYCETNEYGGN-COOH) which had been derived from the CEl2a DNA sequence (amino acids 103-122, cf. Figure 1) was selected as immunogen. The preparation of the antibodies took place as recently described (Kirchhoff et al.
1996). After coupling to KLH (keyhole limpet hemocyanine) the conjugate was injected into hen's eggs, guinea pigs and rabbits. The animals were killed after 120 days' immunization, the immune sera were collected and stored at 4°C using thimorosal as preservative.
The peptide was selected on the basis of its predicted high antigenicity and minimal cross-reactivity with other proteins.
This peptide epitope also appears to be highly preserved between the species (Figure 2b).
Example 6 Protein preparation and Western blot analysis Crude protein extracts were prepared from powdered epididymal tissue, epididymal fluid or washed spermatazoa, and stored in aliquots of equal size at -80°C. Protein extracts of human spermatozoa membranes were prepared as recently described (Alexander and Bearwood, 1984). Probes of 5 to 10 ~l protein extract (depending on the protein concentration or spermatozoa count) were separated on 15a SDS polyacrylamide gels and transferred onto PVDF membranes (Amersham, Brunswick, Germany) using a discontinuous buffer system with a half-dry blotter (Phase, Lizbeck, Germany). Immunodetection was carried out by one-hour blocking of the membrane in to blocking solution (Boehringer-Mannheim), followed by incubation with a guinea pig antiserum (see above, dilution 1 . 5000). Antibody binding was established by a peroxidase-coupled goat-anti-guinea pig anti-body (Sigma, Deisenhofen, Germany). Alternative procedures are known to a person skilled in the art.
Protein extracts of dog epididymal tissue and also protein extracts of epididymal dog spermatozoon were positive for CE12 related antigens (Figure 8a-c). Multiple immuno-reactive bands with a molecular weight in the range from 30 to 35 kDa were identified in every protein probe with the exception of the protein extracts of the spermatozoa from the caput. Thick bands are likewise recorded in human epididymal fluid which was removed from the cauda region, and also in membrane protein preparations of human ejaculated sperm. These reactions were able to be subjected to competition from chemosynthetic peptides (Figure 8).
Example 7 Detection of the HE12 protein is the human parorchis Cryostat sections (8 ~,m) of the epididymis of prostatic carcinoma patients were prepared and incubated for 18 hours at 8~C with the C12-3 antiserum from rabbits. The antibodies with specificity for the HE12 protein were made visible by fluoro-chrome-conjugated antibodies (Cy2-immunofluorescence: Dianova, Amersham). Both antibodies were used in a dilution of 1 . 100.
Cross-sections through the parorchis passage in the distal region are represented_ as immunofluorescence photographs in Figure 9(a) to (i). (a) shows the cross-section through the parorchis passage in the distal region; (b) the comparable region of another patient tissue; (c) and (d) comparable regions of two antiandrogen-treated patients; (e) a negative control in which the parorchis was incubated with a pre-C12-3 serum (1 . 100 diluted).
Figure 10 shows the corresponding result in the case of the analysis of the epididymis of a healthy dog. Prior to incubat-ion with the antibody (designated "pre") a uniform coloration of the epididymis results (LU = lumen, EP = epithelium).
To summarize, it can be established that HE12 is primarily expressed in the epithelial cells and given off into the lumen.
Example 8 Detention of AE12 on human spermatozoa In order to show that HE12 binds to human spermatozoa, LIS
membrane extracts of the spermatozoa were subjected to a two-dimensional electrophoretic separation (2-DE), labelled by the HE12-specific antibodies and made visible. As Fig. 11(a) shows, six different isoforms can be detected by means of this procedure.
In order to obtain more detailed information regarding the nature of the bond, an attempt was made to remove, by washing, the HE12 proteins bound to spermatozoa. The solutions used in each case for the washing were separated via SDS-PAGE
and analysed using the Western blot system for the presence of the HE12. As shown in Fig. 11(b), the greatest part of the spermatozoa-binding protein fraction can be removed by 0.6~M
NaCI washing buffer (Sw.). However, even after this a smaller fraction remains bound on the spermatozoa membrane (Sp) and cannot be removed even by repeated washing (Wt).
Brief description of the Figures Figure 1 cDNA sequences and derived peptide sequences of CE12 variants. a) Sequence of the complete CEl2,a cDNA (top sequence) and predicted peptide sequence (bottom sequence). The arrow points to the probable separ-ation site of the signal peptide. The amino acid sequence, as derived from the first methionine in the reading frame, is shown in grey. The sequence which was used for synthesis of the oligopeptide is under-lined. Two polyadenylation signals are underlined in the 3'UTR. b) Sequences of the CE12 variants at the 5' end, established by inverse PCR (exclusively primers). The 5' regions of the two clones obtained from the library (CEl2a and b) and of the six clones originating in the PCR (Cinv 1 (SEQ ID NO: 6), 4 (SEQ
ID NO: 8), 6 (SEQ ID NO: 7), 8 (SEQ ID NO: 9), 11 (SEQ ID NO: 10), I2 (SEQ ID NO: 11) are compared with each other. c? Comparison of the amino acid sequences of the CE12 cDNA variants. 1-23: signal peptide sequence; 24-45: N-terminal extension of CEl2a. Roman numerals (I-IV) designate Fn2 modules (CEl2b: SEQ ID
NO: 33; CEl2c: SEQ ID NO: 34}.
Short lines show both deletions which were found in CEl2b and c variants and differences in the inter-stices between the Fn2 units. Dotted lines show identical amino acid sequences. The arrow indicates the different N-terminal amino acids.
Figure 2 cDNA and derived amino acid sequence of HE12 compared with homologous sequences from animals. a) Sequence of the complete HE12 cDNA and of the predicted pro-tein sequence. b) Comparison of the predicted human (HE12), dog (CE12), and horse (EQ12) Fn2 module proteins with the amino acid sequence at BSP-A1/A2=PDC-109 (accession number P02784).
Figure 3 Northern blot analyses of the tissue distribution of CE12 mRNAs, shown by non-reactive hybridization. a) Expression patterns of CE12 mRNAs in total RNA
extracts of different dog tissues using a DIG-labelled probe (top panel). Ly - lymph nodes; Mu -muscle; Di = diaphragm; Sp = spleen; Lu = lung; Li -liver; Ov - ovaries; Te - testicle; El - dog epididymis 1; E2 = dog epididymis 2; Du = oviduct; Ut - uterus. Equal loading (10 ~Cg/row} and the integrity of the ribosomal RNA (28S and 18S) was assured by ethidium bromide staining of the gel prior to the blotting (bottom panel). b) Expression patterns of CE12 (1.0 to 1.4 kb) and CE4 (0.7 kb) mRNAs in different regions of the dog epididymis and as a response to the cryptorchidic situation.
Figure 4 Northern blot analyses of the tissue distribution of HE12, shown by non-radioactive hybridization.
Expression of HE12 mRNA was investigated in total RNA
extracts of different human tissues and different regions of the human epididymis using a DIG-labelled HE12 probe (upper panels). Pr - prostate; Sv -seminal vesicles; Ep - epididymis; Te - testicle;
De - decidua; Ki - kidney; Th - thymus; Pi -pituitary gland; Ln - LnCap cells; Ca - caput epididymis; Co - corpus epididymis; Cu - cauda epididymis. Equal loading (10 ~.g/column) and tf~e integrity of .the ribosomal RNA (28S and 18S) was assured by ethidium bromide staining of the gel prior to the blotting (bottom panels).
Figure 5 Northern blot analysis for the determination of the cross-hybridization of CE12 and HE12 cDNA probes between different species. a) Whole extracts of epididymal RNA of nine different species were hybridized with a DIG-labelled CE12 probe under non-stringent conditions. Column I: human; column 2:
stallion; column 3: bovines; column 4: boar; column 5: rat; column 6: mouse; column 7: rabbit; column 8:
guinea pig (degraded?); column 9: dog. b) Columns 1-3: stallion caput, corpus and cauda epididymis;
columns 4-5: boar caput, corpus and cauda epididymis;
column 6: bull; column 7: guinea pig (degraded);
columns 8-10: caput, corpus and cauda epididymis of Macaca mullata (hybridized with HE12 cDNA probe).
Equal loading (10 ug/column) and the integrity of the ribosomal RNA (28S and 18S) was assured by ethidium bromide staining of the gel prior to the blotting (lower panels).
Figure 6 Localization of CE12 transcripts in the ductus epithelium of the dog corpus epididymis by non-radioactive in situ hybridization using DIG-labelled cRNA probes. a) mRNA which hybridizes with CE12 antisense cRNA was detected in tissue sections using an alkaline phosphatase-conjugated anti-DIG antibody in normal bright-light microscopy. b) Adjacent sect-ions which were hybridized with DIG-labelled CE12 sense control cRNA were negative. The bar represents 50 ~.m.
Figure 7 Region-dependent differences in the CE12 transcript concentration in the dog epididymis, shown by in situ hybridization using DIG-labelled CE12 antisense cRNA.
a) Proximal caput epididymis; b) proximal corpus epididymis; c) middle to distal corpus epididymis; d) cauda epididymis. The bar represents 100 ~Cm.
Figure 8 Western blot analysis of antigens related to CE12 using an antipeptide antiserum of guinea pigs (dilution 1 . 500). a) Multiple, immunostained bands were observed between 30 and 35 kDa in crude protein extracts of dog epididymis, which did not react with pre-immunoserum. The staining was subjected to competition from CE12 peptides (20 ~g/ml). P - pre-immunoserum; T= test antiserum; C - competition experiment. A broad immunoreactive band of roughly the same size was observed in the cauda of the dog, but not in epididymal sperm protein extracts of the caput epididymis. Ca - caput sperm extract; Cu -sperm extract. b) A broad band of the same size was also observed in protein extracts from human caudal epididymal fluid, but not of the corpus, and in LIS
extracts of washed human ejaculated sperm. This immunopositive probe was subjected to competition from the chemosynthetic CE12 oligopeptide. Co = fluid from epididymal corpus; Cu - fluid from epididymal cauda; Sp - sperm, no competition; C - competition experiment.
Figure 9 Immunofluorescence detection of the HE12 in the parorchis of patients with prostatic carcinoma; (a) cross-section through the parorchis passage in the distal region; (b) comparable region of another patient tissue; (c) and (d) comparable regions of two antiandrogen-treated patients; (e) negative control, parorchis incubated with a pre-C12-3 serum (diluted 1 100) .
Figure 10 Immunofluorescence detection of the HE12 in the parorchis of a healthy dog; prior to incubation with the antibody ("pre"), LU = lumen; EP = epithelium.
Figure 11 (a): HE12 bound to spermatozoa after two-dimensional electrophoretic separation; (b) SDS-PAGE and Western blot of the washing solutions; 0.6 M NaCl washing buffer Sw; spermatozoa membrane Sp; repeated washing Wt.
References 1. Alexander NJ, Bearwood D. An immunosorption assay for antibodies to spermatozoa: comparison with agglutination and immobilization tests. Fertil Steril 1984; 41: 270-276.
2. Altschul, SF et al. (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs, Nucleic Acids Research 25: 3389-3402.
3. Banyai, L, Tordai H, Patthy L. Structure and domain-domain interactions of the gelatin binding site of human 72-kilodalton type IV collagenase (gelatinase A, matrix metalloproteinase 2). J Biol Chem 1996; 271: 12003-12008.
4. Beiglbock A, Pera I, Ellerbrock K, Kirchhoff C. Canine epididymis-specific mRNA encoding secretory glutathione peroxidase-like protein. J Reprod Fertil 1998; 112: 357-367.
5. Chandonnet L, Roberts KD, Chapdelaine A, Manjunath P.
Identification of heparin-binding proteins in bovine seminal plasma. Mol Reprod Dev 1990; 26: 313-318.
Understanding of the functions of the individual constituents of the epididymal and seminal plasmas is a decisive pre-condition for the ability to intervene in the maturation process of the spermatozoa in the epididymis. Such an inter-vention can for example be necessary to deal with male infertility and for contraception. To date, however, the post-testicular maturation process and the interplay between the individual components of the epididymal/seminal plasma and molecules on the surface of the spermatozoa have been only inadequately researched. In the past, it was possible to ident-ify some proteins which are a constituent of human epididymal fluid and of seminal plasma. These include among others the epididymis-specific proteins HE1 to HE5 (Kirchhoff, C. 1998)).
Most studies in the parorchis have, however, been carried out using animal models. Almost everything that is known comes from work on rats, mice, hamsters, boars, bulls or occasionally apes, the species-specific differences being great. It was possible to identify in the seminal plasma of bovines a family of closely related proteins with two fibronectin type II
domains, which is called the BSP protein family (Desnoyers and Manjunath, 1992; Mizller et al., 1998). BSP proteins display multiple binding properties, among others, they bind to heparin, a glucosaminoglycan which plays an important role in the capacitation of spermatozoa (Parrish et al., 1988, Chandonnet et al., 1990}, and to apolipoprotein-A-I in free form or associated with HDL (Manjunath et al., 1989; Chandonnet et al, 1990). It is_ therefore assumed that BSP proteins accelerate the capacitation of spermatozoa which is induced by heparin and HDL, and that they induce the cholesterol efflux from epididymal spermatozoa (Therien et al., 1998}.
It was not previously possible to also identify in humans similar proteins which are related to the BSP proteins.
Generally, what is learnt from animal tests transfers poorly to humans, because of the high tissue- and species-specificity of the proteins of the epididymal/seminal plasma (T6pfer-Petersen et al. , 1995) . As a result, because deficient knowledge of the factors involved, a targeted influence on the fertilization process is possible only in rare cases today.
The problem underlying the present invention thus resides in providing means by which the epididymal metabolism can be influenced in a targeted manner in humans or in related mammals.
According to one aspect of the invention, this problem is solved by an epididymis-specific protein which is characterized in that it comprises the amino acid sequence shown in the sequence listing under SEQ ID NO: 2.
According to another aspect of the invention, the problem is further solved by proteins which are characterized in that they are derivatives, isoforms or homologues of the aforementioned protein, in which the protein a) has the same epididymis specificity and antigenicity; and b) has a homology of at least 55a compared with the amino acid sequence given in SEQ ID NO: 2.
Knowledge of these novel proteins makes it possible to prepare reversible male contraceptives which have a post-testicular action, i.e. do not act in the testis and do not affect the hormonal balance. Such_ contraceptives are not known in the state of the art.
According to the invention a protein has the same epididymis specificity as the protein with the SEQ ID NO: 2 if it is expressed in the same tissues as this protein. A protein furthermore has the same antigenicity as the protein with the SEQ ID NO: 2 if it is bound by antibodies which also bind to a protein with the SEQ ID NO: 2.
Within the framework of the present invention, it has surprisingly been found that at least in humans, dogs and horses a family of proteins related to HSP proteins exists which are not homologous to the BSP proteins. Therefore it was not possible in the past to discover this -novel protein family according to the invention by means of standard methods. This family is as a rule characterized by four fibronectin type II
modules, although it was possible to also identify by means of RT-PCR individual members having only three or one fibronectin type II (Fn2) modules. Previously, only seminal plasma proteins with two Fn2 modules were known.
According to the invention, the term ~~fibronectin type II
modules~~ refers to collagen-binding sites in proteins which inter olio also occur in fibronectin (Banyai et al., 1985;
Constantine et al., 1990 . According to the invention, all deviations (deletions, insertions, replacements, etc.) in the amino acid sequence are included if the consensus sequence remains.
The consensus sequence of the fibronectin type II module comprises the following sequence (50% consensus):
uss-GssCsFPFpYcG+pYccCTs-GrscshhWCoTTssYDcDc+WGFC.
The capital letters represent amino acids in the single-letter code, the meaning of the remaining characters being represented in Table 1. _ Table 1 C ass Code Amino acs Alcoholic o 5, T
Aliphatic i Z, L, V
Any - A, C, D, E,F, G; ~~ __1'K~~~~' _N'.
P, Q, R, S,T, V, W, Y
Aromatic a F, H, W, Y
Charged c D, E, H, K,R
Hydrophobic h A, C, F, G,H, I, K, L,M, R, T, V, W, Y
Negatively _ D~ E _ charged Polar p C, D, E, H,K, N, Q, R,S, T
Positively + H, K, R
charged Small s A, C, D, G,N, P, S, T,V
Very small a A, G, S
,Freely rotatableT A, C, D, E,G, H, K, N, I I Q. R- s, T-l The Fn2 modules of the proteins according to the invention are similar, but not homologous to known Fn2 modules of previously known seminal plasma proteins of ungulates (see Figure 2b).
The proteins according to the invention are epididymis-specific (see Figure 3), i.e. they are expressed primarily in the epididymis.
All naturally occurring allelic variants and isoforms of the proteins according to the invention are referred to as derivatives within the meaning of this invention, the various forms being able to differ from one another in each case as regards sequence length and also in respect of deletions, substitutions, insertions, replacements or additions of amino acids. In particular, ali naturally occurring variations of the proteins according to the invention are included in which there are differences at the N-terminal.
Within the meaning of the present invention proteins which are derivded from species other than humans are referred to as homologues.
The term ~~proteins or polypeptides having the same antigenicity~~ describes molecules according to the invention which react with the same antibodies.
Compared with the amino acid sequence given in SEQ ID NO: 2, the derivatives according to the invention preferably have a homology of at least 65%, particularly preferably of at least 75o and especially preferred of at least BO%.
According to a preferred embodiment of the invention, the derivatives, isoforms or homologues according to the invention have the same biological activity as the protein with the amino acid sequence given in SEQ ID NO: 2.
According to another preferred embodiment of the invention, the homologues according to the invention are derived from a species selected from humans, primates, dog, horse, bovines, pig.
According to a particularly preferred embodiment, the homologues according to the invention comprise one of the sequences represented in SEQ ID NO: 4, 33 and 34.
The_amino acid sequence shown in SEQ ID NO: 2 represents the amino acid sequence of human HE12, while the amino acid sequences shown in SEQ ID NO: 4 represent the amino acid sequence CEl2a from the dog. The amino acid sequences shown in SEQ ID NO: 33 and 34 are a section cut from the amino acid sequence CEl2b tSEQ ID NO: 33) and CEl2c (SEQ ID NO: ' 3 4 ) f rom the dog .
A subject-matter of the invention relates in particular to derivatives of the proteins according to the invention which comprise at least one fibronectin type II module. The deriv-atives preferably comprise four fibronectin type II modules. No proteins which have four fibronection type II modules have previously been known in the state of the art. An increased number of fibronectin type II modules is expected to lead to a more pronounced binding with the binding partners (see above).
The invention further relates to fragments of the proteins and derivatives according to the invention which have the same biological actuvity and/or antigenicity as the proteins or derivatives according to the invention themselves.
The invention furthermore relates to DNA sequences which code for the proteins, derivatives, isoforms, homologues or fragments according to the invention. These DNA sequences according to the invention are selected in particular from a) the nucleotide sequences given in SEQ ID NO: 1 or SEQ ID NO:
3, 5 and 12, b) sequences homologous to the aforementioned sequences with a degree of homology of at least 55%, preferably 65%, especially preferred 80%, and c) syngenic or complementary sequences of the sequences according to a) and b), or fragments of same, if the fragments code for proteins or polypeptides with the same biological activity and/or antigenicity.
The nucleic acid sequence shown in SEQ ID NO: 1 represents the HE12 sequence from a human, the nucleic acid sequence shown in SEQ ID NO: 3 represents the CEl2a sequence from the dog, _g_ the nucleic acid sequence shown in SEQ ID NO: 5 is the CEl2b sequence from the dog, while the nucleic acid sequence shown in SEQ ID NO: 12 represents a section cut from the coding sequence for the homologue in the horse.
According to the invention the term "syngenic sequence" covers all sequences which were derived from the same or a homologous gene and code for the proteins, derivatives and fragments according to the invention or can be used for the preparation of probes. The term also covers in particular sequences having deviations caused by the degeneration of the genetic code. In particular the term "syngenic sequence" also covers those sequences in which, although the coding region agrees wholly or partly with the coding regions of the sequences represented in SEQ ID NO: 1, 3, 5 and 32, the 5'- and 3'-untranslated regions are different. Further, the term "syngenic sequence" also covers those nucleotide sequences in which the region coding for the mature protein fully or partially complies with the coding regions of the sequences shown in SEQ ID NO: 1, 3, 5 and 12, however in which the regions coding for the signal sequences are different.
According to a preferred embodiment, the nucleic acids according to the invention comprise one of the nucleotide sequences represented in SEQ ID NO: 6, 7, 8, 9, 10 and 11.
The present invention furthermore relates to fragments of the DNA sequences according to the invention which have at least a length of 40 nucleic acids, preferably of 100 nucleic acids, and which in particular are suitable as probes.
The DNA sequences according to the invention can be obtained by searching gene banks comprising epididymis tissue with probes, the probes being fragments of the proteins represented in SEQ
ID NO: 1, 3 and 5. Such procedures are known to a person _g_ skilled in the art {Sambrook et al., 19895. The measurement of the degree of homology is also known to a person skilled in the art (Altschul, et al., 1997).
The present invention also relates to a vector molecule which comprises one or more of the DNA sequences according to the invention. According to a preferred embodiment, the vector molecule according to the invention comprises the DNA sequences according to the invention inserted in a way suitable for expression in suitable host organisms.
Vectors which are suitable within the framework of the present invention to receive the DNA sequences according to the invention include in particular all plasmid and viral vectors that are suitable for the transformation of bacteria, yeasts, insect and mammal cells.
The invention further covers microorganisms which have the registration number DSM 13473 or DSM 13474. These microorganisms have been registered at the German Micro-organisms and Cell Cultures Collection, Mascheroder Weg 1b, D-38124 Brunswick. The microorganism with the registration number DSM 13474 contains a plasmid with a DNA sequence with the nucleotides 156-827 from SEQ ID NO: 1 and thus the complete open reading frame of human HE32-DNA. The microorganism with the registration number DSM 13473 contains a plasmid with a DNA
sequence with the nucleotides 162-917 and thus the complete reading frame for the mature CE12 protein and also for part of the signal sequence.
All prokaryotic or eukaryotic host cells transformed with the vectors according to the invention are a further subject-matter of the invention. Suitable host cells are for example E. coli, Pichia pastoris, Saccharomyces cerevisiae, Sf9, mammal cell lines, primary cell cultures. CHO, BHK or COS cells for example can be used as mammal cell line.
A method of preparing the proteins, derivatives or fragments according to the invention, wherein host cells according to the invention are cultured under conditions which allow the expression of the DNA sequence, and the expression product is obtained from the culture mixture are a further subject of the invention. The conditions to be chosen depend on the vectors and host cells used in each case and are known to a person skilled in the art (Sambrook et al.).
Furthermore, the proteins, derivatives, isafarms, homologues, fragments or DNA sequences according to the invention can also be chemically synthesized by known methods.
The invention also relates to antibodies which are suitable to react in an immunoreaction with the proteins, derivatives, homologues or fragments according to the invention. According to preferred embodiments these antibodies are polyclonal or monoclonal antibodies. The antibodies according to the invention can be prepared with the help of the high-purity proteins according to the invention in a way known to a person skilled in the art. Such antibodies can be prepared on the basis of the complete protein and on the basis of fragments and active derivatives of same if these possess the same antigenicity icf. example 5).
The antibodies can be labelled and serve to detect the proteins, derivatives, homologues and fragments according to the invention in vitro or in viva.
Pharmaceutical compositions which comprise the proteins, derivatives, homologues, fragments, DNA sequences or antibodies according to the invention as active ingredient are a further subject of the present invention. Alongside the active active ingredient, such pharmaceutical compositions regularly further comprise physiologically compatible carriers which are known to a person skilled in the art.
The use of the proteins, derivatives, homologues, fragments, DNA sequences and antibodies according to the invention for the preparation of pharmaceutical compositions for the diagnosis of male reproductive disorders, for the treatment of male reproductive disorders, in particular infertility, and for the preparation of a medicament for contraception is a further subject of the invention.
According to a preferred embodiment, the complementary DNA
sequences according to the invention are used as antisense DNA
molecules for the preparation of the aforementioned pharmaceutical compositions, the antisense molecules being suitable for the suppression of the preparation of the DNA
sequences according to the invention in vivo.
The pharmaceutical compositions according to the invention are preferably used for the modulation of the heparin and apolipoprotein A-I bonds in the epididymis. Their use for the modulation of the capacitation of spermatozoa is especially preferred.
A novel family of epididymis-specific proteins is thus made available with the present invention, which are characterized by the presence of heparin-binding sites. The proteins, DNA
sequences and antibodies according to the invention can be used as a reversible male contraceptive which has a post-testicular action and does not affect the hormonal balance. Such a contraceptive is not known in the state of the art.
The invention is described further in the following by means of figures and examples.
Examples Example 1 Cloning of novel epididymis~specific cDNAs A dog epididymis cDNA library (Ellerbrock et al., 1994) was searched using the subtraction-hybridization strategy described in Kirchhoff et al. (1990). Roughly 10,000 independent cDNA
clones were plated in low density (ca. 500 pfu (plaque forming units) per plate) and searched by means of a mufti-step procedure. 32P-labelled single-strand cDNA pools from dog epididymis were used as probes. 32P-labelled single-strand cDNA
pools from dog liver, testis and lung were used as negative controls. Epididymis-specific plaques were purified by means of gradual dilution, and the incorporated cDNA was obtained by in vivo excision, as recently described (Ellerbrack et al., 1994).
The recombinant plasmid DNAs obtained were amplified and purified, and the cDNA clones were sequenced by means of standard procedures (Sambrook et al., 1989}. This procedure led to the identification of a novel cDNA clone family in the dog, which was named CE12. Two variants with differences in the open reading frame, CEl2a and CEl2b (Figure la to c) were found. Both contained four fibronectin type II (Fn2) modules arranged as a tandem. These modules were similar, but not homologous vis-a-vis known Fn2 modules of other seminal plasma proteins (Figure 2b).
A roughly 400 by cDNA fragment which was common to CEl2a and b was used to search a human epididymis cDNA library (Kirchhoff et al., 1993). The same procedure would be possible with a 750 by PCR product which can be obtained with the following primers: 5'GATCTGTTTACTTCATCTTG3' (SEQ ID NO: 19) and 5'CCTTTATTGGTGGTTATGCC3' (SEQ ID NO: 20). This happened with procedures known to a person skilled in the art (Sambrook et al., 1989). A number of cross-hybridizing clones (0.030 of the library) were isolated, the longest cDNA inserts were sequenced in both directions and the sequences compared. The HE12 cDNA
sequence obtained was almost 80o identical to the CEl2b cDNA.
As neither the CE12 nor the HE12 cDNAs were complete at the 5' end, an inverse PCR technique was carried out in order to obtain the sequence information that was still missing. To this end, a modified technique was used which had recently been described by Gebhardt et al. (1999}. Human and dog epididymis cDNAs which had been prepared from epididymis RNA extracts of other individuals were used. Antisense primers whose sequence was located in the known sequences of the partial cDNA clones were used for the reverse transcription (CE12 cDNA synthesis primer: 5'GTTGTTTTCATCCTCCATGC3' (SEQ ID NO: 13}; HE12 cDNA
synthesis primer: 5' TCCATGCAATCAGAGACCAC3' (SEQ ID NO: 14)).
ug whole epididymis RNA were used. 8uperscript~ Reverse Transcriptase (Gibco-BRL, Karlsruhe, Germany) was used as enzyme. The synthesis of the second strand was carried out with E. coli DNA polymerase after the incorporation of strand breaks with RNase H (both enzymes from Stratagene, Heidelberg, Germany). Smooth ends were prepared with T4 DNA polymerase (Biolabs, Schwalbach/Taunus, Germany} and ligated by means of T4 DNA ligase (Boehringer, Mannheim, Germany), in order to concatemerize and/or circularize the DNA. The inverse PCR was carried out using the following sense primers (CE12:
5'GTGAAACCAATGAGTATGG3' (SEQ ID NO: 15}; HE12: 5'GTGGTGCTCAGT-CACCTCTG3' (SEQ ID NO: 16)) and the corresponding antisense primers: (CE12: 5'GGTAATCGTCTGCCATGC3' (SEQ ID NO: 17); HE12:
5'CGTGGGTAATCTTCACTCTG3' (SEQ ID NO: 18)).
In this way, six independent CE12 and four independent HE12 coding PCR products were subcloned and sequenced. All the HE12 PCR products were essentially co-linear and ended almost at the same 5' site. Combining the sequences of the HE12 cDNA seq-uences obtained from the library and those of the PCR fragments produced an HE12 cDNA clone that was 1025 nucleotides long.
The HE12 clone displays an open reading frame (ORF) of 223 codons with an ATG start codon at nucleotides 156-158 and a TGA
stop codon at nucleotides 825-827 (Figure 2a). The ORF is followed by a 198-nucleotide-long 3' untranslated region which contains an AATAAA polyadenylation signal at nucleotides 1010-1015. The ORF produces a novel human epididymal protein. This comprises an N-terminal signal peptide (amino acid 1-23) which is typical of secretory proteins and which is immediately followed by four Fn2 units arranged in tandem (Figure 2b).
In contrast to this, different CE12 RT PCR fragments formed which resulted from an mRNA length polymorphism. These fragments had different 5' UTRs and extensive deletions or insertions even within their ORFs, which leads to at least two different CE12 proteins (Figure 1b, c). If the obtained sequences are combined, a CEl2a cDNA clone with 1343 nucleotides results iFigure la). The ORF in this long cDNA
variant starts at nucleotide 129 and contains two methionines in the reading frame at positions -39 and -23, the second being homologous to the position of the start methionine of the HE12 cDNA sequence in Figure 2a. Only if this methionine is assumed as start codon can a clear signal peptide sequence be read out from the sequence. In addition, CE12 cDNA clones were obtained which display different 5' UTRs, in which there was only one, namely the second ATG start codon. It is therefore assumed that each of these CE12 sequences have different promoters.
A further polymorphism in the CE12 clones was discovered immed-iately after the assumed signal peptide sequence, which prob-bably led to different N terminals in the CE12 clones. The CEl2a and b clones differ by virtue of 22 codons which are either present in the cDNAs or absent and for which there is no counterpart in the HE12 cDNA sequence (Figure 2). The tv~ro variants in CE12 cDNAs occur with a frequency of 1 _ 3. The N-terminal as it was derived for CEl2a contains a consensus sequence for an N-glycosylation site at asparagine +4 (Asn +4), this site being missing from CEl2b (Figure lc). The amino acids which result from the immediately following codon can be either D or N (Figure lc). The occurrence of smaller, roughly 1 kb CE12 mRNAs in the Northern blot (see below) make it possible that there are additional length polymorphisms also in the 3' untranslated region, although this was not confirmed by the found cDNA clones. However, the 3' UTR of the CE12 mRNA appears to be clearly longer than that of the HE12 mRNA and contains two possible polyadenylation signals, of which only the first is homologous to the human counterpart. On the other hand, the sequences which code for the Fn2 units are identical in all CE12 cDNA variants and also highly homologous to corresponding regions of the HE12 cDNA (roughly 85o sequence identity) (Figure 2b). A comparison of the predicted CE12 and HE12 protein sequences with those of the BSP-A1/PDC-109 and BSP-A3 seminal plasma proteins (accession numbers P02784 and P04557) showed a sequence identity of below 550. This also rules out any cross-hybridization with- the BSPs at nucleic acid level under the stringency conditions used in this study.
Example 2 Analysis of the structure of the epididymal fibronectin type II domain proteins at cDNA level Since proteins which contain four Fn2 units arranged in a tandem were previously unknown, PCR analyses were carried out in order to rule out the possibility that cloning artefacts occurred during the preparation of the libraries.
Oligonucleotide primer pairs were used in the RT-PCR analysis.
The RT-PCR was carried out using standard methods. The following primary pairs were used for the amplification of CE12, HE12 and EQ12 cDNA fragments from 1 ~g of foetal epididymal RNA:
CE12: 750 by product; primers used 5'GATCTGTTTACTTCATCTTG3' (SEQ ID NO: 19} and 5'CCTTTATTGGTGGTTATGCC3' (SEQ ID NO: 20);
HE12: 837 by product; primers used 5'CAACCTTCCTTCTCTATTCC3' (SEQ ID NO: 21} and 5'TCAAGACTCCTTTATTGGTG3' (SEQ ID NO: 22);
EQ12: 752 by product; primers used 5'GGGTCTTCTTACTTCTCTTG3' (SEQ ID NO: 23) and 5'TCCTTTATTGGTGGTTATGCAC3' (SEQ ID NO: 24}.
Each of the products contained those cDNA regions which code for four Fn2 units. As shown, human and dog epididymal cDNAs were used for the PCR amplification. As an additional control, in order to rule out any cDNA probes contamination, a corresponding primer set was also used in the case of the epididymal horse cDNA preparation. In every case an amplified sequence of roughly 800 by was obtained. Subcloning and sequencing confirmed the occurrence of epididymal mRNAs which code for four highly preserved Fn2 units arranged in tandem.
Such proteins occurred not only in humans and dogs but also in horses (see Figure 2c}. During the RT-PCR analyses of the human epididymal cDNA, shorter PCR fragments were also observed, but in much smaller quantities. Subcloning and sequencing of these fragments showed that they originated in variants which contained fewer than four Fn2 motives. These variants most probably contain three or one Fn2 modules, the variant with only one Fn2 module occurring in much smaller quantities.
Corresponding products were not found in dogs and horses.
Example 3 Investigation of the tissue distribution of CE12 and HE12 mRNAs by northern blot analysis Northern blot analyses were carried out with whole RNA extracts of different dog and human tissues in order to investigate the distribution of the CE12 and HE12 mRNAs (Figures 3 and 4). RNA
from different tissues was extracted in 15 to 20 volumes of a chaotropic solution as described in Pera et al. (1996). 5 to ~g of the total RNA/column, depending on the type of experiment, were separated by means of denaturing agarose gel electrophoresis and transferred onto Amersham-Buckler, Brunswick (Germany) nylon membranes. Equal quantities of RNA
were assured by ethidium bromide staining before the transfer.
Digoxigenin (DIG)-labelled cDNA probes were prepared by PCR
amplification of purified fragments according to the instruct-ions provided by the manufacturer (Boehringer, Mannheim, Germany). The sequences of the primers were as follows:
CE12, 404 by product; primers used 5'CTCTCCTTGGTGTGCAACC3' (SEQ
ID NO: 25) and 5'AAGTGGCAGGGAAAGCCAG3' (SEQ ID NO: 26);
HE12, 400 by product; primers used 5'CAACCTTCCTTCTCTATTCC3' (SEQ ID NO: 27) and 5'TCCATGCAATCAGAGACCAC3' (SEQ ID NO: 28);
CE1, 730 by product; primers used 5'AAGCAGCAGAGCTGGAG3' (SEQ ID
NO: 29) and 5'AATGTCACCTTCACCAG3' (SEQ ID NO: 30);
CE4, 370 by product; primers used 5'ACGCAGGAGTGCGTCTCGGA3' (SEQ
ID NO: 31) and 5'AGCGGTCACTTTATTGGTTG3' (SEQ ID NO: 32).
The PCR reactions were carried out as described (Pera. et al.
1996). The hybridization with the non-radioactive probes was carried out using the DTG labelling kit (Boehringer, Mannheim) as recently described (Pera et al., 1996).
It was shown that in both species (human and dog) the mRNAs were present only in the epididymis, but not in the other tissues investigated, _the tissues that were investigated including testis, seminal vesicles and prostate (Figures 3 and 4). A single hybridizing band of roughly 1.1 kb was found in human epididymal extracts, whereas at least two or more hybrid-izing bands were present in dog epididymal RNA preparations.
The bands in the dog mRNA occurred in a wide range from roughly 1.0 to 1.5 kb, which supports the assumption that in the dog a length polymorphism occurs in the case of CE12 mRNA.
The expression pattern of the human and dog mRNAs was investigated along the length of the epididymis. HE12 mRNA was found in all regions of the human epididymis, although a higher content was found in the more proximal parts of the organ (Figure 4). In the dog epididymis CE4 mRNA, which is known to be present in all regions of the organ (Beiglbock et al., 1998;
Gebhardt et al., 1999), served as internal control (Figure 3).
The regionalization of the CE12 expression was clearer in dogs than in humans, and all the CE12-hybridizing mRNAs showed the same pattern with a maximum expression in the corpus region.
Under unilateral-cryptorchidic conditions, in which one testis plus epididymis remains in the abdomen, normal quantities and expression patterns of the mRNA were observed in the scrotal epididymis, which served as control. A reduction in the quantities of CE12 mRNA was found in the abdominal organs. If the different regions of the dog epididymis are considered, the reduction was at its clearest in the caput region, while the quantities of CE12 mRNA in the corpus appeared not to be affected (Figure 3}.
A hybridization under low stringency conditions using hetero-logous DIG-labelled CE12 cDNA as probe showed that cross-hybridizing mRNAs are present in the epididymis of other kinds of mammal including humans, bovines, horses, pigs and apes (Macaca mullata) (Figure 5a, b). Although not investigated in detail, the largest quantities in the pig parorchis were found in the caput region, which is an exception compared with the other mammals. No cross-hybridization with either CE12 or HE12 cDNA probes was found with RNA extracts from rodents, rabbits and guinea pig epididymes.
Example 4 Localization of C812 mRNAs by in situ hybridization The epididymes of two dogs of average size were incubated for 6 hours in Bouin's solution, washed in 70a ethanol and embedded in paraffin wax. 5- to 7-~m sections were prepared and subjected to a non-radioactive hybridization procedure as described (Beiglbock et al., 1998; Gebhardt et al., 1999), using in vitro labelled sense and antisense cRNA probes of sub-cloned Accl/XbaI-digested CE12 cDNA fragments which comprised 404 by of the open reading frame (nucleotides 295-&69, cf.
Figure 1). The labelling was carried out in the presence of dioxigenin UTP (Boehringer-Mannheim). The detection was carried out as suggested by the manufacturer. Alternative methods are known to a person skilled in the art (Kirchhoff et aI. 1991;
Pera et al. 1994; otherwise: see above). Negative control checks were carried out in_ parallel at adjacent sections, using the corresponding dioxigenin-labelled sense strand cRNAs. The sections were analysed by means of light microscopy.
CE12 transcripts were localized in tissue sections of the dog epididymis (see Figure 6a and b). The in situ hybridization showed a strong and highly specific marking of the epithelium of the epididymal duct, while the lumen of the duct which contains the spermatozoa and the tissue between the duct showed no signal. Within the epithelium, the signal was at its strongest in the basal region. In good agreement with the Northern blot results, the maximum coloration was found in the distal caput and in the corpus, whereas the proximal caput appeared to be unmarked and the distal parts of the organ were only weakly marked (Figures 7a-d).
Example 5 Preparation of antibodies A 20-acid oligopeptide (NFi3-SSFDENQQWKYCETNEYGGN-COOH) which had been derived from the CEl2a DNA sequence (amino acids 103-122, cf. Figure 1) was selected as immunogen. The preparation of the antibodies took place as recently described (Kirchhoff et al.
1996). After coupling to KLH (keyhole limpet hemocyanine) the conjugate was injected into hen's eggs, guinea pigs and rabbits. The animals were killed after 120 days' immunization, the immune sera were collected and stored at 4°C using thimorosal as preservative.
The peptide was selected on the basis of its predicted high antigenicity and minimal cross-reactivity with other proteins.
This peptide epitope also appears to be highly preserved between the species (Figure 2b).
Example 6 Protein preparation and Western blot analysis Crude protein extracts were prepared from powdered epididymal tissue, epididymal fluid or washed spermatazoa, and stored in aliquots of equal size at -80°C. Protein extracts of human spermatozoa membranes were prepared as recently described (Alexander and Bearwood, 1984). Probes of 5 to 10 ~l protein extract (depending on the protein concentration or spermatozoa count) were separated on 15a SDS polyacrylamide gels and transferred onto PVDF membranes (Amersham, Brunswick, Germany) using a discontinuous buffer system with a half-dry blotter (Phase, Lizbeck, Germany). Immunodetection was carried out by one-hour blocking of the membrane in to blocking solution (Boehringer-Mannheim), followed by incubation with a guinea pig antiserum (see above, dilution 1 . 5000). Antibody binding was established by a peroxidase-coupled goat-anti-guinea pig anti-body (Sigma, Deisenhofen, Germany). Alternative procedures are known to a person skilled in the art.
Protein extracts of dog epididymal tissue and also protein extracts of epididymal dog spermatozoon were positive for CE12 related antigens (Figure 8a-c). Multiple immuno-reactive bands with a molecular weight in the range from 30 to 35 kDa were identified in every protein probe with the exception of the protein extracts of the spermatozoa from the caput. Thick bands are likewise recorded in human epididymal fluid which was removed from the cauda region, and also in membrane protein preparations of human ejaculated sperm. These reactions were able to be subjected to competition from chemosynthetic peptides (Figure 8).
Example 7 Detection of the HE12 protein is the human parorchis Cryostat sections (8 ~,m) of the epididymis of prostatic carcinoma patients were prepared and incubated for 18 hours at 8~C with the C12-3 antiserum from rabbits. The antibodies with specificity for the HE12 protein were made visible by fluoro-chrome-conjugated antibodies (Cy2-immunofluorescence: Dianova, Amersham). Both antibodies were used in a dilution of 1 . 100.
Cross-sections through the parorchis passage in the distal region are represented_ as immunofluorescence photographs in Figure 9(a) to (i). (a) shows the cross-section through the parorchis passage in the distal region; (b) the comparable region of another patient tissue; (c) and (d) comparable regions of two antiandrogen-treated patients; (e) a negative control in which the parorchis was incubated with a pre-C12-3 serum (1 . 100 diluted).
Figure 10 shows the corresponding result in the case of the analysis of the epididymis of a healthy dog. Prior to incubat-ion with the antibody (designated "pre") a uniform coloration of the epididymis results (LU = lumen, EP = epithelium).
To summarize, it can be established that HE12 is primarily expressed in the epithelial cells and given off into the lumen.
Example 8 Detention of AE12 on human spermatozoa In order to show that HE12 binds to human spermatozoa, LIS
membrane extracts of the spermatozoa were subjected to a two-dimensional electrophoretic separation (2-DE), labelled by the HE12-specific antibodies and made visible. As Fig. 11(a) shows, six different isoforms can be detected by means of this procedure.
In order to obtain more detailed information regarding the nature of the bond, an attempt was made to remove, by washing, the HE12 proteins bound to spermatozoa. The solutions used in each case for the washing were separated via SDS-PAGE
and analysed using the Western blot system for the presence of the HE12. As shown in Fig. 11(b), the greatest part of the spermatozoa-binding protein fraction can be removed by 0.6~M
NaCI washing buffer (Sw.). However, even after this a smaller fraction remains bound on the spermatozoa membrane (Sp) and cannot be removed even by repeated washing (Wt).
Brief description of the Figures Figure 1 cDNA sequences and derived peptide sequences of CE12 variants. a) Sequence of the complete CEl2,a cDNA (top sequence) and predicted peptide sequence (bottom sequence). The arrow points to the probable separ-ation site of the signal peptide. The amino acid sequence, as derived from the first methionine in the reading frame, is shown in grey. The sequence which was used for synthesis of the oligopeptide is under-lined. Two polyadenylation signals are underlined in the 3'UTR. b) Sequences of the CE12 variants at the 5' end, established by inverse PCR (exclusively primers). The 5' regions of the two clones obtained from the library (CEl2a and b) and of the six clones originating in the PCR (Cinv 1 (SEQ ID NO: 6), 4 (SEQ
ID NO: 8), 6 (SEQ ID NO: 7), 8 (SEQ ID NO: 9), 11 (SEQ ID NO: 10), I2 (SEQ ID NO: 11) are compared with each other. c? Comparison of the amino acid sequences of the CE12 cDNA variants. 1-23: signal peptide sequence; 24-45: N-terminal extension of CEl2a. Roman numerals (I-IV) designate Fn2 modules (CEl2b: SEQ ID
NO: 33; CEl2c: SEQ ID NO: 34}.
Short lines show both deletions which were found in CEl2b and c variants and differences in the inter-stices between the Fn2 units. Dotted lines show identical amino acid sequences. The arrow indicates the different N-terminal amino acids.
Figure 2 cDNA and derived amino acid sequence of HE12 compared with homologous sequences from animals. a) Sequence of the complete HE12 cDNA and of the predicted pro-tein sequence. b) Comparison of the predicted human (HE12), dog (CE12), and horse (EQ12) Fn2 module proteins with the amino acid sequence at BSP-A1/A2=PDC-109 (accession number P02784).
Figure 3 Northern blot analyses of the tissue distribution of CE12 mRNAs, shown by non-reactive hybridization. a) Expression patterns of CE12 mRNAs in total RNA
extracts of different dog tissues using a DIG-labelled probe (top panel). Ly - lymph nodes; Mu -muscle; Di = diaphragm; Sp = spleen; Lu = lung; Li -liver; Ov - ovaries; Te - testicle; El - dog epididymis 1; E2 = dog epididymis 2; Du = oviduct; Ut - uterus. Equal loading (10 ~Cg/row} and the integrity of the ribosomal RNA (28S and 18S) was assured by ethidium bromide staining of the gel prior to the blotting (bottom panel). b) Expression patterns of CE12 (1.0 to 1.4 kb) and CE4 (0.7 kb) mRNAs in different regions of the dog epididymis and as a response to the cryptorchidic situation.
Figure 4 Northern blot analyses of the tissue distribution of HE12, shown by non-radioactive hybridization.
Expression of HE12 mRNA was investigated in total RNA
extracts of different human tissues and different regions of the human epididymis using a DIG-labelled HE12 probe (upper panels). Pr - prostate; Sv -seminal vesicles; Ep - epididymis; Te - testicle;
De - decidua; Ki - kidney; Th - thymus; Pi -pituitary gland; Ln - LnCap cells; Ca - caput epididymis; Co - corpus epididymis; Cu - cauda epididymis. Equal loading (10 ~.g/column) and tf~e integrity of .the ribosomal RNA (28S and 18S) was assured by ethidium bromide staining of the gel prior to the blotting (bottom panels).
Figure 5 Northern blot analysis for the determination of the cross-hybridization of CE12 and HE12 cDNA probes between different species. a) Whole extracts of epididymal RNA of nine different species were hybridized with a DIG-labelled CE12 probe under non-stringent conditions. Column I: human; column 2:
stallion; column 3: bovines; column 4: boar; column 5: rat; column 6: mouse; column 7: rabbit; column 8:
guinea pig (degraded?); column 9: dog. b) Columns 1-3: stallion caput, corpus and cauda epididymis;
columns 4-5: boar caput, corpus and cauda epididymis;
column 6: bull; column 7: guinea pig (degraded);
columns 8-10: caput, corpus and cauda epididymis of Macaca mullata (hybridized with HE12 cDNA probe).
Equal loading (10 ug/column) and the integrity of the ribosomal RNA (28S and 18S) was assured by ethidium bromide staining of the gel prior to the blotting (lower panels).
Figure 6 Localization of CE12 transcripts in the ductus epithelium of the dog corpus epididymis by non-radioactive in situ hybridization using DIG-labelled cRNA probes. a) mRNA which hybridizes with CE12 antisense cRNA was detected in tissue sections using an alkaline phosphatase-conjugated anti-DIG antibody in normal bright-light microscopy. b) Adjacent sect-ions which were hybridized with DIG-labelled CE12 sense control cRNA were negative. The bar represents 50 ~.m.
Figure 7 Region-dependent differences in the CE12 transcript concentration in the dog epididymis, shown by in situ hybridization using DIG-labelled CE12 antisense cRNA.
a) Proximal caput epididymis; b) proximal corpus epididymis; c) middle to distal corpus epididymis; d) cauda epididymis. The bar represents 100 ~Cm.
Figure 8 Western blot analysis of antigens related to CE12 using an antipeptide antiserum of guinea pigs (dilution 1 . 500). a) Multiple, immunostained bands were observed between 30 and 35 kDa in crude protein extracts of dog epididymis, which did not react with pre-immunoserum. The staining was subjected to competition from CE12 peptides (20 ~g/ml). P - pre-immunoserum; T= test antiserum; C - competition experiment. A broad immunoreactive band of roughly the same size was observed in the cauda of the dog, but not in epididymal sperm protein extracts of the caput epididymis. Ca - caput sperm extract; Cu -sperm extract. b) A broad band of the same size was also observed in protein extracts from human caudal epididymal fluid, but not of the corpus, and in LIS
extracts of washed human ejaculated sperm. This immunopositive probe was subjected to competition from the chemosynthetic CE12 oligopeptide. Co = fluid from epididymal corpus; Cu - fluid from epididymal cauda; Sp - sperm, no competition; C - competition experiment.
Figure 9 Immunofluorescence detection of the HE12 in the parorchis of patients with prostatic carcinoma; (a) cross-section through the parorchis passage in the distal region; (b) comparable region of another patient tissue; (c) and (d) comparable regions of two antiandrogen-treated patients; (e) negative control, parorchis incubated with a pre-C12-3 serum (diluted 1 100) .
Figure 10 Immunofluorescence detection of the HE12 in the parorchis of a healthy dog; prior to incubation with the antibody ("pre"), LU = lumen; EP = epithelium.
Figure 11 (a): HE12 bound to spermatozoa after two-dimensional electrophoretic separation; (b) SDS-PAGE and Western blot of the washing solutions; 0.6 M NaCl washing buffer Sw; spermatozoa membrane Sp; repeated washing Wt.
References 1. Alexander NJ, Bearwood D. An immunosorption assay for antibodies to spermatozoa: comparison with agglutination and immobilization tests. Fertil Steril 1984; 41: 270-276.
2. Altschul, SF et al. (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs, Nucleic Acids Research 25: 3389-3402.
3. Banyai, L, Tordai H, Patthy L. Structure and domain-domain interactions of the gelatin binding site of human 72-kilodalton type IV collagenase (gelatinase A, matrix metalloproteinase 2). J Biol Chem 1996; 271: 12003-12008.
4. Beiglbock A, Pera I, Ellerbrock K, Kirchhoff C. Canine epididymis-specific mRNA encoding secretory glutathione peroxidase-like protein. J Reprod Fertil 1998; 112: 357-367.
5. Chandonnet L, Roberts KD, Chapdelaine A, Manjunath P.
Identification of heparin-binding proteins in bovine seminal plasma. Mol Reprod Dev 1990; 26: 313-318.
6. Constantine KL, Madrid M, Banyai L, Trexler M, Patchy L, Llinas M. Refined solution structure and ligand-binding properties of PDC-109 domain b. A collagen-binding type II
domain. J Mol Biol 1992; 223: 281-298.
domain. J Mol Biol 1992; 223: 281-298.
7. Desnoyers L, Manjunath P. Major proteins of bovine seminal plasma exhibit novel interactions with phospholipid. J
Biol Chem 1992; 267: 149-155.
Biol Chem 1992; 267: 149-155.
8. Ellerbrock K, Pera I, Hartung S, Ivell R. Gene expression in the dog epididymis: a model for human epididymal function. Int J Androl 1994; 17: 314-323.
9. Gebhardt K, Ellerbrock K, Pera I, Ivell R, Kirchhoff, C.
Differential expression of novel abundant and highly regionalized mRNAs of the canine epididymis. J Reprod Fertil 1999; 116: 391-402.
Differential expression of novel abundant and highly regionalized mRNAs of the canine epididymis. J Reprod Fertil 1999; 116: 391-402.
10. Kirchhoff C, Krull N, Pera I and Ivell R. A major mRNA of the human epididymal principal cells, HE5, encodes the leucocyte differentiation CDw52 antigen peptide backbone_ Mol Regrod Dev 1993; 34: 8-15.
11. Kirchhoff C. Molecular characterization of epididymal proteins. Reviews of Reproduction 1998; 3: 86-95.
12. Manjunath P, Marcel YL, Uma J, Seidah NG, Chretien M, Chapdelaine A. Apolipoprotein A-I binds to a family of bovine seminal plasma proteins. J Biol Chem 1989; 264:
16853-16857.
16853-16857.
13. Muller P, Erlemann KR, Miiller K, Calvete JJ, Topfer-Petersen E, Marienfeld K, Herrmann A. Biophysical characterization of the interaction of bovine seminal plasma protein PDC-109 with phospholipid vesicles. Eur Biophys 1998; 27: 33-41.
14. Parrish JJ, Susko-Parrish J, Winer MA, First NL.
Capacitation of bovine sperm by heparin. Biol Reprod 1988;
38: 1171-1180.
Capacitation of bovine sperm by heparin. Biol Reprod 1988;
38: 1171-1180.
15. Pera I, Ivell R, Kirchhoff C. Regional variation of gene expression in the dog epididymis as revealed by in situ transcript hybridization. Int J Androl 1994; 17: 324-330.
16. Sambrook et al., Molecular Cloning 2nd edition, Cold Spring Harbor Laboratory Press, 1989.
17. Therien I, Moreau R, Manjunath P. Bovine seminal plasma phospholipid-binding proteins stimulate phospholipid efflux from epididymal sperm. Biol Reprod 1999; 61: 590-598.
18. Therien I, Bleau G, Manjunath P. Phosphatidylcholine-binding proteins of bovine seminal plasma modulate capacitation of spermatozoa by heparin. Biol Reprod 1995;
52: 1372-1379.
52: 1372-1379.
19. Topfer-Petersen E, Calvete JJ, Sanz L, Sinowatz F.
Carbohydrate- and heparin-binding proteins in mammalian fertilization. Andrologia 1995; 2?: 303-324.
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Ser ~sn L . ~ A iyr ~ Sn i:ly i.3'SYh l:yS
lc Lt2~ i;In '"2'p 3~ ~a
Carbohydrate- and heparin-binding proteins in mammalian fertilization. Andrologia 1995; 2?: 303-324.
v?-In> '1 V
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Ser ~sn L . ~ A iyr ~ Sn i:ly i.3'SYh l:yS
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Claims (17)
1. Protein, which is a derivative, an isoform or a homologue of a protein comprising the amino acid sequence shown in SEQ ID NO: 2 of the sequence listing, wherein the protein a) comprises the same epididymis specificity and antigenicity and b) has a homology of at least 55% compared with the amino acid sequence given in SEQ ID NO 2.
2. Epididymis-specific protein according to claim 1, characterized in that it comprises the amino acid sequence shown in SEQ ID NO:2.
3. Protein according to claim 1 or 2, characterized in that it is derived from a species of the group consisting of dog, horse, pig, primates and bovines.
4. Protein according to claim 3, characterized in that it has the sequence shown in SEQ ID NOs: 4, 33 or 34.
5. Protein according to claims 1 to 4, characterized in that it comprises at least one fibronectin type II module.
6. Protein according to claim 5, characterized in that it comprises four fibronectin type II modules.
7. Protein, characterized in that it is a fragment of the proteins according to claims 1 to 6 and has the same biological activity and/or antigenicity.
8. DNA sequence, characterized in that it codes for a protein according to claims 1 to 7.
9. DNA sequence according to claim 8, selected from a) the nucleotide sequence shown in SEQ ID NO 1 or SEQ
ID NO 3, 5 or 12, b) sequences homologous to the afore-mentioned sequences with a degree of homology of at least 55%, and c) syngenic or complementary sequences of the sequences according to a) or b), or fragments of same, which code for proteins or polypeptides having the same biological activity and/or antigenicity.
ID NO 3, 5 or 12, b) sequences homologous to the afore-mentioned sequences with a degree of homology of at least 55%, and c) syngenic or complementary sequences of the sequences according to a) or b), or fragments of same, which code for proteins or polypeptides having the same biological activity and/or antigenicity.
10. DNA sequence according to claim 8 or 9, characterized in that it comprises one of the sequences shown in SEQ ID NO:
6 to 11.
6 to 11.
11. Vector comprising one or more of the DNA sequences according to claims 8 to 10.
12. Vector according to claim 11, characterized. in that it comprises the sequences inserted so that their expression can take place in suitable host organisms.
13. Host cell, characterized in that it is transformed with a vector according to claim 11 or 12.
14. Host cell according to claim 13, characterized in that it is a prokaryotic or eukaryotic host cell.
15. Method of preparing a protein according to one of claims 1 to 7, comprising culturing of host cells according to claim 13 or 14 under conditions which allow the expression of the DNA sequence, and optionally isolating the expression product from the culture mixture.
16. Pharmaceutical composition, characterized in that it comprises one or more of the proteins according to claims 1 to 7 or of the DNA sequences according to claims 8 to 10 as active ingredient.
17. Use of the proteins according to claims 1 to 7, of the DNA
sequences according to claims 8 to 10 for the diagnosis of male reproductive disorders, for the treatment of male reproductive disorders or for contraception.
sequences according to claims 8 to 10 for the diagnosis of male reproductive disorders, for the treatment of male reproductive disorders or for contraception.
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DE10028376A DE10028376A1 (en) | 2000-06-08 | 2000-06-08 | Epididymis-specific proteins with fibronectin type II modules |
| DE10028376.4 | 2000-06-08 | ||
| PCT/EP2001/006554 WO2001094387A2 (en) | 2000-06-08 | 2001-06-08 | Epididymis-specific proteins with fibronectin type ii modules |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2411017A1 true CA2411017A1 (en) | 2001-12-13 |
Family
ID=7645110
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002411017A Abandoned CA2411017A1 (en) | 2000-06-08 | 2001-06-08 | Epididymis-specific proteins with fibronectin type ii modules |
Country Status (5)
| Country | Link |
|---|---|
| EP (1) | EP1290015A2 (en) |
| AU (1) | AU2001277505A1 (en) |
| CA (1) | CA2411017A1 (en) |
| DE (1) | DE10028376A1 (en) |
| WO (1) | WO2001094387A2 (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2010025548A1 (en) * | 2008-09-03 | 2010-03-11 | UNIVERSITé LAVAL | Sperm fertility biomarkers and uses thereof |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE4002981C2 (en) * | 1990-02-01 | 1998-03-12 | Ihf Inst Fuer Hormon Und Fortp | Human, epididymis-specific polypeptides and their use for the therapy and diagnosis of male infertility |
-
2000
- 2000-06-08 DE DE10028376A patent/DE10028376A1/en not_active Ceased
-
2001
- 2001-06-08 WO PCT/EP2001/006554 patent/WO2001094387A2/en not_active Application Discontinuation
- 2001-06-08 AU AU2001277505A patent/AU2001277505A1/en not_active Abandoned
- 2001-06-08 EP EP01955303A patent/EP1290015A2/en not_active Withdrawn
- 2001-06-08 CA CA002411017A patent/CA2411017A1/en not_active Abandoned
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2010025548A1 (en) * | 2008-09-03 | 2010-03-11 | UNIVERSITé LAVAL | Sperm fertility biomarkers and uses thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| WO2001094387A2 (en) | 2001-12-13 |
| WO2001094387A3 (en) | 2002-04-25 |
| DE10028376A1 (en) | 2002-03-28 |
| AU2001277505A1 (en) | 2001-12-17 |
| EP1290015A2 (en) | 2003-03-12 |
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